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Inflammation and oxidative stress:

a clinical paradox

A paradox involves contradictory yet interrelated elements that exist

simultaneously and persist over time.

Inflammation

• A defensive immune response

• Innate immune system = vasodilation, vascular leakage and leukocyte emigration

• Heat, redness, pain, swelling

• Pathogen/damage associated molecular patterns (PAMPs/DAMPs) recognised by receptors, e.g. TLRs, NLRs, RAGEs, expressed on macrophages, monocytes, dendritic cells and neutrophils

• Secretion of cytokines and chemokines = further immune cell recruitment and inflammatory meditator production

Inflammation – role in chronic illness

• Products of inflammation can damage tissue and cause further stimulation of immune response

• If the inflammatory response continues unnecessarily, this can lead to accumulative damage

• Thus it follows that poorly regulated, prolonged or inappropriate inflammation, also known as chronic or low-grade inflammation (‘silent inflammation’) increases susceptibility to illness and disease

Resoleomics - the process of inflammation resolution In

flam

mat

ory

res

po

nse

Initiation Resolution Termination

PGE2

LTB4

Eicosanoid switch Stop signal

Time

Pro-inflammatory reduced

Anti-inflammatory increased

Oxidative stress • Reactive oxygen species (ROS) are generated as by-products of cellular

metabolism via the electron transport chain, cytochrome P450 and NADPH oxidases

• Produced in response to infection, exercise, pollutant exposure, UV light, ionising radiation, cellular respiration, inflammation, certain drugs, detoxification of xenobiotics, cigarette smoke…

• In healthy humans, production of ROS/RNS is kept in check by our in-built antioxidant defences

• If delicate balance shifts in favour of

pro-oxidants, oxidative stress results

Oxidative stress

• Conventionally – oxidative stress defined as imbalance between pro-oxidant stress and antioxidant defence

• Recently - disruption of redox signalling important – perhaps more so

• Oxidative stress – an imbalance between oxidants and antioxidants in favour of oxidants, leading to disruption of redox signalling and control and/or molecular damage

Kunwar A et al. Free radicals, oxidative

stress and antioxidants in human

health J Med Allied Sci 2011; 1(2)

Oxidative stress – role in illness

• Uncontrolled/excessive ROS leads to potential damage to all biomolecules - most susceptible being proteins, DNA, lipid membranes - leading to functional impairment and cell death

• Free radical damage to – Carbohydrates = chain breaks in molecules such as hyaluronic acid

– DNA = mutations and strand breaks

– Proteins = affects processing and clearance leading to accumulation and build-up in the brain and tissues

• Antioxidants can become pro-oxidants – in presence of reactive metals

– if subsequent antioxidants in the chain not available

– if levels are too high

Oxidative stress

Cancer

Atherosclerosis

Diabetes

Fatty liver diseaseAgeing

Arthritis

Neurological disease

Interdependence

• Experimental data show simultaneous existence of low-grade chronic inflammation and oxidative stress in – Diabetic complications

– CVD

– Neurodegenerative disease

– Liver disease

– Kidney disease

Inflammation causes oxidative stress

• Production of ROS is central to progression of inflammatory disease

• ROS produced by cells involved in inflammatory response (polymorphonuclear neutrophils) - act as signalling molecules and inflammatory mediator

• At sites of inflammation activated inflammatory cells release ROS & RNS as well as enzymes and chemical mediators, resulting in tissue damage and oxidative stress

• When TLR/NLR/RAGE bind PAMPs = transcription factor activation and proinflammatory gene expression – co-stimulation of several TRLs in the presence of cytokine imbalance results in ROS generation

• INF-γ and LPS synergistically increase ROS production

• Macrophages (M1) produce excessive oxidative stress to eliminate pathogens by inducing cell death via caspase activation and creating an imbalance in glutathione equilibrium

Oxidative stress causes inflammation

• Pro-oxidants can initiate intracellular signalling cascades that enhance proinflammatory gene expression

• NF-ƘB – a key player in the inflammatory cascade is stimulated by oxidative stress and intracellular redox status

• ROS released from damaged mitochondria can activate NLRP3 inflammasomes, leading to IL-1β expression

• Oxidatively damaged DNA induces a signalling cascade that culminates in proinflammatory gene expression and cell accumulation

• 8-isoprostane – an arachidonic acid peroxidation end product and a marker of oxidative stress - increases expression of proinflammatory IL-8

• Oxidation of plasma cysteine triggers monocyte adhesion to the vascular endothelium = activation of NF-ƘB and expression of IL-1β

When oxidative stress appears as a primary disorder inflammation develops as a secondary

disorder and further enhances oxidative stress. On the other hand, inflammation as a primary

disorder can induce oxidative stress as a secondary disorder which can further enhance

inflammation.

