MEDCHEM 562P 2014 - VITAMINS and Minerals

Part 1: Water Soluble Vitamins

Bill Atkins Ph.D. H172Q, 685 0379; winky@u.washington.edu INTRODUCTION I. Who takes vitamin/mineral supplements and why? [Bailey et al. JAMA Intern

Med 173:355 (2013].

• It is estimated that ~50% of the adult population in the US takes some type of dietary supplement, typically to ‘improve/maintain overall health’.

• >75% of products are used without care-provider recommendation.

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II. Multivitamins

It is estimated that ~30% of the adult US population take multivitamins daily. A recent clinical trial of male physicians taking multivitamins concluded there was a

very modest, but (statistically) significant, reduction in total cancers with daily multivitamin use (NEJM, 308:1871 (2012).

Do we need to supplement diets with vitamins/multivitamins? Maybe…. in certain circumstances.

III. When are vitamin supplements worthwhile?

Inadequate intake -- alcoholics, poor, elderly, dieters, poor diet

Increased needs -- pregnancy, lactation, infants, smokers, injury, trauma, recovery from surgery, infection

Poor absorption -- elderly, GI disorders, specific GI surgeries, e.g. gallbladder

removal, gastric bypass, cystic fibrosis, severe diarrhea, drug-induced vitamin deficiencies – e.g. long term antibiotic use, cholestyramine, mineral oil

IV. Definitions

• Vitamins are organic compounds and minerals are chemical elements that are required as nutrients in small amounts by an organism.

• Vitamers are different forms of a particular vitamin, e.g. vitamins K1 and K2, vitamins D2 and D3, retinol and retinal, etc.

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V. Reference Tables for Intake of Vitamins and Elements Intakes DRI Reports are produced by the Food & Nutrition Board of the Institute of

Medicine, National Academies of Science. http://fnic.nal.usda.gov/dietary-guidance/dietary-reference-intakes/dri-tables

Estimated average requirements (EAR): the average daily nutrient intake level estimated to meet the requirements of half of the healthy individuals in a group.

Recommended Dietary Allowance (RDA): the average daily dietary intake level; sufficient to meet the nutrient requirements of nearly all (97-98%) healthy individuals in a group. Calculated from the EAR.

Tolerable Upper Limit (UL): maximum adult daily intake unlikely to cause harm.

RDA= 1.2(EAR)

Daily Values (DVs) are set by the FDA.

http://ods.od.nih.gov/HealthInformation/dailyvalues.aspx • Two groups: Daily Reference Values (DRVs) for energy-producing nutrients,

e/g. fats, carbohydrates, protein etc. and Reference Daily Intakes (RDIs) for vitamins and minerals.

• A DV is often, but not always, similar to one's RDA for that nutrient. • DV is primarily used for labeling purposes. % DV on label is based on 2,000

calorie/day diet for adults and children over 4 yrs.

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VI. Standardization

• Units of biological activity (IU) superceded, where known, by potencies based on weight (µg, mg) of the most active vitamer.

• IoM guidelines use weight. • FDA labels use both.

  VII. History and Discovery 1500 BC Ancient Egyptians used liver - rich in vitamin A - applied to the eye to treat night

blindness.

1536 Jacques Cartier, exploring the St. Lawrence River, uses local native knowledge to save his men from scurvy by boiling the needles from cedar trees to make a vitamin C-rich tea.

1795 British navy adds lemons to sailors' rations, 40 years after a Scottish naval surgeon, James Lind, had urged that citrus fruits be used to prevent scurvy.

1884 Japanese navy eradicates beriberi - vitamin B1 deficiency- by feeding sailors meat and fruit in addition to polished white rice, which lacked the thiamine-rich husks.

1897 Beriberi (a polyneuritis) was induced in birds fed only polished rice and reversed by feeding the rice polishings.

1911 Casimir Funk isolates amine-containing concentrate containing thiamine from rice polishings, which was curative for polyneuritis in pigeons, and names it 'vitamine' for vital amine.

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1912 Xavier Mertz –Antarctic explorer – dies of vitamin A poisoning from ingesting sled dog liver after supplies are lost in a crevasse.

Year discovered Vitamin Source

1912 Vitamin B1 Rice bran

1912 Vitamin C Lemons

1913 Vitamin A Milk/egg yolk

1918 Vitamin D Cod liver oil

1920 Vitamin B2 Eggs

1922 Vitamin E Wheat germ, Seed oils

1926 Vitamin B12 Liver

1929 Vitamin K Alfalfa

1931 Vitamin B5 Liver

1931 Vitamin B7 Liver

1934 Vitamin B6 Rice bran

1936 Vitamin B3 Liver

1941 Vitamin B9 Liver

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VII. WATER SOLUBLE VITAMINS

A. Generalities 1. Metabolism and storage -- only B12 and folate are appreciably stored. In

general, water soluble vitamins are excreted readily and are not stored. As a result, depletion is more of a problem than toxicity.

2. Toxicity -- only niacin and pyridoxine are at all toxic (in high conc.). In general, the water soluble vitamins have few toxicities. The water soluble vitamins are coenzymes for various common biochemical reactions and their status can be readily determined by measuring the appropriate enzyme activities in blood. Typically, the enzyme activity is measured in the absence and the presence of exogenously added coenzyme, to determine whether the patient needs more of the vitamin.

B. Thiamin -- (Vitamin B1) 1. Structure

TMPN SCH2

CH3 N

N

CH3 CH2 CH2OH

Hthiamin

ATP ATPTPP

Chemically, the structure includes a pyrimidine ring and a thiazole ring. The thiazole ring has received enormous attention from mechanistic biochemists - they have pondered the reason for the choice by nature of a sulfur atom in the ring. Imidazole rings and oxazole rings would also, in principle, be capable of catalyzing the relevant chemical reactions.

