Vitamins, coenzymes and cofactors

1. Vitamins, coenzymes and cofactors • Primarily organic molecules • May have inorganic component or cofactor • Some work with/are part of an enzyme • Promote or catalyze biochemical reactions • Most cannot be made by human body • Water-soluble = all B vitamins, vitamin C • Vitamins A, D, E, K are lipid-soluble

2. Analyses for Vitamin Content • Vitamins are structurally diverse, so methods are tailored for particular vitamin • Assay methods include physicochemical methods and microbiological assays • Most approved by AOAC (Association of Official Analytical Chemists) • In most cases, vitamins extracted first • Water-soluble – may be treated with acid/base, heat to hydrolyze linkages • Fat-soluble – often in ester form, extraction w/nonpolar solvent followed by saponification • Antioxidants may be added to prevent oxidation

3. Thiamine (Vitamin B1) • Catalyzes decarboxylation of a-keto acids, a feature of primary metabolism • e.g. pyruvic acid → acetaldehyde in glycolysis • pyruvic acid → acetyl-CoA (Fig.2.15 p23 Dewick) • Helps metabolize carbohydrates • Supports nervous system • Deficiency causes beriberi (neurological) • Found in cereals, wheat germ, beans, nuts, eggs, yeast and vegetables usually found in pyrophosphate form

4. Riboflavin (Vitamin B2) catalyzes dehydrogenation rxns • Involved in redox rxns of C-C bonds • Metabolism of carbs, fat, protein • B2 is widely available in foods, including liver, kidney, dairy products, eggs, yeast,meat, and fresh vegetables • Promotes healthy skin & vision • Deficiency causes eye problems and skin disorders

5. B vitamin determination • Vitamin B1 (thiamin) – thiochrome fluorometric procedure • Sample is treated with HCl, enzymes to hydrolyze phosphate ester • cleanup on ion-exchange column • converted to thiochrome with potassium ferricyanide: • thiochrome measured by fluorescence at 365 nm excitation and 435 nm emission l

6. Chemical tests for B vitamins (cont’d) • Riboflavin (Vitamin B2) can also be determined by fluorescence • sample treated with acid, then oxidized with KMnO4 and peroxide to remove interfering material • Excitation at 440 nm cleaves ribitol, converts B2 to lumiflavin, fluorescence measured at 565 nm

7. Niacin/nicotinic acid (Vitamin B3) • As part of NAD+ system, catalyzes redox rxns of alcohols/carbonyl compounds • Degradation of L-Trp  niacin • (Fig 6.29, p 312 Dewick) • Needed for general metabolism, health of skin, nervous & digestive system • Found in meats, fish, yeast, eggs, dairy, grains, nuts, legumes, roasted coffee • Deficiency causes pellagra • oral lesions, diarrhea, dermatitis, and dementia

8. Microbiological assays for niacin (B3) and folate (B9) • Principle: growth of microorganisms is proportional to requirement for specific vitamins • Bacteria & yeast populations are treated with test sample and quantified by turbidimetry • Results compared to those treated with vitamin standard • Niacin method uses Lactobacillus plantarum • Samples prepared at various concentrations in broth, inoculated with bacteria, incubated for 18 h at 37oC. • Turbidity measured as absorbance @ 540-660 nm, compared to standard curve • Folate assay uses digestion with 3 enzymes (amylase, protease and conjugase) to liberate bound folate, then incubation with Lactobacillus rhamnosus

9. Panthothenic acid (Vitamin B5) is part of Coenzyme A • Synthesis of fatty acids (acetate pathway), some peptides, phenylpropanoids, isoprenoids • Fat, carbs and protein metabolism • Very widely distributed throughout foods,yeast, liver, and cereals provide rich sources. • Deficiency is rare

10. Vitamin B6 (pyridoxamine, pyridoxal phosphate & pyridoxine) • Catalyzes transaminations & decarboxylations of amino acids • Metabolism  energy • In plants, used in biosynthesis of phenylpropanoids from amino acids • Meat, salmon, nuts, potatoes, bananas, and cereals • Can be lost through cooking, though deficiency usually caused by poor absorption • Deficiency causes nervous disorders, skin rash, muscle weakness, anemia pyridoxine