Biswas SK. Does the Interdependence between Oxidative Stress and

Inflammation Explain the Antioxidant Paradox? Oxid Med Cell Longev.

2016;2016:5698931. doi: 10.1155/2016/5698931.

Nutritional approaches to inflammation

The key to regulating inflammation is through the modulation of eicosanoids

• pro-inflammatory eicosanoids drive the immune and inflammatory processes

• anti-inflammatory eicosanoids act to end the process

Overproduction of pro-inflammatory products or reduced production of anti-inflammatory products can result in continued production of inflammatory products – the hallmark of silent inflammation

• Eicosanoids are derived from omega-6 and omega-3 polyunsaturated fats

• The ratio of omega-6 to omega-3 in the diet influences the type of

eicosanoid produced

Arachidonic acid gives rise to key pro-inflammatory mediators (via COX-2) involved in orchestrating crosstalk between cells involved in the regulation of the immune and inflammatory response

Therefore by regulating arachidonic acid levels within cell membranes we can reduce the production of pro-inflammatory eicosanoids and inflammatory mediators

– Prostaglandins

– Thromboxanes

– Leukotrienes

– Cytokines

Omega-6LA

Omega-6GLA

Omega-6DGLA

Omega-3ALA

Omega-3EPA

Omega-3SDA

Anti-inflammatory eicosanoids

Anti-inflammatory eicosanoids

When omega-3 intake is low, the omega-6 pathway converts DGLA to AA, resulting in a corresponding increase in inflammatory

products from AA known as the ‘the arachidonic acid cascade’

INFLAMMATION

Pro-inflammatory leukotriene

Pro-inflammatory thromboxane

Pro-inflammatory prostaglandin

Omega-6 AA

Reduced inflammation

delta-6 desaturase

(FADS2)

delta-5 desaturase(FADS1)

Omega-6LA

Omega-6GLA

Omega-6DGLA

Omega-3ALA

Omega-3EPA

Omega-3SDA

Omega-3DHA

Omega-6AA

delta-6 desaturase(FADS2)

Omega-6 Omega-3

Omega-6LA

Omega-6GLA

Omega-6DGLA

Omega-3ALA

Omega-3EPA

Omega-3SDA

Omega-3DHA

Omega-6AA

delta-6 desaturase(FADS2)

Nutritional approaches to oxidative stress

Antioxidants

• Substances that neutralise free radicals or their actions – endogenous and exogenous sources

• Enzymatic: superoxide dismutase, glutathione peroxidase, glutathione reductase, thioredoxin, thiols and disulfide bonding – act as cellular redox buffers

• Non-enzymatic: α-tocopherol (Vit E), ascorbate (Vit C), carotenoids, flavonoids, polyphenols, α-lipoic acid, glutathione……

• Act at different stages of the process:

1. Prevention – stop the formation of ROS/RNS (e.g. SOD)

2. Interception – mainly free radical scavenging (‘typical’ antioxidants)

3. Repair – reconstitute and repair damaged target molecules (enzymes e.g. methionine-S-sulfoxide reductase A)

Epidemiological studies show inverse correlation between tissue/plasma antioxidant and phytonutrient status and chronic illness and mortality

Omega-3 index above 8% = significant reduction in all-cause mortality

Diet rich in plant matter (>5-a-day) and oily fish is known to confer significant protective health benefits

But – lots of negative results

Inconsistencies arising from omega-3 intervention studies give mixed results and create confusing messages (Von Schacky 2015; Harris 2015)

Poor heterogeneity in study designs, background diets, endpoint definitions, and baseline fish or omega−3 fatty acid intakes cloud meta-analysis outcomes

Patients recruited regardless of their baseline levels and treated with fixed doses

Recent RCTs (virtually all of which have been conducted in European or North American cohorts [low dietary fish intakes]) use relatively low doses (376–850 mg EPA & DHA) which at least partly explains their failure

CVD secondary-prevention populations - include many individuals who are already taking multiple heart medications such as statins, aspirin and ACE inhibitors, which may obscure the effect of omega-3 fatty acids

The inter-individual variability in response to a fixed dose of EPA + DHA has been found to be large, i.e. to vary up to a factor of 13

Not all ‘fish oils’ are the same - addressing quality/concentration and purity

Study design to incorporate use of biomarkers?

Biomed J Vol. 37 No. 3 May -

June 2014

“antioxidants should be beneficial when given to the right subject at the right

time”

Essential role of inflammation

• Wound healing

• Pathogen elimination

• Reduced mobility & pain = protective

• Trigger for adaptive immune response

Essential role of oxidative stress

• Act as signalling molecules e.g. NO.