Space-filling model of Thiamin

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2. Function – used to harvest carbohydrates for energy a) Oxidative decarboxylation of α-keto acids

+

CH3 C CO2HO

TPP

lipoic acidNAD

CoASHpyruvate dehydrogenase complex

CH3 C SCoAO

CO2e.g. + + NADH

e.g. α -ketoglutaric acid TPP succinyl CoA CO2

NADCoASH

++

lipoic acidα-ketoglutarate dehydrogenase complex

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The decarboxylation is accomplished by a mitochondrial enzyme complex as shown below. L = lipoic acid, E = enzyme, TPP = thiamin pyrophosphate. Pyruvate dehydrogenase complex in detail

b) Transfer of α-ketols (pentose phosphate pathway) -- 10% of carbohydrate metabolized this way. This pathway provides pentoses for RNA and DNA synthesis and NADPH for the biosynthesis of fatty acids and other endogenous reactions.

c) Non-coenzyme function – TTP involved in the control of chloride channels in brain and elsewhere in nerve impulse conduction.

E LSH

SH

CoASH

CH3 CSCoAO

NAD+

TCAcycle

E TPP

E TPP

C OH

CH3

(α-hydroxyethyl TPP)

E LS

S

E L SH

S C CH3O

CH3 C COOHO

CO2

NADH

CHOHCHO

CH2OPO3H2

+O=

HOCHCCH2OH

HCOH

HCOH

HCOHCH2OPO3H2

CHOHCHOH

CHOH

CHO

CH2OPO3H2

+O=

HO CHCCH2OH

HC OHCH2OPO3H2

TPPtransketolase

e.g.

xyulose-5-phosphate ribose-5-phosphate

sedoheptulose-7-phosphate

glyceraldehyde phosphate

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3. Mechanism – formation of adduct with C2 of thiazole ring. ‘Classic’ experiments support this mechanism, including facile exchange of solvent D2O at the C-2 position.

4. Deficiency – thiamin needs are proportional to caloric intake and is essential for carbohydrate metabolism. Usually consider requirement as 0.5 mg/1000 calories plus 0.3 mg during pregnancy and lactation. Studies show laboratory evidence of thiamin deficiency (erythrocyte transketolase assay) in 20-30% of elderly patients and 40-50% of chronic alcoholics. <2% of healthy controls showed evidence of deficiency. a) Early signs of deficiency – anorexia, nausea, vomiting, fatigue,

weight loss, nystagmus, tachycardia.

b) Late signs of deficiency – Beriberi cardiac - increased heart size, edema cerebral - depression, irritability, memory loss, lethargy GI tract - vomiting, nausea, weight loss neurological - weakness, polyneuritis, convulsions. Signs and symptoms vary with age of patient, rapidity of onset, and severity of deficiency.

+H

+H

N S

CCH3

OH

NC

S

CHCH3

OH

NC

S

H

H+ + CH3 CHO

α-hydroxy-ethyl-TPP

H+

N S

CCH3 OH

C OO

3 2 H++NC

S

H

NC

S

CH3 C COOH

O

NC

Spyruvate

H+

CH3 C COOH

O

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c) Thiamine and the alcoholic (i) intake low and alcohol blocks conversion of thiamin

→ TPP (ii) ↓ absorption - ↓ active transport (iii) ↓ storage (iv) increased fluid intake and urine flow causes thiamin

washout (v) involved in fetal alcohol syndrome.

d) Wernicke-Korsakoff syndrome – seen in some alcoholics;

neurological disorder resulting in impaired mental functioning → institutionalization for a significant number of patients. Symptoms: confusion, memory loss, confabulation, psychotic behavior; maybe irriversible in part.

e) Factors → B1 deficiency (i) ↑ carbohydrate intake -- TPN, alcoholics (ii) ↓ absorption -- ulcerative colitis, etc., alcoholism (iii) ↓ intake -- poor diet, geriatrics, breast fed infant from B1

deficient mother, etc. (iv) alcoholism.

f) Cellular uptake – Intestinal cells contain a thiamin specific

receptor/transporter (hTHTR) which appears to specifically pump thiamin and not TPP. After cellular uptake, thiamin is converted to TPP. Polymorphisms in the gene encoding hTHTR are known and may contribute to thiamin-responsive megaloblastic anemia. Once in the circulation specific transport proteins may ‘store’ TPP and control its circulation (hypothesized), but most is bound to Albumin. Most ends up in skeletal muscle, brain, heart, liver.

5. Source – present in most tissues (as TPP) and plants (as thiamin); rich sources include: lean meat, especially pork, cereal grains, eggs, yeast, nuts. Thiaminase – in some fish (raw) and shellfish (raw) and ferns. This enzyme can hydrolyze thiamin.

NC

SCH2R

In the milling or processing or processing of rice and flour, the thiamin is lost. ‘Whole’ wheat or rice contains ~10 times as much Thiamin as white wheat or rice. Today in the USA, most white flour, rice and pastas are "enriched" to bring thiamin levels to near original levels. "Enriched" products also have added riboflavin, niacin, iron, and folic acid.

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6. Stability – labile at pH > 4 and when heated (especially at alkaline pH values) prolonged cooking ¯ levels especially at pH > 4.

7. Diagnosis of deficiency state a) ↑ pyruvate and lactate in plasma b) transkelotase activity in RBC – most important technique.

8. Uses

a) deficiency states -- for alcoholics b) thiamin responsive inborn errors of metabolism -- see below c) mosquito repellant -- efficacy? -- for dogs, for humans 50 mg QID

2d before and during exposure is recommended. d) acute alcoholism: give 100 mg IM or IV stat. This is a common

practice. e) Alzheimer’s disease—little evidence for benefit (huge doses used).

9. Thiamin responsive inborn errors of metabolism:

Disease Defect Wernicke – Korsakoff Transketolase Maple Syrup urine disease Failure to decarboxylate branched chain amino acids Thiamin responsive megaloblastic anemia Hyperalanemia ? Hyperpyruvate acidurea Pyruvate dehydrogenase

10. Requirement – 0.5 mg/1000 cal. DV = 1.5 mg. Minimum intake should be at least 1 mg.

11. Toxicity – nontoxic on oral administration; No UL value. Anaphylactic reactions have been observed in patients receiving repetitive parenteral doses.

