維生素的缺乏與檢測英文

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-solubleAnaly

2、ses 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

3、 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 Thiamine (Vitamin B1) Catalyzes decarboxylation of a-keto acids, a feature of primary metabolism e.g. py

4、ruvic 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 vegetablesusually found in pyrophosphate formRiboflavin (Vita

5、min 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 disorde

6、rsB 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 em

7、ission l 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 nmChemical tests for B vitamins (contd)

8、Niacin/nicotinic acid (Vitamin B3)As part of NAD+ system, catalyzes redox rxns of alcohols/carbonyl compoundsDegradation of L-Trp niacin(Fig 6.29, p 312 Dewick)Needed for general metabolism, health of skin, nervous & digestive systemFound in meats, fish, yeast, eggs, dairy, grains, nuts, legumes, ro

9、asted coffeeDeficiency causes pellagraoral lesions, diarrhea, dermatitis, and dementiaMicrobiological 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 quantifi

10、ed 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 Fol

11、ate assay uses digestion with 3 enzymes (amylase, protease and conjugase) to liberate bound folate, then incubation with Lactobacillus rhamnosusPanthothenic acid (Vitamin B5) is part of Coenzyme A Synthesis of fatty acids (acetate pathway), some peptides, phenylpropanoids, isoprenoids Fat, carbs and

12、 protein metabolism Very widely distributed throughout foods,yeast, liver, and cereals provide rich sources. Deficiency is rareVitamin B6 (pyridoxamine, pyridoxal phosphate & pyridoxine) Catalyzes transaminations & decarboxylations of amino acids Metabolism energy In plants, used in biosynthesis of

13、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, anemiapyridoxineOccurs in Krebs cycleVitamin B12 (cobalamins)Involved

14、 in synthesis,of DNA, amino acids, fatty acids, one-C metabolism (methylations)Needed to maintain nerve cells, RBC, genesMicrobial in origin; intestinal flora contribute towards human dietary needs.Stored in the liverFound in meat, shellfish, liver, dairy products and eggsDeficiency causes perniciou

15、s anemiaPoor absorption of B12 is thought to be a complication of agingMethylations such as the conversion of homocysteine to methionine require B12Contains Co(III) coordinated to a corrin ring (R = CN is cyanocobalamin, most common form)R groups vary:CN, OH, H2O, NO2, MeFig 2.29 p 32 DewickHPLC Ana

16、lysis 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 sol

17、vent systems, UV or fluorescence detection Vitamin B12 analysis method uses acetonitrile/ aq. KH2PO4 elution at low pH, with detection possible at 550 nm.Vitamin B9 (folate) Role: Tetrahydrofolate functions in one-C metabolism as a carrier of methyl, methylene or formyl groups Involved in amino acid

18、 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 me

19、nFolate analyses: Microbiological assay HPLC on C18 column withfluorescence detection at350 nm.Relationship between one-C metabolism, B-vitamins and healthOne-C metabolism (methylation) is a key reaction in formation of both primary and secondary metabolites in plants & animals, also involved in DNA

20、 repair and detoxification pathwaysElevated homocysteine levels in humans are an indicator (marker) of faulty one-C metabolismHomocysteine (HCys) is made from methionine (L-Met), but much of the HCys must be cycled back to L-MetDecreased formation of L-Met and SAM result in decreased methylationDecr

21、ease in the methylation pathway is associated with increased risk of cardiovascular disease, Alzheimers disease and some cancers.High HCys levels are also thought to be linked to increased production of reactive oxygen species that damage tissuesAdequate supply of folate (B9), B12 and B6 in diet is

22、necessary to maintain proper HCys metabolism.HSCOOHH2NHomocysteineSelhub, J. 2002. The Journal of Nutrition, Health and Aging, 6: 39-41Vitamin C (ascorbic acid) Antioxidant, strong reducing agent Collagen synthesis, tissue repair, bones & teeth, immune system, iron absorption Cannot be made by human