11. Occurs in Krebs cycle

12. Vitamin B12 (cobalamins) • Involved in synthesis,of DNA, amino acids, fatty acids, one-C metabolism (methylations) • Needed to maintain nerve cells, RBC, genes • Microbial in origin; intestinal flora contribute towards human dietary needs. • Stored in the liver • Found in meat, shellfish, liver, dairy products and eggs • Deficiency causes pernicious anemia • Poor absorption of B12 is thought to be a complication of aging • Methylations such as the conversion of homocysteine to methionine require B12 • Contains Co(III) coordinated to a corrin ring (R = CN is cyanocobalamin, most common form) R groups vary: CN, OH, H2O, NO2, Me Fig 2.29 p 32 Dewick

13. HPLC Analysis • HPLC analysis methods exist for most vitamins • Details in Methods of Analysis for Functional Foods and Nutraceuticals, W. J. Hurst, 2nd ed, CRC Press, 2008 (Table 8.1) • B vitamin analyses generally require acid hydrolysis of sample to free bound vitamin • HPLC on C18 RP columns with variety of solvent systems, UV or fluorescence detection • Vitamin B12 analysis method uses acetonitrile/ aq. KH2PO4 elution at low pH, with detection possible at 550 nm.

14. Vitamin B9 (folate) • Role: Tetrahydrofolate functions in one-C metabolism as a carrier of methyl, methylene or formyl groups • Involved in amino acid and nucleotide metabolism, red blood cell formation • Found in green veggies, yeast, liver, legumes, whole grains, some fruits • Deficiency can cause anemia, neural tube defects in a fetus, cardiovascular problems in adults • Folate intake linked to reduced CVD, colon cancer in women and depression in men • Folate analyses: • Microbiological assay • HPLC on C18 column with • fluorescence detection at • 350 nm.

15. Relationship between one-C metabolism, B-vitamins and health • One-C metabolism (methylation) is a key reaction in formation of both primary and secondary metabolites in plants & animals, also involved in DNA repair and detoxification pathways • Elevated homocysteine levels in humans are an indicator (marker) of faulty one-C metabolism • Homocysteine (HCys) is made from methionine (L-Met), but much of the HCys must be cycled back to L-Met • Decreased formation of L-Met and SAM result in decreased methylation • Decrease in the methylation pathway is associated with increased risk of cardiovascular disease, Alzheimer’s disease and some cancers. • High HCys levels are also thought to be linked to increased production of reactive oxygen species that damage tissues • Adequate supply of folate (B9), B12 and B6 in diet is necessary to maintain proper HCys metabolism.

16. Selhub, J. 2002. The Journal of Nutrition, Health and Aging, 6: 39-41

17. Vitamin C (ascorbic acid) • Antioxidant, strong reducing agent • Collagen synthesis, tissue repair, bones & teeth, immune system, iron absorption • Cannot be made by human body though animals can biosynthesize from glucose • Found in citrus fruits, cruciferous veggies, tomatoes, dark green leafy, berries, mangos, melons • Degraded by cooking • Deficiency causes scurvy, anemia, depression, infection, tooth/gum problems, muscle deterioration, fragile bones, poor wound healing L-dehydroascorbic acid

18. Vitamin C • Highly susceptible to oxidation, so analysis performed in acidic solution, often with EDTA • Indicator 2,6-dichloroindophenol is added to oxidize ascorbate to dehydroascorbate • titration to red endpoint (colored samples tricky, measure at 545 nm) • iodine can also be used to titrate vit.C to oxidation endpt. • HPLC analyses can also be carried out (C18 column with aq. acid elution, monitored at 244 nm; silica column with derivatization by 2,4-DNPH)

19. Lipid-soluble vitamins • Vitamins A, D, E & K are all derived in part through isoprenoid pathway • Vitamins E & K have phenolic moieties derived through shikimate pathway • Toxicity can occur if fat-soluble vitamins are consumed in too high a quantity • Primary role of Vitamin K is blood clotting K supplied by green veggies

20. Vitamin A • Retinoids are isoprenoids(C20 = diterpene) • Retinol (A1) & dehydroretinol (A2) may exist as free alcohols or acetic or palmitic esters • Found only in animal/fish products: eggs, dairy, animal livers, fish liver oils; however plant carotenoids can be precursors • Plays key role in vision, eye health, growth and repair of tissues, bones & teeth, immunity • deficiency causes night blindness, skin thickening • Retinoids are known to act as signalling molecules regulating diverse aspects of cell differentiation, embryonic development, growth, and vision.