• Necessary for stimulating adaptation processes

• Trigger transcription of antioxidant genes

• H2O2 critical for thyroxine synthesis – needed to catalyse binding of iodine to thyroglobulin

• Trigger for apoptosis

• Detoxification via CYP450

• Stimulation of mitochondrial biogenesis

Suppressing inflammation or ROS/oxidative stress too early/aggressively/chronically can

lead to significant exacerbation and/or extension of symptoms and condition

Husson MO, Ley D, Portal C, Gottrand M, Hueso T, Desseyn JL, Gottrand F. Modulation of host defence against bacterial and viral infections by omega-3 polyunsaturated fatty acids. J Infect. 2016 Oct 14. pii: S0163-4453(16)30252-3.

Key messages

• 0.5 g/day EPA + DHA daily improves the outcome of experimental infections caused by opportunistic extracellular pathogens, which induce a strong inflammatory response, including P. aeruginosa, S. aureus, H. pylori, S. pneumonia, E. coli, Streptococcus B in healthy humans

• By contrast, n-3 LC-PUFA supplementation at a 1-2g daily shown to be detrimental in the outcome of C. rodentium or H. hepaticus colitis, and worsened S. aureus infections as skin abscesses (animals)

• In addition, omega-3 supplementation is detrimental in respiratory, systemic, ocular infections with intracellular pathogens such as M. tuberculosis, Influenza A virus, Salmonella spp., L. monocytogenes, and Herpes simplex virus, which need an immune cell response to eradicate infected cells

• In these infections omega-3 are deleterious because of their immunosuppressive properties

• Omega-3 supplementation during infection may prove detrimental, because of the anti-inflammatory properties, when the host inflammatory response is critical for survival

• Host protection against Influenza A virus requires neutrophils, NK cells, T lymphocytes, and secretion of both inflammatory and antiviral cytokines – omega-3, by actively over-suppressing NK cell numbers can lower the immune system’s ability to combat infections

Michael Ristow et al. Antioxidants prevent health-promoting effects of physical

exercise in humans

Recent work suggests that biological context may be key to predicting

whether antioxidants impede or even promote tumorigenesis.

CAUTION!In presence of oxidative stress lipids are

peroxidised – adding high concentration, high dose long-chain, omega-3s to a pro-oxidant

environment is just fanning the flames

Lipid peroxidation• Membrane lipids are highly susceptible to oxidative damage

• When reacted with ROS = chain reaction ‘lipid peroxidation’

• Numerous toxic by-products formed

- can have wide reacting, systemic effects as secondary messengers

- damage is highly detrimental to cell function

• Termination = reaction of the lipid radical with an antioxidant forming a less reactive molecule

Only 1% oxidised DHA was sufficient to reverse protective

effect of DHA and to significantly increase Aβ production.

Results: Lipid peroxidation was greater in MDD than in controls (studies =17, N=857

MDD/782 control, SMD =0.83 [0.56–1.09], z=6.11, P,0.01, I2 =84.0%) and was

correlated with greater depressive symptom severity (B=0.05, df=8, P,0.01).

Antidepressant treatment was associated with a reduction in lipid peroxidation in MDD

patients (studies=5, N=222, SMD=0.71 [0.40–0.97], P,0.01; I 2 =42.5%).

Ensuring safe interventions

DIET!

https://truphys.com/antioxidants-and-training/

Testing

• Omega-3 index and AA: EPA– Igennus Opti-O-3

• Oxidative stress– Mitochondrial function

– Antioxidant status – CoQ10, GSH,

– Enzyme cofactors – Zn, Se, Cu ….

• SNPs– Covered in my July webinar

Q1 Q2 Q3 Q4 Q5

Low Average HighSaturated fat

Myristic acid 14:0 0.17 0.47 0.62 0.85 2.01

Palmitic acid 16:0 15.5 21.55 23.01 24.38 29.26

Stearic acid 18:0 1.45 13.17 14.48 15.62 23.07

Arachidic acid 20:0 0.05 0.14 0.16 0.18 0.75Behenic acid 22:0 0.15 0.36 0.43 0.51 1.13

Lignoceric acid 24:0 0.19 0.51 0.64 0.76 2.36

Monounsaturated fat

Palmitoleic acid n-7 16:1 0.1 0.69 0.92 1.28 3.51

Oleic acid n-9 18:1 12.38 17.96 21.03 32.97 32.97

Eicosenoic acid n-9 20:1 0.08 0.18 0.22 0.26 0.85

Nervonic acid n-9 24:1 0.07 0.4 0.051 0.65 1.69Polyunsaturated fat n-6Linoleic acid (LA) 18:2 11.08 16.83 18.56 21.15 28.74