23、 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 healin

24、gL-dehydroascorbic acidVitamin CHighly susceptible to oxidation, so analysis performed in acidic solution, often with EDTAIndicator 2,6-dichloroindophenol is added to oxidize ascorbate to dehydroascorbatetitration to red endpoint (colored samples tricky, measure at 545 nm)iodine can also be used to

25、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)Lipid-soluble vitamins Vitamins A, D, E & K are all derived in part through isoprenoid pathway Vitamins E & K have phenolic moi

26、eties 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 veggiesVitamin A Retinoids are isoprenoids(C20 = diterpene) Retinol (A1) & dehydroretinol (A2) may exist as free alco

27、hols 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 thickenin

28、g Retinoids are known to act as signalling molecules regulating diverse aspects of cell differentiation, embryonic development, growth, and vision.Retinol and vision For the processes of vision, retinol is first oxidized into the aldehyde all-trans-(11E) retinal and then enzymatically isomerized to

29、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, triggeri

30、ng a nerve impulse to the brain. The absorption of light energy promotes an electron from to MO temporarily destroying double bond character and allowing rotation back to trans All-trans-retinal is then released and the process can repeat itselfDetermination of Vitamin A by HPLC Sensitivity to UV li

31、ght, 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 se

32、parates cis and trans isomers 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 cellmembranes, retinoids, hormones, from oxidation Health:

33、decreases coronary artery lesions Effect on CVD? Observational studies suggest reduced CVD but many clinical trials do not show evidence for reduced mortality (men)Heart benefits for women?A published clinical trial of vitamin Es effects on the heart and blood vessels of women included almost 40,000

34、 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 no

35、nfatal 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 no

36、nfatal heart attack and a 49% decrease in cardiovascular death rates. No effect on cancer incidence reportedSource: NIH Office of Dietary Supplements /factsheets/VitaminE.aspLee, et al, The Womens Health Study: a randomized controlled trial. JAMA 2005;294:56-65. Vitamin E analysi

37、s 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

38、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)Samples dissolved in EtOH with pyrogallol added to prevent oxidation

39、, flush w N2Sample is heated/sonicated 10 min 70 C then 60% KOH added to hydrolyzeHexane extraction with 0.1% BHT added to prevent oxidationDried, filtered and analyzed with HPLC program described belowVitamin DCholecalciferol is a sterol (isoprenoid) formed photochemically in animal skin from 7-deh

40、ydrocholesterol by suns irradiationAlso 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 oilsVitamin D3 is hydroxylated in the body to its active forms, first calcidiol and then to calcitrio

41、l (Figure 5.106, p58 Dewick).Key role: Ca metabolismCalcitriol stimulates the absorption of calcium and phosphate in the intestine and the mobilization of calcium from boneOther physiological functions include a role in immune modulation, hormone secretion, and cell differentiation Vitamin D deficie

42、ncy is common in Northern latitudes due to reduced exposure to sunlightRickets = inability to calcify the collagen matrix of growing bone, causing lack of rigidity in the bones (children) or osteoporosisIn most countries, milk and cereals are fortified with vitamin D3Vitamin D is a product of the me

43、valonate pathway, made from a cholesterol derivative.B ring opens up allowing for rotation and isomerization Vitamin D analysis Difficult to analyze colorimetrically (no strong chromophore) AOAC reference std. method of analysis for many years was a bioassay called the “l(fā)ine test” Young rats deprive

44、d 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 Modern analyses for Vitamin D Current methods are summarized briefly in Lensmeyer, et al, (2006), Clinical Chemistry 52: 1120-2

45、6. 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

46、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 patientsStudies in human populations Epidemiological studies follow a selected population over a defined period of time, mo

47、nitor 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 define

48、d period, then assess the difference in specific measurable endpoints between the populationsRecent 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 highe

49、r 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 Crohns 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