12. Patient Counseling/ Patient Use Issues a) Needed to drive carbohydrates to energy b) Rarely needed as a single supplement. Use a multivitamin to get

needed thiamin. c) Special benefit in alcoholics at higher doses d) Benefit in high doses in rare thiamin-responsive inborn errors of

metabolism e) Uncertain benefit as a mosquito repellant (if true, 1-2 week onset) f) Nontoxic.

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C. Riboflavin (Vitamin B2) Like Vitamin B1, Riboflavin is highly water soluble, and it is difficult to achieve toxic levels in the body – excess vitamin is efficiently eliminated renally. It used as an additive in many enriched foods and can be produced in huge, industrial scale, quantities by expression of the biodynthetic enzymes in fungi or bacteria. These genetically engineered organisms may appear bright yellowish-orange, which is the color of Riboflavin. 1. Structure

riboflavin coenzymesFAD = flavin adenine dianucleotideFMN = riboflavin monophosphate

N

NNH

N

O

OCH3

CH3

CH2 CH CH CH CH2OHOH OH OH

gut mucosa liverriboflavin FMN FADATP ADP ATP ADP

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2. Function – redox, tissue respiration, H transfer as flavin containing enzyme. oxidized – yellow reduced - colorless Examples of enzymes having flavin groups: succinate dehydrogenase (-succinate → fumerate in TCA cycle) fatty acid acyl CoA dehydrogenase (β-oxidation of lipids) glutathione reductase – important in antioxidant activities The following are important flavoproteins (containing FMN): Cytochrome C reductase (electron transport); NADP+ -- cytochrome C reductase; cytochrome P-450 reductase (drug metabolism), flavin monooxygenase (drug metabolism).

3. Deficiency state a) Not usually seen in isolation but occurs in combination with other

B vitamin deficiencies. b) Fatigue, cheilosis, glossitis, vascularization of cornea, dermatitis c) Vegans and teenagers may be low in B2 if dairy intake is low d) Low B2 intake may be a risk factor for cataract development e) Alcoholics are at risk due to low intake and low absorption.

4. Source – milk, meats, leafy vegetables, eggs, yeast, “enriched” products.

5. Stability

a) Usually > 30% destroyed by cooking b) Labile to light

red, colorlessox-yellow

R

NH

NH

HN

RO

O

RN

NNH

N O

O-2H⋅

⋅+2H

NADPH

NADP

FAD

FADH2

2GSH

GSSG

H2O2 or ROOH

2H2O or ROH + H2O

glutatione peroxidaseglutathionereductase

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c) More stable in acid than alkali in absence of light.

6. Use a) Deficiency states. Is a component of most multivitamin mixtures. b) New – may help in migraine headache prevention. c) New – high intake associated with lower risk for cataracts and a

3mg supplement reduced risk.

7. Requirements a) DV = 1.7 mg b) “Average” U.S. diet contains 2 mg for males and 1.5 mg for

females c) Diagnosis – erythrocyte glutathione rreductase activity d) No UL value

8. Patient Counseling/ Patient Use Issues

a) Routine single dose supplementation not needed. Use a

multivitamin to get needed riboflavin. b) Possible use in preventing migraine headaches. Use 400 mg/d. c) Will turn urine bright yellow in doses higher than the DV. d) Nontoxic.

D. Vitamin B6 1. Structure

Pyridoxine is a commonly used term for this vitamin, but all 3 are equally active so vitamin B6 is a better term to use. Three phosphorylated forms are present also: P PO4 PLP

FMNPNP

FMN

pyridoxamine (PN)

N

CH2OHHO

CH3

CH2NH2

N

CH2OHCH2OHHO

CH3

pyridoxine (P)

N

CH2OHHO

CH3

CHO

pyridoxal (PL)

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Coenzyme = pyridoxal-5-phosphate “PLP”

2. Function – participates in over 140 enzymatic reactions by forming a Schiff base with the terminal amino group of lysine in the enzyme. It is estimated that this corresponds to ~4% of all enzymatic reactions known. a) Transamination

e.g. glutamate-aspartate transaminase

N

CH2OP

CH3

HOCH

O

R NH2

holoenzymeNCH3

HO CH2OPCHNR

PLP

PLP dependent enzyme

+

R C COOHO

α-keto acid #1

N

HO CH2OP

CH3

CHNenzyme

N

HO CH2OP

CH3

CHNCHR COOH

N

HO CH2OP

CH3

CHNCR COOHH

N

HO CH2OP

CH3

CHNH2

R CH COOHNH2

amino acid #1

+

+

H2O

+ R´ C COOHNH2

amino acid #2

+ R´ C COOHO

α-keto acid #2N

CH2

NH2

HO

CH3

CH2OP

N

HO

CH3

CH2OPCHO

PLPPNP

PLP

oxaloacetic acid alanineCH3 CH COOH

NH2

transaminasepyruvic acid

HOOC CH2 C COOHO

+

aspartic acid HOOC CH2 CH COOH

NH2+ CH3 C COOH

O

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b) Decarboxylation e.g. e.g. e.g. e.g.

