Mood, Fatigue, Insomia A New Model of Diagnosis and...

78
Mood, Insomnia, Fatigue A New Model of Diagnosis and Treatment Decker Weiss, NMD, FASA Naturopathic Cardiologist Scottsdale Heart Institute Senior Fellow: Artis Research PTSD and Radicalization Researcher, Jordan, Syria, Iraq, Gaza, Israel Financial Disclosure: Nothing to Disclose

Transcript of Mood, Fatigue, Insomia A New Model of Diagnosis and...

Mood Insomnia Fatigue

A New Model of Diagnosisand Treatment

Decker Weiss NMD FASA

Naturopathic Cardiologist

Scottsdale Heart Institute

Senior Fellow Artis Research

PTSD and Radicalization Researcher Jordan Syria Iraq Gaza Israel

Financial Disclosure Nothing to Disclose

Mood

Depression in

America

1 in 10

The diagnosis is INCREASING 20 per year

80 of depressed persons do not seek treatment

Depression varies by state Oklahoma Mississippi Louisiana Alabama W Virginia are amongst the highest

Healthline InfographicPosted as of 29Feb2016 httpwwwhealthlinecomhealthdepressionstatistics-infographic

Depression and Women

ldquoCompared to men women are twice as

likely to develop depressionrdquo

Psychology Today The 7 Reasons Why Depression is More Common in Women Compared to men women are twice as likely to develop depression

Published on May 17 2012 by Neel Burton MD in Hide and Seek

httpwwwpsychologytodaycombloghide-and-seek201205the-7-reasons-why-depression-is-more-common-in-women as reported 30 Sept 2014

Depression in Teens

The Old Model

Educated link to a diagnoses educated

guess to a treatment than repeat

Common Interventions for Depression

Reuptake inhibitors Serotonin

Sertraline

Citalopram

Escitalopram

Reuptake inhibitors

Norepinephrine Dopamine

Bupropion

Venlafaxine

Cyclics

Trazodone

Mirtazapine

Atypical

Ariprazole

Olanzapine

Quetiapine

7

Psychiatry Failure in the Press

The latest edition of psychiatric diagnostic Bible the Diagnostic and Statistical Manual of Mental

Disorders (DSM) shows that US psychiatry has no intention of changing course

Edward Shorter- University of Toronto- WSJ

Waiting on a

Breakthrough

ldquoWe are facing a crisisrdquo the Cornell psychiatrist and New York Times contributor Richard Friedman warned last week In the past few years one pharmaceutical giant after anothermdashGlaxoSmithKline AstraZeneca Novartis Pfizer Merck Sanofimdashhas shrunk or shuttered its neuroscience research facilities

Clinical trials have been halted lines of research abandoned and the new drug pipeline has been allowed to run dry

The New Yorker Posted as of 22Feb 2016

SEPTEMBER 3 2013The Psychiatric Drug Crisis

BY GARY GREENBERG

Fatigue

Fatigue Fatigue is a common complaint with a

prevalence in population-based surveys in Britain

and the United States of between 60 and 75

percent

A cross-sectional survey of United States workers

found the two-week period prevalence of fatigue

to be 38 percent with an estimated annual cost

to employers exceeding $136 billion in lost

productive work time

In addition an estimated 21 to 33 percent of

patients seeking attention in primary care settings

report significant fatigue resulting in

approximately seven million office visits per year in

the United States

Lawrie SM Manders DN Geddes JR Pelosi AJ A population-based incidence study of chronic fatigue Psychol Med 1997 27343Walker EA Katon WJ Jemelka RP Psychiatric disorders and medical care utilization among people in the general population who report fatigue J Gen Intern Med 1993 8436 Ricci JA Chee E Lorandeau AL Berger J Fatigue in the US workforce prevalence and implications for lost productive work time J Occup Environ Med 2007 491

Fatigue

Mayo

Treatment includes

antidepressants

sleeping pills

Pace yourself

Graded Exercise

Psychological counseling

httpwwwmayoclinicorgdiseases-conditionschronic-fatigue-syndromebasicstreatmentcon-20022009 Posted as of Sept 1 20017

Insomnia

httpswwwresearchgatenetfigure26679987_fig1_Fig-1-Prevalence-of-insomnia-symptoms-by-age-and-gender

As reported Sept 1 2017

What Americans Use to Sleep Nearly 9 million US adults resort to prescription sleeping pills mdash and most

are white female educated or 50 or older according to the first

government study of its kind

The CDC estimates that ldquo50-70 million US adults have sleep or

wakefulness disorderrdquo These numbers are striking because they

essentially mean that for every adult with a sleep disorder a doctor is

writing a prescription for sleeping pills

Between 2006 and 2011 sleeping pill prescriptions rose from 47 million to

60 million in the US About 38 million of those prescriptions were for drugs

containing zolpidem (including the brand name Ambien) according

to IMS Health

The United States insomnia pharmacological treatment market is

projected to reach $424 billion by 2021 from $338 billion in 2016

httpwwwnydailynewscomlife-stylehealthcdc-9-million-americans-sleeping-pills-article-11441778

Mood Fatigue Insomnia Theory

Mood Insomnia and Fatigue Issues are

inflammatory issues which are measurable

and reversible even with a poor genomic

outlook

-Decker Weiss

The Three Stooges of Mood Fatigue

and Insomnia

Inflammation causing Neurotransmitter Dysfunction

Inflammation toxicity exposure to free radicals causes Mitochondrial Dysfunction decreasing sex hormone production and energy an defenses

Gut BiomeFood Allergies generates and perpetuates inflammation immune dysfunction and further decreases in neurotransmitter dysfunction

DepressionInsomniaFatigue

GlutamatergicExcess

Many Causes Similar Result

NoradrenergicDeficiency

SerotonergicDeficiency

Belmaker R H and Agam G Major depressive

disorder (1-3-2008) NEnglJMed 358(1) 55-

68

GutBrainDysfunction

Mitochondrial Dysfunction

18

Neurotransmitters

PVN

C1

LC

Pituitary

DR

Thoracic

Lumbar

Cortisol

DHEAEpinephrine

Norepinephrine

CRF

ACTH

Traditional Stress Response

Hypothalamus is the master controller of the HPA axis (endocrine model)

Traditionally the start of the HPA axis

Pituitary gland has two parts that bridge the brain and body

Anterior (adenohypophysis) releases ACTH TSH PRL GH endorphins FSH LH CRF

Posterior (neurohypophysis) releases oxytocin and antidiuretic hormone

Two distinct parts of the adrenal gland produce hormones and neurotransmitters

Adrenal Cortex = cortisol and DHEA

Adrenal Medulla = norepinephrine and epinephrine

Engelmann M et al (2004) Front Neuroendocrinol 25(3-4)132-149Sherwood L (2010) Human Physiology From Cells to Systems 7th ed 698-709

Are you

considering

the adrenal

medulla

20

PVN

C1

LC

Hypothalamus

Pituitary

DR

Cortex

Medulla

Thoracic

Lumbar

CRF

NE

Cortisol

Glutamate

Epinephrine

ACh

Glutamate activates Thoracic pathway to

Adrenals

Sympathetic Chain

Serotonin

21

The ldquoBackward RuleMatch Diagnosis to Labs

Neurotransmitter Subject 1 Subject 2 Subject 3

Serotonin

GABA

Glycine

Glutamate

Histamine

PEA

Dopamine

Norepinephrine

Epinephrine

Cortisol

22

Biomarkers for Early Stage 2

3

23

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Biomarkers for Mid Stage

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Biomarkers for End Stage

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Genomics

COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of

the brain responsible for personality inhibition of behaviors abstract thinking

and emotion1

The COMT val158Met polymorphism has been shown to be associated with phobic

anxiety 2 neuroticism 3 and panic disorders 4

Results indicate a relationship of COMT polymorphism rs4680 with immune

dysregulation in CFS providing a potential link for the association between stress and

infection susceptibility in CFS(chronic fatigue syndrome)

1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype

on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J

Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8

27

17 yo radicalized female with anger

antisocial and rage issues(similar not exact due to security)

Methylene-tetrahydrofolate reductase

(MTHFR SNPrsquos)

Creates methylated folate

the active folate in the

body

Mutations have been

associated with vascular

disease dementia cancer

and autism

Methylation is the genetics

elevated homocysteine is

the genomics 29

End Point of Poor Methylation and

Oxidation

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

bull Superoxide dismutase 1

(SOD1)

bull Superoxide dismutase 2

(SOD2)

bull Superoxide dismutase 3

(SOD3)

bull Glutathione peroxidase 1

(GPx1)

bull Catalase (CAT)

Superoxide Dismutase 1

SOD1 (CuZnSOD)(Encoded Chrom 21)

Found in cytoplasmic and mitochondrial

intermembrane space(inserted)

Converts superoxide radicals (O2-) to hydrogen

peroxide (H2O2)

Needs copper and zinc as cofactors

Apoptosis Oxidative Stress ALS Myocardial

Infarction Macular Degeneration a marker for

heavy Metal toxicity

O2- H2O2

SOD1SOD1

Cu Zn

1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2

2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160

3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034

Superoxide Dismutase 2

SOD2 (MnSOD) (Chrom 6)

Found in mitochondria

Converts superoxide radicals (O2-)

to hydrogen peroxide (H2O2)

Needs manganese as a cofactor

Heart Disease Myocardial

Infarction Tumor metastasis

neurodegenerative diseases

O2- H2O2SOD2SOD2

Mn

Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24

Superoxide Dismutase 3SOD3

Found extracellularly

Converts superoxide radicals (O2-) to

hydrogen peroxide (H2O2)

Needs copper and zinc as cofactors

Chronic skin disorders Diabetic

Neuropathy Folate Metabolism Ischemic

Heart Disease

O2- H2O2SOD3SOD3

Cu Zn

Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004

Glutathione Peroxidase 1

GPx1(Chrom 3)

Found in the cytoplasm of nearly all mammalian tissues

Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)

Uses glutathione an antioxidant as a cofactor

Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression

H2O2H2O O2GPx1GPx1

Glutathione

Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and

Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89

Catalase

CAT(Chrom 11)

Found in the cytoplasm of cells

Needs iron and selenium as cofactors

Reduce hydrogen peroxide (H2O2) to water (H2O)

High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second

Hypertension Type 2 Diabetes Acatalasemia

httpsghrnlmnihgovgeneCATconditions

H2O2 H2O O2CATCAT

Fe Se

Mitochondrial Function Assessment

Mitochondrial Superoxide (O2-)

Plasma Peroxide Assay (PPA)

Mitochondrial Membrane

Potential (MMP) Assay with

Oxidative Challenge

Mitochondrial Superoxide (O2-)

Superoxide is considered to be a major

factor in oxidant toxicity and

mitochondrial MnSOD enzymes

constitute an essential defense against

superoxide

High levels indicate the proanti-oxidant

system is out of balance and oxidative

stress is present

Mitochondria are the major source of

superoxide

Superoxide is the origin of reactive

oxygen (ROS)and nitrogen species (RNS)

and as such causes various redox

related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo

123Hsiu-Chuan Yen

4Ikuo Nakanishi

5Ken-ichiro Matsumoto

5Masato Tamura

6Yumiko Nagano

6Hirofumi Matsui

6Oleg Gusev

789Richard Cornette

9Takashi

Okuda9

Yukiko Minamiyama10

Hiroshi Ichikawa11

Shigeaki Suenaga1

Misato Oki2

Tsuyoshi Sato1

Toshihiko Ozawa12

Daret K St Clair3

and Hideyuki J Majima12dagger

Retrospective Study Sample Distribution

To

tal S

ub

jects

(n

=86)

Lo

w (

n=10)

No

rmal (n

=27)

Ele

vate

d (

n=19)

Hig

h (

n=30)

0

1

2

3

4

5

Mit

oS

OA

R (

RF

U)

3 5

2 2

2 7

1 2

M ito S O A R

R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n

MitoSOARtrade (RFU)

lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects

N 10 27 19 3086

12 31 22 35

57 of the tested population has

superoxide levels in the

elevatedhigh range leading to high

risk for certain clinical conditions

Functional Genetics

CT

CC

0 5

1 0

1 5

2 0

2 5

Mit

oS

OA

R (

RF

U)

S O D 2 H e tro z y g o u s g e n o ty p e (C T )

S O D 2 H o m o z y g o u s g e n o ty p e (C C )

P lt 0 0 5

M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e

(S O D 2 ) P o ly m o rp h is m

Individuals who are homozygous

recessive(CC) for SOD2 may

have impaired SOD2 function

resulting in higher levels of the

free radical superoxide and

require antioxidant support

Mitochondrial Superoxide Levels

Assessment of Redox status

Inflammation

Viral or Bacterial Infection

Monitoring treatment of a

disease or disorder

Borrelia infection

Treatment of Inflammation

Functional Effect

Improvement verifies positive clinical effect

Plasma Peroxide Assay

Low Levels may indicate oxidative stress and

possible need for antioxidants

High levels may indicate immune challenges

Can correlate with uric acid

Potential hypertension risk

Related to lipid peroxides

Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13

Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303

Plasma Peroxidase

To

tal S

ub

jects

(n

=86)

Lo

w (

n=30)

No

rmal (n

=24)

Ele

vate

d (

n=11)

Hig

h (

n=16)

0

2 0

4 0

6 0

8 0

Pla

sm

a P

erix

ida

se

Ac

tiv

ity

(m

Um

l)

2 0

1 4

3 0

3 7

M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s

R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e

S a m p le D is tr ib u tio n

Plasma Peroxidase Activity (mUmL)

lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects

N 30 24 11 1681

37 30 14 20

37 of the population studied had low

peroxidase activity indicating an

impaired ability to defend against free

radicals

34 of the studies population had

elevatedhigh activity potentially

indicating the presence of oxidative

stress

Assessment of Baseline Potential

Starting point for defense of excessive

ROS

Effect of Environmental Insults

Assessment of Capacity to defend against

excessive ROS

Assessment and monitoring treatment of a

disease or disorder

Viral or Bacterial infection

Cardiovascular disease

Mitochondrial Membrane Potential

Oxidative Challenge

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Mood

Depression in

America

1 in 10

The diagnosis is INCREASING 20 per year

80 of depressed persons do not seek treatment

Depression varies by state Oklahoma Mississippi Louisiana Alabama W Virginia are amongst the highest

Healthline InfographicPosted as of 29Feb2016 httpwwwhealthlinecomhealthdepressionstatistics-infographic

Depression and Women

ldquoCompared to men women are twice as

likely to develop depressionrdquo

Psychology Today The 7 Reasons Why Depression is More Common in Women Compared to men women are twice as likely to develop depression

Published on May 17 2012 by Neel Burton MD in Hide and Seek

httpwwwpsychologytodaycombloghide-and-seek201205the-7-reasons-why-depression-is-more-common-in-women as reported 30 Sept 2014

Depression in Teens

The Old Model

Educated link to a diagnoses educated

guess to a treatment than repeat

Common Interventions for Depression

Reuptake inhibitors Serotonin

Sertraline

Citalopram

Escitalopram

Reuptake inhibitors

Norepinephrine Dopamine

Bupropion

Venlafaxine

Cyclics

Trazodone

Mirtazapine

Atypical

Ariprazole

Olanzapine

Quetiapine

7

Psychiatry Failure in the Press

The latest edition of psychiatric diagnostic Bible the Diagnostic and Statistical Manual of Mental

Disorders (DSM) shows that US psychiatry has no intention of changing course

Edward Shorter- University of Toronto- WSJ

Waiting on a

Breakthrough

ldquoWe are facing a crisisrdquo the Cornell psychiatrist and New York Times contributor Richard Friedman warned last week In the past few years one pharmaceutical giant after anothermdashGlaxoSmithKline AstraZeneca Novartis Pfizer Merck Sanofimdashhas shrunk or shuttered its neuroscience research facilities

Clinical trials have been halted lines of research abandoned and the new drug pipeline has been allowed to run dry

The New Yorker Posted as of 22Feb 2016

SEPTEMBER 3 2013The Psychiatric Drug Crisis

BY GARY GREENBERG

Fatigue

Fatigue Fatigue is a common complaint with a

prevalence in population-based surveys in Britain

and the United States of between 60 and 75

percent

A cross-sectional survey of United States workers

found the two-week period prevalence of fatigue

to be 38 percent with an estimated annual cost

to employers exceeding $136 billion in lost

productive work time

In addition an estimated 21 to 33 percent of

patients seeking attention in primary care settings

report significant fatigue resulting in

approximately seven million office visits per year in

the United States

Lawrie SM Manders DN Geddes JR Pelosi AJ A population-based incidence study of chronic fatigue Psychol Med 1997 27343Walker EA Katon WJ Jemelka RP Psychiatric disorders and medical care utilization among people in the general population who report fatigue J Gen Intern Med 1993 8436 Ricci JA Chee E Lorandeau AL Berger J Fatigue in the US workforce prevalence and implications for lost productive work time J Occup Environ Med 2007 491

Fatigue

Mayo

Treatment includes

antidepressants

sleeping pills

Pace yourself

Graded Exercise

Psychological counseling

httpwwwmayoclinicorgdiseases-conditionschronic-fatigue-syndromebasicstreatmentcon-20022009 Posted as of Sept 1 20017

Insomnia

httpswwwresearchgatenetfigure26679987_fig1_Fig-1-Prevalence-of-insomnia-symptoms-by-age-and-gender

As reported Sept 1 2017

What Americans Use to Sleep Nearly 9 million US adults resort to prescription sleeping pills mdash and most

are white female educated or 50 or older according to the first

government study of its kind

The CDC estimates that ldquo50-70 million US adults have sleep or

wakefulness disorderrdquo These numbers are striking because they

essentially mean that for every adult with a sleep disorder a doctor is

writing a prescription for sleeping pills

Between 2006 and 2011 sleeping pill prescriptions rose from 47 million to

60 million in the US About 38 million of those prescriptions were for drugs

containing zolpidem (including the brand name Ambien) according

to IMS Health

The United States insomnia pharmacological treatment market is

projected to reach $424 billion by 2021 from $338 billion in 2016

httpwwwnydailynewscomlife-stylehealthcdc-9-million-americans-sleeping-pills-article-11441778

Mood Fatigue Insomnia Theory

Mood Insomnia and Fatigue Issues are

inflammatory issues which are measurable

and reversible even with a poor genomic

outlook

-Decker Weiss

The Three Stooges of Mood Fatigue

and Insomnia

Inflammation causing Neurotransmitter Dysfunction

Inflammation toxicity exposure to free radicals causes Mitochondrial Dysfunction decreasing sex hormone production and energy an defenses

Gut BiomeFood Allergies generates and perpetuates inflammation immune dysfunction and further decreases in neurotransmitter dysfunction

DepressionInsomniaFatigue

GlutamatergicExcess

Many Causes Similar Result

NoradrenergicDeficiency

SerotonergicDeficiency

Belmaker R H and Agam G Major depressive

disorder (1-3-2008) NEnglJMed 358(1) 55-

68

GutBrainDysfunction

Mitochondrial Dysfunction

18

Neurotransmitters

PVN

C1

LC

Pituitary

DR

Thoracic

Lumbar

Cortisol

DHEAEpinephrine

Norepinephrine

CRF

ACTH

Traditional Stress Response

Hypothalamus is the master controller of the HPA axis (endocrine model)

Traditionally the start of the HPA axis

Pituitary gland has two parts that bridge the brain and body

Anterior (adenohypophysis) releases ACTH TSH PRL GH endorphins FSH LH CRF

Posterior (neurohypophysis) releases oxytocin and antidiuretic hormone

Two distinct parts of the adrenal gland produce hormones and neurotransmitters

Adrenal Cortex = cortisol and DHEA

Adrenal Medulla = norepinephrine and epinephrine

Engelmann M et al (2004) Front Neuroendocrinol 25(3-4)132-149Sherwood L (2010) Human Physiology From Cells to Systems 7th ed 698-709

Are you

considering

the adrenal

medulla

20

PVN

C1

LC

Hypothalamus

Pituitary

DR

Cortex

Medulla

Thoracic

Lumbar

CRF

NE

Cortisol

Glutamate

Epinephrine

ACh

Glutamate activates Thoracic pathway to

Adrenals

Sympathetic Chain

Serotonin

21

The ldquoBackward RuleMatch Diagnosis to Labs

Neurotransmitter Subject 1 Subject 2 Subject 3

Serotonin

GABA

Glycine

Glutamate

Histamine

PEA

Dopamine

Norepinephrine

Epinephrine

Cortisol

22

Biomarkers for Early Stage 2

3

23

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Biomarkers for Mid Stage

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Biomarkers for End Stage

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Genomics

COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of

the brain responsible for personality inhibition of behaviors abstract thinking

and emotion1

The COMT val158Met polymorphism has been shown to be associated with phobic

anxiety 2 neuroticism 3 and panic disorders 4

Results indicate a relationship of COMT polymorphism rs4680 with immune

dysregulation in CFS providing a potential link for the association between stress and

infection susceptibility in CFS(chronic fatigue syndrome)

1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype

on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J

Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8

27

17 yo radicalized female with anger

antisocial and rage issues(similar not exact due to security)

Methylene-tetrahydrofolate reductase

(MTHFR SNPrsquos)

Creates methylated folate

the active folate in the

body

Mutations have been

associated with vascular

disease dementia cancer

and autism

Methylation is the genetics

elevated homocysteine is

the genomics 29

End Point of Poor Methylation and

Oxidation

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

bull Superoxide dismutase 1

(SOD1)

bull Superoxide dismutase 2

(SOD2)

bull Superoxide dismutase 3

(SOD3)

bull Glutathione peroxidase 1

(GPx1)

bull Catalase (CAT)

Superoxide Dismutase 1

SOD1 (CuZnSOD)(Encoded Chrom 21)

Found in cytoplasmic and mitochondrial

intermembrane space(inserted)

Converts superoxide radicals (O2-) to hydrogen

peroxide (H2O2)

Needs copper and zinc as cofactors

Apoptosis Oxidative Stress ALS Myocardial

Infarction Macular Degeneration a marker for

heavy Metal toxicity

O2- H2O2

SOD1SOD1

Cu Zn

1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2

2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160

3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034

Superoxide Dismutase 2

SOD2 (MnSOD) (Chrom 6)

Found in mitochondria

Converts superoxide radicals (O2-)

to hydrogen peroxide (H2O2)

Needs manganese as a cofactor

Heart Disease Myocardial

Infarction Tumor metastasis

neurodegenerative diseases

O2- H2O2SOD2SOD2

Mn

Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24

Superoxide Dismutase 3SOD3

Found extracellularly

Converts superoxide radicals (O2-) to

hydrogen peroxide (H2O2)

Needs copper and zinc as cofactors

Chronic skin disorders Diabetic

Neuropathy Folate Metabolism Ischemic

Heart Disease

O2- H2O2SOD3SOD3

Cu Zn

Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004

Glutathione Peroxidase 1

GPx1(Chrom 3)

Found in the cytoplasm of nearly all mammalian tissues

Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)

Uses glutathione an antioxidant as a cofactor

Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression

H2O2H2O O2GPx1GPx1

Glutathione

Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and

Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89

Catalase

CAT(Chrom 11)

Found in the cytoplasm of cells

Needs iron and selenium as cofactors

Reduce hydrogen peroxide (H2O2) to water (H2O)

High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second

Hypertension Type 2 Diabetes Acatalasemia

httpsghrnlmnihgovgeneCATconditions

H2O2 H2O O2CATCAT

Fe Se

Mitochondrial Function Assessment

Mitochondrial Superoxide (O2-)

Plasma Peroxide Assay (PPA)

Mitochondrial Membrane

Potential (MMP) Assay with

Oxidative Challenge

Mitochondrial Superoxide (O2-)

Superoxide is considered to be a major

factor in oxidant toxicity and

mitochondrial MnSOD enzymes

constitute an essential defense against

superoxide

High levels indicate the proanti-oxidant

system is out of balance and oxidative

stress is present

Mitochondria are the major source of

superoxide

Superoxide is the origin of reactive

oxygen (ROS)and nitrogen species (RNS)

and as such causes various redox

related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo

123Hsiu-Chuan Yen

4Ikuo Nakanishi

5Ken-ichiro Matsumoto

5Masato Tamura

6Yumiko Nagano

6Hirofumi Matsui

6Oleg Gusev

789Richard Cornette

9Takashi

Okuda9

Yukiko Minamiyama10

Hiroshi Ichikawa11

Shigeaki Suenaga1

Misato Oki2

Tsuyoshi Sato1

Toshihiko Ozawa12

Daret K St Clair3

and Hideyuki J Majima12dagger

Retrospective Study Sample Distribution

To

tal S

ub

jects

(n

=86)

Lo

w (

n=10)

No

rmal (n

=27)

Ele

vate

d (

n=19)

Hig

h (

n=30)

0

1

2

3

4

5

Mit

oS

OA

R (

RF

U)

3 5

2 2

2 7

1 2

M ito S O A R

R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n

MitoSOARtrade (RFU)

lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects

N 10 27 19 3086

12 31 22 35

57 of the tested population has

superoxide levels in the

elevatedhigh range leading to high

risk for certain clinical conditions

Functional Genetics

CT

CC

0 5

1 0

1 5

2 0

2 5

Mit

oS

OA

R (

RF

U)

S O D 2 H e tro z y g o u s g e n o ty p e (C T )

S O D 2 H o m o z y g o u s g e n o ty p e (C C )

P lt 0 0 5

M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e

(S O D 2 ) P o ly m o rp h is m

Individuals who are homozygous

recessive(CC) for SOD2 may

have impaired SOD2 function

resulting in higher levels of the

free radical superoxide and

require antioxidant support

Mitochondrial Superoxide Levels

Assessment of Redox status

Inflammation

Viral or Bacterial Infection

Monitoring treatment of a

disease or disorder

Borrelia infection

Treatment of Inflammation

Functional Effect

Improvement verifies positive clinical effect

Plasma Peroxide Assay

Low Levels may indicate oxidative stress and

possible need for antioxidants

High levels may indicate immune challenges

Can correlate with uric acid

Potential hypertension risk

Related to lipid peroxides

Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13

Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303

Plasma Peroxidase

To

tal S

ub

jects

(n

=86)

Lo

w (

n=30)

No

rmal (n

=24)

Ele

vate

d (

n=11)

Hig

h (

n=16)

0

2 0

4 0

6 0

8 0

Pla

sm

a P

erix

ida

se

Ac

tiv

ity

(m

Um

l)

2 0

1 4

3 0

3 7

M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s

R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e

S a m p le D is tr ib u tio n

Plasma Peroxidase Activity (mUmL)

lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects

N 30 24 11 1681

37 30 14 20

37 of the population studied had low

peroxidase activity indicating an

impaired ability to defend against free

radicals

34 of the studies population had

elevatedhigh activity potentially

indicating the presence of oxidative

stress

Assessment of Baseline Potential

Starting point for defense of excessive

ROS

Effect of Environmental Insults

Assessment of Capacity to defend against

excessive ROS

Assessment and monitoring treatment of a

disease or disorder

Viral or Bacterial infection

Cardiovascular disease

Mitochondrial Membrane Potential

Oxidative Challenge

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Depression in

America

1 in 10

The diagnosis is INCREASING 20 per year

80 of depressed persons do not seek treatment

Depression varies by state Oklahoma Mississippi Louisiana Alabama W Virginia are amongst the highest

Healthline InfographicPosted as of 29Feb2016 httpwwwhealthlinecomhealthdepressionstatistics-infographic

Depression and Women

ldquoCompared to men women are twice as

likely to develop depressionrdquo

Psychology Today The 7 Reasons Why Depression is More Common in Women Compared to men women are twice as likely to develop depression

Published on May 17 2012 by Neel Burton MD in Hide and Seek

httpwwwpsychologytodaycombloghide-and-seek201205the-7-reasons-why-depression-is-more-common-in-women as reported 30 Sept 2014

Depression in Teens

The Old Model

Educated link to a diagnoses educated

guess to a treatment than repeat

Common Interventions for Depression

Reuptake inhibitors Serotonin

Sertraline

Citalopram

Escitalopram

Reuptake inhibitors

Norepinephrine Dopamine

Bupropion

Venlafaxine

Cyclics

Trazodone

Mirtazapine

Atypical

Ariprazole

Olanzapine

Quetiapine

7

Psychiatry Failure in the Press

The latest edition of psychiatric diagnostic Bible the Diagnostic and Statistical Manual of Mental

Disorders (DSM) shows that US psychiatry has no intention of changing course

Edward Shorter- University of Toronto- WSJ

Waiting on a

Breakthrough

ldquoWe are facing a crisisrdquo the Cornell psychiatrist and New York Times contributor Richard Friedman warned last week In the past few years one pharmaceutical giant after anothermdashGlaxoSmithKline AstraZeneca Novartis Pfizer Merck Sanofimdashhas shrunk or shuttered its neuroscience research facilities

Clinical trials have been halted lines of research abandoned and the new drug pipeline has been allowed to run dry

The New Yorker Posted as of 22Feb 2016

SEPTEMBER 3 2013The Psychiatric Drug Crisis

BY GARY GREENBERG

Fatigue

Fatigue Fatigue is a common complaint with a

prevalence in population-based surveys in Britain

and the United States of between 60 and 75

percent

A cross-sectional survey of United States workers

found the two-week period prevalence of fatigue

to be 38 percent with an estimated annual cost

to employers exceeding $136 billion in lost

productive work time

In addition an estimated 21 to 33 percent of

patients seeking attention in primary care settings

report significant fatigue resulting in

approximately seven million office visits per year in

the United States

Lawrie SM Manders DN Geddes JR Pelosi AJ A population-based incidence study of chronic fatigue Psychol Med 1997 27343Walker EA Katon WJ Jemelka RP Psychiatric disorders and medical care utilization among people in the general population who report fatigue J Gen Intern Med 1993 8436 Ricci JA Chee E Lorandeau AL Berger J Fatigue in the US workforce prevalence and implications for lost productive work time J Occup Environ Med 2007 491

Fatigue

Mayo

Treatment includes

antidepressants

sleeping pills

Pace yourself

Graded Exercise

Psychological counseling

httpwwwmayoclinicorgdiseases-conditionschronic-fatigue-syndromebasicstreatmentcon-20022009 Posted as of Sept 1 20017

Insomnia

httpswwwresearchgatenetfigure26679987_fig1_Fig-1-Prevalence-of-insomnia-symptoms-by-age-and-gender

As reported Sept 1 2017

What Americans Use to Sleep Nearly 9 million US adults resort to prescription sleeping pills mdash and most

are white female educated or 50 or older according to the first

government study of its kind

The CDC estimates that ldquo50-70 million US adults have sleep or

wakefulness disorderrdquo These numbers are striking because they

essentially mean that for every adult with a sleep disorder a doctor is

writing a prescription for sleeping pills

Between 2006 and 2011 sleeping pill prescriptions rose from 47 million to

60 million in the US About 38 million of those prescriptions were for drugs

containing zolpidem (including the brand name Ambien) according

to IMS Health

The United States insomnia pharmacological treatment market is

projected to reach $424 billion by 2021 from $338 billion in 2016

httpwwwnydailynewscomlife-stylehealthcdc-9-million-americans-sleeping-pills-article-11441778

Mood Fatigue Insomnia Theory

Mood Insomnia and Fatigue Issues are

inflammatory issues which are measurable

and reversible even with a poor genomic

outlook

-Decker Weiss

The Three Stooges of Mood Fatigue

and Insomnia

Inflammation causing Neurotransmitter Dysfunction

Inflammation toxicity exposure to free radicals causes Mitochondrial Dysfunction decreasing sex hormone production and energy an defenses

Gut BiomeFood Allergies generates and perpetuates inflammation immune dysfunction and further decreases in neurotransmitter dysfunction

DepressionInsomniaFatigue

GlutamatergicExcess

Many Causes Similar Result

NoradrenergicDeficiency

SerotonergicDeficiency

Belmaker R H and Agam G Major depressive

disorder (1-3-2008) NEnglJMed 358(1) 55-

68

GutBrainDysfunction

Mitochondrial Dysfunction

18

Neurotransmitters

PVN

C1

LC

Pituitary

DR

Thoracic

Lumbar

Cortisol

DHEAEpinephrine

Norepinephrine

CRF

ACTH

Traditional Stress Response

Hypothalamus is the master controller of the HPA axis (endocrine model)

Traditionally the start of the HPA axis

Pituitary gland has two parts that bridge the brain and body

Anterior (adenohypophysis) releases ACTH TSH PRL GH endorphins FSH LH CRF

Posterior (neurohypophysis) releases oxytocin and antidiuretic hormone

Two distinct parts of the adrenal gland produce hormones and neurotransmitters

Adrenal Cortex = cortisol and DHEA

Adrenal Medulla = norepinephrine and epinephrine

Engelmann M et al (2004) Front Neuroendocrinol 25(3-4)132-149Sherwood L (2010) Human Physiology From Cells to Systems 7th ed 698-709

Are you

considering

the adrenal

medulla

20

PVN

C1

LC

Hypothalamus

Pituitary

DR

Cortex

Medulla

Thoracic

Lumbar

CRF

NE

Cortisol

Glutamate

Epinephrine

ACh

Glutamate activates Thoracic pathway to

Adrenals

Sympathetic Chain

Serotonin

21

The ldquoBackward RuleMatch Diagnosis to Labs

Neurotransmitter Subject 1 Subject 2 Subject 3

Serotonin

GABA

Glycine

Glutamate

Histamine

PEA

Dopamine

Norepinephrine

Epinephrine

Cortisol

22

Biomarkers for Early Stage 2

3

23

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Biomarkers for Mid Stage

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Biomarkers for End Stage

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Genomics

COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of

the brain responsible for personality inhibition of behaviors abstract thinking

and emotion1

The COMT val158Met polymorphism has been shown to be associated with phobic

anxiety 2 neuroticism 3 and panic disorders 4

Results indicate a relationship of COMT polymorphism rs4680 with immune

dysregulation in CFS providing a potential link for the association between stress and

infection susceptibility in CFS(chronic fatigue syndrome)

1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype

on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J

Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8

27

17 yo radicalized female with anger

antisocial and rage issues(similar not exact due to security)

Methylene-tetrahydrofolate reductase

(MTHFR SNPrsquos)

Creates methylated folate

the active folate in the

body

Mutations have been

associated with vascular

disease dementia cancer

and autism

Methylation is the genetics

elevated homocysteine is

the genomics 29

End Point of Poor Methylation and

Oxidation

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

bull Superoxide dismutase 1

(SOD1)

bull Superoxide dismutase 2

(SOD2)

bull Superoxide dismutase 3

(SOD3)

bull Glutathione peroxidase 1

(GPx1)

bull Catalase (CAT)

Superoxide Dismutase 1

SOD1 (CuZnSOD)(Encoded Chrom 21)

Found in cytoplasmic and mitochondrial

intermembrane space(inserted)

Converts superoxide radicals (O2-) to hydrogen

peroxide (H2O2)

Needs copper and zinc as cofactors

Apoptosis Oxidative Stress ALS Myocardial

Infarction Macular Degeneration a marker for

heavy Metal toxicity

O2- H2O2

SOD1SOD1

Cu Zn

1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2

2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160

3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034

Superoxide Dismutase 2

SOD2 (MnSOD) (Chrom 6)

Found in mitochondria

Converts superoxide radicals (O2-)

to hydrogen peroxide (H2O2)

Needs manganese as a cofactor

Heart Disease Myocardial

Infarction Tumor metastasis

neurodegenerative diseases

O2- H2O2SOD2SOD2

Mn

Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24

Superoxide Dismutase 3SOD3

Found extracellularly

Converts superoxide radicals (O2-) to

hydrogen peroxide (H2O2)

Needs copper and zinc as cofactors

Chronic skin disorders Diabetic

Neuropathy Folate Metabolism Ischemic

Heart Disease

O2- H2O2SOD3SOD3

Cu Zn

Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004

Glutathione Peroxidase 1

GPx1(Chrom 3)

Found in the cytoplasm of nearly all mammalian tissues

Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)

Uses glutathione an antioxidant as a cofactor

Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression

H2O2H2O O2GPx1GPx1

Glutathione

Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and

Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89

Catalase

CAT(Chrom 11)

Found in the cytoplasm of cells

Needs iron and selenium as cofactors

Reduce hydrogen peroxide (H2O2) to water (H2O)

High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second

Hypertension Type 2 Diabetes Acatalasemia

httpsghrnlmnihgovgeneCATconditions

H2O2 H2O O2CATCAT

Fe Se

Mitochondrial Function Assessment

Mitochondrial Superoxide (O2-)

Plasma Peroxide Assay (PPA)

Mitochondrial Membrane

Potential (MMP) Assay with

Oxidative Challenge

Mitochondrial Superoxide (O2-)

Superoxide is considered to be a major

factor in oxidant toxicity and

mitochondrial MnSOD enzymes

constitute an essential defense against

superoxide

High levels indicate the proanti-oxidant

system is out of balance and oxidative

stress is present

Mitochondria are the major source of

superoxide

Superoxide is the origin of reactive

oxygen (ROS)and nitrogen species (RNS)

and as such causes various redox

related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo

123Hsiu-Chuan Yen

4Ikuo Nakanishi

5Ken-ichiro Matsumoto

5Masato Tamura

6Yumiko Nagano

6Hirofumi Matsui

6Oleg Gusev

789Richard Cornette

9Takashi

Okuda9

Yukiko Minamiyama10

Hiroshi Ichikawa11

Shigeaki Suenaga1

Misato Oki2

Tsuyoshi Sato1

Toshihiko Ozawa12

Daret K St Clair3

and Hideyuki J Majima12dagger

Retrospective Study Sample Distribution

To

tal S

ub

jects

(n

=86)

Lo

w (

n=10)

No

rmal (n

=27)

Ele

vate

d (

n=19)

Hig

h (

n=30)

0

1

2

3

4

5

Mit

oS

OA

R (

RF

U)

3 5

2 2

2 7

1 2

M ito S O A R

R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n

MitoSOARtrade (RFU)

lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects

N 10 27 19 3086

12 31 22 35

57 of the tested population has

superoxide levels in the

elevatedhigh range leading to high

risk for certain clinical conditions

Functional Genetics

CT

CC

0 5

1 0

1 5

2 0

2 5

Mit

oS

OA

R (

RF

U)

S O D 2 H e tro z y g o u s g e n o ty p e (C T )

S O D 2 H o m o z y g o u s g e n o ty p e (C C )

P lt 0 0 5

M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e

(S O D 2 ) P o ly m o rp h is m

Individuals who are homozygous

recessive(CC) for SOD2 may

have impaired SOD2 function

resulting in higher levels of the

free radical superoxide and

require antioxidant support

Mitochondrial Superoxide Levels

Assessment of Redox status

Inflammation

Viral or Bacterial Infection

Monitoring treatment of a

disease or disorder

Borrelia infection

Treatment of Inflammation

Functional Effect

Improvement verifies positive clinical effect

Plasma Peroxide Assay

Low Levels may indicate oxidative stress and

possible need for antioxidants

High levels may indicate immune challenges

Can correlate with uric acid

Potential hypertension risk

Related to lipid peroxides

Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13

Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303

Plasma Peroxidase

To

tal S

ub

jects

(n

=86)

Lo

w (

n=30)

No

rmal (n

=24)

Ele

vate

d (

n=11)

Hig

h (

n=16)

0

2 0

4 0

6 0

8 0

Pla

sm

a P

erix

ida

se

Ac

tiv

ity

(m

Um

l)

2 0

1 4

3 0

3 7

M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s

R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e

S a m p le D is tr ib u tio n

Plasma Peroxidase Activity (mUmL)

lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects

N 30 24 11 1681

37 30 14 20

37 of the population studied had low

peroxidase activity indicating an

impaired ability to defend against free

radicals

34 of the studies population had

elevatedhigh activity potentially

indicating the presence of oxidative

stress

Assessment of Baseline Potential

Starting point for defense of excessive

ROS

Effect of Environmental Insults

Assessment of Capacity to defend against

excessive ROS

Assessment and monitoring treatment of a

disease or disorder

Viral or Bacterial infection

Cardiovascular disease

Mitochondrial Membrane Potential

Oxidative Challenge

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Depression and Women

ldquoCompared to men women are twice as

likely to develop depressionrdquo

Psychology Today The 7 Reasons Why Depression is More Common in Women Compared to men women are twice as likely to develop depression

Published on May 17 2012 by Neel Burton MD in Hide and Seek

httpwwwpsychologytodaycombloghide-and-seek201205the-7-reasons-why-depression-is-more-common-in-women as reported 30 Sept 2014

Depression in Teens

The Old Model

Educated link to a diagnoses educated

guess to a treatment than repeat

Common Interventions for Depression

Reuptake inhibitors Serotonin

Sertraline

Citalopram

Escitalopram

Reuptake inhibitors

Norepinephrine Dopamine

Bupropion

Venlafaxine

Cyclics

Trazodone

Mirtazapine

Atypical

Ariprazole

Olanzapine

Quetiapine

7

Psychiatry Failure in the Press

The latest edition of psychiatric diagnostic Bible the Diagnostic and Statistical Manual of Mental

Disorders (DSM) shows that US psychiatry has no intention of changing course

Edward Shorter- University of Toronto- WSJ

Waiting on a

Breakthrough

ldquoWe are facing a crisisrdquo the Cornell psychiatrist and New York Times contributor Richard Friedman warned last week In the past few years one pharmaceutical giant after anothermdashGlaxoSmithKline AstraZeneca Novartis Pfizer Merck Sanofimdashhas shrunk or shuttered its neuroscience research facilities

Clinical trials have been halted lines of research abandoned and the new drug pipeline has been allowed to run dry

The New Yorker Posted as of 22Feb 2016

SEPTEMBER 3 2013The Psychiatric Drug Crisis

BY GARY GREENBERG

Fatigue

Fatigue Fatigue is a common complaint with a

prevalence in population-based surveys in Britain

and the United States of between 60 and 75

percent

A cross-sectional survey of United States workers

found the two-week period prevalence of fatigue

to be 38 percent with an estimated annual cost

to employers exceeding $136 billion in lost

productive work time

In addition an estimated 21 to 33 percent of

patients seeking attention in primary care settings

report significant fatigue resulting in

approximately seven million office visits per year in

the United States

Lawrie SM Manders DN Geddes JR Pelosi AJ A population-based incidence study of chronic fatigue Psychol Med 1997 27343Walker EA Katon WJ Jemelka RP Psychiatric disorders and medical care utilization among people in the general population who report fatigue J Gen Intern Med 1993 8436 Ricci JA Chee E Lorandeau AL Berger J Fatigue in the US workforce prevalence and implications for lost productive work time J Occup Environ Med 2007 491

