VITAMIN

1. VITAMIN By: Mohammed Sabah 2013

2. VITAMIN • Minor components of foods • Nutrients that are only needed in small amounts but have powerfull effects • They are required for the normal functioning of every organ and for many important processes such as growth, reproduction and tissue repair • Although vitamin themselves are not a source of energy, they are needed to liberate energy from dietary carbohydrate, protein, fat and alcohol for the body’s cells to use. Some vitamin function as part of coenzyme. • Some vitamins occur in foods as provitamin. Vitamers are members of the vitamin family.

3. Two classes of vitamins • Fat-soluble vitamins : A, D, E and K • have unique functions • occur in oils, dairy products, meats, fish, nuts and grains • can be stored in the body → consuming too much can be toxic and adversely affect the health • stable during cooking and processing • can be destroyed by exposure to air, light or high temperatures

4. 2. Water-soluble vitamins: C and eight B-group Vitamins • disolve in water → excessive amounts of most are removed from the body in the urine • can be lost in the process of washing, soaking or boiling foods and may be destroyed by heat, light or air

5. The sources of vitamins in significant amounts by food group: • Meat, poultry, fish and bean provide thiamin (B1), riboflavin (B2), niacin (B3), pantothenic acid(B5), pyridoxine (B6), biotin and cyanocobalamine (B12) • Milk and milk products provide vitamin A and D, B2, B6, B12 • Bread and cereals provide vitamin B1, B2, B6, niacin, folate, pantothenic acid and biotin • Fruits and vegetables provide vitamin A and K, ascorbic acid (C), B2 and folate • Fats and oils provide vitamins A and E

6. Vitamin analysis • Generally difficult to measure vitamin content of food • chemically heterogeneous group of essential micronutrients • present in very small amounts • often unstable during analysis • Bioassays using humans or animals • Microbiological assays • Physico-chemical assays • titrimetric / spectrophotometric • fluorimetry • chromatographic • enzymatic

7. Example of a bioassay for the analysis of vitamins • Vitamin D • rats fed vit D depleted diet for 18-25 days • groups of rats fed (8 to 11 days) known amounts of vitamin D • other groups of rats fed food sample (8 to 11 days) being analysed • vitamin D potency of the sample is measured as bone calcification level at end of tibia bone compared to calcification in rats fed known amount of vit D

8. Example of a microbiological assay for vitamins • Limited to water soluble vitamins • Growth of specific microorganism in extract of vitamin containing sample is compared with growth of the microorganism in presence of known amounts of the vitamin • Growth measure in terms of • turbidity • acid production • Gravimetry • respiration

9. Niacin microbiological assay procedure • Sample dissolved & autoclaved in 1NH2SO4 • Range of dilutions of sample extract made in assay broth & autoclave (1hr at 121°C) • Range of niacin standards made in same way as sample & autoclave (10 min. at 121°C) • Inoculated with Lactobacillus plantarum, incubated 37oC for 16-18 hr • Measure turbidity by absorbance at any wavelength between 540nm and 660nm. • Compare absorbance of sample incubations with standards to calculate niacin content of original sample Turbidity increases as microorganism numbers increase

10. HPLC analysis of vitamins- vitamin E (Tocopherols & Tocotrienols) • Vitamin E is present in food as eight compounds (alpha, beta, gamma and delta). ) tocopherols and tocotrienols • In order to estimate the Vit E activity  tocopherol equivalent • quantification of each Vit E form is required

11. The Difference Between Tocopherol and Tocotrienol • Vitamin E refers to a group of eight fat-soluble compounds that include both tocopherols and tocotrienols. • Tocopherolis one of the 2 members of the vitamin E family. The other member is known as tocotrienol. Vitamin E incorporates 8 different compounds. These include 4 tocopherols (alpha, beta, gamma and delta) and 4 tocotrienols (alpha, beta, gamma and delta).

12. Tocopherol molecules have a long tail with no double bonds. This inhibits the functional effects of the vitamin within the body. Hence, it has a much lower anti-oxidative capacity than its counterpart. • Tocotrienol molecules have a short tail with 3 double bonds. This unique structure enables the vitamin to perform various functions, especially anti-oxidative, with greater efficacy

13. Structure of vitamin E; Tocopherols & Tocotrienols RI RII

14. Method • saponify • mix with 6% (wt/vol) pyrogallol in ethanol, heat at 70°C & sonicate • add 60% KOH solution digest at 70°C & sonicate • extract with hexane • filter & inject into normal phase HPLC • use fluorescence detector at 290 and 330 nm • quantify by external standard from peak area by linear regression Sonication can be used to speed dissolution, by breaking intermolecular interactions. It is especially useful when it is not possible to stir the sample

17. Vitamin A Chemical name: Retinol or All-Trans-RetinolMolecular formula: C20H30O; Molecular weight: 286.46 g/mole  http://www.natuurlijkerwijs.com/english/vitamins.htm

18. Vitamin A (Retinol) Retinol

19. The pro-vitamin of vitamin A is Beta-carotene (orange dye of carrots). Chemical name: Beta-Carotene Molecular formula: C40H56; Molecular weight: 536.90 g/mole  A provitamin:is a substance that may be converted within the body to a vitamin

20. Vitamin A is a fat-soluble vitamin.  • Retinol is probably formed by an oxidative splitting of the beta-carotene in the small intestine. That is why beta-carotene is also called pro-vitamin A.  • Beta-carotene is an antioxidant and has in this way cancer preventing properties (if you are not a smoker).