Note: B6 contraindicated in levo-DOPA therapy because B6 enhances peripheral decarboxylation of levo-DOPA to dopamine which will not cross Blood Brain Barrier; Larobec® (Roche) contains no pyridoxine and can be used if multivitamin supplementation is desired for patient on l-DOPA. The anti-Parkinsons’s drug Sinemet® contains levo-DOPA and carbidopa (A DOPA

N

HO CH2OP

CH3

CHO

N

HO CH2OP

CH3

CHNCHR COOH

N

HO CH2OP

CH3

CHO

CH2RNH2+

decarboxylase

H2O

amino acid

R CH COOHNH2 + + CO2

HOOC CH2 CH2 C COOHH

NH2PLP

CO2

HOOC CH2 CH2 C HH

NH2

glutamic acid γ-amino butyric acid (GABA)

N

HOC COOHH

NH2

5-hydroxytryptophan

N

HOCH2

NH2

5-hydroxytryptamine (serotonin)

CO2

PLP

PLP

CO2NN

CH2HCH

NH2

histamine

NNCH2

HC COOHNH2

histidine

PLP

CO2 CH2 CH2

OH

NH2

HOHO

CH2HC COOHNH2

OH

DOPA DOPAMINE

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decarboxylase inhibitor) -- therefore, no interaction.

c) B6 and sulfur amino acid metabolism. (Note: elevated homocysteine is an independent risk factor for cardiovascular disease and birth defects.)

d) B6 involvement in methionine formation (and S-adenosyl methionine) makes it indirectly involved in methylation. Hence B6 is indirectly involved in lipid metabolism and nucleic acid formation and immune function.

C COOHCH2

H

NH2

CH2SCH3

OCH2

S CH2NH2

HCH2 COOCH

adenine

OHOH

-

OCH2

CH3 S CH2 CH2 COOCH

adenine

OHOH

H

NH2

-

C COOHCH2

H

NH2

H3C

C COOHCH2

H

NH2

HS

α-ketobutyrate

cysteine

cystathionine

PLP serine

methionine

S-adenosylmethionine

methyl acceptor

S-adenosylhomocysteine

THFA

N5-methylTHFA

methyl B12

hydroxy B12

ATP

C COOHCH2

H

NH2

CH2HS

homocysteine

C COOHCH2

H

NH2

CH2S

CH2 C COOHH

NH2

+

PLPcystathionineγ-lyase

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e) B6 involved in tryptophan metabolism to serotonin and niacin.

f) Other reactions requiring B6: (i) Glycogen phosphorylase (release of glucose in muscle) (ii) Heme biosynthesis

NiacinN

COOH

COOH

NH2NH

tryptophanoxygenase

serotonintryptophan

COOH

NH2NH

HO

N-formylkynurenine

NH2NCHO

COOH

O

3-hydroxykynurenine

NH2

COOH

OHNH2

O

kynureninasePLP

COOH

NH2OH

3-hydroxy anthranilic acid

NNH2

HO

Htryp

decarboxylase

PLP

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(iii) Nucleic acid biosynthesis (via SAM)

SUMMARY of Pyridoxal/pyridoxamine reactions

CH2

N

C

N

HC

HC

N

N

B:

external imine internal imine

HO-

H2C

C

N

HN

OH

external carbinolamine

HC

HC

N

HN

HO-

OH

internal carbinolamine

H2C

N

NH2

OHC

N

O

NH2

hydrolysis of Imine leads to incorporation of ‘oxygen atom’ H2O attacks carbon atom of the imine, that carbon gets an oxygen PLP enzymes control which carbon atom gets the oxygen atom by switching between the external imine and the internal imine.

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3. Deficiency

a) Not seen usually and, if seen, is associated with other vitamin

deficiencies or is iatrogenic; symptoms include rash, peripheral neuritis, anemia and possible seizures. Deficiency diagnosed by low plasma PLP and low transaminase activities (±PLP).

b) Iatrogenic B6 deficiencies (i) Isoniazid – antituberculosis drug – forms Schiff base with

B6. Can get neuritis and convulsions. 25-300 mg/d B6 given to prevent B6 deficiency.

(ii) 4-Deoxypyridoxine – (experimental only)

N

HOCH3

CH2OH

CH3 Symptoms – Skin lesions on face, glossitis, stomatitis, convulsive seizures (↓ GABA?), anemia (↓ heme synthesis?).

(iii) Oral contraceptives.- older high dose Ocs can affect B6 but not a problem now.

4. Source – milk, meats, legumes, tuna, whole grains, beans.

5. Stability – pyridoxine is stable; some loss on cooking, especially with meats, due to Schiff base formation and decrease of the pyridoxal in the foods.

N

CO

NH NH2

N

HCO

CH2OHHO

CH3

+

N

CO

NH N CHO CH2OH

H

CH3

NIsoniazid

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6. Diagnosis of Deficiency – measure erythrocyte transaminases.

7. Use a) Routine use in multivitamin products b) In INH therapy c) Certain inborn errors of metabolism

Name Symptoms Dose of B6 Problem

B6--dependent infantile convulsions

Clonic and tonic seizures

10-25 mg/day

Defective glutamic acid decarboxylase; possible GABA depletion

B6--responsive anemia

Microcytic, hypochromic anemia

100 mg/day

Defective hemoglobin synthesis

Xanthurenic acidurea Mental retardation 25-100 mg/day Defective tryptophan metabolism due to faulty kyureninase, xanthurenic acid spills into urine

Homocystinurea Mental retardation Early heart disease

25-500 mg/day Defective cystathionine synthetase homocysteine appears in urine

Cystathionurea Mental retardation 25-500 mg/day Defective cystathionase d) PMS (50-500 mg/d) -- evidence is uneven. PLP is known to bind

to steroid receptors.

e) Carpal tunnel syndrome -- evidence is uneven. It seems to work for some. A trial of B6 100-200 mg/d for 6 mos. may be worthwhile.

f) Use in lowering homocysteine levels (see sulfur amino acid scheme above). High homocysteine may be an independent risk factor for cardiovascular disease but this is now controversial. Combine with folic acid and B12 for optimum lowering action.

g) Nausea and vomiting in pregnancy-Helpful in high doses. PremesisRx contains 75mg sustained release B6 (plus 12ug B12, 1mg folic acid and 200mg calcium) or 25mg of generic B6 TID is less expensive.

8. Requirement – DV = 2 mg; UL = 100 mg.

9. Toxicity a) > 200 mg/day can decrease prolactin levels b) > 1-2 g/day can cause serious neuropathy by an unknown

mechanism. Recommendation: avoid long term use in doses above

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200 mg.