Fatigue

Mayo

Treatment includes

antidepressants

sleeping pills

Pace yourself

Graded Exercise

Psychological counseling

httpwwwmayoclinicorgdiseases-conditionschronic-fatigue-syndromebasicstreatmentcon-20022009 Posted as of Sept 1 20017

Insomnia

httpswwwresearchgatenetfigure26679987_fig1_Fig-1-Prevalence-of-insomnia-symptoms-by-age-and-gender

As reported Sept 1 2017

What Americans Use to Sleep Nearly 9 million US adults resort to prescription sleeping pills mdash and most

are white female educated or 50 or older according to the first

government study of its kind

The CDC estimates that ldquo50-70 million US adults have sleep or

wakefulness disorderrdquo These numbers are striking because they

essentially mean that for every adult with a sleep disorder a doctor is

writing a prescription for sleeping pills

Between 2006 and 2011 sleeping pill prescriptions rose from 47 million to

60 million in the US About 38 million of those prescriptions were for drugs

containing zolpidem (including the brand name Ambien) according

to IMS Health

The United States insomnia pharmacological treatment market is

projected to reach $424 billion by 2021 from $338 billion in 2016

httpwwwnydailynewscomlife-stylehealthcdc-9-million-americans-sleeping-pills-article-11441778

Mood Fatigue Insomnia Theory

Mood Insomnia and Fatigue Issues are

inflammatory issues which are measurable

and reversible even with a poor genomic

outlook

-Decker Weiss

The Three Stooges of Mood Fatigue

and Insomnia

Inflammation causing Neurotransmitter Dysfunction

Inflammation toxicity exposure to free radicals causes Mitochondrial Dysfunction decreasing sex hormone production and energy an defenses

Gut BiomeFood Allergies generates and perpetuates inflammation immune dysfunction and further decreases in neurotransmitter dysfunction

DepressionInsomniaFatigue

GlutamatergicExcess

Many Causes Similar Result

NoradrenergicDeficiency

SerotonergicDeficiency

Belmaker R H and Agam G Major depressive

disorder (1-3-2008) NEnglJMed 358(1) 55-

68

GutBrainDysfunction

Mitochondrial Dysfunction

18

Neurotransmitters

PVN

C1

LC

Pituitary

DR

Thoracic

Lumbar

Cortisol

DHEAEpinephrine

Norepinephrine

CRF

ACTH

Traditional Stress Response

Hypothalamus is the master controller of the HPA axis (endocrine model)

Traditionally the start of the HPA axis

Pituitary gland has two parts that bridge the brain and body

Anterior (adenohypophysis) releases ACTH TSH PRL GH endorphins FSH LH CRF

Posterior (neurohypophysis) releases oxytocin and antidiuretic hormone

Two distinct parts of the adrenal gland produce hormones and neurotransmitters

Adrenal Cortex = cortisol and DHEA

Adrenal Medulla = norepinephrine and epinephrine

Engelmann M et al (2004) Front Neuroendocrinol 25(3-4)132-149Sherwood L (2010) Human Physiology From Cells to Systems 7th ed 698-709

Are you

considering

the adrenal

medulla

20

PVN

C1

LC

Hypothalamus

Pituitary

DR

Cortex

Medulla

Thoracic

Lumbar

CRF

NE

Cortisol

Glutamate

Epinephrine

ACh

Glutamate activates Thoracic pathway to

Adrenals

Sympathetic Chain

Serotonin

21

The ldquoBackward RuleMatch Diagnosis to Labs

Neurotransmitter Subject 1 Subject 2 Subject 3

Serotonin

GABA

Glycine

Glutamate

Histamine

PEA

Dopamine

Norepinephrine

Epinephrine

Cortisol

22

Biomarkers for Early Stage 2

3

23

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Biomarkers for Mid Stage

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Biomarkers for End Stage

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Genomics

COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of

the brain responsible for personality inhibition of behaviors abstract thinking

and emotion1

The COMT val158Met polymorphism has been shown to be associated with phobic

anxiety 2 neuroticism 3 and panic disorders 4

Results indicate a relationship of COMT polymorphism rs4680 with immune

dysregulation in CFS providing a potential link for the association between stress and

infection susceptibility in CFS(chronic fatigue syndrome)

1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype

on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J

Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8

27

17 yo radicalized female with anger

antisocial and rage issues(similar not exact due to security)

Methylene-tetrahydrofolate reductase

(MTHFR SNPrsquos)

Creates methylated folate

the active folate in the

body

Mutations have been

associated with vascular

disease dementia cancer

and autism

Methylation is the genetics

elevated homocysteine is

the genomics 29

End Point of Poor Methylation and

Oxidation

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

bull Superoxide dismutase 1

(SOD1)

bull Superoxide dismutase 2

(SOD2)

bull Superoxide dismutase 3

(SOD3)

bull Glutathione peroxidase 1

(GPx1)

bull Catalase (CAT)

Superoxide Dismutase 1

SOD1 (CuZnSOD)(Encoded Chrom 21)

Found in cytoplasmic and mitochondrial

intermembrane space(inserted)

Converts superoxide radicals (O2-) to hydrogen

peroxide (H2O2)

Needs copper and zinc as cofactors

Apoptosis Oxidative Stress ALS Myocardial

Infarction Macular Degeneration a marker for

heavy Metal toxicity

O2- H2O2

SOD1SOD1

Cu Zn

1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2

2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160

3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034

Superoxide Dismutase 2

SOD2 (MnSOD) (Chrom 6)

Found in mitochondria

Converts superoxide radicals (O2-)

to hydrogen peroxide (H2O2)

Needs manganese as a cofactor

Heart Disease Myocardial

Infarction Tumor metastasis

neurodegenerative diseases

O2- H2O2SOD2SOD2

Mn

Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24

Superoxide Dismutase 3SOD3

Found extracellularly

Converts superoxide radicals (O2-) to

hydrogen peroxide (H2O2)

Needs copper and zinc as cofactors

Chronic skin disorders Diabetic

Neuropathy Folate Metabolism Ischemic

Heart Disease

O2- H2O2SOD3SOD3

Cu Zn

Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004

Glutathione Peroxidase 1

GPx1(Chrom 3)

Found in the cytoplasm of nearly all mammalian tissues

Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)

Uses glutathione an antioxidant as a cofactor

Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression

H2O2H2O O2GPx1GPx1

Glutathione

Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and

Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89

Catalase

CAT(Chrom 11)

Found in the cytoplasm of cells

Needs iron and selenium as cofactors

Reduce hydrogen peroxide (H2O2) to water (H2O)

High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second

Hypertension Type 2 Diabetes Acatalasemia

httpsghrnlmnihgovgeneCATconditions

H2O2 H2O O2CATCAT

Fe Se

Mitochondrial Function Assessment

Mitochondrial Superoxide (O2-)

Plasma Peroxide Assay (PPA)

Mitochondrial Membrane

Potential (MMP) Assay with

Oxidative Challenge

Mitochondrial Superoxide (O2-)

Superoxide is considered to be a major

factor in oxidant toxicity and

mitochondrial MnSOD enzymes

constitute an essential defense against

superoxide

High levels indicate the proanti-oxidant

system is out of balance and oxidative

stress is present

Mitochondria are the major source of

superoxide

Superoxide is the origin of reactive

oxygen (ROS)and nitrogen species (RNS)

and as such causes various redox

related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo

123Hsiu-Chuan Yen

4Ikuo Nakanishi

5Ken-ichiro Matsumoto

5Masato Tamura

6Yumiko Nagano

6Hirofumi Matsui

6Oleg Gusev

789Richard Cornette

9Takashi

Okuda9

Yukiko Minamiyama10

Hiroshi Ichikawa11

Shigeaki Suenaga1

Misato Oki2

Tsuyoshi Sato1

Toshihiko Ozawa12

Daret K St Clair3

and Hideyuki J Majima12dagger

Retrospective Study Sample Distribution

To

tal S

ub

jects

(n

=86)

Lo

w (

n=10)

No

rmal (n

=27)

Ele

vate

d (

n=19)

Hig

h (

n=30)

0

1

2

3

4

5

Mit

oS

OA

R (

RF

U)

3 5

2 2

2 7

1 2

M ito S O A R

R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n

MitoSOARtrade (RFU)

lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects

N 10 27 19 3086

12 31 22 35

57 of the tested population has

superoxide levels in the

elevatedhigh range leading to high

risk for certain clinical conditions

Functional Genetics

CT

CC

0 5

1 0

1 5

2 0

2 5

Mit

oS

OA

R (

RF

U)

S O D 2 H e tro z y g o u s g e n o ty p e (C T )

S O D 2 H o m o z y g o u s g e n o ty p e (C C )

P lt 0 0 5

M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e

(S O D 2 ) P o ly m o rp h is m

Individuals who are homozygous

recessive(CC) for SOD2 may

have impaired SOD2 function

resulting in higher levels of the

free radical superoxide and

require antioxidant support

Mitochondrial Superoxide Levels

Assessment of Redox status

Inflammation

Viral or Bacterial Infection

Monitoring treatment of a

disease or disorder

Borrelia infection

Treatment of Inflammation

Functional Effect

Improvement verifies positive clinical effect

Plasma Peroxide Assay

Low Levels may indicate oxidative stress and

possible need for antioxidants

High levels may indicate immune challenges

Can correlate with uric acid

Potential hypertension risk

Related to lipid peroxides

Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13

Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303

Plasma Peroxidase

To

tal S

ub

jects

(n

=86)

Lo

w (

n=30)

No

rmal (n

=24)

Ele

vate

d (

n=11)

Hig

h (

n=16)

0

2 0

4 0

6 0

8 0

Pla

sm

a P

erix

ida

se

Ac

tiv

ity

(m

Um

l)

2 0

1 4

3 0

3 7

M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s

R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e

S a m p le D is tr ib u tio n

Plasma Peroxidase Activity (mUmL)

lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects

N 30 24 11 1681

37 30 14 20

37 of the population studied had low

peroxidase activity indicating an

impaired ability to defend against free

radicals

34 of the studies population had

elevatedhigh activity potentially

indicating the presence of oxidative

stress

Assessment of Baseline Potential

Starting point for defense of excessive

ROS

Effect of Environmental Insults

Assessment of Capacity to defend against

excessive ROS

Assessment and monitoring treatment of a

disease or disorder

Viral or Bacterial infection

Cardiovascular disease

Mitochondrial Membrane Potential

Oxidative Challenge

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Depression in Teens

The Old Model

Educated link to a diagnoses educated

guess to a treatment than repeat

Common Interventions for Depression

Reuptake inhibitors Serotonin

Sertraline

Citalopram

Escitalopram

Reuptake inhibitors

Norepinephrine Dopamine

Bupropion

Venlafaxine

Cyclics

Trazodone

Mirtazapine

Atypical

Ariprazole

Olanzapine

Quetiapine

7

Psychiatry Failure in the Press

The latest edition of psychiatric diagnostic Bible the Diagnostic and Statistical Manual of Mental

Disorders (DSM) shows that US psychiatry has no intention of changing course

Edward Shorter- University of Toronto- WSJ

Waiting on a

Breakthrough

ldquoWe are facing a crisisrdquo the Cornell psychiatrist and New York Times contributor Richard Friedman warned last week In the past few years one pharmaceutical giant after anothermdashGlaxoSmithKline AstraZeneca Novartis Pfizer Merck Sanofimdashhas shrunk or shuttered its neuroscience research facilities

Clinical trials have been halted lines of research abandoned and the new drug pipeline has been allowed to run dry

The New Yorker Posted as of 22Feb 2016

SEPTEMBER 3 2013The Psychiatric Drug Crisis

BY GARY GREENBERG

Fatigue

Fatigue Fatigue is a common complaint with a

prevalence in population-based surveys in Britain

and the United States of between 60 and 75

percent

A cross-sectional survey of United States workers

found the two-week period prevalence of fatigue

to be 38 percent with an estimated annual cost

to employers exceeding $136 billion in lost

productive work time

In addition an estimated 21 to 33 percent of

patients seeking attention in primary care settings

report significant fatigue resulting in

approximately seven million office visits per year in

the United States

Lawrie SM Manders DN Geddes JR Pelosi AJ A population-based incidence study of chronic fatigue Psychol Med 1997 27343Walker EA Katon WJ Jemelka RP Psychiatric disorders and medical care utilization among people in the general population who report fatigue J Gen Intern Med 1993 8436 Ricci JA Chee E Lorandeau AL Berger J Fatigue in the US workforce prevalence and implications for lost productive work time J Occup Environ Med 2007 491

Fatigue

Mayo

Treatment includes

antidepressants

sleeping pills

Pace yourself

Graded Exercise

Psychological counseling

httpwwwmayoclinicorgdiseases-conditionschronic-fatigue-syndromebasicstreatmentcon-20022009 Posted as of Sept 1 20017

Insomnia

httpswwwresearchgatenetfigure26679987_fig1_Fig-1-Prevalence-of-insomnia-symptoms-by-age-and-gender

As reported Sept 1 2017

What Americans Use to Sleep Nearly 9 million US adults resort to prescription sleeping pills mdash and most

are white female educated or 50 or older according to the first

government study of its kind

The CDC estimates that ldquo50-70 million US adults have sleep or

wakefulness disorderrdquo These numbers are striking because they

essentially mean that for every adult with a sleep disorder a doctor is

writing a prescription for sleeping pills

Between 2006 and 2011 sleeping pill prescriptions rose from 47 million to

60 million in the US About 38 million of those prescriptions were for drugs

containing zolpidem (including the brand name Ambien) according

to IMS Health

The United States insomnia pharmacological treatment market is

projected to reach $424 billion by 2021 from $338 billion in 2016

httpwwwnydailynewscomlife-stylehealthcdc-9-million-americans-sleeping-pills-article-11441778

Mood Fatigue Insomnia Theory

Mood Insomnia and Fatigue Issues are

inflammatory issues which are measurable

and reversible even with a poor genomic

outlook

-Decker Weiss

The Three Stooges of Mood Fatigue

and Insomnia

Inflammation causing Neurotransmitter Dysfunction

Inflammation toxicity exposure to free radicals causes Mitochondrial Dysfunction decreasing sex hormone production and energy an defenses

Gut BiomeFood Allergies generates and perpetuates inflammation immune dysfunction and further decreases in neurotransmitter dysfunction

DepressionInsomniaFatigue

GlutamatergicExcess

Many Causes Similar Result

NoradrenergicDeficiency

SerotonergicDeficiency

Belmaker R H and Agam G Major depressive

disorder (1-3-2008) NEnglJMed 358(1) 55-

68

GutBrainDysfunction

Mitochondrial Dysfunction

18

Neurotransmitters

PVN

C1

LC

Pituitary

DR

Thoracic

Lumbar

Cortisol

DHEAEpinephrine

Norepinephrine

CRF

ACTH

Traditional Stress Response

Hypothalamus is the master controller of the HPA axis (endocrine model)

Traditionally the start of the HPA axis

Pituitary gland has two parts that bridge the brain and body

Anterior (adenohypophysis) releases ACTH TSH PRL GH endorphins FSH LH CRF

Posterior (neurohypophysis) releases oxytocin and antidiuretic hormone

Two distinct parts of the adrenal gland produce hormones and neurotransmitters

Adrenal Cortex = cortisol and DHEA

Adrenal Medulla = norepinephrine and epinephrine

Engelmann M et al (2004) Front Neuroendocrinol 25(3-4)132-149Sherwood L (2010) Human Physiology From Cells to Systems 7th ed 698-709

Are you

considering

the adrenal

medulla

20

PVN

C1

LC

Hypothalamus

Pituitary

DR

Cortex

Medulla

Thoracic

Lumbar

CRF

NE

Cortisol

Glutamate

Epinephrine

ACh

Glutamate activates Thoracic pathway to

Adrenals

Sympathetic Chain

Serotonin

21

The ldquoBackward RuleMatch Diagnosis to Labs

Neurotransmitter Subject 1 Subject 2 Subject 3

Serotonin

GABA

Glycine

Glutamate

Histamine

PEA

Dopamine

Norepinephrine

Epinephrine

Cortisol

22

Biomarkers for Early Stage 2

3

23

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Biomarkers for Mid Stage

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Biomarkers for End Stage

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Genomics

COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of

the brain responsible for personality inhibition of behaviors abstract thinking

and emotion1

The COMT val158Met polymorphism has been shown to be associated with phobic

anxiety 2 neuroticism 3 and panic disorders 4

Results indicate a relationship of COMT polymorphism rs4680 with immune

dysregulation in CFS providing a potential link for the association between stress and

infection susceptibility in CFS(chronic fatigue syndrome)

1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype

on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J

Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8

27

17 yo radicalized female with anger

antisocial and rage issues(similar not exact due to security)

Methylene-tetrahydrofolate reductase

(MTHFR SNPrsquos)

Creates methylated folate

the active folate in the

body

Mutations have been

associated with vascular

disease dementia cancer

and autism

Methylation is the genetics

elevated homocysteine is

the genomics 29

End Point of Poor Methylation and

Oxidation

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

bull Superoxide dismutase 1

(SOD1)

bull Superoxide dismutase 2

(SOD2)

bull Superoxide dismutase 3

(SOD3)

bull Glutathione peroxidase 1

(GPx1)

bull Catalase (CAT)

Superoxide Dismutase 1

SOD1 (CuZnSOD)(Encoded Chrom 21)

Found in cytoplasmic and mitochondrial

intermembrane space(inserted)

Converts superoxide radicals (O2-) to hydrogen

peroxide (H2O2)

Needs copper and zinc as cofactors

Apoptosis Oxidative Stress ALS Myocardial

Infarction Macular Degeneration a marker for

heavy Metal toxicity

O2- H2O2

SOD1SOD1

Cu Zn

1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2

2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160

3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034

Superoxide Dismutase 2

SOD2 (MnSOD) (Chrom 6)

Found in mitochondria

Converts superoxide radicals (O2-)

to hydrogen peroxide (H2O2)

Needs manganese as a cofactor

Heart Disease Myocardial

Infarction Tumor metastasis

neurodegenerative diseases

O2- H2O2SOD2SOD2

Mn

Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24

Superoxide Dismutase 3SOD3

Found extracellularly

Converts superoxide radicals (O2-) to

hydrogen peroxide (H2O2)

Needs copper and zinc as cofactors

Chronic skin disorders Diabetic

Neuropathy Folate Metabolism Ischemic

Heart Disease

O2- H2O2SOD3SOD3

Cu Zn

Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004

Glutathione Peroxidase 1

GPx1(Chrom 3)

Found in the cytoplasm of nearly all mammalian tissues

Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)

Uses glutathione an antioxidant as a cofactor

Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression

H2O2H2O O2GPx1GPx1

Glutathione

Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and

Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89

Catalase

CAT(Chrom 11)

Found in the cytoplasm of cells

Needs iron and selenium as cofactors

Reduce hydrogen peroxide (H2O2) to water (H2O)

High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second

Hypertension Type 2 Diabetes Acatalasemia

httpsghrnlmnihgovgeneCATconditions

H2O2 H2O O2CATCAT

Fe Se

Mitochondrial Function Assessment

Mitochondrial Superoxide (O2-)

Plasma Peroxide Assay (PPA)

Mitochondrial Membrane

Potential (MMP) Assay with

Oxidative Challenge

Mitochondrial Superoxide (O2-)

Superoxide is considered to be a major

factor in oxidant toxicity and

mitochondrial MnSOD enzymes

constitute an essential defense against

superoxide

High levels indicate the proanti-oxidant

system is out of balance and oxidative

stress is present

Mitochondria are the major source of

superoxide

Superoxide is the origin of reactive

oxygen (ROS)and nitrogen species (RNS)

and as such causes various redox

related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo

123Hsiu-Chuan Yen

4Ikuo Nakanishi

5Ken-ichiro Matsumoto

5Masato Tamura

6Yumiko Nagano

6Hirofumi Matsui

6Oleg Gusev

789Richard Cornette

9Takashi

Okuda9

Yukiko Minamiyama10

Hiroshi Ichikawa11

Shigeaki Suenaga1

Misato Oki2

Tsuyoshi Sato1

Toshihiko Ozawa12

Daret K St Clair3

and Hideyuki J Majima12dagger

Retrospective Study Sample Distribution

To

tal S

ub

jects

(n

=86)

Lo

w (

n=10)

No

rmal (n

=27)

Ele

vate

d (

n=19)

Hig

h (

n=30)

0

1

2

3

4

5

Mit

oS

OA

R (

RF

U)

3 5

2 2

2 7

1 2

M ito S O A R

R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n

MitoSOARtrade (RFU)

lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects

N 10 27 19 3086

12 31 22 35

57 of the tested population has

superoxide levels in the

elevatedhigh range leading to high

risk for certain clinical conditions

Functional Genetics

CT

CC

0 5

1 0

1 5

2 0

2 5

Mit

oS

OA

R (

RF

U)

S O D 2 H e tro z y g o u s g e n o ty p e (C T )

S O D 2 H o m o z y g o u s g e n o ty p e (C C )

P lt 0 0 5

M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e

(S O D 2 ) P o ly m o rp h is m

Individuals who are homozygous

recessive(CC) for SOD2 may

have impaired SOD2 function

resulting in higher levels of the

free radical superoxide and

require antioxidant support

Mitochondrial Superoxide Levels

Assessment of Redox status

Inflammation

Viral or Bacterial Infection

Monitoring treatment of a

disease or disorder

Borrelia infection

Treatment of Inflammation

Functional Effect

Improvement verifies positive clinical effect

Plasma Peroxide Assay

Low Levels may indicate oxidative stress and

possible need for antioxidants

High levels may indicate immune challenges

Can correlate with uric acid

Potential hypertension risk

Related to lipid peroxides

Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13

Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303

Plasma Peroxidase

To

tal S

ub

jects

(n

=86)

Lo

w (

n=30)

No

rmal (n

=24)

Ele

vate

d (

n=11)

Hig

h (

n=16)

0

2 0

4 0

6 0

8 0

Pla

sm

a P

erix

ida

se

Ac

tiv

ity

(m

Um

l)

2 0

1 4

3 0

3 7

M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s

R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e

S a m p le D is tr ib u tio n

Plasma Peroxidase Activity (mUmL)

lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects

N 30 24 11 1681

37 30 14 20

37 of the population studied had low

peroxidase activity indicating an

impaired ability to defend against free

radicals

34 of the studies population had

elevatedhigh activity potentially

indicating the presence of oxidative

stress

Assessment of Baseline Potential

Starting point for defense of excessive

ROS

Effect of Environmental Insults

Assessment of Capacity to defend against

excessive ROS

Assessment and monitoring treatment of a

disease or disorder

Viral or Bacterial infection

Cardiovascular disease

Mitochondrial Membrane Potential

Oxidative Challenge

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

The Old Model

Educated link to a diagnoses educated

guess to a treatment than repeat

Common Interventions for Depression

Reuptake inhibitors Serotonin

Sertraline

Citalopram

Escitalopram

Reuptake inhibitors

Norepinephrine Dopamine

Bupropion

Venlafaxine

Cyclics

Trazodone

Mirtazapine

Atypical

Ariprazole

Olanzapine

Quetiapine

7

Psychiatry Failure in the Press

The latest edition of psychiatric diagnostic Bible the Diagnostic and Statistical Manual of Mental

Disorders (DSM) shows that US psychiatry has no intention of changing course

Edward Shorter- University of Toronto- WSJ

Waiting on a

Breakthrough

ldquoWe are facing a crisisrdquo the Cornell psychiatrist and New York Times contributor Richard Friedman warned last week In the past few years one pharmaceutical giant after anothermdashGlaxoSmithKline AstraZeneca Novartis Pfizer Merck Sanofimdashhas shrunk or shuttered its neuroscience research facilities

Clinical trials have been halted lines of research abandoned and the new drug pipeline has been allowed to run dry

The New Yorker Posted as of 22Feb 2016

SEPTEMBER 3 2013The Psychiatric Drug Crisis

BY GARY GREENBERG

Fatigue

Fatigue Fatigue is a common complaint with a

prevalence in population-based surveys in Britain

and the United States of between 60 and 75

percent

A cross-sectional survey of United States workers

found the two-week period prevalence of fatigue

to be 38 percent with an estimated annual cost

to employers exceeding $136 billion in lost

productive work time

In addition an estimated 21 to 33 percent of

patients seeking attention in primary care settings

report significant fatigue resulting in

approximately seven million office visits per year in

the United States

Lawrie SM Manders DN Geddes JR Pelosi AJ A population-based incidence study of chronic fatigue Psychol Med 1997 27343Walker EA Katon WJ Jemelka RP Psychiatric disorders and medical care utilization among people in the general population who report fatigue J Gen Intern Med 1993 8436 Ricci JA Chee E Lorandeau AL Berger J Fatigue in the US workforce prevalence and implications for lost productive work time J Occup Environ Med 2007 491

Fatigue

Mayo

Treatment includes

antidepressants

sleeping pills

Pace yourself

Graded Exercise

Psychological counseling

httpwwwmayoclinicorgdiseases-conditionschronic-fatigue-syndromebasicstreatmentcon-20022009 Posted as of Sept 1 20017

Insomnia

httpswwwresearchgatenetfigure26679987_fig1_Fig-1-Prevalence-of-insomnia-symptoms-by-age-and-gender

As reported Sept 1 2017

What Americans Use to Sleep Nearly 9 million US adults resort to prescription sleeping pills mdash and most

are white female educated or 50 or older according to the first

government study of its kind

The CDC estimates that ldquo50-70 million US adults have sleep or

wakefulness disorderrdquo These numbers are striking because they

essentially mean that for every adult with a sleep disorder a doctor is

writing a prescription for sleeping pills

Between 2006 and 2011 sleeping pill prescriptions rose from 47 million to

60 million in the US About 38 million of those prescriptions were for drugs

containing zolpidem (including the brand name Ambien) according

to IMS Health

The United States insomnia pharmacological treatment market is

projected to reach $424 billion by 2021 from $338 billion in 2016

httpwwwnydailynewscomlife-stylehealthcdc-9-million-americans-sleeping-pills-article-11441778

Mood Fatigue Insomnia Theory

Mood Insomnia and Fatigue Issues are

inflammatory issues which are measurable

and reversible even with a poor genomic

outlook

-Decker Weiss

The Three Stooges of Mood Fatigue

and Insomnia

Inflammation causing Neurotransmitter Dysfunction

Inflammation toxicity exposure to free radicals causes Mitochondrial Dysfunction decreasing sex hormone production and energy an defenses

Gut BiomeFood Allergies generates and perpetuates inflammation immune dysfunction and further decreases in neurotransmitter dysfunction

DepressionInsomniaFatigue

GlutamatergicExcess

Many Causes Similar Result

NoradrenergicDeficiency

SerotonergicDeficiency

Belmaker R H and Agam G Major depressive

disorder (1-3-2008) NEnglJMed 358(1) 55-

68

GutBrainDysfunction

Mitochondrial Dysfunction

18

Neurotransmitters

PVN

C1

LC

Pituitary

DR

Thoracic

Lumbar

Cortisol

DHEAEpinephrine

Norepinephrine

CRF

ACTH

Traditional Stress Response

Hypothalamus is the master controller of the HPA axis (endocrine model)

Traditionally the start of the HPA axis

Pituitary gland has two parts that bridge the brain and body

Anterior (adenohypophysis) releases ACTH TSH PRL GH endorphins FSH LH CRF

Posterior (neurohypophysis) releases oxytocin and antidiuretic hormone

Two distinct parts of the adrenal gland produce hormones and neurotransmitters

Adrenal Cortex = cortisol and DHEA

Adrenal Medulla = norepinephrine and epinephrine

Engelmann M et al (2004) Front Neuroendocrinol 25(3-4)132-149Sherwood L (2010) Human Physiology From Cells to Systems 7th ed 698-709

Are you

considering

the adrenal

medulla

20

PVN

C1

LC

Hypothalamus

Pituitary

DR

Cortex

Medulla

Thoracic

Lumbar

CRF

NE

Cortisol

Glutamate

Epinephrine

ACh

Glutamate activates Thoracic pathway to

Adrenals

Sympathetic Chain

Serotonin

21

The ldquoBackward RuleMatch Diagnosis to Labs

Neurotransmitter Subject 1 Subject 2 Subject 3

Serotonin

GABA

Glycine

Glutamate

Histamine

PEA

Dopamine

Norepinephrine

Epinephrine

Cortisol

22

Biomarkers for Early Stage 2

3

23

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Biomarkers for Mid Stage

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Biomarkers for End Stage

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Genomics

COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of

the brain responsible for personality inhibition of behaviors abstract thinking

and emotion1

The COMT val158Met polymorphism has been shown to be associated with phobic

anxiety 2 neuroticism 3 and panic disorders 4

Results indicate a relationship of COMT polymorphism rs4680 with immune

dysregulation in CFS providing a potential link for the association between stress and

infection susceptibility in CFS(chronic fatigue syndrome)

1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype

on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J

Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8

27

17 yo radicalized female with anger

antisocial and rage issues(similar not exact due to security)

Methylene-tetrahydrofolate reductase

(MTHFR SNPrsquos)

Creates methylated folate

the active folate in the

body

Mutations have been

associated with vascular

disease dementia cancer

and autism

Methylation is the genetics

elevated homocysteine is

the genomics 29

End Point of Poor Methylation and

Oxidation

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

bull Superoxide dismutase 1

(SOD1)

bull Superoxide dismutase 2

(SOD2)

bull Superoxide dismutase 3

(SOD3)

bull Glutathione peroxidase 1

(GPx1)

bull Catalase (CAT)

Superoxide Dismutase 1

SOD1 (CuZnSOD)(Encoded Chrom 21)

Found in cytoplasmic and mitochondrial

intermembrane space(inserted)

Converts superoxide radicals (O2-) to hydrogen

peroxide (H2O2)

Needs copper and zinc as cofactors

Apoptosis Oxidative Stress ALS Myocardial

Infarction Macular Degeneration a marker for

heavy Metal toxicity

O2- H2O2

SOD1SOD1

Cu Zn

1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2

2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160

3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034

Superoxide Dismutase 2

SOD2 (MnSOD) (Chrom 6)

Found in mitochondria

Converts superoxide radicals (O2-)

to hydrogen peroxide (H2O2)

Needs manganese as a cofactor

Heart Disease Myocardial

Infarction Tumor metastasis

neurodegenerative diseases

O2- H2O2SOD2SOD2

Mn

Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24

Superoxide Dismutase 3SOD3

Found extracellularly

Converts superoxide radicals (O2-) to

hydrogen peroxide (H2O2)

Needs copper and zinc as cofactors

Chronic skin disorders Diabetic

Neuropathy Folate Metabolism Ischemic

Heart Disease

O2- H2O2SOD3SOD3

Cu Zn

Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004

Glutathione Peroxidase 1

GPx1(Chrom 3)

Found in the cytoplasm of nearly all mammalian tissues

Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)

Uses glutathione an antioxidant as a cofactor

Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression

H2O2H2O O2GPx1GPx1

Glutathione

Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and

Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89

Catalase

CAT(Chrom 11)

Found in the cytoplasm of cells

Needs iron and selenium as cofactors

Reduce hydrogen peroxide (H2O2) to water (H2O)

High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second

Hypertension Type 2 Diabetes Acatalasemia

httpsghrnlmnihgovgeneCATconditions

H2O2 H2O O2CATCAT

Fe Se

Mitochondrial Function Assessment

Mitochondrial Superoxide (O2-)

Plasma Peroxide Assay (PPA)

Mitochondrial Membrane

Potential (MMP) Assay with

Oxidative Challenge

Mitochondrial Superoxide (O2-)

Superoxide is considered to be a major

factor in oxidant toxicity and

mitochondrial MnSOD enzymes

constitute an essential defense against

superoxide

High levels indicate the proanti-oxidant

system is out of balance and oxidative

stress is present

Mitochondria are the major source of

superoxide

Superoxide is the origin of reactive

oxygen (ROS)and nitrogen species (RNS)

and as such causes various redox

related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo

123Hsiu-Chuan Yen

4Ikuo Nakanishi

5Ken-ichiro Matsumoto

5Masato Tamura

6Yumiko Nagano

6Hirofumi Matsui

6Oleg Gusev

789Richard Cornette

9Takashi

Okuda9

Yukiko Minamiyama10

Hiroshi Ichikawa11

Shigeaki Suenaga1

Misato Oki2

Tsuyoshi Sato1

Toshihiko Ozawa12

Daret K St Clair3

and Hideyuki J Majima12dagger

Retrospective Study Sample Distribution

To

tal S

ub

jects

(n

=86)

Lo

w (

n=10)

No

rmal (n

=27)

Ele

vate

d (

n=19)

Hig

h (

n=30)

0

1

2

3

4

5

Mit

oS

OA

R (

RF

U)

3 5

2 2

2 7

1 2

M ito S O A R

R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n

MitoSOARtrade (RFU)

lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects

N 10 27 19 3086

12 31 22 35

57 of the tested population has

superoxide levels in the

elevatedhigh range leading to high

risk for certain clinical conditions

Functional Genetics

CT

CC

0 5

1 0

1 5

2 0

2 5

Mit

oS

OA

R (

RF

U)

S O D 2 H e tro z y g o u s g e n o ty p e (C T )

S O D 2 H o m o z y g o u s g e n o ty p e (C C )

P lt 0 0 5

M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e

(S O D 2 ) P o ly m o rp h is m

Individuals who are homozygous

recessive(CC) for SOD2 may

have impaired SOD2 function

resulting in higher levels of the

free radical superoxide and

require antioxidant support

Mitochondrial Superoxide Levels

Assessment of Redox status

Inflammation

Viral or Bacterial Infection

Monitoring treatment of a

disease or disorder

Borrelia infection

Treatment of Inflammation

Functional Effect

Improvement verifies positive clinical effect

Plasma Peroxide Assay

Low Levels may indicate oxidative stress and

possible need for antioxidants

High levels may indicate immune challenges

Can correlate with uric acid

Potential hypertension risk

Related to lipid peroxides

Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13

Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303

Plasma Peroxidase

To

tal S

ub

jects

(n

=86)

Lo

w (

n=30)

No

rmal (n

=24)

Ele

vate

d (

n=11)

Hig

h (

n=16)

0

2 0

4 0

6 0

8 0

Pla

sm

a P

erix

ida

se

Ac

tiv

ity

(m

Um

l)

2 0

1 4

3 0

3 7

M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s

R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e

S a m p le D is tr ib u tio n

Plasma Peroxidase Activity (mUmL)

lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects

N 30 24 11 1681

37 30 14 20

37 of the population studied had low

peroxidase activity indicating an

impaired ability to defend against free

radicals

34 of the studies population had

elevatedhigh activity potentially

indicating the presence of oxidative

stress

Assessment of Baseline Potential

Starting point for defense of excessive

ROS

Effect of Environmental Insults

Assessment of Capacity to defend against

excessive ROS

Assessment and monitoring treatment of a

disease or disorder

Viral or Bacterial infection

Cardiovascular disease

Mitochondrial Membrane Potential

Oxidative Challenge

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Common Interventions for Depression

Reuptake inhibitors Serotonin

Sertraline

Citalopram

Escitalopram

Reuptake inhibitors

Norepinephrine Dopamine

Bupropion

Venlafaxine

Cyclics

Trazodone

Mirtazapine

Atypical

Ariprazole

Olanzapine

Quetiapine

7

Psychiatry Failure in the Press

The latest edition of psychiatric diagnostic Bible the Diagnostic and Statistical Manual of Mental

Disorders (DSM) shows that US psychiatry has no intention of changing course

Edward Shorter- University of Toronto- WSJ

Waiting on a

Breakthrough

ldquoWe are facing a crisisrdquo the Cornell psychiatrist and New York Times contributor Richard Friedman warned last week In the past few years one pharmaceutical giant after anothermdashGlaxoSmithKline AstraZeneca Novartis Pfizer Merck Sanofimdashhas shrunk or shuttered its neuroscience research facilities

Clinical trials have been halted lines of research abandoned and the new drug pipeline has been allowed to run dry

The New Yorker Posted as of 22Feb 2016

SEPTEMBER 3 2013The Psychiatric Drug Crisis

BY GARY GREENBERG

Fatigue

Fatigue Fatigue is a common complaint with a

prevalence in population-based surveys in Britain

and the United States of between 60 and 75

percent

A cross-sectional survey of United States workers

found the two-week period prevalence of fatigue

to be 38 percent with an estimated annual cost

to employers exceeding $136 billion in lost

productive work time

In addition an estimated 21 to 33 percent of

patients seeking attention in primary care settings

report significant fatigue resulting in

approximately seven million office visits per year in

the United States

Lawrie SM Manders DN Geddes JR Pelosi AJ A population-based incidence study of chronic fatigue Psychol Med 1997 27343Walker EA Katon WJ Jemelka RP Psychiatric disorders and medical care utilization among people in the general population who report fatigue J Gen Intern Med 1993 8436 Ricci JA Chee E Lorandeau AL Berger J Fatigue in the US workforce prevalence and implications for lost productive work time J Occup Environ Med 2007 491

Fatigue

Mayo

Treatment includes

antidepressants

sleeping pills

Pace yourself

Graded Exercise

Psychological counseling

httpwwwmayoclinicorgdiseases-conditionschronic-fatigue-syndromebasicstreatmentcon-20022009 Posted as of Sept 1 20017

Insomnia

httpswwwresearchgatenetfigure26679987_fig1_Fig-1-Prevalence-of-insomnia-symptoms-by-age-and-gender

As reported Sept 1 2017

What Americans Use to Sleep Nearly 9 million US adults resort to prescription sleeping pills mdash and most

are white female educated or 50 or older according to the first

government study of its kind

The CDC estimates that ldquo50-70 million US adults have sleep or

wakefulness disorderrdquo These numbers are striking because they

essentially mean that for every adult with a sleep disorder a doctor is

writing a prescription for sleeping pills

Between 2006 and 2011 sleeping pill prescriptions rose from 47 million to

60 million in the US About 38 million of those prescriptions were for drugs

containing zolpidem (including the brand name Ambien) according

to IMS Health

The United States insomnia pharmacological treatment market is

projected to reach $424 billion by 2021 from $338 billion in 2016

httpwwwnydailynewscomlife-stylehealthcdc-9-million-americans-sleeping-pills-article-11441778

Mood Fatigue Insomnia Theory

Mood Insomnia and Fatigue Issues are

inflammatory issues which are measurable

and reversible even with a poor genomic

outlook

-Decker Weiss

The Three Stooges of Mood Fatigue

and Insomnia

Inflammation causing Neurotransmitter Dysfunction

Inflammation toxicity exposure to free radicals causes Mitochondrial Dysfunction decreasing sex hormone production and energy an defenses

Gut BiomeFood Allergies generates and perpetuates inflammation immune dysfunction and further decreases in neurotransmitter dysfunction

DepressionInsomniaFatigue

GlutamatergicExcess

Many Causes Similar Result

NoradrenergicDeficiency

SerotonergicDeficiency

Belmaker R H and Agam G Major depressive

disorder (1-3-2008) NEnglJMed 358(1) 55-

68

GutBrainDysfunction

Mitochondrial Dysfunction

18

Neurotransmitters

PVN

C1

LC

Pituitary

DR

Thoracic

Lumbar

Cortisol

DHEAEpinephrine

Norepinephrine

CRF

ACTH

Traditional Stress Response

Hypothalamus is the master controller of the HPA axis (endocrine model)

Traditionally the start of the HPA axis

Pituitary gland has two parts that bridge the brain and body

Anterior (adenohypophysis) releases ACTH TSH PRL GH endorphins FSH LH CRF

Posterior (neurohypophysis) releases oxytocin and antidiuretic hormone

Two distinct parts of the adrenal gland produce hormones and neurotransmitters

Adrenal Cortex = cortisol and DHEA

Adrenal Medulla = norepinephrine and epinephrine

Engelmann M et al (2004) Front Neuroendocrinol 25(3-4)132-149Sherwood L (2010) Human Physiology From Cells to Systems 7th ed 698-709

Are you

considering

the adrenal

medulla

20

PVN

C1

LC

Hypothalamus

Pituitary

DR

Cortex

Medulla

Thoracic

Lumbar

CRF

NE

Cortisol

Glutamate

Epinephrine

ACh

Glutamate activates Thoracic pathway to

Adrenals

Sympathetic Chain

Serotonin

21

The ldquoBackward RuleMatch Diagnosis to Labs

Neurotransmitter Subject 1 Subject 2 Subject 3

Serotonin

GABA

Glycine

Glutamate

Histamine

PEA

Dopamine

Norepinephrine

Epinephrine

Cortisol

22

Biomarkers for Early Stage 2

3

23

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Biomarkers for Mid Stage

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Biomarkers for End Stage

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Genomics

COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of

the brain responsible for personality inhibition of behaviors abstract thinking

and emotion1

The COMT val158Met polymorphism has been shown to be associated with phobic

anxiety 2 neuroticism 3 and panic disorders 4

Results indicate a relationship of COMT polymorphism rs4680 with immune

dysregulation in CFS providing a potential link for the association between stress and

infection susceptibility in CFS(chronic fatigue syndrome)

1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype

on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J

Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8

27

17 yo radicalized female with anger

antisocial and rage issues(similar not exact due to security)

Methylene-tetrahydrofolate reductase

(MTHFR SNPrsquos)

Creates methylated folate

the active folate in the

body

Mutations have been

associated with vascular

disease dementia cancer

and autism

Methylation is the genetics

elevated homocysteine is

the genomics 29

End Point of Poor Methylation and

Oxidation

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

bull Superoxide dismutase 1

(SOD1)

bull Superoxide dismutase 2

(SOD2)

bull Superoxide dismutase 3

(SOD3)

bull Glutathione peroxidase 1

(GPx1)

bull Catalase (CAT)

Superoxide Dismutase 1

SOD1 (CuZnSOD)(Encoded Chrom 21)

Found in cytoplasmic and mitochondrial

intermembrane space(inserted)

Converts superoxide radicals (O2-) to hydrogen

peroxide (H2O2)

Needs copper and zinc as cofactors

Apoptosis Oxidative Stress ALS Myocardial

Infarction Macular Degeneration a marker for

heavy Metal toxicity

O2- H2O2

SOD1SOD1

Cu Zn

1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2

2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160

3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034

Superoxide Dismutase 2

SOD2 (MnSOD) (Chrom 6)

Found in mitochondria

Converts superoxide radicals (O2-)

to hydrogen peroxide (H2O2)

Needs manganese as a cofactor

Heart Disease Myocardial

Infarction Tumor metastasis

neurodegenerative diseases

O2- H2O2SOD2SOD2

Mn

Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24

Superoxide Dismutase 3SOD3

Found extracellularly

Converts superoxide radicals (O2-) to

hydrogen peroxide (H2O2)