Gamma-linolenic acid (GLA) 18:3 0.02 0.13 0.18 0.27 0.97Eicosadienoic acid (EDA) 20:2 0.10 0.16 0.19 0.22 0.98

Dihomo-gamma linolenic acid (DGLA) 20:3 0.39 0.99 1.23 1.48 2.47

Arachidonic acid (AA) 20:4 2.5 8.56 10.05 11.38 16.51

Docosatetraenoic acid n-6 22:4 0.12 0.64 0.85 1.13 2.58

Docosapentaenoic acid n-6 22:5 0.03 0.14 0.17 0.23 1.53

Polyunsaturated fat n-3Alpha-linolenic acid (ALA) 18:3 0.16 0.34 0.41 0.51 1.4

Eicosapentaenoic acid (EPA) 20:5 0.2 0.86 1.4 2.44 10.68

Docosapentaenoic (DPA) 22:5 0.41 0.89 1.11 1.45 3.97

Docosahexaenoic acid (DHA) 22:6 0.96 2.49 3.35 4.37 8.89

Trans fatTrans palmitoleic acid n-7 16:1 0.11 0.19 0.23 0.28 0.84

Trans oleic acid n-9 18:1 0.01 0.09 0.12 0,18 0.54Trans linoleic acid n-6 18:2 0.07 0.16 0.19 0.23 1.7

Targeted nutrient interventions

Anti-inflammatory eicosanoid production

DGLA

GLA

LA

EPA

ETA

SDA

ALA

Delta -6 desaturase

Delta -5 desaturase

Cyclooxygenase (COX)/lipoxygenase (LOX)

Elongase

Series-2 prostaglandinsSeries-2 thromboxanesSeries-4 leukotrienes Hydroxy fatty acids

AA

COX/LOX

Omega-6 Omega-3 Eicosanoids, including prostaglandins and leukotrienes, are biologically active lipids derived from AA and EPA that have been implicated in various pathological processes, such as inflammation and cancer

The relationship between AA and EPA is therefore significant when considering omega-3 intervention strategies

Key structural role & anti-inflammatory

docosanoid productionResolvins Protectins

DHAElongase &desaturase

Pro-inflammatory eicosanoid production

Series-3 prostaglandinsSeries-3 thromboxanesSeries-5 leukotrienes Hydroxy fatty acids

Resolvins

Primary structural function & anti-inflammatory docosanoid

production

Anti-inflammatory eicosanoid production

REDUCED INFLAMMATION

DHAEPA

Pro-inflammatory eicosanoid production

INFLAMMATION

AA

AA to EPA ratiodirect antagonism

The relationship between the omega-3 index and the AA to EPA ratio

Omega-3 index

Is there an optimal EPA to DHA ratio?

2:1 EPA to DHA proportions demonstrated to be more effective treatments to produce an anti-inflammatory response compared with 1:2 EPA to DHA

6 :1 EPA to DHA ratio may be optimal for correcting omega-3 deficiency, with concomitant positive effects on lipid profiles and on inflammatory indices

EPA in excess of DHA is optimal!

As much as 13% of DHA is retro-converted to EPA

Monitor DHA levels and supplement accordingly

Dasilva G, Pazos M, García-Egido E, Pérez-Jiménez J, Torres JL, Giralt M, Nogués MR, Medina I. Lipiomics to analyse the influence of diets with different ratios of EPA to DHA in the progression of metabolic syndrome using SHTOB rats. Food Chem. 2016 Aug 15;205:196-203.

Shaikh NA, Yantha J, Shaikh S, Rowe W, Laidlaw M, Cockerline C, Ali A, Holub B, Jackowski G: Efficacy of a unique omega-3 formulation on the correction of nutritional deficiency and its effects on cardiovascular disease risk factors in a randomized controlled VASCAZEN((R)) REVEAL Trial. Molecular and cellular biochemistry 2014, 396:9-22.

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Curcumin can modulate various types of signalling molecules including transcription factors, enzymes, growth factors, interleukins, cytokines & chemokines

Ghosh S, Banerjee S, Sil PC. The beneficial role of curcumin on inflammation, diabetes and neurodegenerative disease: A recent update. Food Chem Toxicol. 2015 Sep;83:111-24.