10. Patient Counseling/ Patient Use Considerations a) Routine single dose supplementation usually not needed. Use a

multivitamin to get needed B6. b) Sometimes used, with limited evidence, for carpal tunnel

syndrome, PMS, and depression on OCs. c) Rare use in high doses for inborn errors. d) Sometimes used to prevent neuropathy with isoniazid. e) For nausea and vomiting of pregnancy, 25 mg TID or use

PremensisRx which is FDA approved for this. f) Used with B12 and folic acid in high homocysteine. g) Keep doses < 200 mg/d to avoid neuritis.

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WATER SOLUBLE VITAMINS – PART 2 E. Folic Acid 1. Chemistry

coenzyme

DHFA

DHFA reductaseNADPHPABApteridine ring

Other "conjugates" existthat have more than oneglutamic acid; these havereduced vitamin activity.

N

N

N

NNH2

OHCH2

NH C NH CHO CO2H

CH2

CH2

COOH

N

N

HN

NH

OH

NH2

DHFA reductaseNADPH

= HH

CH2

H

NH R

THFA 2. Nomenclature Folic acid is pteroyl monoglutamic acid. This fully oxidized form is not found

naturally but is what is used in supplements. Reduced polyglutamates are found in animal and plant foods. Folic acid as a supplement or DHFA polyglutamates in foods are readily converted to the active fully reduced polyglutamates unless specific inhibitors are present (e.g. methotrexate).

Common use -- folates means all pteroglutamates having vitamin activity. Absorption, transport, circulation, storage -- dietary folates are enzymatically

cleaved by pancreatic and intestinal mucosal peptidases to the monoglutamate which is absorbed. Synthetic folic acid is well absorbed with a bioavailability of about 85%. Most of circulating folate is 5-methyl tetrahydrofolic acid (5-meTHFA). A considerable amount is excreted in the bile but most is reabsorbed. This continued enterohepatic circulation of folate is important for maintaining adequate levels and is interrupted by alcohol.

Other "conjugates" of DHFA and THFA that have more than one glutamic acid exist in foods; these are not as well absorbed as the oxidized monoglutamate

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3. Function The folate cycle and methylation

(see belowfor detail)

ATP

C22 of purines

NH

NCHO

CH2

HN R

N5 formyl THFA"folinic acid""Leucovoran"

N

N

N

N

O

HCOOH

homocysteine

B12

THFAmethionine

CH2

NH R

H

N

N

HN

HH

NH

OH

C8 of purines

NH

NCH3

CH2

NH R

NH

N CH2

CH2 N R

NADH

N methyl THFA

N5N10methylene THFA

N10 formyl THFA

NH

NCH N R

CH2

NH

HN CH2

N RCHO

-H2O

NADPH2 N5N10 methenyl THFA

pyrimidinesN

NO

OCH3

thymine

822

876

9

105

43

21

(see B6lecture for details on thisimportant reaction)

MTHFR=methylene THFA reductase

OO

O

HN

NH2PO3O

OH OH

OO

O

HN

NH2PO3O

OH OH

CH3

deoxyuridinemonophosphate

deoxythymidinemonophosphate

thymidylate synthesis

N3-N10 methylene-THFA

DHFA

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4. Folate deficiency

• Deficiency results in megabloblastic anemia. Symptoms include headache, fatigue, weight loss, anemia, nausea, anorexia, diarrhea, insomnia, irritability, forgetfulness. Signs are macrocytic red cells and megaloblasts in the bone marrow.

• Deficiency may result in teratogenesis with neural tube defects and possibly orofacial clefts. The importance of adequate folate intake at conception and for the first 3 weeks when the neural tube closes is obvious to few mothers. In 1992 the CDC, FDA, and NIH jointly recommended that "all women of childbearing age who are capable of becoming pregnant should consume 0.4 mg of folic acid per day as folic acid for the purpose of preventing neural tube defects." "Care should be taken to keep folate below 1 mg." “Enriched” grains now fortified with 140mg/100g to help decrease risk for birth defects.

• Defieciency may result in elevated homocysteine (HCS) which is associated with increased risk for coronary disease (and maybe the birth defects). Recent evidence indicates that a genetic polymorphism in the N5N10 methylene THFA reductase (MTHFR) enzyme is involved. Those with a C to T substitution in the gene coding for this enzyme (13%) are at high risk due to an enzyme of higher Km with resulting elevated HCS. This is the so called TT genotype.

• Oral contraceptives and anticonvulsant drugs use may increase folate catabolism.. This possibility seems to be behind the development of Beyaz, a combined oral contraceptive that includes progesterones AND 5-MTHF.

• Folate deficiencies are seen under conditions of poor nutrition, heavy alcohol ingestion and pregnancy and lactation.

• Alcohol decreases enterohepatic circulation of N5 methylTHFA. 5. B12 deficiency in relationship to folate B12 deficiency results in a folate deficiency because folates are not recycled

(see above folate cycle scheme). This is because 5-MeTHFA is not converted back to THFA in the absence of B12. Megabloblastic anemia therefore results from a deficiency of B12. Also, in a B12 deficiency, neurological damage is observed due to lack of B12 (see B12 discussion) which is hard to detect. Therefore high dose folate supplements are risky in cases where there is a possibility of pernicious anemia (B12 deficiency) because folate will mask hematological symptoms while neurological damage goes on unchecked. This is why preparations containing > 0.8 mg of folic acid are on Rx.

6. Folate antagonists a. Methotrexate -- ↓ DHFA reductase ∴ stops all in "S" phase cell cycle. Note: "Leucovoran rescue" technique allows ordinarily lethal dose to be

administered with consequent increased tumor kill. b. Trimethoprim --inhibits bacterial DHFA reductase and, combined with

sulfamethoxazole (Bactrim® Roche, Septra® BW), which is a PABA

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antagonist, a "double hit" against bacterial folate metabolism is affected. There is usually very little effect on mammalian DHFA reductase.

c. Alcohol-affects enterohepatic circulation of folates

d. Nitrous oxide -- continued frequent inhalation has produced fatal megaloblastic hematopoisis and a neuropathy similar to pernicious anemia. The N2O oxidizes the cobalamin to create a B12 deficiency with resulting folate deficiency.

e. Phenytoin – suboptimal folate levels observed with long term therapy and rare megaloblastic anemia. BUT, folic acid (in high does) decreases phenytoin levels with cases of seizures reported.

f. Pyrimethamine- used for parasite infections (e.g. toxoplasma,malaria) as a parasite DHFA reductase target. Folic acid supplements may reduce effect of drug. Leucovorin is OK.