Needs copper and zinc as cofactors

Chronic skin disorders Diabetic

Neuropathy Folate Metabolism Ischemic

Heart Disease

O2- H2O2SOD3SOD3

Cu Zn

Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004

Glutathione Peroxidase 1

GPx1(Chrom 3)

Found in the cytoplasm of nearly all mammalian tissues

Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)

Uses glutathione an antioxidant as a cofactor

Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression

H2O2H2O O2GPx1GPx1

Glutathione

Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and

Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89

Catalase

CAT(Chrom 11)

Found in the cytoplasm of cells

Needs iron and selenium as cofactors

Reduce hydrogen peroxide (H2O2) to water (H2O)

High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second

Hypertension Type 2 Diabetes Acatalasemia

httpsghrnlmnihgovgeneCATconditions

H2O2 H2O O2CATCAT

Fe Se

Mitochondrial Function Assessment

Mitochondrial Superoxide (O2-)

Plasma Peroxide Assay (PPA)

Mitochondrial Membrane

Potential (MMP) Assay with

Oxidative Challenge

Mitochondrial Superoxide (O2-)

Superoxide is considered to be a major

factor in oxidant toxicity and

mitochondrial MnSOD enzymes

constitute an essential defense against

superoxide

High levels indicate the proanti-oxidant

system is out of balance and oxidative

stress is present

Mitochondria are the major source of

superoxide

Superoxide is the origin of reactive

oxygen (ROS)and nitrogen species (RNS)

and as such causes various redox

related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo

123Hsiu-Chuan Yen

4Ikuo Nakanishi

5Ken-ichiro Matsumoto

5Masato Tamura

6Yumiko Nagano

6Hirofumi Matsui

6Oleg Gusev

789Richard Cornette

9Takashi

Okuda9

Yukiko Minamiyama10

Hiroshi Ichikawa11

Shigeaki Suenaga1

Misato Oki2

Tsuyoshi Sato1

Toshihiko Ozawa12

Daret K St Clair3

and Hideyuki J Majima12dagger

Retrospective Study Sample Distribution

To

tal S

ub

jects

(n

=86)

Lo

w (

n=10)

No

rmal (n

=27)

Ele

vate

d (

n=19)

Hig

h (

n=30)

0

1

2

3

4

5

Mit

oS

OA

R (

RF

U)

3 5

2 2

2 7

1 2

M ito S O A R

R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n

MitoSOARtrade (RFU)

lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects

N 10 27 19 3086

12 31 22 35

57 of the tested population has

superoxide levels in the

elevatedhigh range leading to high

risk for certain clinical conditions

Functional Genetics

CT

CC

0 5

1 0

1 5

2 0

2 5

Mit

oS

OA

R (

RF

U)

S O D 2 H e tro z y g o u s g e n o ty p e (C T )

S O D 2 H o m o z y g o u s g e n o ty p e (C C )

P lt 0 0 5

M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e

(S O D 2 ) P o ly m o rp h is m

Individuals who are homozygous

recessive(CC) for SOD2 may

have impaired SOD2 function

resulting in higher levels of the

free radical superoxide and

require antioxidant support

Mitochondrial Superoxide Levels

Assessment of Redox status

Inflammation

Viral or Bacterial Infection

Monitoring treatment of a

disease or disorder

Borrelia infection

Treatment of Inflammation

Functional Effect

Improvement verifies positive clinical effect

Plasma Peroxide Assay

Low Levels may indicate oxidative stress and

possible need for antioxidants

High levels may indicate immune challenges

Can correlate with uric acid

Potential hypertension risk

Related to lipid peroxides

Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13

Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303

Plasma Peroxidase

To

tal S

ub

jects

(n

=86)

Lo

w (

n=30)

No

rmal (n

=24)

Ele

vate

d (

n=11)

Hig

h (

n=16)

0

2 0

4 0

6 0

8 0

Pla

sm

a P

erix

ida

se

Ac

tiv

ity

(m

Um

l)

2 0

1 4

3 0

3 7

M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s

R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e

S a m p le D is tr ib u tio n

Plasma Peroxidase Activity (mUmL)

lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects

N 30 24 11 1681

37 30 14 20

37 of the population studied had low

peroxidase activity indicating an

impaired ability to defend against free

radicals

34 of the studies population had

elevatedhigh activity potentially

indicating the presence of oxidative

stress

Assessment of Baseline Potential

Starting point for defense of excessive

ROS

Effect of Environmental Insults

Assessment of Capacity to defend against

excessive ROS

Assessment and monitoring treatment of a

disease or disorder

Viral or Bacterial infection

Cardiovascular disease

Mitochondrial Membrane Potential

Oxidative Challenge

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Psychiatry Failure in the Press

The latest edition of psychiatric diagnostic Bible the Diagnostic and Statistical Manual of Mental

Disorders (DSM) shows that US psychiatry has no intention of changing course

Edward Shorter- University of Toronto- WSJ

Waiting on a

Breakthrough

ldquoWe are facing a crisisrdquo the Cornell psychiatrist and New York Times contributor Richard Friedman warned last week In the past few years one pharmaceutical giant after anothermdashGlaxoSmithKline AstraZeneca Novartis Pfizer Merck Sanofimdashhas shrunk or shuttered its neuroscience research facilities

Clinical trials have been halted lines of research abandoned and the new drug pipeline has been allowed to run dry

The New Yorker Posted as of 22Feb 2016

SEPTEMBER 3 2013The Psychiatric Drug Crisis

BY GARY GREENBERG

Fatigue

Fatigue Fatigue is a common complaint with a

prevalence in population-based surveys in Britain

and the United States of between 60 and 75

percent

A cross-sectional survey of United States workers

found the two-week period prevalence of fatigue

to be 38 percent with an estimated annual cost

to employers exceeding $136 billion in lost

productive work time

In addition an estimated 21 to 33 percent of

patients seeking attention in primary care settings

report significant fatigue resulting in

approximately seven million office visits per year in

the United States

Lawrie SM Manders DN Geddes JR Pelosi AJ A population-based incidence study of chronic fatigue Psychol Med 1997 27343Walker EA Katon WJ Jemelka RP Psychiatric disorders and medical care utilization among people in the general population who report fatigue J Gen Intern Med 1993 8436 Ricci JA Chee E Lorandeau AL Berger J Fatigue in the US workforce prevalence and implications for lost productive work time J Occup Environ Med 2007 491

Fatigue

Mayo

Treatment includes

antidepressants

sleeping pills

Pace yourself

Graded Exercise

Psychological counseling

httpwwwmayoclinicorgdiseases-conditionschronic-fatigue-syndromebasicstreatmentcon-20022009 Posted as of Sept 1 20017

Insomnia

httpswwwresearchgatenetfigure26679987_fig1_Fig-1-Prevalence-of-insomnia-symptoms-by-age-and-gender

As reported Sept 1 2017

What Americans Use to Sleep Nearly 9 million US adults resort to prescription sleeping pills mdash and most

are white female educated or 50 or older according to the first

government study of its kind

The CDC estimates that ldquo50-70 million US adults have sleep or

wakefulness disorderrdquo These numbers are striking because they

essentially mean that for every adult with a sleep disorder a doctor is

writing a prescription for sleeping pills

Between 2006 and 2011 sleeping pill prescriptions rose from 47 million to

60 million in the US About 38 million of those prescriptions were for drugs

containing zolpidem (including the brand name Ambien) according

to IMS Health

The United States insomnia pharmacological treatment market is

projected to reach $424 billion by 2021 from $338 billion in 2016

httpwwwnydailynewscomlife-stylehealthcdc-9-million-americans-sleeping-pills-article-11441778

Mood Fatigue Insomnia Theory

Mood Insomnia and Fatigue Issues are

inflammatory issues which are measurable

and reversible even with a poor genomic

outlook

-Decker Weiss

The Three Stooges of Mood Fatigue

and Insomnia

Inflammation causing Neurotransmitter Dysfunction

Inflammation toxicity exposure to free radicals causes Mitochondrial Dysfunction decreasing sex hormone production and energy an defenses

Gut BiomeFood Allergies generates and perpetuates inflammation immune dysfunction and further decreases in neurotransmitter dysfunction

DepressionInsomniaFatigue

GlutamatergicExcess

Many Causes Similar Result

NoradrenergicDeficiency

SerotonergicDeficiency

Belmaker R H and Agam G Major depressive

disorder (1-3-2008) NEnglJMed 358(1) 55-

68

GutBrainDysfunction

Mitochondrial Dysfunction

18

Neurotransmitters

PVN

C1

LC

Pituitary

DR

Thoracic

Lumbar

Cortisol

DHEAEpinephrine

Norepinephrine

CRF

ACTH

Traditional Stress Response

Hypothalamus is the master controller of the HPA axis (endocrine model)

Traditionally the start of the HPA axis

Pituitary gland has two parts that bridge the brain and body

Anterior (adenohypophysis) releases ACTH TSH PRL GH endorphins FSH LH CRF

Posterior (neurohypophysis) releases oxytocin and antidiuretic hormone

Two distinct parts of the adrenal gland produce hormones and neurotransmitters

Adrenal Cortex = cortisol and DHEA

Adrenal Medulla = norepinephrine and epinephrine

Engelmann M et al (2004) Front Neuroendocrinol 25(3-4)132-149Sherwood L (2010) Human Physiology From Cells to Systems 7th ed 698-709

Are you

considering

the adrenal

medulla

20

PVN

C1

LC

Hypothalamus

Pituitary

DR

Cortex

Medulla

Thoracic

Lumbar

CRF

NE

Cortisol

Glutamate

Epinephrine

ACh

Glutamate activates Thoracic pathway to

Adrenals

Sympathetic Chain

Serotonin

21

The ldquoBackward RuleMatch Diagnosis to Labs

Neurotransmitter Subject 1 Subject 2 Subject 3

Serotonin

GABA

Glycine

Glutamate

Histamine

PEA

Dopamine

Norepinephrine

Epinephrine

Cortisol

22

Biomarkers for Early Stage 2

3

23

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Biomarkers for Mid Stage

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Biomarkers for End Stage

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Genomics

COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of

the brain responsible for personality inhibition of behaviors abstract thinking

and emotion1

The COMT val158Met polymorphism has been shown to be associated with phobic

anxiety 2 neuroticism 3 and panic disorders 4

Results indicate a relationship of COMT polymorphism rs4680 with immune

dysregulation in CFS providing a potential link for the association between stress and

infection susceptibility in CFS(chronic fatigue syndrome)

1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype

on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J

Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8

27

17 yo radicalized female with anger

antisocial and rage issues(similar not exact due to security)

Methylene-tetrahydrofolate reductase

(MTHFR SNPrsquos)

Creates methylated folate

the active folate in the

body

Mutations have been

associated with vascular

disease dementia cancer

and autism

Methylation is the genetics

elevated homocysteine is

the genomics 29

End Point of Poor Methylation and

Oxidation

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

bull Superoxide dismutase 1

(SOD1)

bull Superoxide dismutase 2

(SOD2)

bull Superoxide dismutase 3

(SOD3)

bull Glutathione peroxidase 1

(GPx1)

bull Catalase (CAT)

Superoxide Dismutase 1

SOD1 (CuZnSOD)(Encoded Chrom 21)

Found in cytoplasmic and mitochondrial

intermembrane space(inserted)

Converts superoxide radicals (O2-) to hydrogen

peroxide (H2O2)

Needs copper and zinc as cofactors

Apoptosis Oxidative Stress ALS Myocardial

Infarction Macular Degeneration a marker for

heavy Metal toxicity

O2- H2O2

SOD1SOD1

Cu Zn

1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2

2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160

3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034

Superoxide Dismutase 2

SOD2 (MnSOD) (Chrom 6)

Found in mitochondria

Converts superoxide radicals (O2-)

to hydrogen peroxide (H2O2)

Needs manganese as a cofactor

Heart Disease Myocardial

Infarction Tumor metastasis

neurodegenerative diseases

O2- H2O2SOD2SOD2

Mn

Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24

Superoxide Dismutase 3SOD3

Found extracellularly

Converts superoxide radicals (O2-) to

hydrogen peroxide (H2O2)

Needs copper and zinc as cofactors

Chronic skin disorders Diabetic

Neuropathy Folate Metabolism Ischemic

Heart Disease

O2- H2O2SOD3SOD3

Cu Zn

Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004

Glutathione Peroxidase 1

GPx1(Chrom 3)

Found in the cytoplasm of nearly all mammalian tissues

Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)

Uses glutathione an antioxidant as a cofactor

Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression

H2O2H2O O2GPx1GPx1

Glutathione

Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and

Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89

Catalase

CAT(Chrom 11)

Found in the cytoplasm of cells

Needs iron and selenium as cofactors

Reduce hydrogen peroxide (H2O2) to water (H2O)

High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second

Hypertension Type 2 Diabetes Acatalasemia

httpsghrnlmnihgovgeneCATconditions

H2O2 H2O O2CATCAT

Fe Se

Mitochondrial Function Assessment

Mitochondrial Superoxide (O2-)

Plasma Peroxide Assay (PPA)

Mitochondrial Membrane

Potential (MMP) Assay with

Oxidative Challenge

Mitochondrial Superoxide (O2-)

Superoxide is considered to be a major

factor in oxidant toxicity and

mitochondrial MnSOD enzymes

constitute an essential defense against

superoxide

High levels indicate the proanti-oxidant

system is out of balance and oxidative

stress is present

Mitochondria are the major source of

superoxide

Superoxide is the origin of reactive

oxygen (ROS)and nitrogen species (RNS)

and as such causes various redox

related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo

123Hsiu-Chuan Yen

4Ikuo Nakanishi

5Ken-ichiro Matsumoto

5Masato Tamura

6Yumiko Nagano

6Hirofumi Matsui

6Oleg Gusev

789Richard Cornette

9Takashi

Okuda9

Yukiko Minamiyama10

Hiroshi Ichikawa11

Shigeaki Suenaga1

Misato Oki2

Tsuyoshi Sato1

Toshihiko Ozawa12

Daret K St Clair3

and Hideyuki J Majima12dagger

Retrospective Study Sample Distribution

To

tal S

ub

jects

(n

=86)

Lo

w (

n=10)

No

rmal (n

=27)

Ele

vate

d (

n=19)

Hig

h (

n=30)

0

1

2

3

4

5

Mit

oS

OA

R (

RF

U)

3 5

2 2

2 7

1 2

M ito S O A R

R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n

MitoSOARtrade (RFU)

lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects

N 10 27 19 3086

12 31 22 35

57 of the tested population has

superoxide levels in the

elevatedhigh range leading to high

risk for certain clinical conditions

Functional Genetics

CT

CC

0 5

1 0

1 5

2 0

2 5

Mit

oS

OA

R (

RF

U)

S O D 2 H e tro z y g o u s g e n o ty p e (C T )

S O D 2 H o m o z y g o u s g e n o ty p e (C C )

P lt 0 0 5

M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e

(S O D 2 ) P o ly m o rp h is m

Individuals who are homozygous

recessive(CC) for SOD2 may

have impaired SOD2 function

resulting in higher levels of the

free radical superoxide and

require antioxidant support

Mitochondrial Superoxide Levels

Assessment of Redox status

Inflammation

Viral or Bacterial Infection

Monitoring treatment of a

disease or disorder

Borrelia infection

Treatment of Inflammation

Functional Effect

Improvement verifies positive clinical effect

Plasma Peroxide Assay

Low Levels may indicate oxidative stress and

possible need for antioxidants

High levels may indicate immune challenges

Can correlate with uric acid

Potential hypertension risk

Related to lipid peroxides

Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13

Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303

Plasma Peroxidase

To

tal S

ub

jects

(n

=86)

Lo

w (

n=30)

No

rmal (n

=24)

Ele

vate

d (

n=11)

Hig

h (

n=16)

0

2 0

4 0

6 0

8 0

Pla

sm

a P

erix

ida

se

Ac

tiv

ity

(m

Um

l)

2 0

1 4

3 0

3 7

M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s

R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e

S a m p le D is tr ib u tio n

Plasma Peroxidase Activity (mUmL)

lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects

N 30 24 11 1681

37 30 14 20

37 of the population studied had low

peroxidase activity indicating an

impaired ability to defend against free

radicals

34 of the studies population had

elevatedhigh activity potentially

indicating the presence of oxidative

stress

Assessment of Baseline Potential

Starting point for defense of excessive

ROS

Effect of Environmental Insults

Assessment of Capacity to defend against

excessive ROS

Assessment and monitoring treatment of a

disease or disorder

Viral or Bacterial infection

Cardiovascular disease

Mitochondrial Membrane Potential

Oxidative Challenge

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Waiting on a

Breakthrough

ldquoWe are facing a crisisrdquo the Cornell psychiatrist and New York Times contributor Richard Friedman warned last week In the past few years one pharmaceutical giant after anothermdashGlaxoSmithKline AstraZeneca Novartis Pfizer Merck Sanofimdashhas shrunk or shuttered its neuroscience research facilities

Clinical trials have been halted lines of research abandoned and the new drug pipeline has been allowed to run dry

The New Yorker Posted as of 22Feb 2016

SEPTEMBER 3 2013The Psychiatric Drug Crisis

BY GARY GREENBERG

Fatigue

Fatigue Fatigue is a common complaint with a

prevalence in population-based surveys in Britain

and the United States of between 60 and 75

percent

A cross-sectional survey of United States workers

found the two-week period prevalence of fatigue

to be 38 percent with an estimated annual cost

to employers exceeding $136 billion in lost

productive work time

In addition an estimated 21 to 33 percent of

patients seeking attention in primary care settings

report significant fatigue resulting in

approximately seven million office visits per year in

the United States

Lawrie SM Manders DN Geddes JR Pelosi AJ A population-based incidence study of chronic fatigue Psychol Med 1997 27343Walker EA Katon WJ Jemelka RP Psychiatric disorders and medical care utilization among people in the general population who report fatigue J Gen Intern Med 1993 8436 Ricci JA Chee E Lorandeau AL Berger J Fatigue in the US workforce prevalence and implications for lost productive work time J Occup Environ Med 2007 491

Fatigue

Mayo

Treatment includes

antidepressants

sleeping pills

Pace yourself

Graded Exercise

Psychological counseling

httpwwwmayoclinicorgdiseases-conditionschronic-fatigue-syndromebasicstreatmentcon-20022009 Posted as of Sept 1 20017

Insomnia

httpswwwresearchgatenetfigure26679987_fig1_Fig-1-Prevalence-of-insomnia-symptoms-by-age-and-gender

As reported Sept 1 2017

What Americans Use to Sleep Nearly 9 million US adults resort to prescription sleeping pills mdash and most

are white female educated or 50 or older according to the first

government study of its kind

The CDC estimates that ldquo50-70 million US adults have sleep or

wakefulness disorderrdquo These numbers are striking because they

essentially mean that for every adult with a sleep disorder a doctor is

writing a prescription for sleeping pills

Between 2006 and 2011 sleeping pill prescriptions rose from 47 million to

60 million in the US About 38 million of those prescriptions were for drugs

containing zolpidem (including the brand name Ambien) according

to IMS Health

The United States insomnia pharmacological treatment market is

projected to reach $424 billion by 2021 from $338 billion in 2016

httpwwwnydailynewscomlife-stylehealthcdc-9-million-americans-sleeping-pills-article-11441778

Mood Fatigue Insomnia Theory

Mood Insomnia and Fatigue Issues are

inflammatory issues which are measurable

and reversible even with a poor genomic

outlook

-Decker Weiss

The Three Stooges of Mood Fatigue

and Insomnia

Inflammation causing Neurotransmitter Dysfunction

Inflammation toxicity exposure to free radicals causes Mitochondrial Dysfunction decreasing sex hormone production and energy an defenses

Gut BiomeFood Allergies generates and perpetuates inflammation immune dysfunction and further decreases in neurotransmitter dysfunction

DepressionInsomniaFatigue

GlutamatergicExcess

Many Causes Similar Result

NoradrenergicDeficiency

SerotonergicDeficiency

Belmaker R H and Agam G Major depressive

disorder (1-3-2008) NEnglJMed 358(1) 55-

68

GutBrainDysfunction

Mitochondrial Dysfunction

18

Neurotransmitters

PVN

C1

LC

Pituitary

DR

Thoracic

Lumbar

Cortisol

DHEAEpinephrine

Norepinephrine

CRF

ACTH

Traditional Stress Response

Hypothalamus is the master controller of the HPA axis (endocrine model)

Traditionally the start of the HPA axis

Pituitary gland has two parts that bridge the brain and body

Anterior (adenohypophysis) releases ACTH TSH PRL GH endorphins FSH LH CRF

Posterior (neurohypophysis) releases oxytocin and antidiuretic hormone

Two distinct parts of the adrenal gland produce hormones and neurotransmitters

Adrenal Cortex = cortisol and DHEA

Adrenal Medulla = norepinephrine and epinephrine

Engelmann M et al (2004) Front Neuroendocrinol 25(3-4)132-149Sherwood L (2010) Human Physiology From Cells to Systems 7th ed 698-709

Are you

considering

the adrenal

medulla

20

PVN

C1

LC

Hypothalamus

Pituitary

DR

Cortex

Medulla

Thoracic

Lumbar

CRF

NE

Cortisol

Glutamate

Epinephrine

ACh

Glutamate activates Thoracic pathway to

Adrenals

Sympathetic Chain

Serotonin

21

The ldquoBackward RuleMatch Diagnosis to Labs

Neurotransmitter Subject 1 Subject 2 Subject 3

Serotonin

GABA

Glycine

Glutamate

Histamine

PEA

Dopamine

Norepinephrine

Epinephrine

Cortisol

22

Biomarkers for Early Stage 2

3

23

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Biomarkers for Mid Stage

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Biomarkers for End Stage

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Genomics

COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of

the brain responsible for personality inhibition of behaviors abstract thinking

and emotion1

The COMT val158Met polymorphism has been shown to be associated with phobic

anxiety 2 neuroticism 3 and panic disorders 4

Results indicate a relationship of COMT polymorphism rs4680 with immune

dysregulation in CFS providing a potential link for the association between stress and

infection susceptibility in CFS(chronic fatigue syndrome)

1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype

on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J

Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8

27

17 yo radicalized female with anger

antisocial and rage issues(similar not exact due to security)

Methylene-tetrahydrofolate reductase

(MTHFR SNPrsquos)

Creates methylated folate

the active folate in the

body

Mutations have been

associated with vascular

disease dementia cancer

and autism

Methylation is the genetics

elevated homocysteine is

the genomics 29

End Point of Poor Methylation and

Oxidation

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

bull Superoxide dismutase 1

(SOD1)

bull Superoxide dismutase 2

(SOD2)

bull Superoxide dismutase 3

(SOD3)

bull Glutathione peroxidase 1

(GPx1)

bull Catalase (CAT)

Superoxide Dismutase 1

SOD1 (CuZnSOD)(Encoded Chrom 21)

Found in cytoplasmic and mitochondrial

intermembrane space(inserted)

Converts superoxide radicals (O2-) to hydrogen

peroxide (H2O2)

Needs copper and zinc as cofactors

Apoptosis Oxidative Stress ALS Myocardial

Infarction Macular Degeneration a marker for

heavy Metal toxicity

O2- H2O2

SOD1SOD1

Cu Zn

1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2

2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160

3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034

Superoxide Dismutase 2

SOD2 (MnSOD) (Chrom 6)

Found in mitochondria

Converts superoxide radicals (O2-)

to hydrogen peroxide (H2O2)

Needs manganese as a cofactor

Heart Disease Myocardial

Infarction Tumor metastasis

neurodegenerative diseases

O2- H2O2SOD2SOD2

Mn

Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24

Superoxide Dismutase 3SOD3

Found extracellularly

Converts superoxide radicals (O2-) to

hydrogen peroxide (H2O2)

Needs copper and zinc as cofactors

Chronic skin disorders Diabetic

Neuropathy Folate Metabolism Ischemic

Heart Disease

O2- H2O2SOD3SOD3

Cu Zn

Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004

Glutathione Peroxidase 1

GPx1(Chrom 3)

Found in the cytoplasm of nearly all mammalian tissues

Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)

Uses glutathione an antioxidant as a cofactor

Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression

H2O2H2O O2GPx1GPx1

Glutathione

Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and

Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89

Catalase

CAT(Chrom 11)

Found in the cytoplasm of cells

Needs iron and selenium as cofactors

Reduce hydrogen peroxide (H2O2) to water (H2O)

High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second

Hypertension Type 2 Diabetes Acatalasemia

httpsghrnlmnihgovgeneCATconditions

H2O2 H2O O2CATCAT

Fe Se

Mitochondrial Function Assessment

Mitochondrial Superoxide (O2-)

Plasma Peroxide Assay (PPA)

Mitochondrial Membrane

Potential (MMP) Assay with

Oxidative Challenge

Mitochondrial Superoxide (O2-)

Superoxide is considered to be a major

factor in oxidant toxicity and

mitochondrial MnSOD enzymes

constitute an essential defense against

superoxide

High levels indicate the proanti-oxidant

system is out of balance and oxidative

stress is present

Mitochondria are the major source of

superoxide

Superoxide is the origin of reactive

oxygen (ROS)and nitrogen species (RNS)

and as such causes various redox

related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo

123Hsiu-Chuan Yen

4Ikuo Nakanishi

5Ken-ichiro Matsumoto

5Masato Tamura

6Yumiko Nagano

6Hirofumi Matsui

6Oleg Gusev

789Richard Cornette

9Takashi

Okuda9

Yukiko Minamiyama10

Hiroshi Ichikawa11

Shigeaki Suenaga1

Misato Oki2

Tsuyoshi Sato1

Toshihiko Ozawa12

Daret K St Clair3

and Hideyuki J Majima12dagger

Retrospective Study Sample Distribution

To

tal S

ub

jects

(n

=86)

Lo

w (

n=10)

No

rmal (n

=27)

Ele

vate

d (

n=19)

Hig

h (

n=30)

0

1

2

3

4

5

Mit

oS

OA

R (

RF

U)

3 5

2 2

2 7

1 2

M ito S O A R

R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n

MitoSOARtrade (RFU)

lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects

N 10 27 19 3086

12 31 22 35

57 of the tested population has

superoxide levels in the

elevatedhigh range leading to high

risk for certain clinical conditions

Functional Genetics

CT

CC

0 5

1 0

1 5

2 0

2 5

Mit

oS

OA

R (

RF

U)

S O D 2 H e tro z y g o u s g e n o ty p e (C T )

S O D 2 H o m o z y g o u s g e n o ty p e (C C )

P lt 0 0 5

M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e

(S O D 2 ) P o ly m o rp h is m

Individuals who are homozygous

recessive(CC) for SOD2 may

have impaired SOD2 function

resulting in higher levels of the

free radical superoxide and

require antioxidant support

Mitochondrial Superoxide Levels

Assessment of Redox status

Inflammation

Viral or Bacterial Infection

Monitoring treatment of a

disease or disorder

Borrelia infection

Treatment of Inflammation

Functional Effect

Improvement verifies positive clinical effect

Plasma Peroxide Assay

Low Levels may indicate oxidative stress and

possible need for antioxidants

High levels may indicate immune challenges

Can correlate with uric acid

Potential hypertension risk

Related to lipid peroxides

Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13

Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303

Plasma Peroxidase

To

tal S

ub

jects

(n

=86)

Lo

w (

n=30)

No

rmal (n

=24)

Ele

vate

d (

n=11)

Hig

h (

n=16)

0

2 0

4 0

6 0

8 0

Pla

sm

a P

erix

ida

se

Ac

tiv

ity

(m

Um

l)

2 0

1 4

3 0

3 7

M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s

R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e

S a m p le D is tr ib u tio n

Plasma Peroxidase Activity (mUmL)

lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects

N 30 24 11 1681

37 30 14 20

37 of the population studied had low

peroxidase activity indicating an

impaired ability to defend against free

radicals

34 of the studies population had

elevatedhigh activity potentially

indicating the presence of oxidative

stress

Assessment of Baseline Potential

Starting point for defense of excessive

ROS

Effect of Environmental Insults

Assessment of Capacity to defend against

excessive ROS

Assessment and monitoring treatment of a

disease or disorder

Viral or Bacterial infection

Cardiovascular disease

Mitochondrial Membrane Potential

Oxidative Challenge

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Fatigue

Fatigue Fatigue is a common complaint with a

prevalence in population-based surveys in Britain

and the United States of between 60 and 75

percent

A cross-sectional survey of United States workers

found the two-week period prevalence of fatigue

to be 38 percent with an estimated annual cost

to employers exceeding $136 billion in lost

productive work time

In addition an estimated 21 to 33 percent of

patients seeking attention in primary care settings

report significant fatigue resulting in

approximately seven million office visits per year in

the United States

Lawrie SM Manders DN Geddes JR Pelosi AJ A population-based incidence study of chronic fatigue Psychol Med 1997 27343Walker EA Katon WJ Jemelka RP Psychiatric disorders and medical care utilization among people in the general population who report fatigue J Gen Intern Med 1993 8436 Ricci JA Chee E Lorandeau AL Berger J Fatigue in the US workforce prevalence and implications for lost productive work time J Occup Environ Med 2007 491

Fatigue

Mayo

Treatment includes

antidepressants

sleeping pills

Pace yourself

Graded Exercise

Psychological counseling

httpwwwmayoclinicorgdiseases-conditionschronic-fatigue-syndromebasicstreatmentcon-20022009 Posted as of Sept 1 20017

Insomnia

httpswwwresearchgatenetfigure26679987_fig1_Fig-1-Prevalence-of-insomnia-symptoms-by-age-and-gender

As reported Sept 1 2017

What Americans Use to Sleep Nearly 9 million US adults resort to prescription sleeping pills mdash and most

are white female educated or 50 or older according to the first

government study of its kind

The CDC estimates that ldquo50-70 million US adults have sleep or

wakefulness disorderrdquo These numbers are striking because they

essentially mean that for every adult with a sleep disorder a doctor is

writing a prescription for sleeping pills

Between 2006 and 2011 sleeping pill prescriptions rose from 47 million to

60 million in the US About 38 million of those prescriptions were for drugs

containing zolpidem (including the brand name Ambien) according

to IMS Health

The United States insomnia pharmacological treatment market is

projected to reach $424 billion by 2021 from $338 billion in 2016

httpwwwnydailynewscomlife-stylehealthcdc-9-million-americans-sleeping-pills-article-11441778

Mood Fatigue Insomnia Theory

Mood Insomnia and Fatigue Issues are

inflammatory issues which are measurable

and reversible even with a poor genomic

outlook

-Decker Weiss

The Three Stooges of Mood Fatigue

and Insomnia

Inflammation causing Neurotransmitter Dysfunction

Inflammation toxicity exposure to free radicals causes Mitochondrial Dysfunction decreasing sex hormone production and energy an defenses

Gut BiomeFood Allergies generates and perpetuates inflammation immune dysfunction and further decreases in neurotransmitter dysfunction

DepressionInsomniaFatigue

GlutamatergicExcess

Many Causes Similar Result

NoradrenergicDeficiency

SerotonergicDeficiency

Belmaker R H and Agam G Major depressive

disorder (1-3-2008) NEnglJMed 358(1) 55-

68

GutBrainDysfunction

Mitochondrial Dysfunction

18

Neurotransmitters

PVN

C1

LC

Pituitary

DR

Thoracic

Lumbar

Cortisol

DHEAEpinephrine

Norepinephrine

CRF

ACTH

Traditional Stress Response

Hypothalamus is the master controller of the HPA axis (endocrine model)

Traditionally the start of the HPA axis

Pituitary gland has two parts that bridge the brain and body

Anterior (adenohypophysis) releases ACTH TSH PRL GH endorphins FSH LH CRF

Posterior (neurohypophysis) releases oxytocin and antidiuretic hormone

Two distinct parts of the adrenal gland produce hormones and neurotransmitters

Adrenal Cortex = cortisol and DHEA

Adrenal Medulla = norepinephrine and epinephrine

Engelmann M et al (2004) Front Neuroendocrinol 25(3-4)132-149Sherwood L (2010) Human Physiology From Cells to Systems 7th ed 698-709

Are you

considering

the adrenal

medulla

20

PVN

C1

LC

Hypothalamus

Pituitary

DR

Cortex

Medulla

Thoracic

Lumbar

CRF

NE

Cortisol

Glutamate

Epinephrine

ACh

Glutamate activates Thoracic pathway to

Adrenals

Sympathetic Chain

Serotonin

21

The ldquoBackward RuleMatch Diagnosis to Labs

Neurotransmitter Subject 1 Subject 2 Subject 3

Serotonin

GABA

Glycine

Glutamate

Histamine

PEA

Dopamine

Norepinephrine

Epinephrine

Cortisol

22

Biomarkers for Early Stage 2

3

23

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Biomarkers for Mid Stage

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Biomarkers for End Stage

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Genomics

COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of

the brain responsible for personality inhibition of behaviors abstract thinking

and emotion1

The COMT val158Met polymorphism has been shown to be associated with phobic

anxiety 2 neuroticism 3 and panic disorders 4

Results indicate a relationship of COMT polymorphism rs4680 with immune

dysregulation in CFS providing a potential link for the association between stress and

infection susceptibility in CFS(chronic fatigue syndrome)

1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype

on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J

Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8

27

17 yo radicalized female with anger

antisocial and rage issues(similar not exact due to security)

Methylene-tetrahydrofolate reductase

(MTHFR SNPrsquos)

Creates methylated folate

the active folate in the

body

Mutations have been

associated with vascular

disease dementia cancer

and autism

Methylation is the genetics

elevated homocysteine is

the genomics 29

End Point of Poor Methylation and

Oxidation

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

bull Superoxide dismutase 1

(SOD1)

bull Superoxide dismutase 2

(SOD2)

bull Superoxide dismutase 3

(SOD3)

bull Glutathione peroxidase 1

(GPx1)

bull Catalase (CAT)

Superoxide Dismutase 1

SOD1 (CuZnSOD)(Encoded Chrom 21)

Found in cytoplasmic and mitochondrial

intermembrane space(inserted)

Converts superoxide radicals (O2-) to hydrogen

peroxide (H2O2)

Needs copper and zinc as cofactors

Apoptosis Oxidative Stress ALS Myocardial

Infarction Macular Degeneration a marker for

heavy Metal toxicity

O2- H2O2

SOD1SOD1

Cu Zn

1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2

2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160

3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034

Superoxide Dismutase 2

SOD2 (MnSOD) (Chrom 6)

Found in mitochondria

Converts superoxide radicals (O2-)

to hydrogen peroxide (H2O2)

Needs manganese as a cofactor

Heart Disease Myocardial

Infarction Tumor metastasis

neurodegenerative diseases

O2- H2O2SOD2SOD2

Mn

Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24

Superoxide Dismutase 3SOD3

Found extracellularly

Converts superoxide radicals (O2-) to

hydrogen peroxide (H2O2)

Needs copper and zinc as cofactors

Chronic skin disorders Diabetic

Neuropathy Folate Metabolism Ischemic

Heart Disease

O2- H2O2SOD3SOD3

Cu Zn

Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004

Glutathione Peroxidase 1

GPx1(Chrom 3)

Found in the cytoplasm of nearly all mammalian tissues

Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)

Uses glutathione an antioxidant as a cofactor

Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression

H2O2H2O O2GPx1GPx1

Glutathione

Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and

Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89

Catalase

CAT(Chrom 11)

Found in the cytoplasm of cells

Needs iron and selenium as cofactors

Reduce hydrogen peroxide (H2O2) to water (H2O)

High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second

Hypertension Type 2 Diabetes Acatalasemia

httpsghrnlmnihgovgeneCATconditions

H2O2 H2O O2CATCAT

Fe Se

Mitochondrial Function Assessment

Mitochondrial Superoxide (O2-)

Plasma Peroxide Assay (PPA)

Mitochondrial Membrane

Potential (MMP) Assay with

Oxidative Challenge

Mitochondrial Superoxide (O2-)

Superoxide is considered to be a major

factor in oxidant toxicity and

mitochondrial MnSOD enzymes

constitute an essential defense against

superoxide

High levels indicate the proanti-oxidant

system is out of balance and oxidative

stress is present

Mitochondria are the major source of

superoxide

Superoxide is the origin of reactive

oxygen (ROS)and nitrogen species (RNS)

and as such causes various redox

related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo

123Hsiu-Chuan Yen

4Ikuo Nakanishi

5Ken-ichiro Matsumoto

5Masato Tamura

6Yumiko Nagano

6Hirofumi Matsui

6Oleg Gusev

789Richard Cornette

9Takashi

Okuda9

Yukiko Minamiyama10

Hiroshi Ichikawa11

Shigeaki Suenaga1

Misato Oki2

Tsuyoshi Sato1

Toshihiko Ozawa12

Daret K St Clair3

and Hideyuki J Majima12dagger

Retrospective Study Sample Distribution

To

tal S

ub

jects

(n

=86)

Lo

w (

n=10)

No

rmal (n

=27)

Ele

vate

d (

n=19)

Hig

h (

n=30)

0

1

2

3

4

5

Mit

oS

OA

R (

RF

U)

3 5

2 2

2 7

1 2

M ito S O A R

R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n

MitoSOARtrade (RFU)

lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects

N 10 27 19 3086

12 31 22 35

57 of the tested population has

superoxide levels in the

elevatedhigh range leading to high

risk for certain clinical conditions

Functional Genetics

CT

CC

0 5

1 0

1 5

2 0

2 5

Mit

oS

OA

R (

RF

U)

S O D 2 H e tro z y g o u s g e n o ty p e (C T )

S O D 2 H o m o z y g o u s g e n o ty p e (C C )

P lt 0 0 5

M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e

(S O D 2 ) P o ly m o rp h is m

Individuals who are homozygous

recessive(CC) for SOD2 may

have impaired SOD2 function

resulting in higher levels of the

free radical superoxide and

require antioxidant support

Mitochondrial Superoxide Levels

Assessment of Redox status

Inflammation

Viral or Bacterial Infection

Monitoring treatment of a

disease or disorder

Borrelia infection

Treatment of Inflammation

Functional Effect

Improvement verifies positive clinical effect

Plasma Peroxide Assay

Low Levels may indicate oxidative stress and

possible need for antioxidants

High levels may indicate immune challenges

Can correlate with uric acid

Potential hypertension risk

Related to lipid peroxides

Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13

Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303

Plasma Peroxidase

To

tal S

ub

jects

(n

=86)

Lo

w (

n=30)

No

rmal (n

=24)

Ele

vate

d (

n=11)

Hig

h (

n=16)

0

2 0

4 0

6 0

8 0

Pla

sm

a P

erix

ida

se

Ac

tiv

ity

(m

Um

l)

2 0

1 4

3 0

3 7

M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s

R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e

S a m p le D is tr ib u tio n

Plasma Peroxidase Activity (mUmL)

lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects

N 30 24 11 1681

37 30 14 20

37 of the population studied had low

peroxidase activity indicating an

impaired ability to defend against free

radicals

34 of the studies population had

elevatedhigh activity potentially

indicating the presence of oxidative

stress

Assessment of Baseline Potential

Starting point for defense of excessive

ROS

Effect of Environmental Insults

Assessment of Capacity to defend against

excessive ROS

Assessment and monitoring treatment of a

disease or disorder

Viral or Bacterial infection

Cardiovascular disease

Mitochondrial Membrane Potential

Oxidative Challenge

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Fatigue Fatigue is a common complaint with a

prevalence in population-based surveys in Britain

and the United States of between 60 and 75

percent

A cross-sectional survey of United States workers

found the two-week period prevalence of fatigue

to be 38 percent with an estimated annual cost

to employers exceeding $136 billion in lost

productive work time

In addition an estimated 21 to 33 percent of

patients seeking attention in primary care settings

report significant fatigue resulting in

approximately seven million office visits per year in

the United States

Lawrie SM Manders DN Geddes JR Pelosi AJ A population-based incidence study of chronic fatigue Psychol Med 1997 27343Walker EA Katon WJ Jemelka RP Psychiatric disorders and medical care utilization among people in the general population who report fatigue J Gen Intern Med 1993 8436 Ricci JA Chee E Lorandeau AL Berger J Fatigue in the US workforce prevalence and implications for lost productive work time J Occup Environ Med 2007 491

Fatigue

Mayo

Treatment includes

antidepressants

sleeping pills

Pace yourself

Graded Exercise

Psychological counseling

httpwwwmayoclinicorgdiseases-conditionschronic-fatigue-syndromebasicstreatmentcon-20022009 Posted as of Sept 1 20017

Insomnia

httpswwwresearchgatenetfigure26679987_fig1_Fig-1-Prevalence-of-insomnia-symptoms-by-age-and-gender

As reported Sept 1 2017

What Americans Use to Sleep Nearly 9 million US adults resort to prescription sleeping pills mdash and most

are white female educated or 50 or older according to the first

government study of its kind

The CDC estimates that ldquo50-70 million US adults have sleep or

wakefulness disorderrdquo These numbers are striking because they

essentially mean that for every adult with a sleep disorder a doctor is

writing a prescription for sleeping pills

Between 2006 and 2011 sleeping pill prescriptions rose from 47 million to

60 million in the US About 38 million of those prescriptions were for drugs

containing zolpidem (including the brand name Ambien) according

to IMS Health

The United States insomnia pharmacological treatment market is

projected to reach $424 billion by 2021 from $338 billion in 2016

httpwwwnydailynewscomlife-stylehealthcdc-9-million-americans-sleeping-pills-article-11441778

Mood Fatigue Insomnia Theory

Mood Insomnia and Fatigue Issues are

inflammatory issues which are measurable

and reversible even with a poor genomic

outlook

-Decker Weiss

The Three Stooges of Mood Fatigue

and Insomnia

Inflammation causing Neurotransmitter Dysfunction

Inflammation toxicity exposure to free radicals causes Mitochondrial Dysfunction decreasing sex hormone production and energy an defenses

Gut BiomeFood Allergies generates and perpetuates inflammation immune dysfunction and further decreases in neurotransmitter dysfunction

DepressionInsomniaFatigue

GlutamatergicExcess

Many Causes Similar Result

NoradrenergicDeficiency

SerotonergicDeficiency

Belmaker R H and Agam G Major depressive

disorder (1-3-2008) NEnglJMed 358(1) 55-

68

GutBrainDysfunction

Mitochondrial Dysfunction

18

Neurotransmitters

PVN

C1

LC

Pituitary

DR

Thoracic

Lumbar

Cortisol

DHEAEpinephrine

Norepinephrine

CRF

ACTH

Traditional Stress Response

Hypothalamus is the master controller of the HPA axis (endocrine model)

Traditionally the start of the HPA axis

Pituitary gland has two parts that bridge the brain and body

Anterior (adenohypophysis) releases ACTH TSH PRL GH endorphins FSH LH CRF

Posterior (neurohypophysis) releases oxytocin and antidiuretic hormone

Two distinct parts of the adrenal gland produce hormones and neurotransmitters

Adrenal Cortex = cortisol and DHEA

Adrenal Medulla = norepinephrine and epinephrine

Engelmann M et al (2004) Front Neuroendocrinol 25(3-4)132-149Sherwood L (2010) Human Physiology From Cells to Systems 7th ed 698-709