21. b - Caroteneand Retinol CH 3 CH CH CH 3 3 3 H C 3 CH 3 CH CH CH 3 3 3 CH 3 Oxidation CH CH O 3 3 H C CH 3 3 C H Retainal CH 3 - 2H CH CH 3 3 H C CH 3 3 CH OH 2 Retinol (Vitamin A) CH 3

22. Vitamin A and b - Carotene Determination Food KOH (Alcoholic) Saponification 3 hrs. at room temperature Ether for Extraction Extract (retinol and b-carotene) HPLC

23. Vitamin D There are two forms of vitamin D, vitamin D3 and D2.  Chemical name: vitamin D3 or CholecalciferolMolecular formula: C27H44O; Molecular weight: 384.6 g/mole

24. Vitamin D2 differs only with one double bond and one methyl group from vitamin D3 (indicated withred).  Chemical name: vitamin D2, Calciferol or Ergosterol Molecular formula: C28H44O; Molecular weight: 396.7 g/mole 

26. Vitamin D2 is also formed from cholesterol, but is vegetable and via another pathway. • Vegetable ergosterol is made from cholesterol and this ergosterol is subsequently converted in ergocalciferol (vitamin D2) under influence of UV-light Vitamin D2 and D3 have biologically the same function. Therefore vitamin D2 is also used as a food supplement.  • Vitamin D is active a hormone in the calcium and phosphate metabolism. • Vitamin D is actually a hormone. As vitaminD it is not active yet. It is made active in subsequent metabolic processes, firstly in the liver and after that in the kidneys. In the liver, an OH group is placed on carbon atom number 25 and in the kidneys also an OH group is placed but now on carbon atom number 1. The synthesised hormone is called 1,25-dihydroxycholecalciferol or 1,25(OH)2D. See the image below. 

28. This hormone stimulates the production of Ca2+binding protein and is in this way responsible for the increased uptake of calcium (Ca) and phosphate (Pi) in the blood. Ca2+-binding protein transports calcium ions and phosphate ions from outside of the cell to the inside of the cell. • Hormones in general and also 1,25-dihydroxycholecalciferol occur in low concentrations in the blood, this also explains why only a little of vitamin D (5 micro grams) is necessarily in our daily diet. • Important foods with vitamin D are: fish (oil), yeast, leaf vegetables, meat and dairy products

29. Vitamin C • Chemical name: L-Ascorbic Acid, L-ascorbate, or E300 Molecular formula: C6H8O6; Molecular weight: 176.12 g/mole  • Vitamin C is a water-soluble vitamin. L-Ascorbic acid has as a redox system a task in the oxidative breakdown of the unsaturated fatty acids (linolenic acid).  • Vitamins C also increase the activity of white blood cells and for this reason promotes a better healing of infections. Vitamin C is easily affected by oxygen (in the presence of heat) and is one of the most sensitive vitamins. Vitamin C deficiency is also the cause of spring tiredness.

30. Careful and toxicity Vitamin C can cause kidney stones and gout. When using high doses some people develop diarrhoea and stomach cramps. Even so this vitamin is considered not harmfully even in very high doses. Very high doses vitamins C can raise the cholesterol levels in the blood, through which the chance to develop heart diseases increase. L-Ascorbic acid is present in many fruits, green plants (leaf vegetables), milk and liver.

31. Reducing agent or converting dehydroascorbic acid to ascorbic acid

32. Acetic Acid (chemical)

33. Vitamin B12 (Cyanocobalamin) • Cyanocobalamin has a complicated chemical structure in which cobalt (III)atom is bound to four nitrogen atoms of a corrin ring as shown in the figure. • Vitamin B12 is a water-soluble dark red crystalline compound which is notstable to acids and alkali. It gets partially destroyed on exposure to sunlight. • Cyanocobalamin is used in the preparation of liquid and dry drug formulationsof all kinds. Cobalamin concentrates are being used in the animal feed industry.

34. Chemical name: CobalaminMolecular formula: C63H88CoN14O14P; Molecular weight: 1355.4 g/mole

35. Vitamin B6 (Pyridoxine hydrochloride) • Pyridoxine is a naturally occurring pyridine derivative. Pyridoxine hydrochloride is a white crystalline powder that is almost colourless. • It iseasily soluble in water and insoluble in 95% ethanol. On prolonged exposureto the sunlight and UV light it changes. The active form of thisvitamin is pyridoxal and a related compound is pyridoxamine.

36. Sterilization of milk done by heating it at high temperature results in the loss ofvitamin B6. It is interesting to know that the vitamin B6 hydrochloride added tomilk or milk preparation is more resistant to thermal sterilization than thevitamin B6 naturally present in the milk. • Loss of vitamin B6 due to thermalsterilization of milk based preparation.