21. Retinol and vision • For the processes of vision, retinol is first oxidized into the aldehyde all-trans-(11E) retinal and then enzymatically isomerized to cis-(11Z) retinal • cis-Retinal is bound to the protein opsin in the retina via a Schiff base linkage to give the red visual pigment rhodopsin • When light hits the retina, it causes isomerization of the cis-retinal to the all-trans form, thus translating the light energy into molecular change, triggering a nerve impulse to the brain. • The absorption of light energy promotes an electron from π to π∗ MO temporarily destroying double bond character and allowing rotation back to trans • All-trans-retinal is then released and the process can repeat itself

22. Determination of Vitamin A by HPLC • Sensitivity to UV light, oxidation, heat limit chemical analysis methods • Vitamin A (retinol) may be esterified with acetic or fatty acids • Samples must be saponified w/ ethanolic KOH, then extracted with hexane & dried • HPLC analysis on silica column (normal-phase) with heptane/isopropanol elution, detection at 340 nm separates cis and trans isomers

23. Vitamin E • Sources: cereals, seed oils, eggs, soybean, corn oil, barley • Chemistry: Free radical scavenger with phenolic ring derived through shikimate pathway and isoprenoid tail • Role: protects lipids in LDL and cell membranes, retinoids, hormones, from oxidation • Health: decreases coronary artery lesions • Effect on CVD??? Observational studies suggest reduced CVD but many clinical trials do not show evidence for reduced mortality (men)

24. Heart benefits for women? • A published clinical trial of vitamin E’s effects on the heart and blood vessels of women included almost 40,000 healthy women ≥45 years of age who were randomly assigned to receive either 600 IU (400 mg) of natural vitamin E on alternate days or placebo and who were followed for an average of 10 years [23]. • The investigators found no significant differences in rates of overall cardiovascular events (combined nonfatal heart attacks, strokes, and cardiovascular deaths) or all-cause mortality between the groups. • However, the study did find two positive and significant results for women taking vitamin E: they had a 24% reduction in cardiovascular death rates, and those ≥65 years of age had a 26% decrease in nonfatal heart attack and a 49% decrease in cardiovascular death rates. • No effect on cancer incidence reported • Source: NIH Office of Dietary Supplements http://ods.od.nih.gov/factsheets/VitaminE.asp • Lee, et al, The Women's Health Study: a randomized controlled trial. JAMA 2005;294:56-65.

25. Vitamin E analysis • Tocopherol present in foods = 8 stereoisomers • (R,R,R)-a-tocopherol is the most bioactive • Saponification (if needed), extraction, drying • HPLC analysis on silica column, elution with 1% isopropanol/hexane, detection at 295 nm • Fluorescence detection is often used, with excitation at 290 nm, emission at 330 nm (see rice bran oil next page) • Cerretani, et al reported new method for analysis of tocopherols on silica “nano-LC” column of 150 x 0.1 mm with uv detection – better sensitivity & “greener” (J.Agric.FoodChem.,58:757-761,2010)

26. Samples dissolved in EtOH with pyrogallol added to prevent oxidation, flush w N2 Sample is heated/sonicated 10 min @ 70 C then 60% KOH added to hydrolyze Hexane extraction with 0.1% BHT added to prevent oxidation Dried, filtered and analyzed with HPLC program described below

27. Vitamin D • Cholecalciferol is a sterol (isoprenoid) formed photochemically in animal skin from 7-dehydrocholesterol by sun’s irradiation • Also found in yeasts and some plants as D2 (ergocalciferol) which only differs in side chain structure • Dietary sources include liver and dairy products, fish liver oils • Vitamin D3 is hydroxylated in the body to its active forms, first calcidiol and then to calcitriol (Figure 5.106, p58 Dewick). • Key role: Ca metabolism • Calcitriol stimulates the absorption of calcium and phosphate in the intestine and the mobilization of calcium from bone • Other physiological functions include a role in immune modulation, hormone secretion, and cell differentiation • Vitamin D deficiency is common in Northern latitudes due to reduced exposure to sunlight • Rickets = inability to calcify the collagen matrix of growing bone, causing lack of rigidity in the bones (children) or osteoporosis • In most countries, milk and cereals are fortified with vitamin D3