Are you

considering

the adrenal

medulla

20

PVN

C1

LC

Hypothalamus

Pituitary

DR

Cortex

Medulla

Thoracic

Lumbar

CRF

NE

Cortisol

Glutamate

Epinephrine

ACh

Glutamate activates Thoracic pathway to

Adrenals

Sympathetic Chain

Serotonin

21

The ldquoBackward RuleMatch Diagnosis to Labs

Neurotransmitter Subject 1 Subject 2 Subject 3

Serotonin

GABA

Glycine

Glutamate

Histamine

PEA

Dopamine

Norepinephrine

Epinephrine

Cortisol

22

Biomarkers for Early Stage 2

3

23

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Biomarkers for Mid Stage

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Biomarkers for End Stage

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Genomics

COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of

the brain responsible for personality inhibition of behaviors abstract thinking

and emotion1

The COMT val158Met polymorphism has been shown to be associated with phobic

anxiety 2 neuroticism 3 and panic disorders 4

Results indicate a relationship of COMT polymorphism rs4680 with immune

dysregulation in CFS providing a potential link for the association between stress and

infection susceptibility in CFS(chronic fatigue syndrome)

1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype

on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J

Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8

27

17 yo radicalized female with anger

antisocial and rage issues(similar not exact due to security)

Methylene-tetrahydrofolate reductase

(MTHFR SNPrsquos)

Creates methylated folate

the active folate in the

body

Mutations have been

associated with vascular

disease dementia cancer

and autism

Methylation is the genetics

elevated homocysteine is

the genomics 29

End Point of Poor Methylation and

Oxidation

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

bull Superoxide dismutase 1

(SOD1)

bull Superoxide dismutase 2

(SOD2)

bull Superoxide dismutase 3

(SOD3)

bull Glutathione peroxidase 1

(GPx1)

bull Catalase (CAT)

Superoxide Dismutase 1

SOD1 (CuZnSOD)(Encoded Chrom 21)

Found in cytoplasmic and mitochondrial

intermembrane space(inserted)

Converts superoxide radicals (O2-) to hydrogen

peroxide (H2O2)

Needs copper and zinc as cofactors

Apoptosis Oxidative Stress ALS Myocardial

Infarction Macular Degeneration a marker for

heavy Metal toxicity

O2- H2O2

SOD1SOD1

Cu Zn

1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2

2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160

3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034

Superoxide Dismutase 2

SOD2 (MnSOD) (Chrom 6)

Found in mitochondria

Converts superoxide radicals (O2-)

to hydrogen peroxide (H2O2)

Needs manganese as a cofactor

Heart Disease Myocardial

Infarction Tumor metastasis

neurodegenerative diseases

O2- H2O2SOD2SOD2

Mn

Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24

Superoxide Dismutase 3SOD3

Found extracellularly

Converts superoxide radicals (O2-) to

hydrogen peroxide (H2O2)

Needs copper and zinc as cofactors

Chronic skin disorders Diabetic

Neuropathy Folate Metabolism Ischemic

Heart Disease

O2- H2O2SOD3SOD3

Cu Zn

Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004

Glutathione Peroxidase 1

GPx1(Chrom 3)

Found in the cytoplasm of nearly all mammalian tissues

Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)

Uses glutathione an antioxidant as a cofactor

Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression

H2O2H2O O2GPx1GPx1

Glutathione

Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and

Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89

Catalase

CAT(Chrom 11)

Found in the cytoplasm of cells

Needs iron and selenium as cofactors

Reduce hydrogen peroxide (H2O2) to water (H2O)

High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second

Hypertension Type 2 Diabetes Acatalasemia

httpsghrnlmnihgovgeneCATconditions

H2O2 H2O O2CATCAT

Fe Se

Mitochondrial Function Assessment

Mitochondrial Superoxide (O2-)

Plasma Peroxide Assay (PPA)

Mitochondrial Membrane

Potential (MMP) Assay with

Oxidative Challenge

Mitochondrial Superoxide (O2-)

Superoxide is considered to be a major

factor in oxidant toxicity and

mitochondrial MnSOD enzymes

constitute an essential defense against

superoxide

High levels indicate the proanti-oxidant

system is out of balance and oxidative

stress is present

Mitochondria are the major source of

superoxide

Superoxide is the origin of reactive

oxygen (ROS)and nitrogen species (RNS)

and as such causes various redox

related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo

123Hsiu-Chuan Yen

4Ikuo Nakanishi

5Ken-ichiro Matsumoto

5Masato Tamura

6Yumiko Nagano

6Hirofumi Matsui

6Oleg Gusev

789Richard Cornette

9Takashi

Okuda9

Yukiko Minamiyama10

Hiroshi Ichikawa11

Shigeaki Suenaga1

Misato Oki2

Tsuyoshi Sato1

Toshihiko Ozawa12

Daret K St Clair3

and Hideyuki J Majima12dagger

Retrospective Study Sample Distribution

To

tal S

ub

jects

(n

=86)

Lo

w (

n=10)

No

rmal (n

=27)

Ele

vate

d (

n=19)

Hig

h (

n=30)

0

1

2

3

4

5

Mit

oS

OA

R (

RF

U)

3 5

2 2

2 7

1 2

M ito S O A R

R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n

MitoSOARtrade (RFU)

lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects

N 10 27 19 3086

12 31 22 35

57 of the tested population has

superoxide levels in the

elevatedhigh range leading to high

risk for certain clinical conditions

Functional Genetics

CT

CC

0 5

1 0

1 5

2 0

2 5

Mit

oS

OA

R (

RF

U)

S O D 2 H e tro z y g o u s g e n o ty p e (C T )

S O D 2 H o m o z y g o u s g e n o ty p e (C C )

P lt 0 0 5

M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e

(S O D 2 ) P o ly m o rp h is m

Individuals who are homozygous

recessive(CC) for SOD2 may

have impaired SOD2 function

resulting in higher levels of the

free radical superoxide and

require antioxidant support

Mitochondrial Superoxide Levels

Assessment of Redox status

Inflammation

Viral or Bacterial Infection

Monitoring treatment of a

disease or disorder

Borrelia infection

Treatment of Inflammation

Functional Effect

Improvement verifies positive clinical effect

Plasma Peroxide Assay

Low Levels may indicate oxidative stress and

possible need for antioxidants

High levels may indicate immune challenges

Can correlate with uric acid

Potential hypertension risk

Related to lipid peroxides

Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13

Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303

Plasma Peroxidase

To

tal S

ub

jects

(n

=86)

Lo

w (

n=30)

No

rmal (n

=24)

Ele

vate

d (

n=11)

Hig

h (

n=16)

0

2 0

4 0

6 0

8 0

Pla

sm

a P

erix

ida

se

Ac

tiv

ity

(m

Um

l)

2 0

1 4

3 0

3 7

M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s

R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e

S a m p le D is tr ib u tio n

Plasma Peroxidase Activity (mUmL)

lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects

N 30 24 11 1681

37 30 14 20

37 of the population studied had low

peroxidase activity indicating an

impaired ability to defend against free

radicals

34 of the studies population had

elevatedhigh activity potentially

indicating the presence of oxidative

stress

Assessment of Baseline Potential

Starting point for defense of excessive

ROS

Effect of Environmental Insults

Assessment of Capacity to defend against

excessive ROS

Assessment and monitoring treatment of a

disease or disorder

Viral or Bacterial infection

Cardiovascular disease

Mitochondrial Membrane Potential

Oxidative Challenge

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Fatigue

Mayo

Treatment includes

antidepressants

sleeping pills

Pace yourself

Graded Exercise

Psychological counseling

httpwwwmayoclinicorgdiseases-conditionschronic-fatigue-syndromebasicstreatmentcon-20022009 Posted as of Sept 1 20017

Insomnia

httpswwwresearchgatenetfigure26679987_fig1_Fig-1-Prevalence-of-insomnia-symptoms-by-age-and-gender

As reported Sept 1 2017

What Americans Use to Sleep Nearly 9 million US adults resort to prescription sleeping pills mdash and most

are white female educated or 50 or older according to the first

government study of its kind

The CDC estimates that ldquo50-70 million US adults have sleep or

wakefulness disorderrdquo These numbers are striking because they

essentially mean that for every adult with a sleep disorder a doctor is

writing a prescription for sleeping pills

Between 2006 and 2011 sleeping pill prescriptions rose from 47 million to

60 million in the US About 38 million of those prescriptions were for drugs

containing zolpidem (including the brand name Ambien) according

to IMS Health

The United States insomnia pharmacological treatment market is

projected to reach $424 billion by 2021 from $338 billion in 2016

httpwwwnydailynewscomlife-stylehealthcdc-9-million-americans-sleeping-pills-article-11441778

Mood Fatigue Insomnia Theory

Mood Insomnia and Fatigue Issues are

inflammatory issues which are measurable

and reversible even with a poor genomic

outlook

-Decker Weiss

The Three Stooges of Mood Fatigue

and Insomnia

Inflammation causing Neurotransmitter Dysfunction

Inflammation toxicity exposure to free radicals causes Mitochondrial Dysfunction decreasing sex hormone production and energy an defenses

Gut BiomeFood Allergies generates and perpetuates inflammation immune dysfunction and further decreases in neurotransmitter dysfunction

DepressionInsomniaFatigue

GlutamatergicExcess

Many Causes Similar Result

NoradrenergicDeficiency

SerotonergicDeficiency

Belmaker R H and Agam G Major depressive

disorder (1-3-2008) NEnglJMed 358(1) 55-

68

GutBrainDysfunction

Mitochondrial Dysfunction

18

Neurotransmitters

PVN

C1

LC

Pituitary

DR

Thoracic

Lumbar

Cortisol

DHEAEpinephrine

Norepinephrine

CRF

ACTH

Traditional Stress Response

Hypothalamus is the master controller of the HPA axis (endocrine model)

Traditionally the start of the HPA axis

Pituitary gland has two parts that bridge the brain and body

Anterior (adenohypophysis) releases ACTH TSH PRL GH endorphins FSH LH CRF

Posterior (neurohypophysis) releases oxytocin and antidiuretic hormone

Two distinct parts of the adrenal gland produce hormones and neurotransmitters

Adrenal Cortex = cortisol and DHEA

Adrenal Medulla = norepinephrine and epinephrine

Engelmann M et al (2004) Front Neuroendocrinol 25(3-4)132-149Sherwood L (2010) Human Physiology From Cells to Systems 7th ed 698-709

Are you

considering

the adrenal

medulla

20

PVN

C1

LC

Hypothalamus

Pituitary

DR

Cortex

Medulla

Thoracic

Lumbar

CRF

NE

Cortisol

Glutamate

Epinephrine

ACh

Glutamate activates Thoracic pathway to

Adrenals

Sympathetic Chain

Serotonin

21

The ldquoBackward RuleMatch Diagnosis to Labs

Neurotransmitter Subject 1 Subject 2 Subject 3

Serotonin

GABA

Glycine

Glutamate

Histamine

PEA

Dopamine

Norepinephrine

Epinephrine

Cortisol

22

Biomarkers for Early Stage 2

3

23

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Biomarkers for Mid Stage

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Biomarkers for End Stage

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Genomics

COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of

the brain responsible for personality inhibition of behaviors abstract thinking

and emotion1

The COMT val158Met polymorphism has been shown to be associated with phobic

anxiety 2 neuroticism 3 and panic disorders 4

Results indicate a relationship of COMT polymorphism rs4680 with immune

dysregulation in CFS providing a potential link for the association between stress and

infection susceptibility in CFS(chronic fatigue syndrome)

1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype

on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J

Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8

27

17 yo radicalized female with anger

antisocial and rage issues(similar not exact due to security)

Methylene-tetrahydrofolate reductase

(MTHFR SNPrsquos)

Creates methylated folate

the active folate in the

body

Mutations have been

associated with vascular

disease dementia cancer

and autism

Methylation is the genetics

elevated homocysteine is

the genomics 29

End Point of Poor Methylation and

Oxidation

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

bull Superoxide dismutase 1

(SOD1)

bull Superoxide dismutase 2

(SOD2)

bull Superoxide dismutase 3

(SOD3)

bull Glutathione peroxidase 1

(GPx1)

bull Catalase (CAT)

Superoxide Dismutase 1

SOD1 (CuZnSOD)(Encoded Chrom 21)

Found in cytoplasmic and mitochondrial

intermembrane space(inserted)

Converts superoxide radicals (O2-) to hydrogen

peroxide (H2O2)

Needs copper and zinc as cofactors

Apoptosis Oxidative Stress ALS Myocardial

Infarction Macular Degeneration a marker for

heavy Metal toxicity

O2- H2O2

SOD1SOD1

Cu Zn

1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2

2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160

3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034

Superoxide Dismutase 2

SOD2 (MnSOD) (Chrom 6)

Found in mitochondria

Converts superoxide radicals (O2-)

to hydrogen peroxide (H2O2)

Needs manganese as a cofactor

Heart Disease Myocardial

Infarction Tumor metastasis

neurodegenerative diseases

O2- H2O2SOD2SOD2

Mn

Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24

Superoxide Dismutase 3SOD3

Found extracellularly

Converts superoxide radicals (O2-) to

hydrogen peroxide (H2O2)

Needs copper and zinc as cofactors

Chronic skin disorders Diabetic

Neuropathy Folate Metabolism Ischemic

Heart Disease

O2- H2O2SOD3SOD3

Cu Zn

Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004

Glutathione Peroxidase 1

GPx1(Chrom 3)

Found in the cytoplasm of nearly all mammalian tissues

Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)

Uses glutathione an antioxidant as a cofactor

Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression

H2O2H2O O2GPx1GPx1

Glutathione

Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and

Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89

Catalase

CAT(Chrom 11)

Found in the cytoplasm of cells

Needs iron and selenium as cofactors

Reduce hydrogen peroxide (H2O2) to water (H2O)

High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second

Hypertension Type 2 Diabetes Acatalasemia

httpsghrnlmnihgovgeneCATconditions

H2O2 H2O O2CATCAT

Fe Se

Mitochondrial Function Assessment

Mitochondrial Superoxide (O2-)

Plasma Peroxide Assay (PPA)

Mitochondrial Membrane

Potential (MMP) Assay with

Oxidative Challenge

Mitochondrial Superoxide (O2-)

Superoxide is considered to be a major

factor in oxidant toxicity and

mitochondrial MnSOD enzymes

constitute an essential defense against

superoxide

High levels indicate the proanti-oxidant

system is out of balance and oxidative

stress is present

Mitochondria are the major source of

superoxide

Superoxide is the origin of reactive

oxygen (ROS)and nitrogen species (RNS)

and as such causes various redox

related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo

123Hsiu-Chuan Yen

4Ikuo Nakanishi

5Ken-ichiro Matsumoto

5Masato Tamura

6Yumiko Nagano

6Hirofumi Matsui

6Oleg Gusev

789Richard Cornette

9Takashi

Okuda9

Yukiko Minamiyama10

Hiroshi Ichikawa11

Shigeaki Suenaga1

Misato Oki2

Tsuyoshi Sato1

Toshihiko Ozawa12

Daret K St Clair3

and Hideyuki J Majima12dagger

Retrospective Study Sample Distribution

To

tal S

ub

jects

(n

=86)

Lo

w (

n=10)

No

rmal (n

=27)

Ele

vate

d (

n=19)

Hig

h (

n=30)

0

1

2

3

4

5

Mit

oS

OA

R (

RF

U)

3 5

2 2

2 7

1 2

M ito S O A R

R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n

MitoSOARtrade (RFU)

lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects

N 10 27 19 3086

12 31 22 35

57 of the tested population has

superoxide levels in the

elevatedhigh range leading to high

risk for certain clinical conditions

Functional Genetics

CT

CC

0 5

1 0

1 5

2 0

2 5

Mit

oS

OA

R (

RF

U)

S O D 2 H e tro z y g o u s g e n o ty p e (C T )

S O D 2 H o m o z y g o u s g e n o ty p e (C C )

P lt 0 0 5

M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e

(S O D 2 ) P o ly m o rp h is m

Individuals who are homozygous

recessive(CC) for SOD2 may

have impaired SOD2 function

resulting in higher levels of the

free radical superoxide and

require antioxidant support

Mitochondrial Superoxide Levels

Assessment of Redox status

Inflammation

Viral or Bacterial Infection

Monitoring treatment of a

disease or disorder

Borrelia infection

Treatment of Inflammation

Functional Effect

Improvement verifies positive clinical effect

Plasma Peroxide Assay

Low Levels may indicate oxidative stress and

possible need for antioxidants

High levels may indicate immune challenges

Can correlate with uric acid

Potential hypertension risk

Related to lipid peroxides

Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13

Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303

Plasma Peroxidase

To

tal S

ub

jects

(n

=86)

Lo

w (

n=30)

No

rmal (n

=24)

Ele

vate

d (

n=11)

Hig

h (

n=16)

0

2 0

4 0

6 0

8 0

Pla

sm

a P

erix

ida

se

Ac

tiv

ity

(m

Um

l)

2 0

1 4

3 0

3 7

M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s

R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e

S a m p le D is tr ib u tio n

Plasma Peroxidase Activity (mUmL)

lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects

N 30 24 11 1681

37 30 14 20

37 of the population studied had low

peroxidase activity indicating an

impaired ability to defend against free

radicals

34 of the studies population had

elevatedhigh activity potentially

indicating the presence of oxidative

stress

Assessment of Baseline Potential

Starting point for defense of excessive

ROS

Effect of Environmental Insults

Assessment of Capacity to defend against

excessive ROS

Assessment and monitoring treatment of a

disease or disorder

Viral or Bacterial infection

Cardiovascular disease

Mitochondrial Membrane Potential

Oxidative Challenge

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Insomnia

httpswwwresearchgatenetfigure26679987_fig1_Fig-1-Prevalence-of-insomnia-symptoms-by-age-and-gender

As reported Sept 1 2017

What Americans Use to Sleep Nearly 9 million US adults resort to prescription sleeping pills mdash and most

are white female educated or 50 or older according to the first

government study of its kind

The CDC estimates that ldquo50-70 million US adults have sleep or

wakefulness disorderrdquo These numbers are striking because they

essentially mean that for every adult with a sleep disorder a doctor is

writing a prescription for sleeping pills

Between 2006 and 2011 sleeping pill prescriptions rose from 47 million to

60 million in the US About 38 million of those prescriptions were for drugs

containing zolpidem (including the brand name Ambien) according

to IMS Health

The United States insomnia pharmacological treatment market is

projected to reach $424 billion by 2021 from $338 billion in 2016

httpwwwnydailynewscomlife-stylehealthcdc-9-million-americans-sleeping-pills-article-11441778

Mood Fatigue Insomnia Theory

Mood Insomnia and Fatigue Issues are

inflammatory issues which are measurable

and reversible even with a poor genomic

outlook

-Decker Weiss

The Three Stooges of Mood Fatigue

and Insomnia

Inflammation causing Neurotransmitter Dysfunction

Inflammation toxicity exposure to free radicals causes Mitochondrial Dysfunction decreasing sex hormone production and energy an defenses

Gut BiomeFood Allergies generates and perpetuates inflammation immune dysfunction and further decreases in neurotransmitter dysfunction

DepressionInsomniaFatigue

GlutamatergicExcess

Many Causes Similar Result

NoradrenergicDeficiency

SerotonergicDeficiency

Belmaker R H and Agam G Major depressive

disorder (1-3-2008) NEnglJMed 358(1) 55-

68

GutBrainDysfunction

Mitochondrial Dysfunction

18

Neurotransmitters

PVN

C1

LC

Pituitary

DR

Thoracic

Lumbar

Cortisol

DHEAEpinephrine

Norepinephrine

CRF

ACTH

Traditional Stress Response

Hypothalamus is the master controller of the HPA axis (endocrine model)

Traditionally the start of the HPA axis

Pituitary gland has two parts that bridge the brain and body

Anterior (adenohypophysis) releases ACTH TSH PRL GH endorphins FSH LH CRF

Posterior (neurohypophysis) releases oxytocin and antidiuretic hormone

Two distinct parts of the adrenal gland produce hormones and neurotransmitters

Adrenal Cortex = cortisol and DHEA

Adrenal Medulla = norepinephrine and epinephrine

Engelmann M et al (2004) Front Neuroendocrinol 25(3-4)132-149Sherwood L (2010) Human Physiology From Cells to Systems 7th ed 698-709

Are you

considering

the adrenal

medulla

20

PVN

C1

LC

Hypothalamus

Pituitary

DR

Cortex

Medulla

Thoracic

Lumbar

CRF

NE

Cortisol

Glutamate

Epinephrine

ACh

Glutamate activates Thoracic pathway to

Adrenals

Sympathetic Chain

Serotonin

21

The ldquoBackward RuleMatch Diagnosis to Labs

Neurotransmitter Subject 1 Subject 2 Subject 3

Serotonin

GABA

Glycine

Glutamate

Histamine

PEA

Dopamine

Norepinephrine

Epinephrine

Cortisol

22

Biomarkers for Early Stage 2

3

23

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Biomarkers for Mid Stage

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Biomarkers for End Stage

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Genomics

COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of

the brain responsible for personality inhibition of behaviors abstract thinking

and emotion1

The COMT val158Met polymorphism has been shown to be associated with phobic

anxiety 2 neuroticism 3 and panic disorders 4

Results indicate a relationship of COMT polymorphism rs4680 with immune

dysregulation in CFS providing a potential link for the association between stress and

infection susceptibility in CFS(chronic fatigue syndrome)

1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype

on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J

Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8

27

17 yo radicalized female with anger

antisocial and rage issues(similar not exact due to security)

Methylene-tetrahydrofolate reductase

(MTHFR SNPrsquos)

Creates methylated folate

the active folate in the

body

Mutations have been

associated with vascular

disease dementia cancer

and autism

Methylation is the genetics

elevated homocysteine is

the genomics 29

End Point of Poor Methylation and

Oxidation

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

bull Superoxide dismutase 1

(SOD1)

bull Superoxide dismutase 2

(SOD2)

bull Superoxide dismutase 3

(SOD3)

bull Glutathione peroxidase 1

(GPx1)

bull Catalase (CAT)

Superoxide Dismutase 1

SOD1 (CuZnSOD)(Encoded Chrom 21)

Found in cytoplasmic and mitochondrial

intermembrane space(inserted)

Converts superoxide radicals (O2-) to hydrogen

peroxide (H2O2)

Needs copper and zinc as cofactors

Apoptosis Oxidative Stress ALS Myocardial

Infarction Macular Degeneration a marker for

heavy Metal toxicity

O2- H2O2

SOD1SOD1

Cu Zn

1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2

2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160

3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034

Superoxide Dismutase 2

SOD2 (MnSOD) (Chrom 6)

Found in mitochondria

Converts superoxide radicals (O2-)

to hydrogen peroxide (H2O2)

Needs manganese as a cofactor

Heart Disease Myocardial

Infarction Tumor metastasis

neurodegenerative diseases

O2- H2O2SOD2SOD2

Mn

Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24

Superoxide Dismutase 3SOD3

Found extracellularly

Converts superoxide radicals (O2-) to

hydrogen peroxide (H2O2)

Needs copper and zinc as cofactors

Chronic skin disorders Diabetic

Neuropathy Folate Metabolism Ischemic

Heart Disease

O2- H2O2SOD3SOD3

Cu Zn

Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004

Glutathione Peroxidase 1

GPx1(Chrom 3)

Found in the cytoplasm of nearly all mammalian tissues

Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)

Uses glutathione an antioxidant as a cofactor

Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression

H2O2H2O O2GPx1GPx1

Glutathione

Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and

Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89

Catalase

CAT(Chrom 11)

Found in the cytoplasm of cells

Needs iron and selenium as cofactors

Reduce hydrogen peroxide (H2O2) to water (H2O)

High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second

Hypertension Type 2 Diabetes Acatalasemia

httpsghrnlmnihgovgeneCATconditions

H2O2 H2O O2CATCAT

Fe Se

Mitochondrial Function Assessment

Mitochondrial Superoxide (O2-)

Plasma Peroxide Assay (PPA)

Mitochondrial Membrane

Potential (MMP) Assay with

Oxidative Challenge

Mitochondrial Superoxide (O2-)

Superoxide is considered to be a major

factor in oxidant toxicity and

mitochondrial MnSOD enzymes

constitute an essential defense against

superoxide

High levels indicate the proanti-oxidant

system is out of balance and oxidative

stress is present

Mitochondria are the major source of

superoxide

Superoxide is the origin of reactive

oxygen (ROS)and nitrogen species (RNS)

and as such causes various redox

related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo

123Hsiu-Chuan Yen

4Ikuo Nakanishi

5Ken-ichiro Matsumoto

5Masato Tamura

6Yumiko Nagano

6Hirofumi Matsui

6Oleg Gusev

789Richard Cornette

9Takashi

Okuda9

Yukiko Minamiyama10

Hiroshi Ichikawa11

Shigeaki Suenaga1

Misato Oki2

Tsuyoshi Sato1

Toshihiko Ozawa12

Daret K St Clair3

and Hideyuki J Majima12dagger

Retrospective Study Sample Distribution

To

tal S

ub

jects

(n

=86)

Lo

w (

n=10)

No

rmal (n

=27)

Ele

vate

d (

n=19)

Hig

h (

n=30)

0

1

2

3

4

5

Mit

oS

OA

R (

RF

U)

3 5

2 2

2 7

1 2

M ito S O A R

R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n

MitoSOARtrade (RFU)

lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects

N 10 27 19 3086

12 31 22 35

57 of the tested population has

superoxide levels in the

elevatedhigh range leading to high

risk for certain clinical conditions

Functional Genetics

CT

CC

0 5

1 0

1 5

2 0

2 5

Mit

oS

OA

R (

RF

U)

S O D 2 H e tro z y g o u s g e n o ty p e (C T )

S O D 2 H o m o z y g o u s g e n o ty p e (C C )

P lt 0 0 5

M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e

(S O D 2 ) P o ly m o rp h is m

Individuals who are homozygous

recessive(CC) for SOD2 may

have impaired SOD2 function

resulting in higher levels of the

free radical superoxide and

require antioxidant support

Mitochondrial Superoxide Levels

Assessment of Redox status

Inflammation

Viral or Bacterial Infection

Monitoring treatment of a

disease or disorder

Borrelia infection

Treatment of Inflammation

Functional Effect

Improvement verifies positive clinical effect

Plasma Peroxide Assay

Low Levels may indicate oxidative stress and

possible need for antioxidants

High levels may indicate immune challenges

Can correlate with uric acid

Potential hypertension risk

Related to lipid peroxides

Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13

Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303

Plasma Peroxidase

To

tal S

ub

jects

(n

=86)

Lo

w (

n=30)

No

rmal (n

=24)

Ele

vate

d (

n=11)

Hig

h (

n=16)

0

2 0

4 0

6 0

8 0

Pla

sm

a P

erix

ida

se

Ac

tiv

ity

(m

Um

l)

2 0

1 4

3 0

3 7

M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s

R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e

S a m p le D is tr ib u tio n

Plasma Peroxidase Activity (mUmL)

lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects

N 30 24 11 1681

37 30 14 20

37 of the population studied had low

peroxidase activity indicating an

impaired ability to defend against free

radicals

34 of the studies population had

elevatedhigh activity potentially

indicating the presence of oxidative

stress

Assessment of Baseline Potential

Starting point for defense of excessive

ROS

Effect of Environmental Insults

Assessment of Capacity to defend against

excessive ROS

Assessment and monitoring treatment of a

disease or disorder

Viral or Bacterial infection

Cardiovascular disease

Mitochondrial Membrane Potential

Oxidative Challenge

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

httpswwwresearchgatenetfigure26679987_fig1_Fig-1-Prevalence-of-insomnia-symptoms-by-age-and-gender

As reported Sept 1 2017

What Americans Use to Sleep Nearly 9 million US adults resort to prescription sleeping pills mdash and most

are white female educated or 50 or older according to the first

government study of its kind

The CDC estimates that ldquo50-70 million US adults have sleep or

wakefulness disorderrdquo These numbers are striking because they

essentially mean that for every adult with a sleep disorder a doctor is

writing a prescription for sleeping pills

Between 2006 and 2011 sleeping pill prescriptions rose from 47 million to

60 million in the US About 38 million of those prescriptions were for drugs

containing zolpidem (including the brand name Ambien) according

to IMS Health

The United States insomnia pharmacological treatment market is

projected to reach $424 billion by 2021 from $338 billion in 2016

httpwwwnydailynewscomlife-stylehealthcdc-9-million-americans-sleeping-pills-article-11441778

Mood Fatigue Insomnia Theory

Mood Insomnia and Fatigue Issues are

inflammatory issues which are measurable

and reversible even with a poor genomic

outlook

-Decker Weiss

The Three Stooges of Mood Fatigue

and Insomnia

Inflammation causing Neurotransmitter Dysfunction

Inflammation toxicity exposure to free radicals causes Mitochondrial Dysfunction decreasing sex hormone production and energy an defenses

Gut BiomeFood Allergies generates and perpetuates inflammation immune dysfunction and further decreases in neurotransmitter dysfunction

DepressionInsomniaFatigue

GlutamatergicExcess

Many Causes Similar Result

NoradrenergicDeficiency

SerotonergicDeficiency

Belmaker R H and Agam G Major depressive

disorder (1-3-2008) NEnglJMed 358(1) 55-

68

GutBrainDysfunction

Mitochondrial Dysfunction

18

Neurotransmitters

PVN

C1

LC

Pituitary

DR

Thoracic

Lumbar

Cortisol

DHEAEpinephrine

Norepinephrine

CRF

ACTH

Traditional Stress Response

Hypothalamus is the master controller of the HPA axis (endocrine model)

Traditionally the start of the HPA axis

Pituitary gland has two parts that bridge the brain and body

Anterior (adenohypophysis) releases ACTH TSH PRL GH endorphins FSH LH CRF

Posterior (neurohypophysis) releases oxytocin and antidiuretic hormone

Two distinct parts of the adrenal gland produce hormones and neurotransmitters

Adrenal Cortex = cortisol and DHEA

Adrenal Medulla = norepinephrine and epinephrine

Engelmann M et al (2004) Front Neuroendocrinol 25(3-4)132-149Sherwood L (2010) Human Physiology From Cells to Systems 7th ed 698-709

Are you

considering

the adrenal

medulla

20

PVN

C1

LC

Hypothalamus

Pituitary

DR

Cortex

Medulla

Thoracic

Lumbar

CRF

NE

Cortisol

Glutamate

Epinephrine

ACh

Glutamate activates Thoracic pathway to

Adrenals

Sympathetic Chain

Serotonin

21

The ldquoBackward RuleMatch Diagnosis to Labs

Neurotransmitter Subject 1 Subject 2 Subject 3

Serotonin

GABA

Glycine

Glutamate

Histamine

PEA

Dopamine

Norepinephrine

Epinephrine

Cortisol

22

Biomarkers for Early Stage 2

3

23

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Biomarkers for Mid Stage

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Biomarkers for End Stage

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Genomics

COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of

the brain responsible for personality inhibition of behaviors abstract thinking

and emotion1

The COMT val158Met polymorphism has been shown to be associated with phobic

anxiety 2 neuroticism 3 and panic disorders 4

Results indicate a relationship of COMT polymorphism rs4680 with immune

dysregulation in CFS providing a potential link for the association between stress and

infection susceptibility in CFS(chronic fatigue syndrome)

1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype

on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J

Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8

27

17 yo radicalized female with anger

antisocial and rage issues(similar not exact due to security)

Methylene-tetrahydrofolate reductase

(MTHFR SNPrsquos)

Creates methylated folate

the active folate in the

body

Mutations have been

associated with vascular

disease dementia cancer

and autism

Methylation is the genetics

elevated homocysteine is

the genomics 29

End Point of Poor Methylation and

Oxidation

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

bull Superoxide dismutase 1

(SOD1)

bull Superoxide dismutase 2

(SOD2)

bull Superoxide dismutase 3

(SOD3)

bull Glutathione peroxidase 1

(GPx1)

bull Catalase (CAT)

Superoxide Dismutase 1

SOD1 (CuZnSOD)(Encoded Chrom 21)

Found in cytoplasmic and mitochondrial

intermembrane space(inserted)

Converts superoxide radicals (O2-) to hydrogen

peroxide (H2O2)

Needs copper and zinc as cofactors

Apoptosis Oxidative Stress ALS Myocardial

Infarction Macular Degeneration a marker for

heavy Metal toxicity

O2- H2O2

SOD1SOD1

Cu Zn

1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2

2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160

3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034

Superoxide Dismutase 2

SOD2 (MnSOD) (Chrom 6)

Found in mitochondria

Converts superoxide radicals (O2-)

to hydrogen peroxide (H2O2)

Needs manganese as a cofactor

Heart Disease Myocardial

Infarction Tumor metastasis

neurodegenerative diseases

O2- H2O2SOD2SOD2

Mn

Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24

Superoxide Dismutase 3SOD3

Found extracellularly

Converts superoxide radicals (O2-) to

hydrogen peroxide (H2O2)

Needs copper and zinc as cofactors

Chronic skin disorders Diabetic

Neuropathy Folate Metabolism Ischemic

Heart Disease

O2- H2O2SOD3SOD3

Cu Zn

Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004

Glutathione Peroxidase 1

GPx1(Chrom 3)

Found in the cytoplasm of nearly all mammalian tissues

Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)

Uses glutathione an antioxidant as a cofactor

Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression

H2O2H2O O2GPx1GPx1

Glutathione

Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and

Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89

Catalase

CAT(Chrom 11)

Found in the cytoplasm of cells

Needs iron and selenium as cofactors

Reduce hydrogen peroxide (H2O2) to water (H2O)

High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second

Hypertension Type 2 Diabetes Acatalasemia

httpsghrnlmnihgovgeneCATconditions

H2O2 H2O O2CATCAT

Fe Se

Mitochondrial Function Assessment

Mitochondrial Superoxide (O2-)

Plasma Peroxide Assay (PPA)

Mitochondrial Membrane

Potential (MMP) Assay with

Oxidative Challenge

Mitochondrial Superoxide (O2-)

Superoxide is considered to be a major

factor in oxidant toxicity and

mitochondrial MnSOD enzymes

constitute an essential defense against

superoxide

High levels indicate the proanti-oxidant

system is out of balance and oxidative

stress is present

Mitochondria are the major source of

superoxide

Superoxide is the origin of reactive

oxygen (ROS)and nitrogen species (RNS)

and as such causes various redox

related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo

123Hsiu-Chuan Yen

4Ikuo Nakanishi

5Ken-ichiro Matsumoto

5Masato Tamura

6Yumiko Nagano

6Hirofumi Matsui

6Oleg Gusev

789Richard Cornette

9Takashi

Okuda9

Yukiko Minamiyama10

Hiroshi Ichikawa11

Shigeaki Suenaga1

Misato Oki2

Tsuyoshi Sato1

Toshihiko Ozawa12

Daret K St Clair3

and Hideyuki J Majima12dagger

Retrospective Study Sample Distribution

To

tal S

ub

jects

(n

=86)

Lo

w (

n=10)

No

rmal (n

=27)

Ele

vate

d (

n=19)

Hig

h (

n=30)

0

1

2

3

4

5

Mit

oS

OA

R (

RF

U)

3 5

2 2

2 7

1 2

M ito S O A R

R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n

MitoSOARtrade (RFU)

lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects

N 10 27 19 3086

12 31 22 35

57 of the tested population has

superoxide levels in the

elevatedhigh range leading to high

risk for certain clinical conditions

Functional Genetics

CT

CC

0 5

1 0

1 5

2 0

2 5

Mit

oS

OA

R (

RF

U)

S O D 2 H e tro z y g o u s g e n o ty p e (C T )

S O D 2 H o m o z y g o u s g e n o ty p e (C C )

P lt 0 0 5

M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e

(S O D 2 ) P o ly m o rp h is m

Individuals who are homozygous

recessive(CC) for SOD2 may

have impaired SOD2 function

resulting in higher levels of the

free radical superoxide and

require antioxidant support

Mitochondrial Superoxide Levels

Assessment of Redox status

Inflammation

Viral or Bacterial Infection

Monitoring treatment of a

disease or disorder

Borrelia infection

Treatment of Inflammation

Functional Effect

Improvement verifies positive clinical effect

Plasma Peroxide Assay

Low Levels may indicate oxidative stress and

possible need for antioxidants

High levels may indicate immune challenges

Can correlate with uric acid

Potential hypertension risk

Related to lipid peroxides

Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13

Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303

Plasma Peroxidase

To

tal S

ub

jects

(n

=86)

Lo

w (

n=30)

No

rmal (n

=24)

Ele

vate

d (

n=11)

Hig

h (

n=16)

0

2 0

4 0

6 0

8 0

Pla

sm

a P

erix

ida

se

Ac

tiv

ity

(m

Um

l)

2 0

1 4

3 0

3 7

M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s

R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e

S a m p le D is tr ib u tio n

Plasma Peroxidase Activity (mUmL)

lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects

N 30 24 11 1681

37 30 14 20

37 of the population studied had low

peroxidase activity indicating an

impaired ability to defend against free

radicals

34 of the studies population had

elevatedhigh activity potentially

indicating the presence of oxidative

stress

Assessment of Baseline Potential

Starting point for defense of excessive

ROS

Effect of Environmental Insults

Assessment of Capacity to defend against

excessive ROS

Assessment and monitoring treatment of a

disease or disorder

Viral or Bacterial infection

Cardiovascular disease

Mitochondrial Membrane Potential

Oxidative Challenge

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

What Americans Use to Sleep Nearly 9 million US adults resort to prescription sleeping pills mdash and most

are white female educated or 50 or older according to the first

government study of its kind

The CDC estimates that ldquo50-70 million US adults have sleep or

wakefulness disorderrdquo These numbers are striking because they

essentially mean that for every adult with a sleep disorder a doctor is

writing a prescription for sleeping pills

Between 2006 and 2011 sleeping pill prescriptions rose from 47 million to

60 million in the US About 38 million of those prescriptions were for drugs

containing zolpidem (including the brand name Ambien) according

to IMS Health

The United States insomnia pharmacological treatment market is

projected to reach $424 billion by 2021 from $338 billion in 2016

httpwwwnydailynewscomlife-stylehealthcdc-9-million-americans-sleeping-pills-article-11441778

Mood Fatigue Insomnia Theory

Mood Insomnia and Fatigue Issues are

inflammatory issues which are measurable

and reversible even with a poor genomic

outlook

-Decker Weiss

The Three Stooges of Mood Fatigue

and Insomnia

Inflammation causing Neurotransmitter Dysfunction

Inflammation toxicity exposure to free radicals causes Mitochondrial Dysfunction decreasing sex hormone production and energy an defenses

Gut BiomeFood Allergies generates and perpetuates inflammation immune dysfunction and further decreases in neurotransmitter dysfunction

DepressionInsomniaFatigue

GlutamatergicExcess

Many Causes Similar Result

NoradrenergicDeficiency

SerotonergicDeficiency

Belmaker R H and Agam G Major depressive

disorder (1-3-2008) NEnglJMed 358(1) 55-

68

GutBrainDysfunction

Mitochondrial Dysfunction

18

Neurotransmitters

PVN

C1

LC

Pituitary

DR

Thoracic

Lumbar

Cortisol

DHEAEpinephrine

Norepinephrine

CRF

ACTH

Traditional Stress Response

Hypothalamus is the master controller of the HPA axis (endocrine model)

Traditionally the start of the HPA axis

Pituitary gland has two parts that bridge the brain and body

Anterior (adenohypophysis) releases ACTH TSH PRL GH endorphins FSH LH CRF

Posterior (neurohypophysis) releases oxytocin and antidiuretic hormone

Two distinct parts of the adrenal gland produce hormones and neurotransmitters

Adrenal Cortex = cortisol and DHEA

Adrenal Medulla = norepinephrine and epinephrine

Engelmann M et al (2004) Front Neuroendocrinol 25(3-4)132-149Sherwood L (2010) Human Physiology From Cells to Systems 7th ed 698-709

Are you

considering

the adrenal

medulla

20

PVN

C1

LC

Hypothalamus

Pituitary

DR

Cortex

Medulla

Thoracic

Lumbar

CRF

NE

Cortisol

Glutamate

Epinephrine

ACh

Glutamate activates Thoracic pathway to

Adrenals

Sympathetic Chain

Serotonin

21

The ldquoBackward RuleMatch Diagnosis to Labs

Neurotransmitter Subject 1 Subject 2 Subject 3

Serotonin

GABA

Glycine

Glutamate

Histamine

PEA

Dopamine

Norepinephrine

Epinephrine

Cortisol

22

Biomarkers for Early Stage 2

3

23

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Biomarkers for Mid Stage

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Biomarkers for End Stage

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Genomics

COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of

the brain responsible for personality inhibition of behaviors abstract thinking

and emotion1

The COMT val158Met polymorphism has been shown to be associated with phobic

anxiety 2 neuroticism 3 and panic disorders 4

Results indicate a relationship of COMT polymorphism rs4680 with immune

dysregulation in CFS providing a potential link for the association between stress and

infection susceptibility in CFS(chronic fatigue syndrome)

1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype

on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J

Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8

27

17 yo radicalized female with anger

antisocial and rage issues(similar not exact due to security)

Methylene-tetrahydrofolate reductase

(MTHFR SNPrsquos)

Creates methylated folate

the active folate in the

body

Mutations have been

associated with vascular

disease dementia cancer

and autism

Methylation is the genetics

elevated homocysteine is

the genomics 29

End Point of Poor Methylation and

Oxidation

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

bull Superoxide dismutase 1

(SOD1)

bull Superoxide dismutase 2

(SOD2)

bull Superoxide dismutase 3

(SOD3)

bull Glutathione peroxidase 1

(GPx1)

bull Catalase (CAT)

Superoxide Dismutase 1

SOD1 (CuZnSOD)(Encoded Chrom 21)

Found in cytoplasmic and mitochondrial

intermembrane space(inserted)

Converts superoxide radicals (O2-) to hydrogen

peroxide (H2O2)

Needs copper and zinc as cofactors

Apoptosis Oxidative Stress ALS Myocardial

Infarction Macular Degeneration a marker for

heavy Metal toxicity

O2- H2O2

SOD1SOD1

Cu Zn

1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2

2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160

3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034

Superoxide Dismutase 2

SOD2 (MnSOD) (Chrom 6)

Found in mitochondria

Converts superoxide radicals (O2-)

to hydrogen peroxide (H2O2)