7. Use

a) Deficiency-use with oral contraceptives and during pregnancy and lactation.

b) To prevent neural tube defects in the unborn, women contemplating

pregnancy should take a supplement containing 0.4 folic acid, otherwise women of childbearing potential should assure that they are consuming at least 0.4 mg/day of synthetic folic acid (I suggest a multivitamin supplement).

c) Cervical dysplasia, brochial squamous dysplasia, and dysplasia of

colonic tissue in ulcerative colitis patients -- studies show elevated risk is associated with low folate.

d) Colon cancer - low folate , high alcohol, and low methionine

intakes as well as family history give an elevated risk. Long term use of multivitamins associated with lower risk (folate?).

e) breast cancer – high dietary intake decreases risk

f) Alzheimer’s disease. Preliminary evidence shows low folate levels

associated with increased risk.

g) coronary heart disease and stroke – elevated homocysteine associated with an increased risk but efficacy of folic acid, B12 and B6 to lower adverse outcomes are not well established. A modest effect may be evident (see B6 notes and discussion) especially for stroke. Those with preexisting disease show no benefit on outcomes for supplementation. Ongoing trials may clarify the situation.

8. Source

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("Folic") leafy vegetables, fruit juices, beans; meats and most fruits are low.. "Enriched" flour now contains 140 µg/100 g of folic acid.

9. Stability Labile to light, heat, storage. Should try to eat some "fresh" vegetables.

Cooking losses are high (80-90%). 10. Dose

• Preparations containing greater than 0.8 mg are on Rx only. • DV = 0.4 mg of folate • The Food and Nutrition Board has set the requirement in DFE (dietary

folate equivalents) (see supplemental table). 1 DFE=1ug of food folate; 1ug of supplement folic acid =1.7DFE. On an empty stomach it is 2DFE.

11. Toxicity: essentially non-toxic. UL=1000ug folic acid

12. Patient Counseling/ patient use issues

a. A very important vitamin because inadequate intakes can increase risks for cancers, heart disease and stroke.

b. Rarely needed as a single supplement. Use a multivitamin to get needed folic acid. Benefit seen at levels in most multivitamins( 400ug/d) and the folic acid is very well absorbed. Folate rich foods (from which folate is less well absorbed) can supply the rest of intake.

c. Special benefit in alcoholics d. Use for those with elevated HCS in doses 0.8mg and higher. Use

together with B12 and B6. e. Intake of 400ug/d of supplement (multivitamin) is very important for

women of child bearing potential in order to help prevent birth defects.

f. Folic acid intake of >800ug/d is of concern in older populations due to the risk of masking pernicious anemia

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F. Vitamin B12 1. Structure -- synthetic material is cyanocobalamin Coenzymes = 5 deoxyadenosine or methyl group replacing the CN; Hydroxy

cobalamin is also active.

Co

CN

base (5,6-dimethyl -benzimidazole)

1 amino-2-propanol

ribose

N CNN

N NCo

H3CH2NCOCH2

CH2CONH2CH3

CH2

H2CCH3

H2NCOCH2

HH3C

H2NCOCH2CH2CH3

CH3

CH3

CH3

HN

O PO

OO HO

O

N

N

CH3

CH3

HO

CH3

O

CH2CONH2

CH2CH2CONH2

Vitamin B12

29

2. Function a. Methyl transfer reactions

MethionineHomocysteine

N-5 methyl THFATHFA

methylcobalamin cobalamin

i.e. needed for recycling of THFA b. Metabolism of odd chain fatty acids

CH3 CH2 CSCoAO

CH3 CH C SCoA

O

O C O

H+

CO2

propionylCoA carboxylasebiotin

CH3 CH CSCoAO

COOHmethyl malonyl CoA

HOOC CH2 CH2 CSCoAO

succinyl CoA

B12mutase

excreted in urine asmethyl malonic acid in B12 deficiency

TCA cycle Note: 5-deoxyadenosyl cobalamin is the coenzyme form of B12 for this

reaction c. Myelin synthesis Mechanism of B12 involvement may relate to the buildup of methyl

malonic acid with a resulting decrease in myelin synthesis. B12 deficiency results in demyelination and neurological damage.

3. Deficiency -- pernicious anemia Symptoms related to inadequate myelin synthesis and megabloblastic anemia

due to failure to recycle folates; i.e., numbness, poor coordination, poor

30

memory, confusion, depression; at least 2-5 mg stored in liver and turnover is only 0.1% per day, therefore deficiency takes years to develop. Deficiency is rarely diet based, although vegans are at risk.

4. Absorption -- B12 absorption HCL in stomach splits B12 from peptide links in food; intrinsic factor (a

glycoprotein) secreted by stomach mucosa; required for transport of B12 across ileum wall (also requires Ca++ and a pH > 6 and releasing enzyme). Most of pernicious anemia is due to lack of synthesis of intrinsic factor and not due to dietary deficiency of B12. A simple B12 deficiency may be seen in older populations due to decrease in gastric HCl.

5. Source -- meats, especially liver and yeast; microorganisms are ultimate

sources of B12. 6. Production -- by fermentation. 7. Stability -- Stable at pH 4-7; labile to light. 8. DV = 6 µg. 9. Use In pernicious anemia, give 100 µg IM q 4 weeks; Studies indicate that 1

mg/day P.O. will work also. Sublingual 1mg “dots”may be better. A nasal solution 0.5mg/0.1ml) is marketed and is convenient. High IM doses are used for methylmalonic acidurea, an inborn error of metabolism.