28. Vitamin D is a product of the mevalonate pathway, made from a cholesterol derivative. B ring opens up allowing for rotation and isomerization

29. Vitamin D analysis • Difficult to analyze colorimetrically (no strong chromophore) • AOAC reference std. method of analysis for many years was a bioassay called the “line test” • Young rats deprived of Vit. D to simulate rickets for 3 weeks, then fed sample or stds containing Vit. D for 8-10 days • Bone calcification is measured by removing and staining bone with AgNO3

30. Modern analyses for Vitamin D • Current methods are summarized briefly in Lensmeyer, et al, (2006), Clinical Chemistry 52: 1120-26. • Competitive protein-binding assays (CPBAs) for 25(OH)D are commercially available • Enzyme immunoassays (ELISA) and radioimmunoassay (RIA) kits available but require the instrumentation • LC-MS/MS methods expensive; potential coelution of inactive 25(OH)-epi-D3 • HPLC analysis on silica or C18 columns with UV detection (275 nm) may be most economical route • HPLC method reported here uses a cyanopropyl-bonded silica column and is suitable for analysis of serum Vit. D levels in patients

31. Studies in human populations • Epidemiological studies follow a selected population over a defined period of time, monitor intake of the test substance as well as other health factors, habits, lifestyle and assess the effect of these parameters on the number and type of disease-related incidents. • Clinical trials usually use a randomized, placebo-controlled, double-blind design to administer treatment over a defined period, then assess the difference in specific measurable endpoints between the populations

32. Recent findings on Vitamin D and health • Cancer: Vitamin D thought to improve cancer survival – high levels associated with 40% less colon cancer mortality (Dana Farber study) • Incidence of certain cancers higher in Northern latitudes • CVD: patients age 50+ with Vit. D deficiency were 45-78% more likely to develop cardiovascular diseases & mortality (Utah study) • Diabetes: Vitamin D supplementation reduced insulin resistance in a New Zealand study • Crohn’s disease: Vit.D induced expression of NOD2 gene for defense against inflammatory bacteria • Bone health: a study of n = 70,000 showed Vit D + Ca supplement reduced fractures by < 16% (BMJ study); Vit.D (600 IU) + Ca (1200 mg) with weight-bearing exercise recommended to prevent osteoporosis.

33. Vitamin D and immunity • An 2009 study published by researchers at Oregon State University reveals that Vitamin D is so crucial to the functioning of your immune system that the ability of vitamin D to boost immune function and destroy invading microorganisms has been conserved in the genome for over 60 million years. • In primates, this action of "turning on" an optimal response to microbial attack only works properly in the presence of adequate vitamin D, which is produced as a result of sun exposure, and is available in smaller amounts from dietary sources. • Vitamin D prevents the "adaptive" immune response from over-reacting, thereby reducing inflammation, and appears to suppress that immune response. At the same time vitamin D boosts the innate immune response by turning on production of antimicrobial peptides. The overall effect may help to prevent the immune system from overreacting. • http://www.naturalnews.com/027231_Vitamin_D_immune_system_vaccines.html • Also check out “The miracle of Vitamin D” at http://www.westonaprice.org/The-Miracle-of-Vitamin-D.html

34. in vitro study: Vitamin D2 induces apoptosis in human leukemia cells • Chen, W-J et al, Induction of Apoptosis by Vitamin D2, Ergocalciferol, via Reactive Oxygen Species Generation, Glutathione Depletion, and Caspase Activation in Human Leukemia Cells. J. Agric. Food Chem. 2008, 56, 2996–3005 • Investigated the ability of D2 to induce apoptosis (programmed cell death) in HL-60 leukemia cells and the mechanistic pathways involved • Found that D2 induced apoptosis primarily through an oxidative pathway involving reactions in the mitochondria (caspase activation and modulation of regulatory proteins)