Needs manganese as a cofactor

Heart Disease Myocardial

Infarction Tumor metastasis

neurodegenerative diseases

O2- H2O2SOD2SOD2

Mn

Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24

Superoxide Dismutase 3SOD3

Found extracellularly

Converts superoxide radicals (O2-) to

hydrogen peroxide (H2O2)

Needs copper and zinc as cofactors

Chronic skin disorders Diabetic

Neuropathy Folate Metabolism Ischemic

Heart Disease

O2- H2O2SOD3SOD3

Cu Zn

Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004

Glutathione Peroxidase 1

GPx1(Chrom 3)

Found in the cytoplasm of nearly all mammalian tissues

Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)

Uses glutathione an antioxidant as a cofactor

Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression

H2O2H2O O2GPx1GPx1

Glutathione

Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and

Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89

Catalase

CAT(Chrom 11)

Found in the cytoplasm of cells

Needs iron and selenium as cofactors

Reduce hydrogen peroxide (H2O2) to water (H2O)

High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second

Hypertension Type 2 Diabetes Acatalasemia

httpsghrnlmnihgovgeneCATconditions

H2O2 H2O O2CATCAT

Fe Se

Mitochondrial Function Assessment

Mitochondrial Superoxide (O2-)

Plasma Peroxide Assay (PPA)

Mitochondrial Membrane

Potential (MMP) Assay with

Oxidative Challenge

Mitochondrial Superoxide (O2-)

Superoxide is considered to be a major

factor in oxidant toxicity and

mitochondrial MnSOD enzymes

constitute an essential defense against

superoxide

High levels indicate the proanti-oxidant

system is out of balance and oxidative

stress is present

Mitochondria are the major source of

superoxide

Superoxide is the origin of reactive

oxygen (ROS)and nitrogen species (RNS)

and as such causes various redox

related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo

123Hsiu-Chuan Yen

4Ikuo Nakanishi

5Ken-ichiro Matsumoto

5Masato Tamura

6Yumiko Nagano

6Hirofumi Matsui

6Oleg Gusev

789Richard Cornette

9Takashi

Okuda9

Yukiko Minamiyama10

Hiroshi Ichikawa11

Shigeaki Suenaga1

Misato Oki2

Tsuyoshi Sato1

Toshihiko Ozawa12

Daret K St Clair3

and Hideyuki J Majima12dagger

Retrospective Study Sample Distribution

To

tal S

ub

jects

(n

=86)

Lo

w (

n=10)

No

rmal (n

=27)

Ele

vate

d (

n=19)

Hig

h (

n=30)

0

1

2

3

4

5

Mit

oS

OA

R (

RF

U)

3 5

2 2

2 7

1 2

M ito S O A R

R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n

MitoSOARtrade (RFU)

lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects

N 10 27 19 3086

12 31 22 35

57 of the tested population has

superoxide levels in the

elevatedhigh range leading to high

risk for certain clinical conditions

Functional Genetics

CT

CC

0 5

1 0

1 5

2 0

2 5

Mit

oS

OA

R (

RF

U)

S O D 2 H e tro z y g o u s g e n o ty p e (C T )

S O D 2 H o m o z y g o u s g e n o ty p e (C C )

P lt 0 0 5

M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e

(S O D 2 ) P o ly m o rp h is m

Individuals who are homozygous

recessive(CC) for SOD2 may

have impaired SOD2 function

resulting in higher levels of the

free radical superoxide and

require antioxidant support

Mitochondrial Superoxide Levels

Assessment of Redox status

Inflammation

Viral or Bacterial Infection

Monitoring treatment of a

disease or disorder

Borrelia infection

Treatment of Inflammation

Functional Effect

Improvement verifies positive clinical effect

Plasma Peroxide Assay

Low Levels may indicate oxidative stress and

possible need for antioxidants

High levels may indicate immune challenges

Can correlate with uric acid

Potential hypertension risk

Related to lipid peroxides

Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13

Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303

Plasma Peroxidase

To

tal S

ub

jects

(n

=86)

Lo

w (

n=30)

No

rmal (n

=24)

Ele

vate

d (

n=11)

Hig

h (

n=16)

0

2 0

4 0

6 0

8 0

Pla

sm

a P

erix

ida

se

Ac

tiv

ity

(m

Um

l)

2 0

1 4

3 0

3 7

M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s

R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e

S a m p le D is tr ib u tio n

Plasma Peroxidase Activity (mUmL)

lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects

N 30 24 11 1681

37 30 14 20

37 of the population studied had low

peroxidase activity indicating an

impaired ability to defend against free

radicals

34 of the studies population had

elevatedhigh activity potentially

indicating the presence of oxidative

stress

Assessment of Baseline Potential

Starting point for defense of excessive

ROS

Effect of Environmental Insults

Assessment of Capacity to defend against

excessive ROS

Assessment and monitoring treatment of a

disease or disorder

Viral or Bacterial infection

Cardiovascular disease

Mitochondrial Membrane Potential

Oxidative Challenge

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Mood Fatigue Insomnia Theory

Mood Insomnia and Fatigue Issues are

inflammatory issues which are measurable

and reversible even with a poor genomic

outlook

-Decker Weiss

The Three Stooges of Mood Fatigue

and Insomnia

Inflammation causing Neurotransmitter Dysfunction

Inflammation toxicity exposure to free radicals causes Mitochondrial Dysfunction decreasing sex hormone production and energy an defenses

Gut BiomeFood Allergies generates and perpetuates inflammation immune dysfunction and further decreases in neurotransmitter dysfunction

DepressionInsomniaFatigue

GlutamatergicExcess

Many Causes Similar Result

NoradrenergicDeficiency

SerotonergicDeficiency

Belmaker R H and Agam G Major depressive

disorder (1-3-2008) NEnglJMed 358(1) 55-

68

GutBrainDysfunction

Mitochondrial Dysfunction

18

Neurotransmitters

PVN

C1

LC

Pituitary

DR

Thoracic

Lumbar

Cortisol

DHEAEpinephrine

Norepinephrine

CRF

ACTH

Traditional Stress Response

Hypothalamus is the master controller of the HPA axis (endocrine model)

Traditionally the start of the HPA axis

Pituitary gland has two parts that bridge the brain and body

Anterior (adenohypophysis) releases ACTH TSH PRL GH endorphins FSH LH CRF

Posterior (neurohypophysis) releases oxytocin and antidiuretic hormone

Two distinct parts of the adrenal gland produce hormones and neurotransmitters

Adrenal Cortex = cortisol and DHEA

Adrenal Medulla = norepinephrine and epinephrine

Engelmann M et al (2004) Front Neuroendocrinol 25(3-4)132-149Sherwood L (2010) Human Physiology From Cells to Systems 7th ed 698-709

Are you

considering

the adrenal

medulla

20

PVN

C1

LC

Hypothalamus

Pituitary

DR

Cortex

Medulla

Thoracic

Lumbar

CRF

NE

Cortisol

Glutamate

Epinephrine

ACh

Glutamate activates Thoracic pathway to

Adrenals

Sympathetic Chain

Serotonin

21

The ldquoBackward RuleMatch Diagnosis to Labs

Neurotransmitter Subject 1 Subject 2 Subject 3

Serotonin

GABA

Glycine

Glutamate

Histamine

PEA

Dopamine

Norepinephrine

Epinephrine

Cortisol

22

Biomarkers for Early Stage 2

3

23

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Biomarkers for Mid Stage

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Biomarkers for End Stage

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Genomics

COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of

the brain responsible for personality inhibition of behaviors abstract thinking

and emotion1

The COMT val158Met polymorphism has been shown to be associated with phobic

anxiety 2 neuroticism 3 and panic disorders 4

Results indicate a relationship of COMT polymorphism rs4680 with immune

dysregulation in CFS providing a potential link for the association between stress and

infection susceptibility in CFS(chronic fatigue syndrome)

1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype

on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J

Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8

27

17 yo radicalized female with anger

antisocial and rage issues(similar not exact due to security)

Methylene-tetrahydrofolate reductase

(MTHFR SNPrsquos)

Creates methylated folate

the active folate in the

body

Mutations have been

associated with vascular

disease dementia cancer

and autism

Methylation is the genetics

elevated homocysteine is

the genomics 29

End Point of Poor Methylation and

Oxidation

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

bull Superoxide dismutase 1

(SOD1)

bull Superoxide dismutase 2

(SOD2)

bull Superoxide dismutase 3

(SOD3)

bull Glutathione peroxidase 1

(GPx1)

bull Catalase (CAT)

Superoxide Dismutase 1

SOD1 (CuZnSOD)(Encoded Chrom 21)

Found in cytoplasmic and mitochondrial

intermembrane space(inserted)

Converts superoxide radicals (O2-) to hydrogen

peroxide (H2O2)

Needs copper and zinc as cofactors

Apoptosis Oxidative Stress ALS Myocardial

Infarction Macular Degeneration a marker for

heavy Metal toxicity

O2- H2O2

SOD1SOD1

Cu Zn

1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2

2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160

3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034

Superoxide Dismutase 2

SOD2 (MnSOD) (Chrom 6)

Found in mitochondria

Converts superoxide radicals (O2-)

to hydrogen peroxide (H2O2)

Needs manganese as a cofactor

Heart Disease Myocardial

Infarction Tumor metastasis

neurodegenerative diseases

O2- H2O2SOD2SOD2

Mn

Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24

Superoxide Dismutase 3SOD3

Found extracellularly

Converts superoxide radicals (O2-) to

hydrogen peroxide (H2O2)

Needs copper and zinc as cofactors

Chronic skin disorders Diabetic

Neuropathy Folate Metabolism Ischemic

Heart Disease

O2- H2O2SOD3SOD3

Cu Zn

Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004

Glutathione Peroxidase 1

GPx1(Chrom 3)

Found in the cytoplasm of nearly all mammalian tissues

Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)

Uses glutathione an antioxidant as a cofactor

Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression

H2O2H2O O2GPx1GPx1

Glutathione

Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and

Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89

Catalase

CAT(Chrom 11)

Found in the cytoplasm of cells

Needs iron and selenium as cofactors

Reduce hydrogen peroxide (H2O2) to water (H2O)

High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second

Hypertension Type 2 Diabetes Acatalasemia

httpsghrnlmnihgovgeneCATconditions

H2O2 H2O O2CATCAT

Fe Se

Mitochondrial Function Assessment

Mitochondrial Superoxide (O2-)

Plasma Peroxide Assay (PPA)

Mitochondrial Membrane

Potential (MMP) Assay with

Oxidative Challenge

Mitochondrial Superoxide (O2-)

Superoxide is considered to be a major

factor in oxidant toxicity and

mitochondrial MnSOD enzymes

constitute an essential defense against

superoxide

High levels indicate the proanti-oxidant

system is out of balance and oxidative

stress is present

Mitochondria are the major source of

superoxide

Superoxide is the origin of reactive

oxygen (ROS)and nitrogen species (RNS)

and as such causes various redox

related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo

123Hsiu-Chuan Yen

4Ikuo Nakanishi

5Ken-ichiro Matsumoto

5Masato Tamura

6Yumiko Nagano

6Hirofumi Matsui

6Oleg Gusev

789Richard Cornette

9Takashi

Okuda9

Yukiko Minamiyama10

Hiroshi Ichikawa11

Shigeaki Suenaga1

Misato Oki2

Tsuyoshi Sato1

Toshihiko Ozawa12

Daret K St Clair3

and Hideyuki J Majima12dagger

Retrospective Study Sample Distribution

To

tal S

ub

jects

(n

=86)

Lo

w (

n=10)

No

rmal (n

=27)

Ele

vate

d (

n=19)

Hig

h (

n=30)

0

1

2

3

4

5

Mit

oS

OA

R (

RF

U)

3 5

2 2

2 7

1 2

M ito S O A R

R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n

MitoSOARtrade (RFU)

lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects

N 10 27 19 3086

12 31 22 35

57 of the tested population has

superoxide levels in the

elevatedhigh range leading to high

risk for certain clinical conditions

Functional Genetics

CT

CC

0 5

1 0

1 5

2 0

2 5

Mit

oS

OA

R (

RF

U)

S O D 2 H e tro z y g o u s g e n o ty p e (C T )

S O D 2 H o m o z y g o u s g e n o ty p e (C C )

P lt 0 0 5

M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e

(S O D 2 ) P o ly m o rp h is m

Individuals who are homozygous

recessive(CC) for SOD2 may

have impaired SOD2 function

resulting in higher levels of the

free radical superoxide and

require antioxidant support

Mitochondrial Superoxide Levels

Assessment of Redox status

Inflammation

Viral or Bacterial Infection

Monitoring treatment of a

disease or disorder

Borrelia infection

Treatment of Inflammation

Functional Effect

Improvement verifies positive clinical effect

Plasma Peroxide Assay

Low Levels may indicate oxidative stress and

possible need for antioxidants

High levels may indicate immune challenges

Can correlate with uric acid

Potential hypertension risk

Related to lipid peroxides

Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13

Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303

Plasma Peroxidase

To

tal S

ub

jects

(n

=86)

Lo

w (

n=30)

No

rmal (n

=24)

Ele

vate

d (

n=11)

Hig

h (

n=16)

0

2 0

4 0

6 0

8 0

Pla

sm

a P

erix

ida

se

Ac

tiv

ity

(m

Um

l)

2 0

1 4

3 0

3 7

M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s

R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e

S a m p le D is tr ib u tio n

Plasma Peroxidase Activity (mUmL)

lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects

N 30 24 11 1681

37 30 14 20

37 of the population studied had low

peroxidase activity indicating an

impaired ability to defend against free

radicals

34 of the studies population had

elevatedhigh activity potentially

indicating the presence of oxidative

stress

Assessment of Baseline Potential

Starting point for defense of excessive

ROS

Effect of Environmental Insults

Assessment of Capacity to defend against

excessive ROS

Assessment and monitoring treatment of a

disease or disorder

Viral or Bacterial infection

Cardiovascular disease

Mitochondrial Membrane Potential

Oxidative Challenge

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

The Three Stooges of Mood Fatigue

and Insomnia

Inflammation causing Neurotransmitter Dysfunction

Inflammation toxicity exposure to free radicals causes Mitochondrial Dysfunction decreasing sex hormone production and energy an defenses

Gut BiomeFood Allergies generates and perpetuates inflammation immune dysfunction and further decreases in neurotransmitter dysfunction

DepressionInsomniaFatigue

GlutamatergicExcess

Many Causes Similar Result

NoradrenergicDeficiency

SerotonergicDeficiency

Belmaker R H and Agam G Major depressive

disorder (1-3-2008) NEnglJMed 358(1) 55-

68

GutBrainDysfunction

Mitochondrial Dysfunction

18

Neurotransmitters

PVN

C1

LC

Pituitary

DR

Thoracic

Lumbar

Cortisol

DHEAEpinephrine

Norepinephrine

CRF

ACTH

Traditional Stress Response

Hypothalamus is the master controller of the HPA axis (endocrine model)

Traditionally the start of the HPA axis

Pituitary gland has two parts that bridge the brain and body

Anterior (adenohypophysis) releases ACTH TSH PRL GH endorphins FSH LH CRF

Posterior (neurohypophysis) releases oxytocin and antidiuretic hormone

Two distinct parts of the adrenal gland produce hormones and neurotransmitters

Adrenal Cortex = cortisol and DHEA

Adrenal Medulla = norepinephrine and epinephrine

Engelmann M et al (2004) Front Neuroendocrinol 25(3-4)132-149Sherwood L (2010) Human Physiology From Cells to Systems 7th ed 698-709

Are you

considering

the adrenal

medulla

20

PVN

C1

LC

Hypothalamus

Pituitary

DR

Cortex

Medulla

Thoracic

Lumbar

CRF

NE

Cortisol

Glutamate

Epinephrine

ACh

Glutamate activates Thoracic pathway to

Adrenals

Sympathetic Chain

Serotonin

21

The ldquoBackward RuleMatch Diagnosis to Labs

Neurotransmitter Subject 1 Subject 2 Subject 3

Serotonin

GABA

Glycine

Glutamate

Histamine

PEA

Dopamine

Norepinephrine

Epinephrine

Cortisol

22

Biomarkers for Early Stage 2

3

23

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Biomarkers for Mid Stage

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Biomarkers for End Stage

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Genomics

COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of

the brain responsible for personality inhibition of behaviors abstract thinking

and emotion1

The COMT val158Met polymorphism has been shown to be associated with phobic

anxiety 2 neuroticism 3 and panic disorders 4

Results indicate a relationship of COMT polymorphism rs4680 with immune

dysregulation in CFS providing a potential link for the association between stress and

infection susceptibility in CFS(chronic fatigue syndrome)

1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype

on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J

Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8

27

17 yo radicalized female with anger

antisocial and rage issues(similar not exact due to security)

Methylene-tetrahydrofolate reductase

(MTHFR SNPrsquos)

Creates methylated folate

the active folate in the

body

Mutations have been

associated with vascular

disease dementia cancer

and autism

Methylation is the genetics

elevated homocysteine is

the genomics 29

End Point of Poor Methylation and

Oxidation

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

bull Superoxide dismutase 1

(SOD1)

bull Superoxide dismutase 2

(SOD2)

bull Superoxide dismutase 3

(SOD3)

bull Glutathione peroxidase 1

(GPx1)

bull Catalase (CAT)

Superoxide Dismutase 1

SOD1 (CuZnSOD)(Encoded Chrom 21)

Found in cytoplasmic and mitochondrial

intermembrane space(inserted)

Converts superoxide radicals (O2-) to hydrogen

peroxide (H2O2)

Needs copper and zinc as cofactors

Apoptosis Oxidative Stress ALS Myocardial

Infarction Macular Degeneration a marker for

heavy Metal toxicity

O2- H2O2

SOD1SOD1

Cu Zn

1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2

2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160

3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034

Superoxide Dismutase 2

SOD2 (MnSOD) (Chrom 6)

Found in mitochondria

Converts superoxide radicals (O2-)

to hydrogen peroxide (H2O2)

Needs manganese as a cofactor

Heart Disease Myocardial

Infarction Tumor metastasis

neurodegenerative diseases

O2- H2O2SOD2SOD2

Mn

Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24

Superoxide Dismutase 3SOD3

Found extracellularly

Converts superoxide radicals (O2-) to

hydrogen peroxide (H2O2)

Needs copper and zinc as cofactors

Chronic skin disorders Diabetic

Neuropathy Folate Metabolism Ischemic

Heart Disease

O2- H2O2SOD3SOD3

Cu Zn

Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004

Glutathione Peroxidase 1

GPx1(Chrom 3)

Found in the cytoplasm of nearly all mammalian tissues

Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)

Uses glutathione an antioxidant as a cofactor

Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression

H2O2H2O O2GPx1GPx1

Glutathione

Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and

Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89

Catalase

CAT(Chrom 11)

Found in the cytoplasm of cells

Needs iron and selenium as cofactors

Reduce hydrogen peroxide (H2O2) to water (H2O)

High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second

Hypertension Type 2 Diabetes Acatalasemia

httpsghrnlmnihgovgeneCATconditions

H2O2 H2O O2CATCAT

Fe Se

Mitochondrial Function Assessment

Mitochondrial Superoxide (O2-)

Plasma Peroxide Assay (PPA)

Mitochondrial Membrane

Potential (MMP) Assay with

Oxidative Challenge

Mitochondrial Superoxide (O2-)

Superoxide is considered to be a major

factor in oxidant toxicity and

mitochondrial MnSOD enzymes

constitute an essential defense against

superoxide

High levels indicate the proanti-oxidant

system is out of balance and oxidative

stress is present

Mitochondria are the major source of

superoxide

Superoxide is the origin of reactive

oxygen (ROS)and nitrogen species (RNS)

and as such causes various redox

related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo

123Hsiu-Chuan Yen

4Ikuo Nakanishi

5Ken-ichiro Matsumoto

5Masato Tamura

6Yumiko Nagano

6Hirofumi Matsui

6Oleg Gusev

789Richard Cornette

9Takashi

Okuda9

Yukiko Minamiyama10

Hiroshi Ichikawa11

Shigeaki Suenaga1

Misato Oki2

Tsuyoshi Sato1

Toshihiko Ozawa12

Daret K St Clair3

and Hideyuki J Majima12dagger

Retrospective Study Sample Distribution

To

tal S

ub

jects

(n

=86)

Lo

w (

n=10)

No

rmal (n

=27)

Ele

vate

d (

n=19)

Hig

h (

n=30)

0

1

2

3

4

5

Mit

oS

OA

R (

RF

U)

3 5

2 2

2 7

1 2

M ito S O A R

R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n

MitoSOARtrade (RFU)

lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects

N 10 27 19 3086

12 31 22 35

57 of the tested population has

superoxide levels in the

elevatedhigh range leading to high

risk for certain clinical conditions

Functional Genetics

CT

CC

0 5

1 0

1 5

2 0

2 5

Mit

oS

OA

R (

RF

U)

S O D 2 H e tro z y g o u s g e n o ty p e (C T )

S O D 2 H o m o z y g o u s g e n o ty p e (C C )

P lt 0 0 5

M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e

(S O D 2 ) P o ly m o rp h is m

Individuals who are homozygous

recessive(CC) for SOD2 may

have impaired SOD2 function

resulting in higher levels of the

free radical superoxide and

require antioxidant support

Mitochondrial Superoxide Levels

Assessment of Redox status

Inflammation

Viral or Bacterial Infection

Monitoring treatment of a

disease or disorder

Borrelia infection

Treatment of Inflammation

Functional Effect

Improvement verifies positive clinical effect

Plasma Peroxide Assay

Low Levels may indicate oxidative stress and

possible need for antioxidants

High levels may indicate immune challenges

Can correlate with uric acid

Potential hypertension risk

Related to lipid peroxides

Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13

Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303

Plasma Peroxidase

To

tal S

ub

jects

(n

=86)

Lo

w (

n=30)

No

rmal (n

=24)

Ele

vate

d (

n=11)

Hig

h (

n=16)

0

2 0

4 0

6 0

8 0

Pla

sm

a P

erix

ida

se

Ac

tiv

ity

(m

Um

l)

2 0

1 4

3 0

3 7

M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s

R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e

S a m p le D is tr ib u tio n

Plasma Peroxidase Activity (mUmL)

lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects

N 30 24 11 1681

37 30 14 20

37 of the population studied had low

peroxidase activity indicating an

impaired ability to defend against free

radicals

34 of the studies population had

elevatedhigh activity potentially

indicating the presence of oxidative

stress

Assessment of Baseline Potential

Starting point for defense of excessive

ROS

Effect of Environmental Insults

Assessment of Capacity to defend against

excessive ROS

Assessment and monitoring treatment of a

disease or disorder

Viral or Bacterial infection

Cardiovascular disease

Mitochondrial Membrane Potential

Oxidative Challenge

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

DepressionInsomniaFatigue

GlutamatergicExcess

Many Causes Similar Result

NoradrenergicDeficiency

SerotonergicDeficiency

Belmaker R H and Agam G Major depressive

disorder (1-3-2008) NEnglJMed 358(1) 55-

68

GutBrainDysfunction

Mitochondrial Dysfunction

18

Neurotransmitters

PVN

C1

LC

Pituitary

DR

Thoracic

Lumbar

Cortisol

DHEAEpinephrine

Norepinephrine

CRF

ACTH

Traditional Stress Response

Hypothalamus is the master controller of the HPA axis (endocrine model)

Traditionally the start of the HPA axis

Pituitary gland has two parts that bridge the brain and body

Anterior (adenohypophysis) releases ACTH TSH PRL GH endorphins FSH LH CRF

Posterior (neurohypophysis) releases oxytocin and antidiuretic hormone

Two distinct parts of the adrenal gland produce hormones and neurotransmitters

Adrenal Cortex = cortisol and DHEA

Adrenal Medulla = norepinephrine and epinephrine

Engelmann M et al (2004) Front Neuroendocrinol 25(3-4)132-149Sherwood L (2010) Human Physiology From Cells to Systems 7th ed 698-709

Are you

considering

the adrenal

medulla

20

PVN

C1

LC

Hypothalamus

Pituitary

DR

Cortex

Medulla

Thoracic

Lumbar

CRF

NE

Cortisol

Glutamate

Epinephrine

ACh

Glutamate activates Thoracic pathway to

Adrenals

Sympathetic Chain

Serotonin

21

The ldquoBackward RuleMatch Diagnosis to Labs

Neurotransmitter Subject 1 Subject 2 Subject 3

Serotonin

GABA

Glycine

Glutamate

Histamine

PEA

Dopamine

Norepinephrine

Epinephrine

Cortisol

22

Biomarkers for Early Stage 2

3

23

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Biomarkers for Mid Stage

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Biomarkers for End Stage

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Genomics

COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of

the brain responsible for personality inhibition of behaviors abstract thinking

and emotion1

The COMT val158Met polymorphism has been shown to be associated with phobic

anxiety 2 neuroticism 3 and panic disorders 4

Results indicate a relationship of COMT polymorphism rs4680 with immune

dysregulation in CFS providing a potential link for the association between stress and

infection susceptibility in CFS(chronic fatigue syndrome)

1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype

on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J

Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8

27

17 yo radicalized female with anger

antisocial and rage issues(similar not exact due to security)

Methylene-tetrahydrofolate reductase

(MTHFR SNPrsquos)

Creates methylated folate

the active folate in the

body

Mutations have been

associated with vascular

disease dementia cancer

and autism

Methylation is the genetics

elevated homocysteine is

the genomics 29

End Point of Poor Methylation and

Oxidation

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

bull Superoxide dismutase 1

(SOD1)

bull Superoxide dismutase 2

(SOD2)

bull Superoxide dismutase 3

(SOD3)

bull Glutathione peroxidase 1

(GPx1)

bull Catalase (CAT)

Superoxide Dismutase 1

SOD1 (CuZnSOD)(Encoded Chrom 21)

Found in cytoplasmic and mitochondrial

intermembrane space(inserted)

Converts superoxide radicals (O2-) to hydrogen

peroxide (H2O2)

Needs copper and zinc as cofactors

Apoptosis Oxidative Stress ALS Myocardial

Infarction Macular Degeneration a marker for

heavy Metal toxicity

O2- H2O2

SOD1SOD1

Cu Zn

1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2

2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160

3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034

Superoxide Dismutase 2

SOD2 (MnSOD) (Chrom 6)

Found in mitochondria

Converts superoxide radicals (O2-)

to hydrogen peroxide (H2O2)

Needs manganese as a cofactor

Heart Disease Myocardial

Infarction Tumor metastasis

neurodegenerative diseases

O2- H2O2SOD2SOD2

Mn

Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24

Superoxide Dismutase 3SOD3

Found extracellularly

Converts superoxide radicals (O2-) to

hydrogen peroxide (H2O2)

Needs copper and zinc as cofactors

Chronic skin disorders Diabetic

Neuropathy Folate Metabolism Ischemic

Heart Disease

O2- H2O2SOD3SOD3

Cu Zn

Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004

Glutathione Peroxidase 1

GPx1(Chrom 3)

Found in the cytoplasm of nearly all mammalian tissues

Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)

Uses glutathione an antioxidant as a cofactor

Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression

H2O2H2O O2GPx1GPx1

Glutathione

Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and

Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89

Catalase

CAT(Chrom 11)

Found in the cytoplasm of cells

Needs iron and selenium as cofactors

Reduce hydrogen peroxide (H2O2) to water (H2O)

High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second

Hypertension Type 2 Diabetes Acatalasemia

httpsghrnlmnihgovgeneCATconditions

H2O2 H2O O2CATCAT

Fe Se

Mitochondrial Function Assessment

Mitochondrial Superoxide (O2-)

Plasma Peroxide Assay (PPA)

Mitochondrial Membrane

Potential (MMP) Assay with

Oxidative Challenge

Mitochondrial Superoxide (O2-)

Superoxide is considered to be a major

factor in oxidant toxicity and

mitochondrial MnSOD enzymes

constitute an essential defense against

superoxide

High levels indicate the proanti-oxidant

system is out of balance and oxidative

stress is present

Mitochondria are the major source of

superoxide

Superoxide is the origin of reactive

oxygen (ROS)and nitrogen species (RNS)

and as such causes various redox

related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo

123Hsiu-Chuan Yen

4Ikuo Nakanishi

5Ken-ichiro Matsumoto

5Masato Tamura

6Yumiko Nagano

6Hirofumi Matsui

6Oleg Gusev

789Richard Cornette

9Takashi

Okuda9

Yukiko Minamiyama10

Hiroshi Ichikawa11

Shigeaki Suenaga1

Misato Oki2

Tsuyoshi Sato1

Toshihiko Ozawa12

Daret K St Clair3

and Hideyuki J Majima12dagger

Retrospective Study Sample Distribution

To

tal S

ub

jects

(n

=86)

Lo

w (

n=10)

No

rmal (n

=27)

Ele

vate

d (

n=19)

Hig

h (

n=30)

0

1

2

3

4

5

Mit

oS

OA

R (

RF

U)

3 5

2 2

2 7

1 2

M ito S O A R

R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n

MitoSOARtrade (RFU)

lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects

N 10 27 19 3086

12 31 22 35

57 of the tested population has

superoxide levels in the

elevatedhigh range leading to high

risk for certain clinical conditions

Functional Genetics

CT

CC

0 5

1 0

1 5

2 0

2 5

Mit

oS

OA

R (

RF

U)

S O D 2 H e tro z y g o u s g e n o ty p e (C T )

S O D 2 H o m o z y g o u s g e n o ty p e (C C )

P lt 0 0 5

M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e

(S O D 2 ) P o ly m o rp h is m

Individuals who are homozygous

recessive(CC) for SOD2 may

have impaired SOD2 function

resulting in higher levels of the

free radical superoxide and

require antioxidant support

Mitochondrial Superoxide Levels

Assessment of Redox status

Inflammation

Viral or Bacterial Infection

Monitoring treatment of a

disease or disorder

Borrelia infection

Treatment of Inflammation

Functional Effect

Improvement verifies positive clinical effect

Plasma Peroxide Assay

Low Levels may indicate oxidative stress and

possible need for antioxidants

High levels may indicate immune challenges

Can correlate with uric acid

Potential hypertension risk

Related to lipid peroxides

Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13

Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303

Plasma Peroxidase

To

tal S

ub

jects

(n

=86)

Lo

w (

n=30)

No

rmal (n

=24)

Ele

vate

d (

n=11)

Hig

h (

n=16)

0

2 0

4 0

6 0

8 0

Pla

sm

a P

erix

ida

se

Ac

tiv

ity

(m

Um

l)

2 0

1 4

3 0

3 7

M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s

R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e

S a m p le D is tr ib u tio n

Plasma Peroxidase Activity (mUmL)

lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects

N 30 24 11 1681

37 30 14 20

37 of the population studied had low

peroxidase activity indicating an

impaired ability to defend against free

radicals

34 of the studies population had

elevatedhigh activity potentially

indicating the presence of oxidative

stress

Assessment of Baseline Potential

Starting point for defense of excessive

ROS

Effect of Environmental Insults

Assessment of Capacity to defend against

excessive ROS

Assessment and monitoring treatment of a

disease or disorder

Viral or Bacterial infection

Cardiovascular disease

Mitochondrial Membrane Potential

Oxidative Challenge

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Neurotransmitters

PVN

C1

LC

Pituitary

DR

Thoracic

Lumbar

Cortisol

DHEAEpinephrine

Norepinephrine

CRF

ACTH

Traditional Stress Response

Hypothalamus is the master controller of the HPA axis (endocrine model)

Traditionally the start of the HPA axis

Pituitary gland has two parts that bridge the brain and body

Anterior (adenohypophysis) releases ACTH TSH PRL GH endorphins FSH LH CRF

Posterior (neurohypophysis) releases oxytocin and antidiuretic hormone

Two distinct parts of the adrenal gland produce hormones and neurotransmitters

Adrenal Cortex = cortisol and DHEA

Adrenal Medulla = norepinephrine and epinephrine

Engelmann M et al (2004) Front Neuroendocrinol 25(3-4)132-149Sherwood L (2010) Human Physiology From Cells to Systems 7th ed 698-709

Are you

considering

the adrenal

medulla

20

PVN

C1

LC

Hypothalamus

Pituitary

DR

Cortex

Medulla

Thoracic

Lumbar

CRF

NE

Cortisol

Glutamate

Epinephrine

ACh

Glutamate activates Thoracic pathway to

Adrenals

Sympathetic Chain

Serotonin

21

The ldquoBackward RuleMatch Diagnosis to Labs

Neurotransmitter Subject 1 Subject 2 Subject 3

Serotonin

GABA

Glycine

Glutamate

Histamine

PEA

Dopamine

Norepinephrine

Epinephrine

Cortisol

22

Biomarkers for Early Stage 2

3

23

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Biomarkers for Mid Stage

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Biomarkers for End Stage

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Genomics

COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of

the brain responsible for personality inhibition of behaviors abstract thinking

and emotion1

The COMT val158Met polymorphism has been shown to be associated with phobic

anxiety 2 neuroticism 3 and panic disorders 4

Results indicate a relationship of COMT polymorphism rs4680 with immune

dysregulation in CFS providing a potential link for the association between stress and

infection susceptibility in CFS(chronic fatigue syndrome)

1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype

on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J

Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8

27

17 yo radicalized female with anger

antisocial and rage issues(similar not exact due to security)

Methylene-tetrahydrofolate reductase

(MTHFR SNPrsquos)

Creates methylated folate

the active folate in the

body

Mutations have been

associated with vascular

disease dementia cancer

and autism

Methylation is the genetics

elevated homocysteine is

the genomics 29

End Point of Poor Methylation and

Oxidation

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

bull Superoxide dismutase 1

(SOD1)

bull Superoxide dismutase 2

(SOD2)

bull Superoxide dismutase 3

(SOD3)

bull Glutathione peroxidase 1

(GPx1)

bull Catalase (CAT)

Superoxide Dismutase 1

SOD1 (CuZnSOD)(Encoded Chrom 21)

Found in cytoplasmic and mitochondrial

intermembrane space(inserted)

Converts superoxide radicals (O2-) to hydrogen

peroxide (H2O2)

Needs copper and zinc as cofactors

Apoptosis Oxidative Stress ALS Myocardial

Infarction Macular Degeneration a marker for

heavy Metal toxicity

O2- H2O2

SOD1SOD1

Cu Zn

1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2

2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160

3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034

Superoxide Dismutase 2

SOD2 (MnSOD) (Chrom 6)

Found in mitochondria

Converts superoxide radicals (O2-)

to hydrogen peroxide (H2O2)

Needs manganese as a cofactor

Heart Disease Myocardial

Infarction Tumor metastasis

neurodegenerative diseases

O2- H2O2SOD2SOD2

Mn

Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24

Superoxide Dismutase 3SOD3

Found extracellularly

Converts superoxide radicals (O2-) to

hydrogen peroxide (H2O2)

Needs copper and zinc as cofactors

Chronic skin disorders Diabetic

Neuropathy Folate Metabolism Ischemic

Heart Disease

O2- H2O2SOD3SOD3

Cu Zn

Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004

Glutathione Peroxidase 1

GPx1(Chrom 3)

Found in the cytoplasm of nearly all mammalian tissues

Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)

Uses glutathione an antioxidant as a cofactor

Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression

H2O2H2O O2GPx1GPx1

Glutathione

Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and

Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89

Catalase

CAT(Chrom 11)

Found in the cytoplasm of cells

Needs iron and selenium as cofactors

Reduce hydrogen peroxide (H2O2) to water (H2O)

High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second

Hypertension Type 2 Diabetes Acatalasemia

httpsghrnlmnihgovgeneCATconditions

H2O2 H2O O2CATCAT

Fe Se

Mitochondrial Function Assessment

Mitochondrial Superoxide (O2-)

Plasma Peroxide Assay (PPA)

Mitochondrial Membrane

Potential (MMP) Assay with

Oxidative Challenge

Mitochondrial Superoxide (O2-)

Superoxide is considered to be a major

factor in oxidant toxicity and

mitochondrial MnSOD enzymes

constitute an essential defense against

superoxide

High levels indicate the proanti-oxidant

system is out of balance and oxidative

stress is present

Mitochondria are the major source of

superoxide

Superoxide is the origin of reactive

oxygen (ROS)and nitrogen species (RNS)

and as such causes various redox

related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo

123Hsiu-Chuan Yen

4Ikuo Nakanishi

5Ken-ichiro Matsumoto

5Masato Tamura

6Yumiko Nagano

6Hirofumi Matsui

6Oleg Gusev

789Richard Cornette

9Takashi

Okuda9

Yukiko Minamiyama10

Hiroshi Ichikawa11

Shigeaki Suenaga1

Misato Oki2

Tsuyoshi Sato1

Toshihiko Ozawa12

Daret K St Clair3

and Hideyuki J Majima12dagger

Retrospective Study Sample Distribution

To

tal S

ub

jects

(n

=86)

Lo

w (

n=10)

No

rmal (n

=27)

Ele

vate

d (

n=19)

Hig

h (

n=30)

0

1

2

3

4

5

Mit

oS

OA

R (

RF

U)

3 5

2 2

2 7

1 2

M ito S O A R

R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n

MitoSOARtrade (RFU)

lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects

N 10 27 19 3086

12 31 22 35

57 of the tested population has

superoxide levels in the

elevatedhigh range leading to high

risk for certain clinical conditions

Functional Genetics

CT

CC

0 5

1 0

1 5

2 0

2 5

Mit

oS

OA

R (

RF

U)

S O D 2 H e tro z y g o u s g e n o ty p e (C T )

S O D 2 H o m o z y g o u s g e n o ty p e (C C )

P lt 0 0 5

M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e

(S O D 2 ) P o ly m o rp h is m

Individuals who are homozygous

recessive(CC) for SOD2 may

have impaired SOD2 function

resulting in higher levels of the

free radical superoxide and

require antioxidant support

Mitochondrial Superoxide Levels

Assessment of Redox status

Inflammation

Viral or Bacterial Infection

Monitoring treatment of a

disease or disorder

Borrelia infection

Treatment of Inflammation

Functional Effect

Improvement verifies positive clinical effect

Plasma Peroxide Assay

Low Levels may indicate oxidative stress and

possible need for antioxidants

High levels may indicate immune challenges

Can correlate with uric acid

Potential hypertension risk

Related to lipid peroxides

Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13

Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303

Plasma Peroxidase

To

tal S

ub

jects

(n

=86)

Lo

w (

n=30)

No

rmal (n

=24)

Ele

vate

d (

n=11)

Hig

h (

n=16)

0

2 0

4 0

6 0

8 0

Pla

sm

a P

erix

ida

se

Ac

tiv

ity

(m

Um

l)

2 0

1 4

3 0

3 7

M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s

R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e

S a m p le D is tr ib u tio n

Plasma Peroxidase Activity (mUmL)

lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects

N 30 24 11 1681

37 30 14 20

37 of the population studied had low

peroxidase activity indicating an

impaired ability to defend against free

radicals

34 of the studies population had

elevatedhigh activity potentially

indicating the presence of oxidative

stress

Assessment of Baseline Potential

Starting point for defense of excessive

ROS

Effect of Environmental Insults

Assessment of Capacity to defend against

excessive ROS

Assessment and monitoring treatment of a

disease or disorder

Viral or Bacterial infection

Cardiovascular disease

Mitochondrial Membrane Potential

Oxidative Challenge

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

PVN

C1

LC

Pituitary

DR

Thoracic

Lumbar

Cortisol

DHEAEpinephrine

Norepinephrine

CRF

ACTH

Traditional Stress Response

Hypothalamus is the master controller of the HPA axis (endocrine model)

Traditionally the start of the HPA axis

Pituitary gland has two parts that bridge the brain and body

Anterior (adenohypophysis) releases ACTH TSH PRL GH endorphins FSH LH CRF

Posterior (neurohypophysis) releases oxytocin and antidiuretic hormone

Two distinct parts of the adrenal gland produce hormones and neurotransmitters

Adrenal Cortex = cortisol and DHEA

Adrenal Medulla = norepinephrine and epinephrine

Engelmann M et al (2004) Front Neuroendocrinol 25(3-4)132-149Sherwood L (2010) Human Physiology From Cells to Systems 7th ed 698-709

Are you

considering

the adrenal

medulla

20

PVN

C1

LC

Hypothalamus

Pituitary

DR

Cortex

Medulla

Thoracic

Lumbar

CRF

NE

Cortisol

Glutamate

Epinephrine

ACh

Glutamate activates Thoracic pathway to

Adrenals

Sympathetic Chain

Serotonin

21

The ldquoBackward RuleMatch Diagnosis to Labs

Neurotransmitter Subject 1 Subject 2 Subject 3

Serotonin

GABA

Glycine

Glutamate

Histamine

PEA

Dopamine

Norepinephrine

Epinephrine

Cortisol

22

Biomarkers for Early Stage 2

3

23

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Biomarkers for Mid Stage

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Biomarkers for End Stage

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Genomics

COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of

the brain responsible for personality inhibition of behaviors abstract thinking

and emotion1

The COMT val158Met polymorphism has been shown to be associated with phobic

anxiety 2 neuroticism 3 and panic disorders 4

Results indicate a relationship of COMT polymorphism rs4680 with immune

dysregulation in CFS providing a potential link for the association between stress and

infection susceptibility in CFS(chronic fatigue syndrome)

1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype

on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J

Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8

27

17 yo radicalized female with anger

antisocial and rage issues(similar not exact due to security)

Methylene-tetrahydrofolate reductase

(MTHFR SNPrsquos)

Creates methylated folate

the active folate in the

body

Mutations have been

associated with vascular

disease dementia cancer

and autism

Methylation is the genetics

elevated homocysteine is

the genomics 29

End Point of Poor Methylation and

Oxidation

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

bull Superoxide dismutase 1

(SOD1)

bull Superoxide dismutase 2

(SOD2)

bull Superoxide dismutase 3

(SOD3)

bull Glutathione peroxidase 1

(GPx1)

bull Catalase (CAT)

Superoxide Dismutase 1

SOD1 (CuZnSOD)(Encoded Chrom 21)

Found in cytoplasmic and mitochondrial

intermembrane space(inserted)

Converts superoxide radicals (O2-) to hydrogen

peroxide (H2O2)

Needs copper and zinc as cofactors

Apoptosis Oxidative Stress ALS Myocardial

Infarction Macular Degeneration a marker for

heavy Metal toxicity

O2- H2O2

SOD1SOD1

Cu Zn

1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2

2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160

3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034

Superoxide Dismutase 2

SOD2 (MnSOD) (Chrom 6)

Found in mitochondria

Converts superoxide radicals (O2-)

to hydrogen peroxide (H2O2)

Needs manganese as a cofactor

Heart Disease Myocardial

Infarction Tumor metastasis

neurodegenerative diseases

O2- H2O2SOD2SOD2

Mn

Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24

Superoxide Dismutase 3SOD3

Found extracellularly

Converts superoxide radicals (O2-) to

hydrogen peroxide (H2O2)