Increasing interest in the role of B12 in keeping folic acid levels up and

homocysteine levels low. See folic acid lecture for the implications of high homocysteine and low folic acid.

Vegans should take supplements Those over 65 should take a supplement (multivit) due to decreasing HCl

needed for absorption of B12 from foods 10. Diagnosis: Schillings test (labeled B12) or better, measure methyl malonic

acid in plasma. Accurate diagnosis is important for rational therapy of anemias. 11. Preparations available In case of pernicious anemia usually given IM because patients often become

refractory to the intrinsic factor in oral preparations. High oral doses (>500ug/d), sublingual or nasal spray may obviate the need for IM injections. If problem is lack of HCl, po doses are OK. Long-acting form is hydroxycobalamin (Alpha redisol® MSD) -- dose is 100 µg/mo.

11. Toxicity -- essentially nontoxic

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12. Patient Counseling/ patient use issues a. An important vitamin especially for older adults who may not absorb

B12 from foods efficiently due to low stomach acid or have pernicious anemia

b. B12 is important for maintaining adequate levels of folic acid in the body

c. Needed, together with B6 and folic acid, to keep HCS low d. An injection or special oral dosage forms needed to treat pernicious

anemia e. For most, the amounts contained in a good multivitamin will be

sufficient

G. Pantothenic Acid -- (Vitamin B5) 1. Chemistry

CH C NH CH2 CH2 CO2HOH

CHOH2CH3C O

H3C

d−α−hydroxy−β−β−dimethyl butyric acid

β−alanine

"CoASH"

β−mercaptoethylamine

O CH2 C C C NH CH2

H3C OH O

H3C

CH2ADP

Coenzyme = CoA

C NH CH2 CH2 SHO

Only the d-enantiomer has biological activity 2. Function -- as a thioester bond –A component of coenzyme A.- high energy

bond; 8,800 ca/mole; more than ATP -- used in transfer of acyl groups.

pyruvate acetyl CoA

NADTPPCoASH

3. Deficiency -- rare -- intestinal synthesis probably important, as well as,

widespread occurrence in foods. Symptoms are fatigue, numbness in extremities, cramps.

4. Use -- deficiency states -- topically for ulcers and sores, e.g. Panthoderm. 5. Requirements -- 10 mg = DV.

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6. Source -- widespread in foods; liver, meat, eggs, potatoes are rich sources. 7. Properties -- alcohol or calcium salt somewhat more stable, therefore used in

vitamin preparations, stable at neutral pH, but not in acid or alkali.

8. Toxicity -- essentially nontoxic

9. Other—a dimmer (P-S-S-P) called pantethine is used in Europe as a drug to lower cholesterol. It is available in the USA as a dietary supplement. Seems safe and has mild effects in decreasing total cholesterol. Controversial use in treating acne.

10. Patient Counseling/ patient use issues Nothing special; deficiencies are very rare; is a component of most multivitamins

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H. Biotin 1.

CHCH

CHN

O

S

NH

CHCH (CH2)4 CO2H

bound to enzymes through ε amino of lysine; dietary proteins are digested to lysine-biotin (biocytin) which is hydrolyzed by biotinase to release biotin. An inborn error with a defect in biotinase is known.

2. Function -- carboxylation reactions

e.g., acetyl CoAacetyl CoA carboxylase, biotin

malonyl CoA + Pi

lipids

CO2, ATP

e.g. pyruvate carboxylase- gluconeogenesis e.g. propionyl-CoA carboxylase – see B12 lecture carboxybiotin = active species

O C NC

NHO

O

3. Deficiency -- rare; rash, hair loss, fatty deposits on face, depression. 4. Requirement -- avidin (protein from egg whites) can precipitate deficiency

state → dermatitis, muscle pain, etc.; synthesized by intestinal flora. 5. Stability -- very stable. 6. Source -- eggs, meat, nuts are rich sources. 7. DV = 0.3 mg. 8. Use -- rarely used alone; several biotin responsive inborn errors of metabolism

are known, the most common being a defective biotinidase. Some use for brittle nails (mg doses and together with selenium (Diachrome) for improved glucose tolerance in diabetics

9. Patient Counseling/ patient use issues - nothing special; deficiencies are very rare; is a component of most multivitamins;

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I. Niacin (Vitamin B3) 1. Chemistry

N

CO2H

niacinN

CNH2

O

niacinamide

O

HO

OH2C

OH

N

CNH2

O

coenzyme form is NAD or NADPADPorATP

(nicotinic acid)

2. Function -- >150 enzymes require NAD or NADP. a. redox and electron transport

H HCNH2

NR

O

R C C R´

H H

H H N

HCNH2

O

R

H++R C C R´H

H

+

b. ribosylation of proteins in cell signaling and DNA replication and repair

NAD + protein niacin + ADP-protein 3. Deficiency state - Pellagra - "4D's" dermatitis, diarrhea, dementia, death; red

tongue and pigmentation = common signs; seen in "corn belt" in U.S. during early 1900's, reason -- lack of available nicotinic acid and tryptophan in corn.

4. Biosynthesis -- See B6 section for biosynthesis of niacin from tryptophan. It is

estimated that 60 mg of tryptophan gives 1 mg of niacin. Isoniazid therapy can precipitate some symptoms of pellagra by binding up PLP and stopping the conversion of tryp to niacin.

5. Source -- meat, fish, whole grain cereals, peanuts. Ingested in foods as NAD

or NADP then hydrolyzed in the intestinal mucosa. 6. Stability -- very stable, but much is lost if cooking water is discarded. 7. Requirements -- DV = 20 mg, but requirement will depend on tryptophan

intake. UL=35mg

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8. Use -- Component of multivitamin preps also used in high doses for its

pharmacological effects as described below. a. For improving serum lipids -- use Niacin (NA); Niacinamide is not

effective; NA used in doses up to 10 q/d (usually 3-5g), will lower LDL 5-25%, triglycerides 20-50% and raise HDL 15-35%. Combines well with statin drugs. Side effects are significant, but decrease with time. Mechanism: exact mechanism is unknown but does lower production of VLDLP and activates lipoprotein lipase. Niacin and some sustained release products are OTC. The extended release product (Niaspan) is Rx.

b. Schizophrenia – use of high dose niacin has been popular but is of

unproven efficacy.

c. Peripheral Vasodilator -- usefulness is questionable. d. Diabetes—there is interest in niacinamide in high doses to prevent type 1

diabetes in high risk kids and for type 2 adults. Niacinamide may help protect pancreatic beta cells but results showing benefit are preliminary.