NF-κB in chronic disease – a target for nutritional intervention

Potent antioxidant effects– curcumin’s antioxidant mechanisms protect cells against oxidative damage

Improves liver function – curcumin regulates the activity of a number of key enzymes and antioxidants essential for optimal detoxification

Cardiovascular health – curcumin promotes cardiovascular health and function, and protects low density lipoprotein (LDL) from oxidation

Immune health – curcumin improves and supports immune function

Joint health – curcumin significantly improves joint health by reducing inflammation and promoting joint comfort and flexibility

Digestive health – curcumin stimulates bile production and promotes healthy digestive function

Anti-cancer benefits – curcumin offers protective benefits against the main hallmarks of cancer including angiogenesis, proliferation, metastasis, inflammation and apoptosis

Curcumin clinical benefits

• CoQ10 is a powerful, fat-soluble, vitamin-like substance

• Two main functions:• energy production – cellular respiration• antioxidant recycling

CoQ10 - as ubiquinol

• Ubiquinone – oxidised form

• Ubiquinol – reduced form

• 96% of CoQ10 within the body is in the form of ubiquinol

• Ubiquinol is the active form of CoQ10

A free radical has an electron missing from its outer shell

X

Ubiquinol donates anelectron to a free radical

X

Ubiquinol donates electrons to other

antioxidants

‘Recharged’ antioxidants (i.e. vitamins C & E, lipoic acid) can

donate electrons to free radical

Free radical damage and

oxidative stressSkin

LungsInflammation

Cardiovascular

Brain

Immunity

Organs

Ubiquinol deficiency alters mitochondria function and lowers antioxidant status, leading to increased free radical generation

Ubiquinol routinely outperforms ubiquinone

Supplementation with 150 mg/ day ubiquinol for 14 days reduces inflammatory processes via gene expression

Oral intake of ubiquinol increased its proportion significantly (P < 0.001), with the highest increase in those persons having a low basal serum ubiquinol content (<92.3%)

Ubiquinol status significantly correlated to the concentration of the inflammation marker monocyte chemotactic protein 1 (involved in the accumulation of inflammatory cells).CoQ10 redox state predicts the concentration of C-reactive protein (CRP)

People with lower ubiquinol status, higher BMI, and low-grade inflammation may benefit from ubiquinol supplementation

Fischer A1, Onur S1, Niklowitz P2, Menke T2, Laudes M3, Döring F1. Coenzyme Q10 redox state predicts the concentration of c-reactive protein in a large Caucasian cohort. Biofactors. 2016 Feb 23. doi: 10.1002/biof.1269. [Epub ahead of print]

Fischer A, Onur S, Schmelzer C, Döring F. Ubiquinol decreases monocytic expression and DNA methylation of the pro-inflammatory chemokine ligand 2 gene in humans. BMC Res Notes. 2012 Oct 1;5:540. doi: 10.1186/1756-0500-5-540.

CoQ10 supplementation significantly reduced the levels of circulating

CRP (P = 0.022), IL-6 (P = 0.002) and TNF- (P = 0.027). The results of

meta-regression showed that the changes of CRP were independent of

baseline CRP, treatment duration, dosage, and patient characteristics. In

the meta-regression analyses, a higher baseline IL-6 level was

significantly associated with greater effects of CoQ10 on IL-6 levels.

Resveratrol

Combination therapies

Studies are beginning to show that targeted combination therapies elicit greater benefits than single nutrients

Addressing multiple factors/pathways involved in disease onset and progression simultaneously helps overcome clinical paradox effect

Other things to consider

• Remove the root cause

- Importance of finding the triggers

- Removing mediators

• Address collateral damage

– Once the body is primed to react any exposure could re-trigger disease/symptoms

– Managing and maintaining inflammatory balance and anti-oxidant status vital

Triggers and mediators

• Systems under stress – Gut pathology/permeability

– Food intolerance

– Infections

– Hormone imbalance

– Liver function

• Environment and lifestyle– Nutrient imbalance

– Heavy metal toxicity

– Stress

– Poor sleep

Summary – key messages

• Human beings are multi-system organisms

• The complexity of each biological process and how they relate is still not fully understood

• Multi-pronged approach to disease and dysfunction necessary to elicit benefits

• Understanding of mechanisms of action and inter-individual variation essential for therapeutic success

Summary – plan of action

• Know your environment - oxidative stress/inflammation -severity

• What are you combating – specific condition, dysfunction, system

• Identify need – testing

• Lay the foundations – dietary modification

• Targeted interventions – choose proven nutrients for specific, identified issues

• Low dose, high bioavailability/quality

• Co-supplementation for ‘bigger picture’ support

• Ongoing management to address ATMs

Education Technical

Sophie TullyNutrition Education Manager

sophiet@igennus.com

Dr Nina Bailey Head of Nutrition

ninab@igennus.comTwitter @DrNinaBailey