Needs copper and zinc as cofactors

Chronic skin disorders Diabetic

Neuropathy Folate Metabolism Ischemic

Heart Disease

O2- H2O2SOD3SOD3

Cu Zn

Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004

Glutathione Peroxidase 1

GPx1(Chrom 3)

Found in the cytoplasm of nearly all mammalian tissues

Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)

Uses glutathione an antioxidant as a cofactor

Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression

H2O2H2O O2GPx1GPx1

Glutathione

Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and

Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89

Catalase

CAT(Chrom 11)

Found in the cytoplasm of cells

Needs iron and selenium as cofactors

Reduce hydrogen peroxide (H2O2) to water (H2O)

High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second

Hypertension Type 2 Diabetes Acatalasemia

httpsghrnlmnihgovgeneCATconditions

H2O2 H2O O2CATCAT

Fe Se

Mitochondrial Function Assessment

Mitochondrial Superoxide (O2-)

Plasma Peroxide Assay (PPA)

Mitochondrial Membrane

Potential (MMP) Assay with

Oxidative Challenge

Mitochondrial Superoxide (O2-)

Superoxide is considered to be a major

factor in oxidant toxicity and

mitochondrial MnSOD enzymes

constitute an essential defense against

superoxide

High levels indicate the proanti-oxidant

system is out of balance and oxidative

stress is present

Mitochondria are the major source of

superoxide

Superoxide is the origin of reactive

oxygen (ROS)and nitrogen species (RNS)

and as such causes various redox

related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo

123Hsiu-Chuan Yen

4Ikuo Nakanishi

5Ken-ichiro Matsumoto

5Masato Tamura

6Yumiko Nagano

6Hirofumi Matsui

6Oleg Gusev

789Richard Cornette

9Takashi

Okuda9

Yukiko Minamiyama10

Hiroshi Ichikawa11

Shigeaki Suenaga1

Misato Oki2

Tsuyoshi Sato1

Toshihiko Ozawa12

Daret K St Clair3

and Hideyuki J Majima12dagger

Retrospective Study Sample Distribution

To

tal S

ub

jects

(n

=86)

Lo

w (

n=10)

No

rmal (n

=27)

Ele

vate

d (

n=19)

Hig

h (

n=30)

0

1

2

3

4

5

Mit

oS

OA

R (

RF

U)

3 5

2 2

2 7

1 2

M ito S O A R

R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n

MitoSOARtrade (RFU)

lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects

N 10 27 19 3086

12 31 22 35

57 of the tested population has

superoxide levels in the

elevatedhigh range leading to high

risk for certain clinical conditions

Functional Genetics

CT

CC

0 5

1 0

1 5

2 0

2 5

Mit

oS

OA

R (

RF

U)

S O D 2 H e tro z y g o u s g e n o ty p e (C T )

S O D 2 H o m o z y g o u s g e n o ty p e (C C )

P lt 0 0 5

M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e

(S O D 2 ) P o ly m o rp h is m

Individuals who are homozygous

recessive(CC) for SOD2 may

have impaired SOD2 function

resulting in higher levels of the

free radical superoxide and

require antioxidant support

Mitochondrial Superoxide Levels

Assessment of Redox status

Inflammation

Viral or Bacterial Infection

Monitoring treatment of a

disease or disorder

Borrelia infection

Treatment of Inflammation

Functional Effect

Improvement verifies positive clinical effect

Plasma Peroxide Assay

Low Levels may indicate oxidative stress and

possible need for antioxidants

High levels may indicate immune challenges

Can correlate with uric acid

Potential hypertension risk

Related to lipid peroxides

Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13

Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303

Plasma Peroxidase

To

tal S

ub

jects

(n

=86)

Lo

w (

n=30)

No

rmal (n

=24)

Ele

vate

d (

n=11)

Hig

h (

n=16)

0

2 0

4 0

6 0

8 0

Pla

sm

a P

erix

ida

se

Ac

tiv

ity

(m

Um

l)

2 0

1 4

3 0

3 7

M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s

R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e

S a m p le D is tr ib u tio n

Plasma Peroxidase Activity (mUmL)

lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects

N 30 24 11 1681

37 30 14 20

37 of the population studied had low

peroxidase activity indicating an

impaired ability to defend against free

radicals

34 of the studies population had

elevatedhigh activity potentially

indicating the presence of oxidative

stress

Assessment of Baseline Potential

Starting point for defense of excessive

ROS

Effect of Environmental Insults

Assessment of Capacity to defend against

excessive ROS

Assessment and monitoring treatment of a

disease or disorder

Viral or Bacterial infection

Cardiovascular disease

Mitochondrial Membrane Potential

Oxidative Challenge

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

PVN

C1

LC

Hypothalamus

Pituitary

DR

Cortex

Medulla

Thoracic

Lumbar

CRF

NE

Cortisol

Glutamate

Epinephrine

ACh

Glutamate activates Thoracic pathway to

Adrenals

Sympathetic Chain

Serotonin

21

The ldquoBackward RuleMatch Diagnosis to Labs

Neurotransmitter Subject 1 Subject 2 Subject 3

Serotonin

GABA

Glycine

Glutamate

Histamine

PEA

Dopamine

Norepinephrine

Epinephrine

Cortisol

22

Biomarkers for Early Stage 2

3

23

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Biomarkers for Mid Stage

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Biomarkers for End Stage

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Genomics

COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of

the brain responsible for personality inhibition of behaviors abstract thinking

and emotion1

The COMT val158Met polymorphism has been shown to be associated with phobic

anxiety 2 neuroticism 3 and panic disorders 4

Results indicate a relationship of COMT polymorphism rs4680 with immune

dysregulation in CFS providing a potential link for the association between stress and

infection susceptibility in CFS(chronic fatigue syndrome)

1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype

on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J

Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8

27

17 yo radicalized female with anger

antisocial and rage issues(similar not exact due to security)

Methylene-tetrahydrofolate reductase

(MTHFR SNPrsquos)

Creates methylated folate

the active folate in the

body

Mutations have been

associated with vascular

disease dementia cancer

and autism

Methylation is the genetics

elevated homocysteine is

the genomics 29

End Point of Poor Methylation and

Oxidation

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

bull Superoxide dismutase 1

(SOD1)

bull Superoxide dismutase 2

(SOD2)

bull Superoxide dismutase 3

(SOD3)

bull Glutathione peroxidase 1

(GPx1)

bull Catalase (CAT)

Superoxide Dismutase 1

SOD1 (CuZnSOD)(Encoded Chrom 21)

Found in cytoplasmic and mitochondrial

intermembrane space(inserted)

Converts superoxide radicals (O2-) to hydrogen

peroxide (H2O2)

Needs copper and zinc as cofactors

Apoptosis Oxidative Stress ALS Myocardial

Infarction Macular Degeneration a marker for

heavy Metal toxicity

O2- H2O2

SOD1SOD1

Cu Zn

1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2

2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160

3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034

Superoxide Dismutase 2

SOD2 (MnSOD) (Chrom 6)

Found in mitochondria

Converts superoxide radicals (O2-)

to hydrogen peroxide (H2O2)

Needs manganese as a cofactor

Heart Disease Myocardial

Infarction Tumor metastasis

neurodegenerative diseases

O2- H2O2SOD2SOD2

Mn

Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24

Superoxide Dismutase 3SOD3

Found extracellularly

Converts superoxide radicals (O2-) to

hydrogen peroxide (H2O2)

Needs copper and zinc as cofactors

Chronic skin disorders Diabetic

Neuropathy Folate Metabolism Ischemic

Heart Disease

O2- H2O2SOD3SOD3

Cu Zn

Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004

Glutathione Peroxidase 1

GPx1(Chrom 3)

Found in the cytoplasm of nearly all mammalian tissues

Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)

Uses glutathione an antioxidant as a cofactor

Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression

H2O2H2O O2GPx1GPx1

Glutathione

Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and

Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89

Catalase

CAT(Chrom 11)

Found in the cytoplasm of cells

Needs iron and selenium as cofactors

Reduce hydrogen peroxide (H2O2) to water (H2O)

High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second

Hypertension Type 2 Diabetes Acatalasemia

httpsghrnlmnihgovgeneCATconditions

H2O2 H2O O2CATCAT

Fe Se

Mitochondrial Function Assessment

Mitochondrial Superoxide (O2-)

Plasma Peroxide Assay (PPA)

Mitochondrial Membrane

Potential (MMP) Assay with

Oxidative Challenge

Mitochondrial Superoxide (O2-)

Superoxide is considered to be a major

factor in oxidant toxicity and

mitochondrial MnSOD enzymes

constitute an essential defense against

superoxide

High levels indicate the proanti-oxidant

system is out of balance and oxidative

stress is present

Mitochondria are the major source of

superoxide

Superoxide is the origin of reactive

oxygen (ROS)and nitrogen species (RNS)

and as such causes various redox

related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo

123Hsiu-Chuan Yen

4Ikuo Nakanishi

5Ken-ichiro Matsumoto

5Masato Tamura

6Yumiko Nagano

6Hirofumi Matsui

6Oleg Gusev

789Richard Cornette

9Takashi

Okuda9

Yukiko Minamiyama10

Hiroshi Ichikawa11

Shigeaki Suenaga1

Misato Oki2

Tsuyoshi Sato1

Toshihiko Ozawa12

Daret K St Clair3

and Hideyuki J Majima12dagger

Retrospective Study Sample Distribution

To

tal S

ub

jects

(n

=86)

Lo

w (

n=10)

No

rmal (n

=27)

Ele

vate

d (

n=19)

Hig

h (

n=30)

0

1

2

3

4

5

Mit

oS

OA

R (

RF

U)

3 5

2 2

2 7

1 2

M ito S O A R

R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n

MitoSOARtrade (RFU)

lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects

N 10 27 19 3086

12 31 22 35

57 of the tested population has

superoxide levels in the

elevatedhigh range leading to high

risk for certain clinical conditions

Functional Genetics

CT

CC

0 5

1 0

1 5

2 0

2 5

Mit

oS

OA

R (

RF

U)

S O D 2 H e tro z y g o u s g e n o ty p e (C T )

S O D 2 H o m o z y g o u s g e n o ty p e (C C )

P lt 0 0 5

M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e

(S O D 2 ) P o ly m o rp h is m

Individuals who are homozygous

recessive(CC) for SOD2 may

have impaired SOD2 function

resulting in higher levels of the

free radical superoxide and

require antioxidant support

Mitochondrial Superoxide Levels

Assessment of Redox status

Inflammation

Viral or Bacterial Infection

Monitoring treatment of a

disease or disorder

Borrelia infection

Treatment of Inflammation

Functional Effect

Improvement verifies positive clinical effect

Plasma Peroxide Assay

Low Levels may indicate oxidative stress and

possible need for antioxidants

High levels may indicate immune challenges

Can correlate with uric acid

Potential hypertension risk

Related to lipid peroxides

Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13

Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303

Plasma Peroxidase

To

tal S

ub

jects

(n

=86)

Lo

w (

n=30)

No

rmal (n

=24)

Ele

vate

d (

n=11)

Hig

h (

n=16)

0

2 0

4 0

6 0

8 0

Pla

sm

a P

erix

ida

se

Ac

tiv

ity

(m

Um

l)

2 0

1 4

3 0

3 7

M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s

R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e

S a m p le D is tr ib u tio n

Plasma Peroxidase Activity (mUmL)

lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects

N 30 24 11 1681

37 30 14 20

37 of the population studied had low

peroxidase activity indicating an

impaired ability to defend against free

radicals

34 of the studies population had

elevatedhigh activity potentially

indicating the presence of oxidative

stress

Assessment of Baseline Potential

Starting point for defense of excessive

ROS

Effect of Environmental Insults

Assessment of Capacity to defend against

excessive ROS

Assessment and monitoring treatment of a

disease or disorder

Viral or Bacterial infection

Cardiovascular disease

Mitochondrial Membrane Potential

Oxidative Challenge

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

The ldquoBackward RuleMatch Diagnosis to Labs

Neurotransmitter Subject 1 Subject 2 Subject 3

Serotonin

GABA

Glycine

Glutamate

Histamine

PEA

Dopamine

Norepinephrine

Epinephrine

Cortisol

22

Biomarkers for Early Stage 2

3

23

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Biomarkers for Mid Stage

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Biomarkers for End Stage

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Genomics

COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of

the brain responsible for personality inhibition of behaviors abstract thinking

and emotion1

The COMT val158Met polymorphism has been shown to be associated with phobic

anxiety 2 neuroticism 3 and panic disorders 4

Results indicate a relationship of COMT polymorphism rs4680 with immune

dysregulation in CFS providing a potential link for the association between stress and

infection susceptibility in CFS(chronic fatigue syndrome)

1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype

on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J

Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8

27

17 yo radicalized female with anger

antisocial and rage issues(similar not exact due to security)

Methylene-tetrahydrofolate reductase

(MTHFR SNPrsquos)

Creates methylated folate

the active folate in the

body

Mutations have been

associated with vascular

disease dementia cancer

and autism

Methylation is the genetics

elevated homocysteine is

the genomics 29

End Point of Poor Methylation and

Oxidation

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

bull Superoxide dismutase 1

(SOD1)

bull Superoxide dismutase 2

(SOD2)

bull Superoxide dismutase 3

(SOD3)

bull Glutathione peroxidase 1

(GPx1)

bull Catalase (CAT)

Superoxide Dismutase 1

SOD1 (CuZnSOD)(Encoded Chrom 21)

Found in cytoplasmic and mitochondrial

intermembrane space(inserted)

Converts superoxide radicals (O2-) to hydrogen

peroxide (H2O2)

Needs copper and zinc as cofactors

Apoptosis Oxidative Stress ALS Myocardial

Infarction Macular Degeneration a marker for

heavy Metal toxicity

O2- H2O2

SOD1SOD1

Cu Zn

1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2

2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160

3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034

Superoxide Dismutase 2

SOD2 (MnSOD) (Chrom 6)

Found in mitochondria

Converts superoxide radicals (O2-)

to hydrogen peroxide (H2O2)

Needs manganese as a cofactor

Heart Disease Myocardial

Infarction Tumor metastasis

neurodegenerative diseases

O2- H2O2SOD2SOD2

Mn

Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24

Superoxide Dismutase 3SOD3

Found extracellularly

Converts superoxide radicals (O2-) to

hydrogen peroxide (H2O2)

Needs copper and zinc as cofactors

Chronic skin disorders Diabetic

Neuropathy Folate Metabolism Ischemic

Heart Disease

O2- H2O2SOD3SOD3

Cu Zn

Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004

Glutathione Peroxidase 1

GPx1(Chrom 3)

Found in the cytoplasm of nearly all mammalian tissues

Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)

Uses glutathione an antioxidant as a cofactor

Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression

H2O2H2O O2GPx1GPx1

Glutathione

Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and

Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89

Catalase

CAT(Chrom 11)

Found in the cytoplasm of cells

Needs iron and selenium as cofactors

Reduce hydrogen peroxide (H2O2) to water (H2O)

High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second

Hypertension Type 2 Diabetes Acatalasemia

httpsghrnlmnihgovgeneCATconditions

H2O2 H2O O2CATCAT

Fe Se

Mitochondrial Function Assessment

Mitochondrial Superoxide (O2-)

Plasma Peroxide Assay (PPA)

Mitochondrial Membrane

Potential (MMP) Assay with

Oxidative Challenge

Mitochondrial Superoxide (O2-)

Superoxide is considered to be a major

factor in oxidant toxicity and

mitochondrial MnSOD enzymes

constitute an essential defense against

superoxide

High levels indicate the proanti-oxidant

system is out of balance and oxidative

stress is present

Mitochondria are the major source of

superoxide

Superoxide is the origin of reactive

oxygen (ROS)and nitrogen species (RNS)

and as such causes various redox

related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo

123Hsiu-Chuan Yen

4Ikuo Nakanishi

5Ken-ichiro Matsumoto

5Masato Tamura

6Yumiko Nagano

6Hirofumi Matsui

6Oleg Gusev

789Richard Cornette

9Takashi

Okuda9

Yukiko Minamiyama10

Hiroshi Ichikawa11

Shigeaki Suenaga1

Misato Oki2

Tsuyoshi Sato1

Toshihiko Ozawa12

Daret K St Clair3

and Hideyuki J Majima12dagger

Retrospective Study Sample Distribution

To

tal S

ub

jects

(n

=86)

Lo

w (

n=10)

No

rmal (n

=27)

Ele

vate

d (

n=19)

Hig

h (

n=30)

0

1

2

3

4

5

Mit

oS

OA

R (

RF

U)

3 5

2 2

2 7

1 2

M ito S O A R

R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n

MitoSOARtrade (RFU)

lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects

N 10 27 19 3086

12 31 22 35

57 of the tested population has

superoxide levels in the

elevatedhigh range leading to high

risk for certain clinical conditions

Functional Genetics

CT

CC

0 5

1 0

1 5

2 0

2 5

Mit

oS

OA

R (

RF

U)

S O D 2 H e tro z y g o u s g e n o ty p e (C T )

S O D 2 H o m o z y g o u s g e n o ty p e (C C )

P lt 0 0 5

M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e

(S O D 2 ) P o ly m o rp h is m

Individuals who are homozygous

recessive(CC) for SOD2 may

have impaired SOD2 function

resulting in higher levels of the

free radical superoxide and

require antioxidant support

Mitochondrial Superoxide Levels

Assessment of Redox status

Inflammation

Viral or Bacterial Infection

Monitoring treatment of a

disease or disorder

Borrelia infection

Treatment of Inflammation

Functional Effect

Improvement verifies positive clinical effect

Plasma Peroxide Assay

Low Levels may indicate oxidative stress and

possible need for antioxidants

High levels may indicate immune challenges

Can correlate with uric acid

Potential hypertension risk

Related to lipid peroxides

Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13

Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303

Plasma Peroxidase

To

tal S

ub

jects

(n

=86)

Lo

w (

n=30)

No

rmal (n

=24)

Ele

vate

d (

n=11)

Hig

h (

n=16)

0

2 0

4 0

6 0

8 0

Pla

sm

a P

erix

ida

se

Ac

tiv

ity

(m

Um

l)

2 0

1 4

3 0

3 7

M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s

R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e

S a m p le D is tr ib u tio n

Plasma Peroxidase Activity (mUmL)

lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects

N 30 24 11 1681

37 30 14 20

37 of the population studied had low

peroxidase activity indicating an

impaired ability to defend against free

radicals

34 of the studies population had

elevatedhigh activity potentially

indicating the presence of oxidative

stress

Assessment of Baseline Potential

Starting point for defense of excessive

ROS

Effect of Environmental Insults

Assessment of Capacity to defend against

excessive ROS

Assessment and monitoring treatment of a

disease or disorder

Viral or Bacterial infection

Cardiovascular disease

Mitochondrial Membrane Potential

Oxidative Challenge

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Biomarkers for Early Stage 2

3

23

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Clinical Presentation19 year old Israeli Soldier during OPE

OverstimulationAnxietyInsomniaViolence Homicidal Suicidal Behaviors

Biomarkers for Mid Stage

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Biomarkers for End Stage

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Genomics

COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of

the brain responsible for personality inhibition of behaviors abstract thinking

and emotion1

The COMT val158Met polymorphism has been shown to be associated with phobic

anxiety 2 neuroticism 3 and panic disorders 4

Results indicate a relationship of COMT polymorphism rs4680 with immune

dysregulation in CFS providing a potential link for the association between stress and

infection susceptibility in CFS(chronic fatigue syndrome)

1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype

on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J

Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8

27

17 yo radicalized female with anger

antisocial and rage issues(similar not exact due to security)

Methylene-tetrahydrofolate reductase

(MTHFR SNPrsquos)

Creates methylated folate

the active folate in the

body

Mutations have been

associated with vascular

disease dementia cancer

and autism

Methylation is the genetics

elevated homocysteine is

the genomics 29

End Point of Poor Methylation and

Oxidation

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

bull Superoxide dismutase 1

(SOD1)

bull Superoxide dismutase 2

(SOD2)

bull Superoxide dismutase 3

(SOD3)

bull Glutathione peroxidase 1

(GPx1)

bull Catalase (CAT)

Superoxide Dismutase 1

SOD1 (CuZnSOD)(Encoded Chrom 21)

Found in cytoplasmic and mitochondrial

intermembrane space(inserted)

Converts superoxide radicals (O2-) to hydrogen

peroxide (H2O2)

Needs copper and zinc as cofactors

Apoptosis Oxidative Stress ALS Myocardial

Infarction Macular Degeneration a marker for

heavy Metal toxicity

O2- H2O2

SOD1SOD1

Cu Zn

1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2

2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160

3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034

Superoxide Dismutase 2

SOD2 (MnSOD) (Chrom 6)

Found in mitochondria

Converts superoxide radicals (O2-)

to hydrogen peroxide (H2O2)

Needs manganese as a cofactor

Heart Disease Myocardial

Infarction Tumor metastasis

neurodegenerative diseases

O2- H2O2SOD2SOD2

Mn

Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24

Superoxide Dismutase 3SOD3

Found extracellularly

Converts superoxide radicals (O2-) to

hydrogen peroxide (H2O2)

Needs copper and zinc as cofactors

Chronic skin disorders Diabetic

Neuropathy Folate Metabolism Ischemic

Heart Disease

O2- H2O2SOD3SOD3

Cu Zn

Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004

Glutathione Peroxidase 1

GPx1(Chrom 3)

Found in the cytoplasm of nearly all mammalian tissues

Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)

Uses glutathione an antioxidant as a cofactor

Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression

H2O2H2O O2GPx1GPx1

Glutathione

Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and

Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89

Catalase

CAT(Chrom 11)

Found in the cytoplasm of cells

Needs iron and selenium as cofactors

Reduce hydrogen peroxide (H2O2) to water (H2O)

High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second

Hypertension Type 2 Diabetes Acatalasemia

httpsghrnlmnihgovgeneCATconditions

H2O2 H2O O2CATCAT

Fe Se

Mitochondrial Function Assessment

Mitochondrial Superoxide (O2-)

Plasma Peroxide Assay (PPA)

Mitochondrial Membrane

Potential (MMP) Assay with

Oxidative Challenge

Mitochondrial Superoxide (O2-)

Superoxide is considered to be a major

factor in oxidant toxicity and

mitochondrial MnSOD enzymes

constitute an essential defense against

superoxide

High levels indicate the proanti-oxidant

system is out of balance and oxidative

stress is present

Mitochondria are the major source of

superoxide

Superoxide is the origin of reactive

oxygen (ROS)and nitrogen species (RNS)

and as such causes various redox

related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo

123Hsiu-Chuan Yen

4Ikuo Nakanishi

5Ken-ichiro Matsumoto

5Masato Tamura

6Yumiko Nagano

6Hirofumi Matsui

6Oleg Gusev

789Richard Cornette

9Takashi

Okuda9

Yukiko Minamiyama10

Hiroshi Ichikawa11

Shigeaki Suenaga1

Misato Oki2

Tsuyoshi Sato1

Toshihiko Ozawa12

Daret K St Clair3

and Hideyuki J Majima12dagger

Retrospective Study Sample Distribution

To

tal S

ub

jects

(n

=86)

Lo

w (

n=10)

No

rmal (n

=27)

Ele

vate

d (

n=19)

Hig

h (

n=30)

0

1

2

3

4

5

Mit

oS

OA

R (

RF

U)

3 5

2 2

2 7

1 2

M ito S O A R

R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n

MitoSOARtrade (RFU)

lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects

N 10 27 19 3086

12 31 22 35

57 of the tested population has

superoxide levels in the

elevatedhigh range leading to high

risk for certain clinical conditions

Functional Genetics

CT

CC

0 5

1 0

1 5

2 0

2 5

Mit

oS

OA

R (

RF

U)

S O D 2 H e tro z y g o u s g e n o ty p e (C T )

S O D 2 H o m o z y g o u s g e n o ty p e (C C )

P lt 0 0 5

M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e

(S O D 2 ) P o ly m o rp h is m

Individuals who are homozygous

recessive(CC) for SOD2 may

have impaired SOD2 function

resulting in higher levels of the

free radical superoxide and

require antioxidant support

Mitochondrial Superoxide Levels

Assessment of Redox status

Inflammation

Viral or Bacterial Infection

Monitoring treatment of a

disease or disorder

Borrelia infection

Treatment of Inflammation

Functional Effect

Improvement verifies positive clinical effect

Plasma Peroxide Assay

Low Levels may indicate oxidative stress and

possible need for antioxidants

High levels may indicate immune challenges

Can correlate with uric acid

Potential hypertension risk

Related to lipid peroxides

Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13

Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303

Plasma Peroxidase

To

tal S

ub

jects

(n

=86)

Lo

w (

n=30)

No

rmal (n

=24)

Ele

vate

d (

n=11)

Hig

h (

n=16)

0

2 0

4 0

6 0

8 0

Pla

sm

a P

erix

ida

se

Ac

tiv

ity

(m

Um

l)

2 0

1 4

3 0

3 7

M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s

R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e

S a m p le D is tr ib u tio n

Plasma Peroxidase Activity (mUmL)

lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects

N 30 24 11 1681

37 30 14 20

37 of the population studied had low

peroxidase activity indicating an

impaired ability to defend against free

radicals

34 of the studies population had

elevatedhigh activity potentially

indicating the presence of oxidative

stress

Assessment of Baseline Potential

Starting point for defense of excessive

ROS

Effect of Environmental Insults

Assessment of Capacity to defend against

excessive ROS

Assessment and monitoring treatment of a

disease or disorder

Viral or Bacterial infection

Cardiovascular disease

Mitochondrial Membrane Potential

Oxidative Challenge

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Biomarkers for Mid Stage

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Clinical Presentation36 Year old Israeli Nurse during OPE

Wired but TiredDepressionInsomnia

Biomarkers for End Stage

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Genomics

COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of

the brain responsible for personality inhibition of behaviors abstract thinking

and emotion1

The COMT val158Met polymorphism has been shown to be associated with phobic

anxiety 2 neuroticism 3 and panic disorders 4

Results indicate a relationship of COMT polymorphism rs4680 with immune

dysregulation in CFS providing a potential link for the association between stress and

infection susceptibility in CFS(chronic fatigue syndrome)

1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype

on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J

Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8

27

17 yo radicalized female with anger

antisocial and rage issues(similar not exact due to security)

Methylene-tetrahydrofolate reductase

(MTHFR SNPrsquos)

Creates methylated folate

the active folate in the

body

Mutations have been

associated with vascular

disease dementia cancer

and autism

Methylation is the genetics

elevated homocysteine is

the genomics 29

End Point of Poor Methylation and

Oxidation

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

bull Superoxide dismutase 1

(SOD1)

bull Superoxide dismutase 2

(SOD2)

bull Superoxide dismutase 3

(SOD3)

bull Glutathione peroxidase 1

(GPx1)

bull Catalase (CAT)

Superoxide Dismutase 1

SOD1 (CuZnSOD)(Encoded Chrom 21)

Found in cytoplasmic and mitochondrial

intermembrane space(inserted)

Converts superoxide radicals (O2-) to hydrogen

peroxide (H2O2)

Needs copper and zinc as cofactors

Apoptosis Oxidative Stress ALS Myocardial

Infarction Macular Degeneration a marker for

heavy Metal toxicity

O2- H2O2

SOD1SOD1

Cu Zn

1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2

2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160

3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034

Superoxide Dismutase 2

SOD2 (MnSOD) (Chrom 6)

Found in mitochondria

Converts superoxide radicals (O2-)

to hydrogen peroxide (H2O2)

Needs manganese as a cofactor

Heart Disease Myocardial

Infarction Tumor metastasis

neurodegenerative diseases

O2- H2O2SOD2SOD2

Mn

Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24

Superoxide Dismutase 3SOD3

Found extracellularly

Converts superoxide radicals (O2-) to

hydrogen peroxide (H2O2)

Needs copper and zinc as cofactors

Chronic skin disorders Diabetic

Neuropathy Folate Metabolism Ischemic

Heart Disease

O2- H2O2SOD3SOD3

Cu Zn

Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004

Glutathione Peroxidase 1

GPx1(Chrom 3)

Found in the cytoplasm of nearly all mammalian tissues

Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)

Uses glutathione an antioxidant as a cofactor

Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression

H2O2H2O O2GPx1GPx1

Glutathione

Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and

Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89

Catalase

CAT(Chrom 11)

Found in the cytoplasm of cells

Needs iron and selenium as cofactors

Reduce hydrogen peroxide (H2O2) to water (H2O)

High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second

Hypertension Type 2 Diabetes Acatalasemia

httpsghrnlmnihgovgeneCATconditions

H2O2 H2O O2CATCAT

Fe Se

Mitochondrial Function Assessment

Mitochondrial Superoxide (O2-)

Plasma Peroxide Assay (PPA)

Mitochondrial Membrane

Potential (MMP) Assay with

Oxidative Challenge

Mitochondrial Superoxide (O2-)

Superoxide is considered to be a major

factor in oxidant toxicity and

mitochondrial MnSOD enzymes

constitute an essential defense against

superoxide

High levels indicate the proanti-oxidant

system is out of balance and oxidative

stress is present

Mitochondria are the major source of

superoxide

Superoxide is the origin of reactive

oxygen (ROS)and nitrogen species (RNS)

and as such causes various redox

related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo

123Hsiu-Chuan Yen

4Ikuo Nakanishi

5Ken-ichiro Matsumoto

5Masato Tamura

6Yumiko Nagano

6Hirofumi Matsui

6Oleg Gusev

789Richard Cornette

9Takashi

Okuda9

Yukiko Minamiyama10

Hiroshi Ichikawa11

Shigeaki Suenaga1

Misato Oki2

Tsuyoshi Sato1

Toshihiko Ozawa12

Daret K St Clair3

and Hideyuki J Majima12dagger

Retrospective Study Sample Distribution

To

tal S

ub

jects

(n

=86)

Lo

w (

n=10)

No

rmal (n

=27)

Ele

vate

d (

n=19)

Hig

h (

n=30)

0

1

2

3

4

5

Mit

oS

OA

R (

RF

U)

3 5

2 2

2 7

1 2

M ito S O A R

R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n

MitoSOARtrade (RFU)

lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects

N 10 27 19 3086

12 31 22 35

57 of the tested population has

superoxide levels in the

elevatedhigh range leading to high

risk for certain clinical conditions

Functional Genetics

CT

CC

0 5

1 0

1 5

2 0

2 5

Mit

oS

OA

R (

RF

U)

S O D 2 H e tro z y g o u s g e n o ty p e (C T )

S O D 2 H o m o z y g o u s g e n o ty p e (C C )

P lt 0 0 5

M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e

(S O D 2 ) P o ly m o rp h is m

Individuals who are homozygous

recessive(CC) for SOD2 may

have impaired SOD2 function

resulting in higher levels of the

free radical superoxide and

require antioxidant support

Mitochondrial Superoxide Levels

Assessment of Redox status

Inflammation

Viral or Bacterial Infection

Monitoring treatment of a

disease or disorder

Borrelia infection

Treatment of Inflammation

Functional Effect

Improvement verifies positive clinical effect

Plasma Peroxide Assay

Low Levels may indicate oxidative stress and

possible need for antioxidants

High levels may indicate immune challenges

Can correlate with uric acid

Potential hypertension risk

Related to lipid peroxides

Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13

Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303

Plasma Peroxidase

To

tal S

ub

jects

(n

=86)

Lo

w (

n=30)

No

rmal (n

=24)

Ele

vate

d (

n=11)

Hig

h (

n=16)

0

2 0

4 0

6 0

8 0

Pla

sm

a P

erix

ida

se

Ac

tiv

ity

(m

Um

l)

2 0

1 4

3 0

3 7

M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s

R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e

S a m p le D is tr ib u tio n

Plasma Peroxidase Activity (mUmL)

lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects

N 30 24 11 1681

37 30 14 20

37 of the population studied had low

peroxidase activity indicating an

impaired ability to defend against free

radicals

34 of the studies population had

elevatedhigh activity potentially

indicating the presence of oxidative

stress

Assessment of Baseline Potential

Starting point for defense of excessive

ROS

Effect of Environmental Insults

Assessment of Capacity to defend against

excessive ROS

Assessment and monitoring treatment of a

disease or disorder

Viral or Bacterial infection

Cardiovascular disease

Mitochondrial Membrane Potential

Oxidative Challenge

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Biomarkers for End Stage

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Clinical Presentation

ExhaustionDifficulty dealing with stressFatigueCognitive declineViolent Suicidal Behaviors

Palestinian with suicidal homicidal thoughts considered ldquointractablerdquo

Genomics

COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of

the brain responsible for personality inhibition of behaviors abstract thinking

and emotion1

The COMT val158Met polymorphism has been shown to be associated with phobic

anxiety 2 neuroticism 3 and panic disorders 4

Results indicate a relationship of COMT polymorphism rs4680 with immune

dysregulation in CFS providing a potential link for the association between stress and

infection susceptibility in CFS(chronic fatigue syndrome)

1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype

on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J

Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8

27

17 yo radicalized female with anger

antisocial and rage issues(similar not exact due to security)

Methylene-tetrahydrofolate reductase

(MTHFR SNPrsquos)

Creates methylated folate

the active folate in the

body

Mutations have been

associated with vascular

disease dementia cancer

and autism

Methylation is the genetics

elevated homocysteine is

the genomics 29

End Point of Poor Methylation and

Oxidation

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

bull Superoxide dismutase 1

(SOD1)

bull Superoxide dismutase 2

(SOD2)

bull Superoxide dismutase 3

(SOD3)

bull Glutathione peroxidase 1

(GPx1)

bull Catalase (CAT)

Superoxide Dismutase 1

SOD1 (CuZnSOD)(Encoded Chrom 21)

Found in cytoplasmic and mitochondrial

intermembrane space(inserted)

Converts superoxide radicals (O2-) to hydrogen

peroxide (H2O2)

Needs copper and zinc as cofactors

Apoptosis Oxidative Stress ALS Myocardial

Infarction Macular Degeneration a marker for

heavy Metal toxicity

O2- H2O2

SOD1SOD1

Cu Zn

1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2

2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160

3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034

Superoxide Dismutase 2

SOD2 (MnSOD) (Chrom 6)

Found in mitochondria

Converts superoxide radicals (O2-)

to hydrogen peroxide (H2O2)

Needs manganese as a cofactor

Heart Disease Myocardial

Infarction Tumor metastasis

neurodegenerative diseases

O2- H2O2SOD2SOD2

Mn

Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24

Superoxide Dismutase 3SOD3

Found extracellularly

Converts superoxide radicals (O2-) to

hydrogen peroxide (H2O2)

Needs copper and zinc as cofactors

Chronic skin disorders Diabetic

Neuropathy Folate Metabolism Ischemic

Heart Disease

O2- H2O2SOD3SOD3

Cu Zn

Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004

Glutathione Peroxidase 1

GPx1(Chrom 3)

Found in the cytoplasm of nearly all mammalian tissues

Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)

Uses glutathione an antioxidant as a cofactor

Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression

H2O2H2O O2GPx1GPx1

Glutathione

Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and

Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89

Catalase

CAT(Chrom 11)

Found in the cytoplasm of cells

Needs iron and selenium as cofactors

Reduce hydrogen peroxide (H2O2) to water (H2O)

High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second

Hypertension Type 2 Diabetes Acatalasemia

httpsghrnlmnihgovgeneCATconditions

H2O2 H2O O2CATCAT

Fe Se

Mitochondrial Function Assessment

Mitochondrial Superoxide (O2-)

Plasma Peroxide Assay (PPA)

Mitochondrial Membrane

Potential (MMP) Assay with

Oxidative Challenge

Mitochondrial Superoxide (O2-)

Superoxide is considered to be a major

factor in oxidant toxicity and

mitochondrial MnSOD enzymes

constitute an essential defense against

superoxide

High levels indicate the proanti-oxidant

system is out of balance and oxidative

stress is present

Mitochondria are the major source of

superoxide

Superoxide is the origin of reactive

oxygen (ROS)and nitrogen species (RNS)

and as such causes various redox

related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo

123Hsiu-Chuan Yen

4Ikuo Nakanishi

5Ken-ichiro Matsumoto

5Masato Tamura

6Yumiko Nagano

6Hirofumi Matsui

6Oleg Gusev

789Richard Cornette

9Takashi

Okuda9

Yukiko Minamiyama10

Hiroshi Ichikawa11

Shigeaki Suenaga1

Misato Oki2

Tsuyoshi Sato1

Toshihiko Ozawa12

Daret K St Clair3

and Hideyuki J Majima12dagger

Retrospective Study Sample Distribution

To

tal S

ub

jects

(n

=86)

Lo

w (

n=10)

No

rmal (n

=27)

Ele

vate

d (

n=19)

Hig

h (

n=30)

0

1

2

3

4

5

Mit

oS

OA

R (

RF

U)

3 5

2 2

2 7

1 2

M ito S O A R

R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n

MitoSOARtrade (RFU)

lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects

N 10 27 19 3086

12 31 22 35

57 of the tested population has

superoxide levels in the

elevatedhigh range leading to high

risk for certain clinical conditions

Functional Genetics

CT

CC

0 5

1 0

1 5

2 0

2 5

Mit

oS

OA

R (

RF

U)

S O D 2 H e tro z y g o u s g e n o ty p e (C T )

S O D 2 H o m o z y g o u s g e n o ty p e (C C )

P lt 0 0 5

M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e

(S O D 2 ) P o ly m o rp h is m

Individuals who are homozygous

recessive(CC) for SOD2 may

have impaired SOD2 function

resulting in higher levels of the

free radical superoxide and

require antioxidant support

Mitochondrial Superoxide Levels

Assessment of Redox status

Inflammation

Viral or Bacterial Infection

Monitoring treatment of a

disease or disorder

Borrelia infection

Treatment of Inflammation

Functional Effect

Improvement verifies positive clinical effect

Plasma Peroxide Assay

Low Levels may indicate oxidative stress and

possible need for antioxidants

High levels may indicate immune challenges

Can correlate with uric acid

Potential hypertension risk

Related to lipid peroxides

Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13

Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303

Plasma Peroxidase

To

tal S

ub

jects

(n

=86)

Lo

w (

n=30)

No

rmal (n

=24)

Ele

vate

d (

n=11)

Hig

h (

n=16)

0

2 0

4 0

6 0

8 0

Pla

sm

a P

erix

ida

se

Ac

tiv

ity

(m

Um

l)

2 0

1 4

3 0

3 7

M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s

R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e

S a m p le D is tr ib u tio n

Plasma Peroxidase Activity (mUmL)

lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects

N 30 24 11 1681

37 30 14 20

37 of the population studied had low

peroxidase activity indicating an

impaired ability to defend against free

radicals

34 of the studies population had

elevatedhigh activity potentially

indicating the presence of oxidative

stress

Assessment of Baseline Potential

Starting point for defense of excessive

ROS

Effect of Environmental Insults

Assessment of Capacity to defend against

excessive ROS

Assessment and monitoring treatment of a

disease or disorder

Viral or Bacterial infection

Cardiovascular disease

Mitochondrial Membrane Potential

Oxidative Challenge

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Genomics

COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of

the brain responsible for personality inhibition of behaviors abstract thinking

and emotion1

The COMT val158Met polymorphism has been shown to be associated with phobic

anxiety 2 neuroticism 3 and panic disorders 4

Results indicate a relationship of COMT polymorphism rs4680 with immune

dysregulation in CFS providing a potential link for the association between stress and

infection susceptibility in CFS(chronic fatigue syndrome)

1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype

on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J

Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8

27

17 yo radicalized female with anger

antisocial and rage issues(similar not exact due to security)

Methylene-tetrahydrofolate reductase

(MTHFR SNPrsquos)

Creates methylated folate

the active folate in the

body

Mutations have been

associated with vascular

disease dementia cancer

and autism

Methylation is the genetics

elevated homocysteine is

the genomics 29

End Point of Poor Methylation and

Oxidation

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

bull Superoxide dismutase 1

(SOD1)

bull Superoxide dismutase 2

(SOD2)

bull Superoxide dismutase 3

(SOD3)

bull Glutathione peroxidase 1

(GPx1)

bull Catalase (CAT)

Superoxide Dismutase 1

SOD1 (CuZnSOD)(Encoded Chrom 21)

Found in cytoplasmic and mitochondrial

intermembrane space(inserted)

Converts superoxide radicals (O2-) to hydrogen

peroxide (H2O2)

Needs copper and zinc as cofactors

Apoptosis Oxidative Stress ALS Myocardial

Infarction Macular Degeneration a marker for

heavy Metal toxicity

O2- H2O2

SOD1SOD1

Cu Zn

1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2

2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160

3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034

Superoxide Dismutase 2

SOD2 (MnSOD) (Chrom 6)

Found in mitochondria

Converts superoxide radicals (O2-)

to hydrogen peroxide (H2O2)

Needs manganese as a cofactor

Heart Disease Myocardial

Infarction Tumor metastasis

neurodegenerative diseases

O2- H2O2SOD2SOD2

Mn

Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24

Superoxide Dismutase 3SOD3

Found extracellularly

Converts superoxide radicals (O2-) to

hydrogen peroxide (H2O2)

Needs copper and zinc as cofactors

Chronic skin disorders Diabetic

Neuropathy Folate Metabolism Ischemic

Heart Disease

O2- H2O2SOD3SOD3

Cu Zn

Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004

Glutathione Peroxidase 1

GPx1(Chrom 3)

Found in the cytoplasm of nearly all mammalian tissues

Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)

Uses glutathione an antioxidant as a cofactor

Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression

H2O2H2O O2GPx1GPx1

Glutathione

Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and

Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89

Catalase

CAT(Chrom 11)

Found in the cytoplasm of cells

Needs iron and selenium as cofactors

Reduce hydrogen peroxide (H2O2) to water (H2O)

High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second

Hypertension Type 2 Diabetes Acatalasemia

httpsghrnlmnihgovgeneCATconditions

H2O2 H2O O2CATCAT

Fe Se

Mitochondrial Function Assessment

Mitochondrial Superoxide (O2-)

Plasma Peroxide Assay (PPA)

Mitochondrial Membrane

Potential (MMP) Assay with

Oxidative Challenge

Mitochondrial Superoxide (O2-)

Superoxide is considered to be a major

factor in oxidant toxicity and

mitochondrial MnSOD enzymes

constitute an essential defense against

superoxide

High levels indicate the proanti-oxidant

system is out of balance and oxidative

stress is present

Mitochondria are the major source of

superoxide

Superoxide is the origin of reactive

oxygen (ROS)and nitrogen species (RNS)

and as such causes various redox

related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo

123Hsiu-Chuan Yen

4Ikuo Nakanishi

5Ken-ichiro Matsumoto

5Masato Tamura

6Yumiko Nagano

6Hirofumi Matsui

6Oleg Gusev

789Richard Cornette

9Takashi

Okuda9

Yukiko Minamiyama10

Hiroshi Ichikawa11

Shigeaki Suenaga1

Misato Oki2

Tsuyoshi Sato1

Toshihiko Ozawa12

Daret K St Clair3

and Hideyuki J Majima12dagger

Retrospective Study Sample Distribution

To

tal S

ub

jects

(n

=86)