9. Toxicity ( in doses over UL) -- peripheral vasodilation, flushing, GI upset,

ulcers, diarrhea, impaired glucose tolerance, liver damage (reversible?) and increased gout. These effects are seen with high doses (gram quantities) and decrease the usefulness of this vitamin for treating hyperlipidemias. Hepatitis has been associated more with the sustained release preparations of NA but not as much with the “extended release” product (Niaspan). The vasodilation and GI upset decreases after a few weeks on the drug. Aspirin and NSAIDS help. Niacinamide in high doses has significant associated adverse effects also but not the flushing reaction.

10. Patient counseling Issues

a) Single use is not necessary for nutritional purposes. Take a multivitamin.

b) High dose (gram quantities or Niaspan) are not for self medication because niacin can cause toxicity at doses needed for favorably affecting blood lipids.

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J. Vitamin C (ascorbic acid) 1. Structure

(kidney stone risk?)

OO

OO

OHOH

dehydroascorbic acid

COHC

OHO

O

+ other metabolites

oxalic acid

OO

OO

OHOH

vitamin C oxidizedOO

OHOH

OHOH

vitamin C

–1e –1e

metabolism

2. Function -- true coenzyme function is not well understood; ascorbic acid is an

electron donor and facilitates the following reactions: a. Dopamine → norepinephrine b. Proline → hydroxyproline (this is a major component of

collagen and many of the signs of scurvy are due to impaired collagen synthesis).

c. Lysine hydroxylase → collagen. d. Folic acid → THFA (this explains the macrocytic anemia seen in

scurvy). e. Involved in absorption of iron (keeps in ferrous form for better

absorption). e. General water soluble antioxidant/free radical scavenger. We will

discuss this aspect in more detail later. a. Keeps LDL from being oxidized b. Possible regeneration of reduced vitamin E c. Prevent generation of mutagenic compounds in gastric juices and

elsewhere

Vitamin C as a free radical scavenger

37

3. Deficiency state → Scurvy Symptoms – hemorrhages, lassitude, weight loss, bone weakening, anemia,

edema, tooth loss. Biological lesion -- impaired collagen and connective tissue synthesis due to

lack of hydroxyproline, hydroxylysine. Also low THFA. 4. Utility of C a. In surgery and fractures -- to increase collagen synthesis. Probably helps. b. Common cold prophylaxis -- The books by Linus Pauling ("Vitamin C

and the Common Cold") and others have advocated that C has profound beneficial effects in preventing the common cold. Numerous clinical trials since 1970 show, at most, a slight beneficial effect. Ascorbic acid seems to cause a slight reduction in severity of colds, but the results are inconsistent from investigator-to-investigator. Gram quantities are not necessary, 100-500 mg/day will saturate tissues.

c. Cancer –diets with >200mg/d vitamin C are associated with lower

cancer risks, especially esophagus, stomach, colon and lung but studies using Vit C supplements have led to conflicting results.

38

d. heart disease—low dietary levels and low blood levels are associated

with an increased risk. Vitamin C supplements can modestly lower BP but no clear effect on outcomes in intervention studies. The benefit of supplements is unclear unless one is deficient in Vitamin C

e. ataracts-higher intakes show lower risks but the benefit of intervention with supplements is not proven.

5. Storage -- is present in most tissues at low levels; there is a threshold level

above which excess is excreted. This is about 200mg/d. 6. Source -- richest sources are not citrus juices, but broccoli, brussel sprouts,

peppers; other items high in ascorbate are citrus products, potatoes, and tomatoes. Cereal products, grains, and meats contain very little; if 5 fruits or vegetables are eaten daily, the intake would be 250 mg., average intake is about 75 mg./day.

7. Stability -- in solution is relatively unstable being oxidized by air and being

photolabile, therefore, much C can be destroyed in cooking foods. 8. Toxicity -- Essentially nontoxic; in gram doses may increase oxalate urine

concentrations and subsequent increased risk for urinary stones. Will make urine tests for sugar unreliable (false positive) because C will reduce the copper in Clinitest and Benedicts solution; In doses over 250mg/d can make false negative haemoccult test for blood in stool; contraindicated when have iron overload.

9. Requirements -- DV = 60 mg. but is this intake optimum for best health??

New RDA is 75mg for females and 90mg for males and 125mg for smokers. UL=2g/d

10. Bioavailability of various products. AUC-PO/AUC-IV is true bioavailability studies should be done at steady state 200 mg. = 80% bioavailability 500 mg. = 63% bioavailability 1250 mg. = 46% bioavailabililty at > 500 mg/d all of the absorbed ascorbate was excreted in the urine conclusions: best dose is 200-500 mg/d timed release products, Ester-C (calcium ascorbate) or esters of vitamin C are

not worthwhile 11. Patient Counseling Issues

a) The benefits of taking high doses (>200mg) of vitamin C are not established

b) Most multivitamins provide 60mg/dose which may be too low for optimum benefit

c) Daily consumption of a glass of rehydrated frozen OJ will, when taken with a

multivitamin, will give saturation levels (~200mg) of C d) Studies to date indicate the value of eating ample fruits and vegetables

to

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help prevent cancers and heart disease. The use of a multivitamin may also

help. e) synthetic vitamin C is the same as natural vitamin C f) Use of high doses to prevent or treat the common cold is unwarranted.

Doses of ~200mg may slightly reduce severity of the cold.