Lo

w (

n=10)

No

rmal (n

=27)

Ele

vate

d (

n=19)

Hig

h (

n=30)

0

1

2

3

4

5

Mit

oS

OA

R (

RF

U)

3 5

2 2

2 7

1 2

M ito S O A R

R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n

MitoSOARtrade (RFU)

lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects

N 10 27 19 3086

12 31 22 35

57 of the tested population has

superoxide levels in the

elevatedhigh range leading to high

risk for certain clinical conditions

Functional Genetics

CT

CC

0 5

1 0

1 5

2 0

2 5

Mit

oS

OA

R (

RF

U)

S O D 2 H e tro z y g o u s g e n o ty p e (C T )

S O D 2 H o m o z y g o u s g e n o ty p e (C C )

P lt 0 0 5

M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e

(S O D 2 ) P o ly m o rp h is m

Individuals who are homozygous

recessive(CC) for SOD2 may

have impaired SOD2 function

resulting in higher levels of the

free radical superoxide and

require antioxidant support

Mitochondrial Superoxide Levels

Assessment of Redox status

Inflammation

Viral or Bacterial Infection

Monitoring treatment of a

disease or disorder

Borrelia infection

Treatment of Inflammation

Functional Effect

Improvement verifies positive clinical effect

Plasma Peroxide Assay

Low Levels may indicate oxidative stress and

possible need for antioxidants

High levels may indicate immune challenges

Can correlate with uric acid

Potential hypertension risk

Related to lipid peroxides

Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13

Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303

Plasma Peroxidase

To

tal S

ub

jects

(n

=86)

Lo

w (

n=30)

No

rmal (n

=24)

Ele

vate

d (

n=11)

Hig

h (

n=16)

0

2 0

4 0

6 0

8 0

Pla

sm

a P

erix

ida

se

Ac

tiv

ity

(m

Um

l)

2 0

1 4

3 0

3 7

M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s

R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e

S a m p le D is tr ib u tio n

Plasma Peroxidase Activity (mUmL)

lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects

N 30 24 11 1681

37 30 14 20

37 of the population studied had low

peroxidase activity indicating an

impaired ability to defend against free

radicals

34 of the studies population had

elevatedhigh activity potentially

indicating the presence of oxidative

stress

Assessment of Baseline Potential

Starting point for defense of excessive

ROS

Effect of Environmental Insults

Assessment of Capacity to defend against

excessive ROS

Assessment and monitoring treatment of a

disease or disorder

Viral or Bacterial infection

Cardiovascular disease

Mitochondrial Membrane Potential

Oxidative Challenge

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

COMT ConnectionCOMT helps maintain appropriate levels of neurotransmitters in the prefrontal cortex of

the brain responsible for personality inhibition of behaviors abstract thinking

and emotion1

The COMT val158Met polymorphism has been shown to be associated with phobic

anxiety 2 neuroticism 3 and panic disorders 4

Results indicate a relationship of COMT polymorphism rs4680 with immune

dysregulation in CFS providing a potential link for the association between stress and

infection susceptibility in CFS(chronic fatigue syndrome)

1 Yager JD Catechol-O-methyltransferase characteristics polymorphisms and role in breast cancer Drug Discov Today Dis Mech 2012 Jun 19(1-2)e41-e462 Domschke K Ohrmann P Braun M Suslow T Bauer J Hohoff C Kersting AEngelien A Arolt V Heindel W Deckert J Kugel H Influence of the catechol-O-methyltransferase val158met genotype

on amygdala and prefrontal cortex emotional processing in panic disorder Psychiatry Res 2008 May 30163(1)13-203 McGrath M Kawachi I Ascherio A Colditz GA Hunter DJ De Vivo I Association between catechol-O-methyltransferase and phobic anxiety Am J Psychiatry 2004 Sep161(9)1703-54 Eley TC Tahir E Angleitner A Harriss K McClay J Plomin R et al (2003) Association analysis of MAOA and COMT with neuroticism assessed by peers Am J Med Genet 120B 90ndash965 Domschke K Freitag CM Kuhlenbaumlumer G Schirmacher A Sand P Nyhuis P Jacob C Fritze J Franke P Rietschel M Garritsen HS Fimmers R Noumlthen MM Lesch KP Stoumlgbauer F Deckert J

Association of the functional V158M catechol-O-methyl-transferase polymorphism with panic disorder in women Int J Neuropsychopharmacol 2004 Jun7(2)183-8

27

17 yo radicalized female with anger

antisocial and rage issues(similar not exact due to security)

Methylene-tetrahydrofolate reductase

(MTHFR SNPrsquos)

Creates methylated folate

the active folate in the

body

Mutations have been

associated with vascular

disease dementia cancer

and autism

Methylation is the genetics

elevated homocysteine is

the genomics 29

End Point of Poor Methylation and

Oxidation

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

bull Superoxide dismutase 1

(SOD1)

bull Superoxide dismutase 2

(SOD2)

bull Superoxide dismutase 3

(SOD3)

bull Glutathione peroxidase 1

(GPx1)

bull Catalase (CAT)

Superoxide Dismutase 1

SOD1 (CuZnSOD)(Encoded Chrom 21)

Found in cytoplasmic and mitochondrial

intermembrane space(inserted)

Converts superoxide radicals (O2-) to hydrogen

peroxide (H2O2)

Needs copper and zinc as cofactors

Apoptosis Oxidative Stress ALS Myocardial

Infarction Macular Degeneration a marker for

heavy Metal toxicity

O2- H2O2

SOD1SOD1

Cu Zn

1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2

2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160

3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034

Superoxide Dismutase 2

SOD2 (MnSOD) (Chrom 6)

Found in mitochondria

Converts superoxide radicals (O2-)

to hydrogen peroxide (H2O2)

Needs manganese as a cofactor

Heart Disease Myocardial

Infarction Tumor metastasis

neurodegenerative diseases

O2- H2O2SOD2SOD2

Mn

Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24

Superoxide Dismutase 3SOD3

Found extracellularly

Converts superoxide radicals (O2-) to

hydrogen peroxide (H2O2)

Needs copper and zinc as cofactors

Chronic skin disorders Diabetic

Neuropathy Folate Metabolism Ischemic

Heart Disease

O2- H2O2SOD3SOD3

Cu Zn

Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004

Glutathione Peroxidase 1

GPx1(Chrom 3)

Found in the cytoplasm of nearly all mammalian tissues

Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)

Uses glutathione an antioxidant as a cofactor

Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression

H2O2H2O O2GPx1GPx1

Glutathione

Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and

Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89

Catalase

CAT(Chrom 11)

Found in the cytoplasm of cells

Needs iron and selenium as cofactors

Reduce hydrogen peroxide (H2O2) to water (H2O)

High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second

Hypertension Type 2 Diabetes Acatalasemia

httpsghrnlmnihgovgeneCATconditions

H2O2 H2O O2CATCAT

Fe Se

Mitochondrial Function Assessment

Mitochondrial Superoxide (O2-)

Plasma Peroxide Assay (PPA)

Mitochondrial Membrane

Potential (MMP) Assay with

Oxidative Challenge

Mitochondrial Superoxide (O2-)

Superoxide is considered to be a major

factor in oxidant toxicity and

mitochondrial MnSOD enzymes

constitute an essential defense against

superoxide

High levels indicate the proanti-oxidant

system is out of balance and oxidative

stress is present

Mitochondria are the major source of

superoxide

Superoxide is the origin of reactive

oxygen (ROS)and nitrogen species (RNS)

and as such causes various redox

related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo

123Hsiu-Chuan Yen

4Ikuo Nakanishi

5Ken-ichiro Matsumoto

5Masato Tamura

6Yumiko Nagano

6Hirofumi Matsui

6Oleg Gusev

789Richard Cornette

9Takashi

Okuda9

Yukiko Minamiyama10

Hiroshi Ichikawa11

Shigeaki Suenaga1

Misato Oki2

Tsuyoshi Sato1

Toshihiko Ozawa12

Daret K St Clair3

and Hideyuki J Majima12dagger

Retrospective Study Sample Distribution

To

tal S

ub

jects

(n

=86)

Lo

w (

n=10)

No

rmal (n

=27)

Ele

vate

d (

n=19)

Hig

h (

n=30)

0

1

2

3

4

5

Mit

oS

OA

R (

RF

U)

3 5

2 2

2 7

1 2

M ito S O A R

R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n

MitoSOARtrade (RFU)

lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects

N 10 27 19 3086

12 31 22 35

57 of the tested population has

superoxide levels in the

elevatedhigh range leading to high

risk for certain clinical conditions

Functional Genetics

CT

CC

0 5

1 0

1 5

2 0

2 5

Mit

oS

OA

R (

RF

U)

S O D 2 H e tro z y g o u s g e n o ty p e (C T )

S O D 2 H o m o z y g o u s g e n o ty p e (C C )

P lt 0 0 5

M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e

(S O D 2 ) P o ly m o rp h is m

Individuals who are homozygous

recessive(CC) for SOD2 may

have impaired SOD2 function

resulting in higher levels of the

free radical superoxide and

require antioxidant support

Mitochondrial Superoxide Levels

Assessment of Redox status

Inflammation

Viral or Bacterial Infection

Monitoring treatment of a

disease or disorder

Borrelia infection

Treatment of Inflammation

Functional Effect

Improvement verifies positive clinical effect

Plasma Peroxide Assay

Low Levels may indicate oxidative stress and

possible need for antioxidants

High levels may indicate immune challenges

Can correlate with uric acid

Potential hypertension risk

Related to lipid peroxides

Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13

Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303

Plasma Peroxidase

To

tal S

ub

jects

(n

=86)

Lo

w (

n=30)

No

rmal (n

=24)

Ele

vate

d (

n=11)

Hig

h (

n=16)

0

2 0

4 0

6 0

8 0

Pla

sm

a P

erix

ida

se

Ac

tiv

ity

(m

Um

l)

2 0

1 4

3 0

3 7

M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s

R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e

S a m p le D is tr ib u tio n

Plasma Peroxidase Activity (mUmL)

lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects

N 30 24 11 1681

37 30 14 20

37 of the population studied had low

peroxidase activity indicating an

impaired ability to defend against free

radicals

34 of the studies population had

elevatedhigh activity potentially

indicating the presence of oxidative

stress

Assessment of Baseline Potential

Starting point for defense of excessive

ROS

Effect of Environmental Insults

Assessment of Capacity to defend against

excessive ROS

Assessment and monitoring treatment of a

disease or disorder

Viral or Bacterial infection

Cardiovascular disease

Mitochondrial Membrane Potential

Oxidative Challenge

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

17 yo radicalized female with anger

antisocial and rage issues(similar not exact due to security)

Methylene-tetrahydrofolate reductase

(MTHFR SNPrsquos)

Creates methylated folate

the active folate in the

body

Mutations have been

associated with vascular

disease dementia cancer

and autism

Methylation is the genetics

elevated homocysteine is

the genomics 29

End Point of Poor Methylation and

Oxidation

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

bull Superoxide dismutase 1

(SOD1)

bull Superoxide dismutase 2

(SOD2)

bull Superoxide dismutase 3

(SOD3)

bull Glutathione peroxidase 1

(GPx1)

bull Catalase (CAT)

Superoxide Dismutase 1

SOD1 (CuZnSOD)(Encoded Chrom 21)

Found in cytoplasmic and mitochondrial

intermembrane space(inserted)

Converts superoxide radicals (O2-) to hydrogen

peroxide (H2O2)

Needs copper and zinc as cofactors

Apoptosis Oxidative Stress ALS Myocardial

Infarction Macular Degeneration a marker for

heavy Metal toxicity

O2- H2O2

SOD1SOD1

Cu Zn

1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2

2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160

3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034

Superoxide Dismutase 2

SOD2 (MnSOD) (Chrom 6)

Found in mitochondria

Converts superoxide radicals (O2-)

to hydrogen peroxide (H2O2)

Needs manganese as a cofactor

Heart Disease Myocardial

Infarction Tumor metastasis

neurodegenerative diseases

O2- H2O2SOD2SOD2

Mn

Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24

Superoxide Dismutase 3SOD3

Found extracellularly

Converts superoxide radicals (O2-) to

hydrogen peroxide (H2O2)

Needs copper and zinc as cofactors

Chronic skin disorders Diabetic

Neuropathy Folate Metabolism Ischemic

Heart Disease

O2- H2O2SOD3SOD3

Cu Zn

Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004

Glutathione Peroxidase 1

GPx1(Chrom 3)

Found in the cytoplasm of nearly all mammalian tissues

Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)

Uses glutathione an antioxidant as a cofactor

Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression

H2O2H2O O2GPx1GPx1

Glutathione

Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and

Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89

Catalase

CAT(Chrom 11)

Found in the cytoplasm of cells

Needs iron and selenium as cofactors

Reduce hydrogen peroxide (H2O2) to water (H2O)

High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second

Hypertension Type 2 Diabetes Acatalasemia

httpsghrnlmnihgovgeneCATconditions

H2O2 H2O O2CATCAT

Fe Se

Mitochondrial Function Assessment

Mitochondrial Superoxide (O2-)

Plasma Peroxide Assay (PPA)

Mitochondrial Membrane

Potential (MMP) Assay with

Oxidative Challenge

Mitochondrial Superoxide (O2-)

Superoxide is considered to be a major

factor in oxidant toxicity and

mitochondrial MnSOD enzymes

constitute an essential defense against

superoxide

High levels indicate the proanti-oxidant

system is out of balance and oxidative

stress is present

Mitochondria are the major source of

superoxide

Superoxide is the origin of reactive

oxygen (ROS)and nitrogen species (RNS)

and as such causes various redox

related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo

123Hsiu-Chuan Yen

4Ikuo Nakanishi

5Ken-ichiro Matsumoto

5Masato Tamura

6Yumiko Nagano

6Hirofumi Matsui

6Oleg Gusev

789Richard Cornette

9Takashi

Okuda9

Yukiko Minamiyama10

Hiroshi Ichikawa11

Shigeaki Suenaga1

Misato Oki2

Tsuyoshi Sato1

Toshihiko Ozawa12

Daret K St Clair3

and Hideyuki J Majima12dagger

Retrospective Study Sample Distribution

To

tal S

ub

jects

(n

=86)

Lo

w (

n=10)

No

rmal (n

=27)

Ele

vate

d (

n=19)

Hig

h (

n=30)

0

1

2

3

4

5

Mit

oS

OA

R (

RF

U)

3 5

2 2

2 7

1 2

M ito S O A R

R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n

MitoSOARtrade (RFU)

lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects

N 10 27 19 3086

12 31 22 35

57 of the tested population has

superoxide levels in the

elevatedhigh range leading to high

risk for certain clinical conditions

Functional Genetics

CT

CC

0 5

1 0

1 5

2 0

2 5

Mit

oS

OA

R (

RF

U)

S O D 2 H e tro z y g o u s g e n o ty p e (C T )

S O D 2 H o m o z y g o u s g e n o ty p e (C C )

P lt 0 0 5

M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e

(S O D 2 ) P o ly m o rp h is m

Individuals who are homozygous

recessive(CC) for SOD2 may

have impaired SOD2 function

resulting in higher levels of the

free radical superoxide and

require antioxidant support

Mitochondrial Superoxide Levels

Assessment of Redox status

Inflammation

Viral or Bacterial Infection

Monitoring treatment of a

disease or disorder

Borrelia infection

Treatment of Inflammation

Functional Effect

Improvement verifies positive clinical effect

Plasma Peroxide Assay

Low Levels may indicate oxidative stress and

possible need for antioxidants

High levels may indicate immune challenges

Can correlate with uric acid

Potential hypertension risk

Related to lipid peroxides

Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13

Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303

Plasma Peroxidase

To

tal S

ub

jects

(n

=86)

Lo

w (

n=30)

No

rmal (n

=24)

Ele

vate

d (

n=11)

Hig

h (

n=16)

0

2 0

4 0

6 0

8 0

Pla

sm

a P

erix

ida

se

Ac

tiv

ity

(m

Um

l)

2 0

1 4

3 0

3 7

M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s

R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e

S a m p le D is tr ib u tio n

Plasma Peroxidase Activity (mUmL)

lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects

N 30 24 11 1681

37 30 14 20

37 of the population studied had low

peroxidase activity indicating an

impaired ability to defend against free

radicals

34 of the studies population had

elevatedhigh activity potentially

indicating the presence of oxidative

stress

Assessment of Baseline Potential

Starting point for defense of excessive

ROS

Effect of Environmental Insults

Assessment of Capacity to defend against

excessive ROS

Assessment and monitoring treatment of a

disease or disorder

Viral or Bacterial infection

Cardiovascular disease

Mitochondrial Membrane Potential

Oxidative Challenge

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Methylene-tetrahydrofolate reductase

(MTHFR SNPrsquos)

Creates methylated folate

the active folate in the

body

Mutations have been

associated with vascular

disease dementia cancer

and autism

Methylation is the genetics

elevated homocysteine is

the genomics 29

End Point of Poor Methylation and

Oxidation

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

bull Superoxide dismutase 1

(SOD1)

bull Superoxide dismutase 2

(SOD2)

bull Superoxide dismutase 3

(SOD3)

bull Glutathione peroxidase 1

(GPx1)

bull Catalase (CAT)

Superoxide Dismutase 1

SOD1 (CuZnSOD)(Encoded Chrom 21)

Found in cytoplasmic and mitochondrial

intermembrane space(inserted)

Converts superoxide radicals (O2-) to hydrogen

peroxide (H2O2)

Needs copper and zinc as cofactors

Apoptosis Oxidative Stress ALS Myocardial

Infarction Macular Degeneration a marker for

heavy Metal toxicity

O2- H2O2

SOD1SOD1

Cu Zn

1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2

2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160

3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034

Superoxide Dismutase 2

SOD2 (MnSOD) (Chrom 6)

Found in mitochondria

Converts superoxide radicals (O2-)

to hydrogen peroxide (H2O2)

Needs manganese as a cofactor

Heart Disease Myocardial

Infarction Tumor metastasis

neurodegenerative diseases

O2- H2O2SOD2SOD2

Mn

Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24

Superoxide Dismutase 3SOD3

Found extracellularly

Converts superoxide radicals (O2-) to

hydrogen peroxide (H2O2)

Needs copper and zinc as cofactors

Chronic skin disorders Diabetic

Neuropathy Folate Metabolism Ischemic

Heart Disease

O2- H2O2SOD3SOD3

Cu Zn

Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004

Glutathione Peroxidase 1

GPx1(Chrom 3)

Found in the cytoplasm of nearly all mammalian tissues

Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)

Uses glutathione an antioxidant as a cofactor

Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression

H2O2H2O O2GPx1GPx1

Glutathione

Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and

Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89

Catalase

CAT(Chrom 11)

Found in the cytoplasm of cells

Needs iron and selenium as cofactors

Reduce hydrogen peroxide (H2O2) to water (H2O)

High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second

Hypertension Type 2 Diabetes Acatalasemia

httpsghrnlmnihgovgeneCATconditions

H2O2 H2O O2CATCAT

Fe Se

Mitochondrial Function Assessment

Mitochondrial Superoxide (O2-)

Plasma Peroxide Assay (PPA)

Mitochondrial Membrane

Potential (MMP) Assay with

Oxidative Challenge

Mitochondrial Superoxide (O2-)

Superoxide is considered to be a major

factor in oxidant toxicity and

mitochondrial MnSOD enzymes

constitute an essential defense against

superoxide

High levels indicate the proanti-oxidant

system is out of balance and oxidative

stress is present

Mitochondria are the major source of

superoxide

Superoxide is the origin of reactive

oxygen (ROS)and nitrogen species (RNS)

and as such causes various redox

related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo

123Hsiu-Chuan Yen

4Ikuo Nakanishi

5Ken-ichiro Matsumoto

5Masato Tamura

6Yumiko Nagano

6Hirofumi Matsui

6Oleg Gusev

789Richard Cornette

9Takashi

Okuda9

Yukiko Minamiyama10

Hiroshi Ichikawa11

Shigeaki Suenaga1

Misato Oki2

Tsuyoshi Sato1

Toshihiko Ozawa12

Daret K St Clair3

and Hideyuki J Majima12dagger

Retrospective Study Sample Distribution

To

tal S

ub

jects

(n

=86)

Lo

w (

n=10)

No

rmal (n

=27)

Ele

vate

d (

n=19)

Hig

h (

n=30)

0

1

2

3

4

5

Mit

oS

OA

R (

RF

U)

3 5

2 2

2 7

1 2

M ito S O A R

R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n

MitoSOARtrade (RFU)

lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects

N 10 27 19 3086

12 31 22 35

57 of the tested population has

superoxide levels in the

elevatedhigh range leading to high

risk for certain clinical conditions

Functional Genetics

CT

CC

0 5

1 0

1 5

2 0

2 5

Mit

oS

OA

R (

RF

U)

S O D 2 H e tro z y g o u s g e n o ty p e (C T )

S O D 2 H o m o z y g o u s g e n o ty p e (C C )

P lt 0 0 5

M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e

(S O D 2 ) P o ly m o rp h is m

Individuals who are homozygous

recessive(CC) for SOD2 may

have impaired SOD2 function

resulting in higher levels of the

free radical superoxide and

require antioxidant support

Mitochondrial Superoxide Levels

Assessment of Redox status

Inflammation

Viral or Bacterial Infection

Monitoring treatment of a

disease or disorder

Borrelia infection

Treatment of Inflammation

Functional Effect

Improvement verifies positive clinical effect

Plasma Peroxide Assay

Low Levels may indicate oxidative stress and

possible need for antioxidants

High levels may indicate immune challenges

Can correlate with uric acid

Potential hypertension risk

Related to lipid peroxides

Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13

Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303

Plasma Peroxidase

To

tal S

ub

jects

(n

=86)

Lo

w (

n=30)

No

rmal (n

=24)

Ele

vate

d (

n=11)

Hig

h (

n=16)

0

2 0

4 0

6 0

8 0

Pla

sm

a P

erix

ida

se

Ac

tiv

ity

(m

Um

l)

2 0

1 4

3 0

3 7

M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s

R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e

S a m p le D is tr ib u tio n

Plasma Peroxidase Activity (mUmL)

lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects

N 30 24 11 1681

37 30 14 20

37 of the population studied had low

peroxidase activity indicating an

impaired ability to defend against free

radicals

34 of the studies population had

elevatedhigh activity potentially

indicating the presence of oxidative

stress

Assessment of Baseline Potential

Starting point for defense of excessive

ROS

Effect of Environmental Insults

Assessment of Capacity to defend against

excessive ROS

Assessment and monitoring treatment of a

disease or disorder

Viral or Bacterial infection

Cardiovascular disease

Mitochondrial Membrane Potential

Oxidative Challenge

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

End Point of Poor Methylation and

Oxidation

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

bull Superoxide dismutase 1

(SOD1)

bull Superoxide dismutase 2

(SOD2)

bull Superoxide dismutase 3

(SOD3)

bull Glutathione peroxidase 1

(GPx1)

bull Catalase (CAT)

Superoxide Dismutase 1

SOD1 (CuZnSOD)(Encoded Chrom 21)

Found in cytoplasmic and mitochondrial

intermembrane space(inserted)

Converts superoxide radicals (O2-) to hydrogen

peroxide (H2O2)

Needs copper and zinc as cofactors

Apoptosis Oxidative Stress ALS Myocardial

Infarction Macular Degeneration a marker for

heavy Metal toxicity

O2- H2O2

SOD1SOD1

Cu Zn

1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2

2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160

3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034

Superoxide Dismutase 2

SOD2 (MnSOD) (Chrom 6)

Found in mitochondria

Converts superoxide radicals (O2-)

to hydrogen peroxide (H2O2)

Needs manganese as a cofactor

Heart Disease Myocardial

Infarction Tumor metastasis

neurodegenerative diseases

O2- H2O2SOD2SOD2

Mn

Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24

Superoxide Dismutase 3SOD3

Found extracellularly

Converts superoxide radicals (O2-) to

hydrogen peroxide (H2O2)

Needs copper and zinc as cofactors

Chronic skin disorders Diabetic

Neuropathy Folate Metabolism Ischemic

Heart Disease

O2- H2O2SOD3SOD3

Cu Zn

Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004

Glutathione Peroxidase 1

GPx1(Chrom 3)

Found in the cytoplasm of nearly all mammalian tissues

Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)

Uses glutathione an antioxidant as a cofactor

Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression

H2O2H2O O2GPx1GPx1

Glutathione

Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and

Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89

Catalase

CAT(Chrom 11)

Found in the cytoplasm of cells

Needs iron and selenium as cofactors

Reduce hydrogen peroxide (H2O2) to water (H2O)

High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second

Hypertension Type 2 Diabetes Acatalasemia

httpsghrnlmnihgovgeneCATconditions

H2O2 H2O O2CATCAT

Fe Se

Mitochondrial Function Assessment

Mitochondrial Superoxide (O2-)

Plasma Peroxide Assay (PPA)

Mitochondrial Membrane

Potential (MMP) Assay with

Oxidative Challenge

Mitochondrial Superoxide (O2-)

Superoxide is considered to be a major

factor in oxidant toxicity and

mitochondrial MnSOD enzymes

constitute an essential defense against

superoxide

High levels indicate the proanti-oxidant

system is out of balance and oxidative

stress is present

Mitochondria are the major source of

superoxide

Superoxide is the origin of reactive

oxygen (ROS)and nitrogen species (RNS)

and as such causes various redox

related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo

123Hsiu-Chuan Yen

4Ikuo Nakanishi

5Ken-ichiro Matsumoto

5Masato Tamura

6Yumiko Nagano

6Hirofumi Matsui

6Oleg Gusev

789Richard Cornette

9Takashi

Okuda9

Yukiko Minamiyama10

Hiroshi Ichikawa11

Shigeaki Suenaga1

Misato Oki2

Tsuyoshi Sato1

Toshihiko Ozawa12

Daret K St Clair3

and Hideyuki J Majima12dagger

Retrospective Study Sample Distribution

To

tal S

ub

jects

(n

=86)

Lo

w (

n=10)

No

rmal (n

=27)

Ele

vate

d (

n=19)

Hig

h (

n=30)

0

1

2

3

4

5

Mit

oS

OA

R (

RF

U)

3 5

2 2

2 7

1 2

M ito S O A R

R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n

MitoSOARtrade (RFU)

lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects

N 10 27 19 3086

12 31 22 35

57 of the tested population has

superoxide levels in the

elevatedhigh range leading to high

risk for certain clinical conditions

Functional Genetics

CT

CC

0 5

1 0

1 5

2 0

2 5

Mit

oS

OA

R (

RF

U)

S O D 2 H e tro z y g o u s g e n o ty p e (C T )

S O D 2 H o m o z y g o u s g e n o ty p e (C C )

P lt 0 0 5

M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e

(S O D 2 ) P o ly m o rp h is m

Individuals who are homozygous

recessive(CC) for SOD2 may

have impaired SOD2 function

resulting in higher levels of the

free radical superoxide and

require antioxidant support

Mitochondrial Superoxide Levels

Assessment of Redox status

Inflammation

Viral or Bacterial Infection

Monitoring treatment of a

disease or disorder

Borrelia infection

Treatment of Inflammation

Functional Effect

Improvement verifies positive clinical effect

Plasma Peroxide Assay

Low Levels may indicate oxidative stress and

possible need for antioxidants

High levels may indicate immune challenges

Can correlate with uric acid

Potential hypertension risk

Related to lipid peroxides

Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13

Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303

Plasma Peroxidase

To

tal S

ub

jects

(n

=86)

Lo

w (

n=30)

No

rmal (n

=24)

Ele

vate

d (

n=11)

Hig

h (

n=16)

0

2 0

4 0

6 0

8 0

Pla

sm

a P

erix

ida

se

Ac

tiv

ity

(m

Um

l)

2 0

1 4

3 0

3 7

M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s

R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e

S a m p le D is tr ib u tio n

Plasma Peroxidase Activity (mUmL)

lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects

N 30 24 11 1681

37 30 14 20

37 of the population studied had low

peroxidase activity indicating an

impaired ability to defend against free

radicals

34 of the studies population had

elevatedhigh activity potentially

indicating the presence of oxidative

stress

Assessment of Baseline Potential

Starting point for defense of excessive

ROS

Effect of Environmental Insults

Assessment of Capacity to defend against

excessive ROS

Assessment and monitoring treatment of a

disease or disorder

Viral or Bacterial infection

Cardiovascular disease

Mitochondrial Membrane Potential

Oxidative Challenge

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

Genetic Expression on Mitochondrial

Function

(ROS Degradation)

bull Superoxide dismutase 1

(SOD1)

bull Superoxide dismutase 2

(SOD2)

bull Superoxide dismutase 3

(SOD3)

bull Glutathione peroxidase 1

(GPx1)

bull Catalase (CAT)

Superoxide Dismutase 1

SOD1 (CuZnSOD)(Encoded Chrom 21)

Found in cytoplasmic and mitochondrial

intermembrane space(inserted)

Converts superoxide radicals (O2-) to hydrogen

peroxide (H2O2)

Needs copper and zinc as cofactors

Apoptosis Oxidative Stress ALS Myocardial

Infarction Macular Degeneration a marker for

heavy Metal toxicity

O2- H2O2

SOD1SOD1

Cu Zn

1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2

2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160

3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034

Superoxide Dismutase 2

SOD2 (MnSOD) (Chrom 6)

Found in mitochondria

Converts superoxide radicals (O2-)

to hydrogen peroxide (H2O2)

Needs manganese as a cofactor

Heart Disease Myocardial

Infarction Tumor metastasis

neurodegenerative diseases

O2- H2O2SOD2SOD2

Mn

Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24

Superoxide Dismutase 3SOD3

Found extracellularly

Converts superoxide radicals (O2-) to

hydrogen peroxide (H2O2)

Needs copper and zinc as cofactors

Chronic skin disorders Diabetic

Neuropathy Folate Metabolism Ischemic

Heart Disease

O2- H2O2SOD3SOD3

Cu Zn

Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004

Glutathione Peroxidase 1

GPx1(Chrom 3)

Found in the cytoplasm of nearly all mammalian tissues

Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)

Uses glutathione an antioxidant as a cofactor

Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression

H2O2H2O O2GPx1GPx1

Glutathione

Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and

Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89

Catalase

CAT(Chrom 11)

Found in the cytoplasm of cells

Needs iron and selenium as cofactors

Reduce hydrogen peroxide (H2O2) to water (H2O)

High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second

Hypertension Type 2 Diabetes Acatalasemia

httpsghrnlmnihgovgeneCATconditions

H2O2 H2O O2CATCAT

Fe Se

Mitochondrial Function Assessment

Mitochondrial Superoxide (O2-)

Plasma Peroxide Assay (PPA)

Mitochondrial Membrane

Potential (MMP) Assay with

Oxidative Challenge

Mitochondrial Superoxide (O2-)

Superoxide is considered to be a major

factor in oxidant toxicity and

mitochondrial MnSOD enzymes

constitute an essential defense against

superoxide

High levels indicate the proanti-oxidant

system is out of balance and oxidative

stress is present

Mitochondria are the major source of

superoxide

Superoxide is the origin of reactive

oxygen (ROS)and nitrogen species (RNS)

and as such causes various redox

related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo

123Hsiu-Chuan Yen

4Ikuo Nakanishi

5Ken-ichiro Matsumoto

5Masato Tamura

6Yumiko Nagano

6Hirofumi Matsui

6Oleg Gusev

789Richard Cornette

9Takashi

Okuda9

Yukiko Minamiyama10

Hiroshi Ichikawa11

Shigeaki Suenaga1

Misato Oki2

Tsuyoshi Sato1

Toshihiko Ozawa12

Daret K St Clair3

and Hideyuki J Majima12dagger

Retrospective Study Sample Distribution

To

tal S

ub

jects

(n

=86)

Lo

w (

n=10)

No

rmal (n

=27)

Ele

vate

d (

n=19)

Hig

h (

n=30)

0

1

2

3

4

5

Mit

oS

OA

R (

RF

U)

3 5

2 2

2 7

1 2

M ito S O A R

R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n

MitoSOARtrade (RFU)

lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects

N 10 27 19 3086

12 31 22 35

57 of the tested population has

superoxide levels in the

elevatedhigh range leading to high

risk for certain clinical conditions

Functional Genetics

CT

CC

0 5

1 0

1 5

2 0

2 5

Mit

oS

OA

R (

RF

U)

S O D 2 H e tro z y g o u s g e n o ty p e (C T )

S O D 2 H o m o z y g o u s g e n o ty p e (C C )

P lt 0 0 5

M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e

(S O D 2 ) P o ly m o rp h is m

Individuals who are homozygous

recessive(CC) for SOD2 may

have impaired SOD2 function

resulting in higher levels of the

free radical superoxide and

require antioxidant support

Mitochondrial Superoxide Levels

Assessment of Redox status

Inflammation

Viral or Bacterial Infection

Monitoring treatment of a

disease or disorder

Borrelia infection

Treatment of Inflammation

Functional Effect

Improvement verifies positive clinical effect

Plasma Peroxide Assay

Low Levels may indicate oxidative stress and

possible need for antioxidants

High levels may indicate immune challenges

Can correlate with uric acid

Potential hypertension risk

Related to lipid peroxides

Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13

Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303

Plasma Peroxidase

To

tal S

ub

jects

(n

=86)

Lo

w (

n=30)

No

rmal (n

=24)

Ele

vate

d (

n=11)

Hig

h (

n=16)

0

2 0

4 0

6 0

8 0

Pla

sm

a P

erix

ida

se

Ac

tiv

ity

(m

Um

l)

2 0

1 4

3 0

3 7

M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s

R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e

S a m p le D is tr ib u tio n

Plasma Peroxidase Activity (mUmL)

lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects

N 30 24 11 1681

37 30 14 20

37 of the population studied had low

peroxidase activity indicating an

impaired ability to defend against free

radicals

34 of the studies population had

elevatedhigh activity potentially

indicating the presence of oxidative

stress

Assessment of Baseline Potential

Starting point for defense of excessive

ROS

Effect of Environmental Insults

Assessment of Capacity to defend against

excessive ROS

Assessment and monitoring treatment of a

disease or disorder

Viral or Bacterial infection

Cardiovascular disease

Mitochondrial Membrane Potential

Oxidative Challenge

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Superoxide Dismutase 1

SOD1 (CuZnSOD)(Encoded Chrom 21)

Found in cytoplasmic and mitochondrial

intermembrane space(inserted)

Converts superoxide radicals (O2-) to hydrogen

peroxide (H2O2)

Needs copper and zinc as cofactors

Apoptosis Oxidative Stress ALS Myocardial

Infarction Macular Degeneration a marker for

heavy Metal toxicity

O2- H2O2

SOD1SOD1

Cu Zn

1Murray CJ Lopez AD (May 1997) Alternative projections of mortality and disability by cause 1990-2020 Global Burden of Disease Study Lancet 349 (9064) 1498ndash504 PMID 9167458 doi101016S0140-6736(96)07492-2

2Jump up ^ Braunwald E Kloner RA (November 1985) Myocardial reperfusion a double-edged sword The Journal of Clinical Investigation 76 (5) 1713ndash9 PMC 424191 PMID 4056048 doi101172JCI112160

3uller FL Lustgarten MS Jang Y Richardson A Van Remmen H (August 2007) Trends in oxidative aging theories Free Radical Biology amp Medicine 43 (4) 477ndash503 PMID 17640558 doi101016jfreeradbiomed200703034

Superoxide Dismutase 2

SOD2 (MnSOD) (Chrom 6)

Found in mitochondria

Converts superoxide radicals (O2-)

to hydrogen peroxide (H2O2)

Needs manganese as a cofactor

Heart Disease Myocardial

Infarction Tumor metastasis

neurodegenerative diseases

O2- H2O2SOD2SOD2

Mn

Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24

Superoxide Dismutase 3SOD3

Found extracellularly

Converts superoxide radicals (O2-) to

hydrogen peroxide (H2O2)

Needs copper and zinc as cofactors

Chronic skin disorders Diabetic

Neuropathy Folate Metabolism Ischemic

Heart Disease

O2- H2O2SOD3SOD3

Cu Zn

Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004

Glutathione Peroxidase 1

GPx1(Chrom 3)

Found in the cytoplasm of nearly all mammalian tissues

Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)

Uses glutathione an antioxidant as a cofactor

Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression

H2O2H2O O2GPx1GPx1

Glutathione

Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and

Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89

Catalase

CAT(Chrom 11)

Found in the cytoplasm of cells

Needs iron and selenium as cofactors

Reduce hydrogen peroxide (H2O2) to water (H2O)

High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second

Hypertension Type 2 Diabetes Acatalasemia

httpsghrnlmnihgovgeneCATconditions

H2O2 H2O O2CATCAT

Fe Se

Mitochondrial Function Assessment

Mitochondrial Superoxide (O2-)

Plasma Peroxide Assay (PPA)

Mitochondrial Membrane

Potential (MMP) Assay with

Oxidative Challenge

Mitochondrial Superoxide (O2-)

Superoxide is considered to be a major

factor in oxidant toxicity and

mitochondrial MnSOD enzymes

constitute an essential defense against

superoxide

High levels indicate the proanti-oxidant

system is out of balance and oxidative

stress is present

Mitochondria are the major source of

superoxide

Superoxide is the origin of reactive

oxygen (ROS)and nitrogen species (RNS)

and as such causes various redox

related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo

123Hsiu-Chuan Yen

4Ikuo Nakanishi

5Ken-ichiro Matsumoto

5Masato Tamura

6Yumiko Nagano

6Hirofumi Matsui

6Oleg Gusev

789Richard Cornette

9Takashi

Okuda9

Yukiko Minamiyama10

Hiroshi Ichikawa11

Shigeaki Suenaga1

Misato Oki2

Tsuyoshi Sato1

Toshihiko Ozawa12

Daret K St Clair3

and Hideyuki J Majima12dagger

Retrospective Study Sample Distribution

To

tal S

ub

jects

(n

=86)

Lo

w (

n=10)

No

rmal (n

=27)

Ele

vate

d (

n=19)

Hig

h (

n=30)

0

1

2

3

4

5

Mit

oS

OA

R (

RF

U)

3 5

2 2

2 7

1 2

M ito S O A R

R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n

MitoSOARtrade (RFU)

lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects

N 10 27 19 3086

12 31 22 35

57 of the tested population has

superoxide levels in the

elevatedhigh range leading to high

risk for certain clinical conditions

Functional Genetics

CT

CC

0 5

1 0

1 5

2 0

2 5

Mit

oS

OA

R (

RF

U)

S O D 2 H e tro z y g o u s g e n o ty p e (C T )

S O D 2 H o m o z y g o u s g e n o ty p e (C C )

P lt 0 0 5

M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e

(S O D 2 ) P o ly m o rp h is m

Individuals who are homozygous

recessive(CC) for SOD2 may

have impaired SOD2 function

resulting in higher levels of the

free radical superoxide and

require antioxidant support

Mitochondrial Superoxide Levels

Assessment of Redox status

Inflammation

Viral or Bacterial Infection

Monitoring treatment of a

disease or disorder

Borrelia infection

Treatment of Inflammation

Functional Effect

Improvement verifies positive clinical effect

Plasma Peroxide Assay

Low Levels may indicate oxidative stress and

possible need for antioxidants

High levels may indicate immune challenges

Can correlate with uric acid

Potential hypertension risk

Related to lipid peroxides

Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13

Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303

Plasma Peroxidase

To

tal S

ub

jects

(n

=86)

Lo

w (

n=30)

No

rmal (n

=24)

Ele

vate

d (

n=11)

Hig

h (

n=16)

0

2 0

4 0

6 0

8 0

Pla

sm

a P

erix

ida

se

Ac

tiv

ity

(m

Um

l)

2 0

1 4

3 0

3 7

M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s

R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e

S a m p le D is tr ib u tio n

Plasma Peroxidase Activity (mUmL)

lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects

N 30 24 11 1681

37 30 14 20

37 of the population studied had low

peroxidase activity indicating an

impaired ability to defend against free

radicals

34 of the studies population had

elevatedhigh activity potentially

indicating the presence of oxidative

stress

Assessment of Baseline Potential

Starting point for defense of excessive

ROS

Effect of Environmental Insults

Assessment of Capacity to defend against

excessive ROS

Assessment and monitoring treatment of a

disease or disorder

Viral or Bacterial infection

Cardiovascular disease

Mitochondrial Membrane Potential

Oxidative Challenge

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Superoxide Dismutase 2

SOD2 (MnSOD) (Chrom 6)

Found in mitochondria

Converts superoxide radicals (O2-)

to hydrogen peroxide (H2O2)

Needs manganese as a cofactor

Heart Disease Myocardial

Infarction Tumor metastasis

neurodegenerative diseases

O2- H2O2SOD2SOD2

Mn

Pias EK Ekshyyan OY Rhoads CA Fuseler J Harrison L Aw TY (Apr 2003) Differential effects of superoxide dismutase isoform expression on hydroperoxide-induced apoptosis in PC-12 cells The Journal of Biological Chemistry 278 (15) 13294ndash301Perry JJ Hearn AS Cabelli DE Nick HS Tainer JA Silverman DN (Apr 2009) Contribution of human manganese superoxide dismutase tyrosine 34 to structure and catalysis Biochemistry 48 (15) 3417ndash24

Superoxide Dismutase 3SOD3

Found extracellularly

Converts superoxide radicals (O2-) to

hydrogen peroxide (H2O2)

Needs copper and zinc as cofactors

Chronic skin disorders Diabetic

Neuropathy Folate Metabolism Ischemic

Heart Disease

O2- H2O2SOD3SOD3

Cu Zn

Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004

Glutathione Peroxidase 1

GPx1(Chrom 3)

Found in the cytoplasm of nearly all mammalian tissues

Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)

Uses glutathione an antioxidant as a cofactor

Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression

H2O2H2O O2GPx1GPx1

Glutathione

Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and

Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89

Catalase

CAT(Chrom 11)

Found in the cytoplasm of cells

Needs iron and selenium as cofactors

Reduce hydrogen peroxide (H2O2) to water (H2O)

High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second

Hypertension Type 2 Diabetes Acatalasemia

httpsghrnlmnihgovgeneCATconditions

H2O2 H2O O2CATCAT

Fe Se

Mitochondrial Function Assessment

Mitochondrial Superoxide (O2-)

Plasma Peroxide Assay (PPA)

Mitochondrial Membrane

Potential (MMP) Assay with

Oxidative Challenge

Mitochondrial Superoxide (O2-)

Superoxide is considered to be a major

factor in oxidant toxicity and

mitochondrial MnSOD enzymes

constitute an essential defense against

superoxide

High levels indicate the proanti-oxidant

system is out of balance and oxidative

stress is present

Mitochondria are the major source of

superoxide

Superoxide is the origin of reactive

oxygen (ROS)and nitrogen species (RNS)

and as such causes various redox

related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo

123Hsiu-Chuan Yen

4Ikuo Nakanishi

5Ken-ichiro Matsumoto

5Masato Tamura

6Yumiko Nagano

6Hirofumi Matsui

6Oleg Gusev

789Richard Cornette

9Takashi

Okuda9

Yukiko Minamiyama10

Hiroshi Ichikawa11

Shigeaki Suenaga1

Misato Oki2

Tsuyoshi Sato1

Toshihiko Ozawa12

Daret K St Clair3

and Hideyuki J Majima12dagger

Retrospective Study Sample Distribution

To

tal S

ub

jects

(n

=86)

Lo

w (

n=10)

No

rmal (n

=27)

Ele

vate

d (

n=19)

Hig

h (

n=30)

0

1

2

3

4

5

Mit

oS

OA

R (

RF

U)

3 5

2 2

2 7

1 2

M ito S O A R

R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n

MitoSOARtrade (RFU)

lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects

N 10 27 19 3086

12 31 22 35

57 of the tested population has

superoxide levels in the

elevatedhigh range leading to high

risk for certain clinical conditions

Functional Genetics

CT

CC

0 5

1 0

1 5

2 0

2 5

Mit

oS

OA

R (

RF

U)

S O D 2 H e tro z y g o u s g e n o ty p e (C T )

S O D 2 H o m o z y g o u s g e n o ty p e (C C )

P lt 0 0 5

M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e

(S O D 2 ) P o ly m o rp h is m

Individuals who are homozygous

recessive(CC) for SOD2 may

have impaired SOD2 function

resulting in higher levels of the

free radical superoxide and

require antioxidant support

Mitochondrial Superoxide Levels

Assessment of Redox status

Inflammation

Viral or Bacterial Infection

Monitoring treatment of a

disease or disorder

Borrelia infection

Treatment of Inflammation

Functional Effect

Improvement verifies positive clinical effect

Plasma Peroxide Assay

Low Levels may indicate oxidative stress and

possible need for antioxidants

High levels may indicate immune challenges

Can correlate with uric acid

Potential hypertension risk

Related to lipid peroxides

Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13

Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303

Plasma Peroxidase

To

tal S

ub

jects

(n

=86)

Lo

w (

n=30)

No

rmal (n

=24)

Ele

vate

d (

n=11)

Hig

h (

n=16)

0

2 0

4 0

6 0

8 0

Pla

sm

a P

erix

ida

se

Ac

tiv

ity

(m

Um

l)

2 0

1 4

3 0

3 7

M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s

R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e

S a m p le D is tr ib u tio n

Plasma Peroxidase Activity (mUmL)

lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects

N 30 24 11 1681

37 30 14 20

37 of the population studied had low

peroxidase activity indicating an

impaired ability to defend against free

radicals

34 of the studies population had

elevatedhigh activity potentially

indicating the presence of oxidative

stress

Assessment of Baseline Potential

Starting point for defense of excessive

ROS

Effect of Environmental Insults

Assessment of Capacity to defend against

excessive ROS

Assessment and monitoring treatment of a

disease or disorder

Viral or Bacterial infection

Cardiovascular disease

Mitochondrial Membrane Potential

Oxidative Challenge

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Superoxide Dismutase 3SOD3

Found extracellularly

Converts superoxide radicals (O2-) to

hydrogen peroxide (H2O2)

Needs copper and zinc as cofactors

Chronic skin disorders Diabetic

Neuropathy Folate Metabolism Ischemic

Heart Disease

O2- H2O2SOD3SOD3

Cu Zn

Extracellular superoxide dismutase for the treatment of inflammatory skin diseasesSunghwan Kim amp Tae-Yoon KimExpert Review of Dermatology Vol 8 Iss 62013Juul K Tybjaerg-Hansen A Marklund S Heegaard N H H Steffensen R Sillesen H Jensen G Nordestgaard B G Genetically reduced antioxidative protection and increased ischemic heart disease risk the Copenhagen City Heart Study Circulation 109 59-65 2004

Glutathione Peroxidase 1

GPx1(Chrom 3)

Found in the cytoplasm of nearly all mammalian tissues

Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)

Uses glutathione an antioxidant as a cofactor

Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression

H2O2H2O O2GPx1GPx1

Glutathione

Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and

Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89

Catalase

CAT(Chrom 11)

Found in the cytoplasm of cells

Needs iron and selenium as cofactors

Reduce hydrogen peroxide (H2O2) to water (H2O)

High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second

Hypertension Type 2 Diabetes Acatalasemia

httpsghrnlmnihgovgeneCATconditions

H2O2 H2O O2CATCAT

Fe Se

Mitochondrial Function Assessment

Mitochondrial Superoxide (O2-)

Plasma Peroxide Assay (PPA)

Mitochondrial Membrane

Potential (MMP) Assay with

Oxidative Challenge

Mitochondrial Superoxide (O2-)

Superoxide is considered to be a major

factor in oxidant toxicity and

mitochondrial MnSOD enzymes

constitute an essential defense against

superoxide

High levels indicate the proanti-oxidant

system is out of balance and oxidative

stress is present

Mitochondria are the major source of

superoxide

Superoxide is the origin of reactive

oxygen (ROS)and nitrogen species (RNS)

and as such causes various redox

related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo

123Hsiu-Chuan Yen

4Ikuo Nakanishi

5Ken-ichiro Matsumoto

5Masato Tamura

6Yumiko Nagano

6Hirofumi Matsui

6Oleg Gusev

789Richard Cornette

9Takashi

Okuda9

Yukiko Minamiyama10

Hiroshi Ichikawa11

Shigeaki Suenaga1

Misato Oki2

Tsuyoshi Sato1

Toshihiko Ozawa12

Daret K St Clair3

and Hideyuki J Majima12dagger

Retrospective Study Sample Distribution

To

tal S

ub

jects

(n

=86)

Lo

w (

n=10)

No

rmal (n

=27)

Ele

vate

d (

n=19)

Hig

h (

n=30)

0

1

2

3

4

5

Mit

oS

OA

R (

RF

U)

3 5

2 2

2 7

1 2

M ito S O A R

R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n

MitoSOARtrade (RFU)

lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects

N 10 27 19 3086

12 31 22 35

57 of the tested population has

superoxide levels in the

elevatedhigh range leading to high

risk for certain clinical conditions

Functional Genetics

CT

CC

0 5

1 0

1 5

2 0

2 5

Mit

oS

OA

R (

RF

U)

S O D 2 H e tro z y g o u s g e n o ty p e (C T )

S O D 2 H o m o z y g o u s g e n o ty p e (C C )

P lt 0 0 5

M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e

(S O D 2 ) P o ly m o rp h is m

Individuals who are homozygous

recessive(CC) for SOD2 may

have impaired SOD2 function

resulting in higher levels of the

free radical superoxide and

require antioxidant support

Mitochondrial Superoxide Levels

Assessment of Redox status

Inflammation

Viral or Bacterial Infection

Monitoring treatment of a

disease or disorder

Borrelia infection

Treatment of Inflammation

Functional Effect

Improvement verifies positive clinical effect

Plasma Peroxide Assay

Low Levels may indicate oxidative stress and

possible need for antioxidants

High levels may indicate immune challenges

Can correlate with uric acid

Potential hypertension risk

Related to lipid peroxides

Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13

Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303

Plasma Peroxidase

To

tal S

ub

jects

(n

=86)

Lo

w (

n=30)

No

rmal (n

=24)

Ele

vate

d (

n=11)

Hig

h (

n=16)

0

2 0

4 0

6 0

8 0

Pla

sm

a P

erix

ida

se

Ac

tiv

ity

(m

Um

l)

2 0

1 4

3 0

3 7

M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s

R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e

S a m p le D is tr ib u tio n

Plasma Peroxidase Activity (mUmL)

lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects

N 30 24 11 1681

37 30 14 20

37 of the population studied had low

peroxidase activity indicating an

impaired ability to defend against free

radicals

34 of the studies population had

elevatedhigh activity potentially

indicating the presence of oxidative

stress

Assessment of Baseline Potential

Starting point for defense of excessive

ROS

Effect of Environmental Insults

Assessment of Capacity to defend against

excessive ROS

Assessment and monitoring treatment of a

disease or disorder

Viral or Bacterial infection

Cardiovascular disease

Mitochondrial Membrane Potential

Oxidative Challenge

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Glutathione Peroxidase 1

GPx1(Chrom 3)

Found in the cytoplasm of nearly all mammalian tissues

Reduce hydrogen peroxide (H2O2) to water (H2O) and oxygen (O2)

Uses glutathione an antioxidant as a cofactor

Increased breast cancer risk esophageal cancer Diabetes (2) teleomere expression

H2O2H2O O2GPx1GPx1

Glutathione

Entrez Gene GPX1 glutathione peroxidase 1 Vats P Sagar N Singh TP Banerjee M (Jan 2015) Association of Superoxide dismutases (SOD1 and SOD2) and

Glutathione peroxidase 1 (GPx1) gene polymorphisms with type 2 diabetes mellitus Free Radical Research 49 (1) 17ndash24 Szebeni A Szebeni K DiPeri T Chandley MJ Crawford JD Stockmeier CA Ordway GA (Oct 2014) Shortened telomere length in white matter oligodendrocytes in major depression potential role of oxidative stress The International Journal of Neuropsychopharmacology 17 (10) 1579ndash89

Catalase

CAT(Chrom 11)

Found in the cytoplasm of cells

Needs iron and selenium as cofactors

Reduce hydrogen peroxide (H2O2) to water (H2O)

High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second

Hypertension Type 2 Diabetes Acatalasemia

httpsghrnlmnihgovgeneCATconditions

H2O2 H2O O2CATCAT

Fe Se

Mitochondrial Function Assessment

Mitochondrial Superoxide (O2-)

Plasma Peroxide Assay (PPA)

Mitochondrial Membrane

Potential (MMP) Assay with

Oxidative Challenge

Mitochondrial Superoxide (O2-)

Superoxide is considered to be a major

factor in oxidant toxicity and

mitochondrial MnSOD enzymes

constitute an essential defense against

superoxide

High levels indicate the proanti-oxidant

system is out of balance and oxidative

stress is present

Mitochondria are the major source of

superoxide

Superoxide is the origin of reactive

oxygen (ROS)and nitrogen species (RNS)

and as such causes various redox

related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo

123Hsiu-Chuan Yen

4Ikuo Nakanishi

5Ken-ichiro Matsumoto

5Masato Tamura

6Yumiko Nagano

6Hirofumi Matsui

6Oleg Gusev

789Richard Cornette

9Takashi

Okuda9

Yukiko Minamiyama10

Hiroshi Ichikawa11

Shigeaki Suenaga1

Misato Oki2

Tsuyoshi Sato1

Toshihiko Ozawa12

Daret K St Clair3

and Hideyuki J Majima12dagger

Retrospective Study Sample Distribution

To

tal S

ub

jects

(n

=86)

Lo

w (

n=10)

No

rmal (n

=27)

Ele

vate

d (

n=19)

Hig

h (

n=30)

0

1

2

3

4

5

Mit

oS

OA

R (

RF

U)

3 5

2 2

2 7

1 2

M ito S O A R

R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n

MitoSOARtrade (RFU)

lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects

N 10 27 19 3086

12 31 22 35

57 of the tested population has

superoxide levels in the

elevatedhigh range leading to high

risk for certain clinical conditions

Functional Genetics

CT

CC

0 5

1 0

1 5

2 0

2 5

Mit

oS

OA

R (

RF

U)

S O D 2 H e tro z y g o u s g e n o ty p e (C T )

S O D 2 H o m o z y g o u s g e n o ty p e (C C )

P lt 0 0 5

M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e

(S O D 2 ) P o ly m o rp h is m

Individuals who are homozygous

recessive(CC) for SOD2 may

have impaired SOD2 function

resulting in higher levels of the

free radical superoxide and

require antioxidant support

Mitochondrial Superoxide Levels

Assessment of Redox status

Inflammation

Viral or Bacterial Infection

Monitoring treatment of a

disease or disorder

Borrelia infection

Treatment of Inflammation

Functional Effect

Improvement verifies positive clinical effect

Plasma Peroxide Assay

Low Levels may indicate oxidative stress and

possible need for antioxidants

High levels may indicate immune challenges

Can correlate with uric acid

Potential hypertension risk

Related to lipid peroxides

Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13

Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303

Plasma Peroxidase

To

tal S

ub

jects

(n

=86)

Lo

w (

n=30)

No

rmal (n

=24)

Ele

vate

d (

n=11)

Hig

h (

n=16)

0

2 0

4 0

6 0

8 0

Pla

sm

a P

erix

ida

se

Ac

tiv

ity

(m

Um

l)

2 0

1 4

3 0

3 7

M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s

R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e

S a m p le D is tr ib u tio n

Plasma Peroxidase Activity (mUmL)

lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects

N 30 24 11 1681

37 30 14 20

37 of the population studied had low

peroxidase activity indicating an

impaired ability to defend against free

radicals

34 of the studies population had

elevatedhigh activity potentially

indicating the presence of oxidative

stress

Assessment of Baseline Potential

Starting point for defense of excessive

ROS

Effect of Environmental Insults

Assessment of Capacity to defend against

excessive ROS

Assessment and monitoring treatment of a

disease or disorder

Viral or Bacterial infection

Cardiovascular disease

Mitochondrial Membrane Potential

Oxidative Challenge

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Catalase

CAT(Chrom 11)

Found in the cytoplasm of cells

Needs iron and selenium as cofactors

Reduce hydrogen peroxide (H2O2) to water (H2O)

High turnover coverts millions of molecules of H2O2 to H2O and oxygen (O2) each second

Hypertension Type 2 Diabetes Acatalasemia

httpsghrnlmnihgovgeneCATconditions

H2O2 H2O O2CATCAT

Fe Se

Mitochondrial Function Assessment

Mitochondrial Superoxide (O2-)

Plasma Peroxide Assay (PPA)

Mitochondrial Membrane

Potential (MMP) Assay with

Oxidative Challenge

Mitochondrial Superoxide (O2-)

Superoxide is considered to be a major

factor in oxidant toxicity and

mitochondrial MnSOD enzymes

constitute an essential defense against

superoxide

High levels indicate the proanti-oxidant

system is out of balance and oxidative

stress is present

Mitochondria are the major source of

superoxide

Superoxide is the origin of reactive

oxygen (ROS)and nitrogen species (RNS)

and as such causes various redox

related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo

123Hsiu-Chuan Yen

4Ikuo Nakanishi

5Ken-ichiro Matsumoto

5Masato Tamura

6Yumiko Nagano

6Hirofumi Matsui

6Oleg Gusev

789Richard Cornette

9Takashi

Okuda9

Yukiko Minamiyama10

Hiroshi Ichikawa11

Shigeaki Suenaga1

Misato Oki2

Tsuyoshi Sato1

Toshihiko Ozawa12

Daret K St Clair3

and Hideyuki J Majima12dagger

Retrospective Study Sample Distribution

To

tal S

ub

jects

(n

=86)

Lo

w (

n=10)

No

rmal (n

=27)

Ele

vate

d (

n=19)

Hig

h (

n=30)

0

1

2

3

4

5

Mit

oS

OA

R (

RF

U)

3 5

2 2

2 7

1 2

M ito S O A R

R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n

MitoSOARtrade (RFU)

lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects

N 10 27 19 3086

12 31 22 35

57 of the tested population has

superoxide levels in the

elevatedhigh range leading to high

risk for certain clinical conditions

Functional Genetics

CT

CC

0 5

1 0

1 5

2 0

2 5

Mit

oS

OA

R (

RF

U)

S O D 2 H e tro z y g o u s g e n o ty p e (C T )

S O D 2 H o m o z y g o u s g e n o ty p e (C C )

P lt 0 0 5

M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e

(S O D 2 ) P o ly m o rp h is m

Individuals who are homozygous

recessive(CC) for SOD2 may

have impaired SOD2 function

resulting in higher levels of the

free radical superoxide and

require antioxidant support

Mitochondrial Superoxide Levels

Assessment of Redox status

Inflammation

Viral or Bacterial Infection

Monitoring treatment of a

disease or disorder

Borrelia infection

Treatment of Inflammation

Functional Effect

Improvement verifies positive clinical effect

Plasma Peroxide Assay

Low Levels may indicate oxidative stress and

possible need for antioxidants

High levels may indicate immune challenges

Can correlate with uric acid

Potential hypertension risk

Related to lipid peroxides

Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13

Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303

Plasma Peroxidase

To

tal S

ub

jects

(n

=86)

Lo

w (

n=30)

No

rmal (n

=24)

Ele

vate

d (

n=11)

Hig

h (

n=16)

0

2 0

4 0

6 0

8 0

Pla

sm

a P

erix

ida

se

Ac

tiv

ity

(m

Um

l)

2 0

1 4

3 0

3 7

M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s

R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e

S a m p le D is tr ib u tio n

Plasma Peroxidase Activity (mUmL)

lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects

N 30 24 11 1681

37 30 14 20

37 of the population studied had low

peroxidase activity indicating an

impaired ability to defend against free

radicals

34 of the studies population had

elevatedhigh activity potentially

indicating the presence of oxidative

stress

Assessment of Baseline Potential

Starting point for defense of excessive

ROS

Effect of Environmental Insults

Assessment of Capacity to defend against

excessive ROS

Assessment and monitoring treatment of a

disease or disorder

Viral or Bacterial infection

Cardiovascular disease

Mitochondrial Membrane Potential

Oxidative Challenge

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Mitochondrial Function Assessment

Mitochondrial Superoxide (O2-)

Plasma Peroxide Assay (PPA)

Mitochondrial Membrane

Potential (MMP) Assay with

Oxidative Challenge

Mitochondrial Superoxide (O2-)

Superoxide is considered to be a major

factor in oxidant toxicity and

mitochondrial MnSOD enzymes

constitute an essential defense against

superoxide

High levels indicate the proanti-oxidant

system is out of balance and oxidative

stress is present

Mitochondria are the major source of

superoxide

Superoxide is the origin of reactive

oxygen (ROS)and nitrogen species (RNS)

and as such causes various redox

related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo

123Hsiu-Chuan Yen

4Ikuo Nakanishi

5Ken-ichiro Matsumoto

5Masato Tamura

6Yumiko Nagano

6Hirofumi Matsui

6Oleg Gusev

789Richard Cornette

9Takashi

Okuda9

Yukiko Minamiyama10

Hiroshi Ichikawa11

Shigeaki Suenaga1

Misato Oki2

Tsuyoshi Sato1

Toshihiko Ozawa12

Daret K St Clair3

and Hideyuki J Majima12dagger

Retrospective Study Sample Distribution

To

tal S

ub

jects

(n

=86)

Lo

w (

n=10)

No

rmal (n

=27)

Ele

vate

d (

n=19)

Hig

h (

n=30)

0

1

2

3

4

5

Mit

oS

OA

R (

RF

U)

3 5

2 2

2 7

1 2

M ito S O A R

R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n

MitoSOARtrade (RFU)

lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects

N 10 27 19 3086

12 31 22 35

57 of the tested population has

superoxide levels in the

elevatedhigh range leading to high

risk for certain clinical conditions

Functional Genetics

CT

CC

0 5

1 0

1 5

2 0

2 5

Mit

oS

OA

R (

RF

U)

S O D 2 H e tro z y g o u s g e n o ty p e (C T )

S O D 2 H o m o z y g o u s g e n o ty p e (C C )

P lt 0 0 5

M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e

(S O D 2 ) P o ly m o rp h is m

Individuals who are homozygous

recessive(CC) for SOD2 may

have impaired SOD2 function

resulting in higher levels of the

free radical superoxide and

require antioxidant support

Mitochondrial Superoxide Levels

Assessment of Redox status

Inflammation

Viral or Bacterial Infection

Monitoring treatment of a

disease or disorder

Borrelia infection

Treatment of Inflammation

Functional Effect

Improvement verifies positive clinical effect

Plasma Peroxide Assay

Low Levels may indicate oxidative stress and

possible need for antioxidants

High levels may indicate immune challenges

Can correlate with uric acid

Potential hypertension risk

Related to lipid peroxides

Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13

Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303

Plasma Peroxidase

To

tal S

ub

jects

(n

=86)

Lo

w (

n=30)

No

rmal (n

=24)

Ele

vate

d (

n=11)

Hig

h (

n=16)

0

2 0

4 0

6 0

8 0

Pla

sm

a P

erix

ida

se

Ac

tiv

ity

(m

Um

l)

2 0

1 4

3 0

3 7

M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s

R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e

S a m p le D is tr ib u tio n

Plasma Peroxidase Activity (mUmL)

lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects

N 30 24 11 1681

37 30 14 20

37 of the population studied had low

peroxidase activity indicating an

impaired ability to defend against free

radicals

34 of the studies population had

elevatedhigh activity potentially

indicating the presence of oxidative

stress

Assessment of Baseline Potential

Starting point for defense of excessive

ROS

Effect of Environmental Insults

Assessment of Capacity to defend against

excessive ROS

Assessment and monitoring treatment of a

disease or disorder

Viral or Bacterial infection

Cardiovascular disease

Mitochondrial Membrane Potential

Oxidative Challenge

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Mitochondrial Superoxide (O2-)

Superoxide is considered to be a major

factor in oxidant toxicity and

mitochondrial MnSOD enzymes

constitute an essential defense against

superoxide

High levels indicate the proanti-oxidant

system is out of balance and oxidative

stress is present

Mitochondria are the major source of

superoxide

Superoxide is the origin of reactive

oxygen (ROS)and nitrogen species (RNS)

and as such causes various redox

related diseases and agingJ Clin Biochem Nutr 2015 Jan 56(1) 1ndash7 Published online 2014 Dec 23 doi 103164jcbn14-42 PMCID PMC4306659 A mitochondrial superoxide theory for oxidative stress diseases and aging Hiroko P Indo

123Hsiu-Chuan Yen

4Ikuo Nakanishi

5Ken-ichiro Matsumoto

5Masato Tamura

6Yumiko Nagano

6Hirofumi Matsui

6Oleg Gusev

789Richard Cornette

9Takashi

Okuda9

Yukiko Minamiyama10

Hiroshi Ichikawa11

Shigeaki Suenaga1

Misato Oki2

Tsuyoshi Sato1

Toshihiko Ozawa12

Daret K St Clair3

and Hideyuki J Majima12dagger

Retrospective Study Sample Distribution

To

tal S

ub

jects

(n

=86)

Lo

w (

n=10)

No

rmal (n

=27)

Ele

vate

d (

n=19)

Hig

h (

n=30)

0

1

2

3

4

5

Mit

oS

OA

R (

RF

U)

3 5

2 2

2 7

1 2

M ito S O A R

R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n

MitoSOARtrade (RFU)

lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects

N 10 27 19 3086

12 31 22 35

57 of the tested population has

superoxide levels in the

elevatedhigh range leading to high

risk for certain clinical conditions

Functional Genetics

CT

CC

0 5

1 0

1 5

2 0

2 5

Mit

oS

OA

R (

RF

U)

S O D 2 H e tro z y g o u s g e n o ty p e (C T )

S O D 2 H o m o z y g o u s g e n o ty p e (C C )

P lt 0 0 5

M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e

(S O D 2 ) P o ly m o rp h is m

Individuals who are homozygous

recessive(CC) for SOD2 may

have impaired SOD2 function

resulting in higher levels of the

free radical superoxide and

require antioxidant support

Mitochondrial Superoxide Levels

Assessment of Redox status

Inflammation

Viral or Bacterial Infection

Monitoring treatment of a

disease or disorder

Borrelia infection

Treatment of Inflammation

Functional Effect

Improvement verifies positive clinical effect

Plasma Peroxide Assay

Low Levels may indicate oxidative stress and

possible need for antioxidants

High levels may indicate immune challenges

Can correlate with uric acid

Potential hypertension risk

Related to lipid peroxides

Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13

Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303

Plasma Peroxidase

To

tal S

ub

jects

(n

=86)

Lo

w (

n=30)

No

rmal (n

=24)

Ele

vate

d (

n=11)

Hig

h (

n=16)

0

2 0

4 0

6 0

8 0

Pla

sm

a P

erix

ida

se

Ac

tiv

ity

(m

Um

l)

2 0

1 4

3 0

3 7

M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s

R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e

S a m p le D is tr ib u tio n

Plasma Peroxidase Activity (mUmL)

lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects

N 30 24 11 1681

37 30 14 20

37 of the population studied had low

peroxidase activity indicating an

impaired ability to defend against free

radicals

34 of the studies population had

elevatedhigh activity potentially

indicating the presence of oxidative

stress

Assessment of Baseline Potential

Starting point for defense of excessive

ROS

Effect of Environmental Insults

Assessment of Capacity to defend against

excessive ROS

Assessment and monitoring treatment of a

disease or disorder

Viral or Bacterial infection

Cardiovascular disease

Mitochondrial Membrane Potential

Oxidative Challenge

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Retrospective Study Sample Distribution

To

tal S

ub

jects

(n

=86)

Lo

w (

n=10)

No

rmal (n

=27)

Ele

vate

d (

n=19)

Hig

h (

n=30)

0

1

2

3

4

5

Mit

oS

OA

R (

RF

U)

3 5

2 2

2 7

1 2

M ito S O A R

R e tro s p e c t iv e S tu d y S a m p le D is tr ib u tio n

MitoSOARtrade (RFU)

lt102 (Low) 102-141 (Normal) 141-167 (Elevated) gt167 (High) Total Subjects

N 10 27 19 3086

12 31 22 35

57 of the tested population has

superoxide levels in the

elevatedhigh range leading to high

risk for certain clinical conditions

Functional Genetics

CT

CC

0 5

1 0

1 5

2 0

2 5

Mit

oS

OA

R (

RF

U)

S O D 2 H e tro z y g o u s g e n o ty p e (C T )

S O D 2 H o m o z y g o u s g e n o ty p e (C C )

P lt 0 0 5

M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e

(S O D 2 ) P o ly m o rp h is m

Individuals who are homozygous

recessive(CC) for SOD2 may

have impaired SOD2 function

resulting in higher levels of the

free radical superoxide and

require antioxidant support

Mitochondrial Superoxide Levels

Assessment of Redox status

Inflammation

Viral or Bacterial Infection

Monitoring treatment of a

disease or disorder

Borrelia infection

Treatment of Inflammation

Functional Effect

Improvement verifies positive clinical effect

Plasma Peroxide Assay

Low Levels may indicate oxidative stress and

possible need for antioxidants

High levels may indicate immune challenges

Can correlate with uric acid

Potential hypertension risk

Related to lipid peroxides

Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13

Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303

Plasma Peroxidase

To

tal S

ub

jects

(n

=86)

Lo

w (

n=30)

No

rmal (n

=24)

Ele

vate

d (

n=11)

Hig

h (

n=16)

0

2 0

4 0

6 0

8 0

Pla

sm

a P

erix

ida

se

Ac

tiv

ity

(m

Um

l)

2 0

1 4

3 0

3 7

M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s

R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e

S a m p le D is tr ib u tio n

Plasma Peroxidase Activity (mUmL)

lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects

N 30 24 11 1681

37 30 14 20

37 of the population studied had low

peroxidase activity indicating an

impaired ability to defend against free

radicals

34 of the studies population had

elevatedhigh activity potentially

indicating the presence of oxidative

stress

Assessment of Baseline Potential

Starting point for defense of excessive

ROS

Effect of Environmental Insults

Assessment of Capacity to defend against

excessive ROS

Assessment and monitoring treatment of a

disease or disorder

Viral or Bacterial infection

Cardiovascular disease

Mitochondrial Membrane Potential

Oxidative Challenge

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Functional Genetics

CT

CC

0 5

1 0

1 5

2 0

2 5

Mit

oS

OA

R (

RF

U)

S O D 2 H e tro z y g o u s g e n o ty p e (C T )

S O D 2 H o m o z y g o u s g e n o ty p e (C C )

P lt 0 0 5

M ito S O A R a n d M ito c h o n d r ia l S u p e ro x id e D is m u ta s e

(S O D 2 ) P o ly m o rp h is m

Individuals who are homozygous

recessive(CC) for SOD2 may

have impaired SOD2 function

resulting in higher levels of the

free radical superoxide and

require antioxidant support

Mitochondrial Superoxide Levels

Assessment of Redox status

Inflammation

Viral or Bacterial Infection

Monitoring treatment of a

disease or disorder

Borrelia infection

Treatment of Inflammation

Functional Effect

Improvement verifies positive clinical effect

Plasma Peroxide Assay

Low Levels may indicate oxidative stress and

possible need for antioxidants

High levels may indicate immune challenges

Can correlate with uric acid

Potential hypertension risk

Related to lipid peroxides

Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13

Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303

Plasma Peroxidase

To

tal S

ub

jects

(n

=86)

Lo

w (

n=30)

No

rmal (n

=24)

Ele

vate

d (

n=11)

Hig

h (

n=16)

0

2 0

4 0

6 0

8 0

Pla

sm

a P

erix

ida

se

Ac

tiv

ity

(m

Um

l)

2 0

1 4

3 0

3 7

M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s

R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e

S a m p le D is tr ib u tio n

Plasma Peroxidase Activity (mUmL)

lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects

N 30 24 11 1681

37 30 14 20

37 of the population studied had low

peroxidase activity indicating an

impaired ability to defend against free

radicals

34 of the studies population had

elevatedhigh activity potentially

indicating the presence of oxidative

stress

Assessment of Baseline Potential

Starting point for defense of excessive

ROS

Effect of Environmental Insults

Assessment of Capacity to defend against

excessive ROS

Assessment and monitoring treatment of a

disease or disorder

Viral or Bacterial infection

Cardiovascular disease

Mitochondrial Membrane Potential

Oxidative Challenge

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Mitochondrial Superoxide Levels

Assessment of Redox status

Inflammation

Viral or Bacterial Infection

Monitoring treatment of a

disease or disorder

Borrelia infection

Treatment of Inflammation

Functional Effect

Improvement verifies positive clinical effect

Plasma Peroxide Assay

Low Levels may indicate oxidative stress and

possible need for antioxidants

High levels may indicate immune challenges

Can correlate with uric acid

Potential hypertension risk

Related to lipid peroxides

Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13

Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303

Plasma Peroxidase

To

tal S

ub

jects

(n

=86)

Lo

w (

n=30)

No

rmal (n

=24)

Ele

vate

d (

n=11)

Hig

h (

n=16)

0

2 0

4 0

6 0

8 0

Pla

sm

a P

erix

ida

se

Ac

tiv

ity

(m

Um

l)

2 0

1 4

3 0

3 7

M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s

R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e

S a m p le D is tr ib u tio n

Plasma Peroxidase Activity (mUmL)

lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects

N 30 24 11 1681

37 30 14 20

37 of the population studied had low

peroxidase activity indicating an

impaired ability to defend against free

radicals

34 of the studies population had

elevatedhigh activity potentially

indicating the presence of oxidative

stress

Assessment of Baseline Potential

Starting point for defense of excessive

ROS

Effect of Environmental Insults

Assessment of Capacity to defend against

excessive ROS

Assessment and monitoring treatment of a

disease or disorder

Viral or Bacterial infection

Cardiovascular disease

Mitochondrial Membrane Potential

Oxidative Challenge

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Plasma Peroxide Assay

Low Levels may indicate oxidative stress and

possible need for antioxidants

High levels may indicate immune challenges

Can correlate with uric acid

Potential hypertension risk

Related to lipid peroxides

Plasma hydrogen peroxide production in hypertensives and normotensive subjects at genetic risk of hypertensionLacy Fred1 OConnor Daniel T2 Schmid-Schoumlnbein Geert W13

Journal of Hypertension March 1998 - Volume 16 - Issue 3 - p 291ndash303

Plasma Peroxidase

To

tal S

ub

jects

(n

=86)

Lo

w (

n=30)

No

rmal (n

=24)

Ele

vate

d (

n=11)

Hig

h (

n=16)

0

2 0

4 0

6 0

8 0

Pla

sm

a P

erix

ida

se

Ac

tiv

ity

(m

Um

l)

2 0

1 4

3 0

3 7

M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s

R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e

S a m p le D is tr ib u tio n

Plasma Peroxidase Activity (mUmL)

lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects

N 30 24 11 1681

37 30 14 20

37 of the population studied had low

peroxidase activity indicating an

impaired ability to defend against free

radicals

34 of the studies population had

elevatedhigh activity potentially

indicating the presence of oxidative

stress

Assessment of Baseline Potential

Starting point for defense of excessive

ROS

Effect of Environmental Insults

Assessment of Capacity to defend against

excessive ROS

Assessment and monitoring treatment of a

disease or disorder

Viral or Bacterial infection

Cardiovascular disease

Mitochondrial Membrane Potential

Oxidative Challenge

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Plasma Peroxidase

To

tal S

ub

jects

(n

=86)

Lo

w (

n=30)

No

rmal (n

=24)

Ele

vate

d (

n=11)

Hig

h (

n=16)

0

2 0

4 0

6 0

8 0

Pla

sm

a P

erix

ida

se

Ac

tiv

ity

(m

Um

l)

2 0

1 4

3 0

3 7

M ito c h o n d r ia l F u n c tio n O x id a t iv e S tr e s s

R e s tro s p e c tiv e S tu d y P la s m a P e r o x id a s e

S a m p le D is tr ib u tio n

Plasma Peroxidase Activity (mUmL)

lt1162 (Low) 1162 - 1939 (Normal) 1939 - 2455 (Elevated) gt2455 (High) Total Subjects

N 30 24 11 1681

37 30 14 20

37 of the population studied had low

peroxidase activity indicating an

impaired ability to defend against free

radicals

34 of the studies population had

elevatedhigh activity potentially

indicating the presence of oxidative

stress

Assessment of Baseline Potential

Starting point for defense of excessive

ROS

Effect of Environmental Insults

Assessment of Capacity to defend against

excessive ROS

Assessment and monitoring treatment of a

disease or disorder

Viral or Bacterial infection

Cardiovascular disease

Mitochondrial Membrane Potential

Oxidative Challenge

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Assessment of Baseline Potential

Starting point for defense of excessive

ROS

Effect of Environmental Insults

Assessment of Capacity to defend against

excessive ROS

Assessment and monitoring treatment of a

disease or disorder

Viral or Bacterial infection

Cardiovascular disease

Mitochondrial Membrane Potential

Oxidative Challenge

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Mitochondrial Membrane

PotentialJC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

JC-1 is a highly specific probe for detecting changes in mitochondrial membrane potential

JC-1 forms red aggregates in healthy and intact mitochondria while green fluorescence is due to the formation of JC-1 monomers at low mitochondrial membrane potential

Membrane potential is lost upon challenge with excessive ROS (ie Hydrogen Peroxide)

Fibroblast cells loaded with JC-1

A Healthy unchallenged mitochondria

B Membrane potential collapse after

challenge with ROS (Hydrogen

peroxide)httpwwwmedunipgitimagelabmitochhtml

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Mitochondrial Membrane PotentialNovel Biomarker of Environmental Oxidative

Stress

N o n S m o k e r s (n = 1 3 ) S m o k e r s (n = 8 )

7 5

8 0

8 5

9 0

9 5

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

()

bull Assessed baseline potential between non-smokers and current smokers

bull Significantly different of p lt 0005

Low baseline mitochondrial membrane

potential results in a decreased capacity

to defend against excessive ROS and

may lead to Oxidative Stress

Similar results to published findingsMuriel Vayssier-Taussat Environmental Health Perspectives

2002

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Mitochondrial Membrane Potential Biological Variability

S u b je c t- 1 S u b je c t- 2 S u b je c t- 3 S u b je c t- 4 S u b je c t- 5

5 0

6 0

7 0

8 0

9 0

1 0 0

B io lo g ic a l V a r ia b ility

I n tr a -I n d iv id u a l B a se lin e (3 -d a y s )

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

ntia

l (

)Subject Day 1 Day 2 Day 3 Mean SD CV

1 91 91 91 91 0 0

2 88 91 88 89 2 2

3 90 93 90 91 1 2

4 93 93 92 93 1 1

5 90 91 92 91 1 2

There is very little variability

between health subjects

indicating a good marker

for clinical utility

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Mitochondrial Membrane Potential

Reference Interval

Copyright 1991-2012 Data Innovations LLC

Page 2MMPMitochondrial Membrane Potential Printed 02 Feb 2016 112710

EP Evaluator 1000517

Baseline

Laboratory -- Pharmasan Labs Inc Reference Interval 60 and 90 Feb 201(1)

Central 95 Interval(N = 48)

Lower UpperValue 90 CI Value 90 CI Ratio

Confidence

Parametric 8139 7984 to 8294 9627 9472 to 9783 021

Alternatives

Nonparametric Index 8090 lt8090 to 8360 9631 9420 to gt9640 gt016

Transformed Parametric -- -- -- -- --

Confidence Limits for Nonparametric Index method computed by exact formula

True Gaussian SDI

3210-1-2-3

Ba

selin

e (

)

95

90

85

Nonparametric 90 CI Parametric

(Original Data)

Probability Plot

No transformation found

that significantly improves

fit to Gaussian distribution

(Transformed Data)

Probability Plot

Reference Interval Estimation Combined

Selection Criteria

Bounds None

Filter None

Statistics

Mean 88831

SD 3797

Median 89250

Range 8090 to 9640

N 48 of 48

Distinct Values 42

Zeroes 0

Central 95 Index 12 to 478

Analyst BNP

Expt Date 02 Feb 2016

Baseline ()

110100908070

25

20

15

10

5

0

Histogram

Normalizing Transformation

Exponent --

Constant --

Accepted by

DateSignature

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Mitochondrial Membrane Potential

Novel Biomarker of CVD-Derived Oxidative

Stressbull Males and Females age

45-65

bull All diagnosed with Cardiovascular disease

bull Non-Diabetic and within the past 90-days bull NO Chemotherapeutics

NO antioxidants NO Tanning or Excessive Sun

bull Non-Smokers and NO Excessive use of Alcohol

bull Fatigue was a major complaint

7 5

8 0

8 5

9 0

9 5

B a se l in e

Mit

oc

ho

nd

ria

l M

em

br

an

e P

ote

nti

al

() H e a lth y (5 5 )

C a rd ia c (1 3 )

Maack and Bohm 2011 Madamanchi et al 2005

Patients with CVD

have low

membrane

potential The

integrity of their

mitochondria has

been

compromised by

oxidative stress

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Mitochondrial Membrane

Potential Assessment and

Treatment Monitoringbull The influence of HIV infection

and antiretroviral therapy on the mitochondrial membrane potential

bull Increase of Membrane Potential post Anti-Retroviral Therapy is significant at p lt 002

Schmid Antivir Ther 2007

Mitochondrial membrane potential

can be improved and can be

used to monitor treatment

effectiveness

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

BrainGut or GutBrain

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Signs and Symptoms of Food

Intolerance

Colitis

Gastrointestinal issues

Irritable Bowel Syndrome

Bloating

Ear infections

Arthritis

Chronic inflammation

Chronic pain

Autism

Fatigue

Frequent illness

Eczema

Sinusitis

Dermatitis

Weight gain

Anxiety

Headaches

Hypersensitivity

Hyperactivity

Attention difficulties

Sleep difficulties

Depression

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Itrsquos all About the Gut

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Pro-Inflammatory Cytokines

Several pro-inflammatory cytokines directly increase intestinal permeability

IL-6

TNF-alpha

IL-17

Heyman Mamp Desjeux JF(2000)Cytokine-induced alteration of the epithelial barrier to food antigens in disease Ann NY Acad Sci 915 304minus311

HeymanMDarmonNDupont C Dugas B Hirribaren A Blaton MA et al(1994)Mononuclear cells from infants allergic to cows milk secrete tumor necrosis factor alpha altering intestinal function Gastroenterology 1061514minus1523

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Clinical Pearl

ldquoIgG food sensitivity symptoms are typically

delayed up to 72 hours after ingesting the

offending foodrdquo

ldquoA need to know for reintroduction symptomsrdquo

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Case Analysis

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Case Study 56 year old male

Primary complaints

Severe insomnia

Irritability

Mild anxiety

Joint pain

Gas and Bloating

Multivitamin

Fish omegas

58

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Individual Lab Results

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

SOD1

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

SOD 1 (another)

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

SOD 2

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

SOD 3

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

GPX-1

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

CAT

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

bull Strategybull Modified paleo diet

with allergens removed

bull 90 days to six months

bull Alkalinization

bull Consider SIBOFODMAP

reset

bull Consider detox

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

PLAN First 90 days

Waking N-A-C 600 mgs am and noon to support Glutathione synthesis

Ubiquinol 100 mgs am and noon to support mitochondria

Catecholamine support product with tyrosine Macuna DLPA (PEA) and Bacopa

for free radical scavenging (Get inflammation under control)

BanabaPhosphatidylserine combo 4 caps at bed with 600 N-A-C and a combo

fat soluble antioxidant

Concentrated Theanine 2 at bedtime (push down Glutamate )

Taurine 1000 mgs at bedtime

AM exercise

Implement exercise plan

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

90 Day Follow Up

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Wrap Up

Within 3 weeks sleep arrived

Joint pain almost resolved

Occasional anxiety (resolves with

glutamate reduction lozenge)

Needed testosterone to increase

stamina and energy

Dependent on antioxidants

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Case Study

FatigueDepression

49 year old Female

Menopause

Depression

Fatigue

Stopped SSRI due to

ineffectiveness

Bio-identical hormones

improved Vaginal dryness

but nothing else

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Clinical Pearl

Sex hormones can cover

up neurotransmitter

depletion symptoms

such as mood issues

and hot flashes

Menopause hormone

reduction exposes the

issue

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Clinical Pearl

Mitochondrial

dysfunction directly

decreases sex hormone

and neurotransmitter

production

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

90 Day Protocol Modified SIBOFODMAP diet

Catecholamine support am and noon (NT report)

Ubiquinol am and noon (SOD 2 support)

Stop hormones except a testestriol suppository

5-HTP lozenge as needed to cool off

SerotoninGABAMelatonin support at bedtime

Concentrated Theanine at bedtime

3 week liver detox with colon hydrotherapy X 6

IV antixidants X 6

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

90 DAY Follow UP

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

ldquoThe Fog is Liftedrdquo

Patient states that the

ldquofog liftedrdquo in two weeks

Started exercise on her

own

Sex drive slowly returning

True story new hair-style

and new boyfriend

Questions

Questions