Vitamin biological potency

No unequivocal unique function for vitamin E has been defined. However, it does act as a hpid-soluble antioxidant in cell membranes, where many of its functions can be provided by synthetic antioxidants. Vitamin E is the generic descriptor for two famihes of compounds, the tocopherols and the tocotrienols (Figure 45—5). The different vitamers (compounds having similar vitamin activity) have different biologic potencies the most active is D-a-tocopherol, and it is usual to express vitamin E intake in milhgrams of D-a-tocoph-erol equivalents. Synthetic DL-a-tocopherol does not have the same biologic potency as the namrally occurring compound. 

Vitamin D3 (C27H440, MW = 384.62) and vitamin D2 (C28H440), MW = 396.63) differ structurally only in the C-17 side chain, which in vitamin D2 has a double bond and an additional methyl group (Fig. 4). Both compounds occur naturally with 5,6 double bond in the cis configuration. The biological potencies of vitamins D2 and D3 in humans are essentially equal. 

Vitamin E is a generic term that represents four tocopherols and four tocotrienols of varying biological potency. The term tocopherol correctly refers to the methyl-substituted derivatives of to-col and is not synonymous with the term vitamin E. The tocopherols and tocotrienols may be referred to collectively as tocochromanols. Many of the diverse deficiency syndromes observed in animals experimentally deprived of vitamin E can be explained by the vitamin s acting as an antioxidant in stabilizing unsaturated lipids in biological membranes. 

It has been known for some time that vitamin E can act as an antioxidant within the body [21, 61] and that the biological potency of the tocopherols is proportional to their antioxidant activity [62], Synthetic antioxidants, which often have structures unrelated to that of the vitamin, are also capable of preventing the symptoms of vitamin E deficiency [23, 24, 63, 64]. The general proposal [63, 65], therefore, is that the function of vitamin E is one of an in vivo antioxidant, protecting membrane phospholipids from attack by free radicals generated within the cell. 

Based on biological assay in vitamin E-deficient rats, the vitamers have widely varying biological activity. The original international unit (iu) of vitamin E potency was equated with the activity of 1 mg of (synthetic) dl- -tocopherol acetate on this basis, pure D-a-tocopherol (i i i -a-tocopherol, the most potent vitamer) is 1.49 iu per mg. The precise mixture of stereoisomers in this original standard is unknown, and the different stereoisomers have very different biological activities, so that different preparations may differ considerably. 

In intestinal mucosal cells, all vitamers of vitamin E cue incorporated into chylomicrons, and tissues take up some vitamin E from chylomicrons. Most, however, goes to the liver in chylomicron remnants, a -Tocopherol, which binds to the liver a-tocopherol transfer protein, is then exported in very low-density lipoprotein (VLDL) and is available for tissue uptake (Traber and Aral, 1999 Stocker and Azzi, 2000). Later, it appears in low-density Upoprotein (LDL) and high-density lipoprotein, as a result of metabolism of VLDL in the circulation. The other vitamers, which do not bind well to the a-tocopherol transfer protein, are not incorporated into VLDL, but are metabolized in the Uver and excreted. This explains thelower biological potency of the othervitcimers,despitesimilar, or higher, in vitro antioxidant activity. 

The Vitamin A potency of butter is in part due to Vitamin A as such and in part to carotene, which is partially converted to the vitamin in the human body. The Vitamin A content of butter is usually within the range of 6-12 mg/g, and the carotene content is in the range of 2-10 mg/g (33) 1 lU of Vitamin A is defined as the amount possessing the biological activity of 0.6 pg of pure (3-carotene. 

We have used the growth effects and pathologies associated with L-ascorbic acid deficiency as a basis for the determination of the biological potency of related compounds (Table I). At a dietary concentration of 0.5 mM, L-ascorbic acid and dehydroascorbic acid were fully active, as well as some ester derivatives including the 6-myristate and 2-phosphate compounds. The insect may be metabolically like the guinea pig because both were able to utilize those esters (17), Carboxylesterases and phosphatases probably converted those derivatives to the free vitamin (18). The 6-bromo compound was less active and apparently cannot be metabolized to L-ascorbic acid or only poorly so. 

The vitamin C activity of L-ascorbic acid or reduced ascorbic acid (RAA) and its oxidized form, dehydroascorbic acid (DHA) is essentially the same, while D-ascorbic acid (isoascorbic acid or erythroascorbic acid) has little of the vitamin s biological potency (1). The readiness with which RAA is reversibly oxidized to DHA is the basis of its physiological activity, and of its use as an antioxidant in food systems. 

Vitamin A2 is found in vertebrates that live nr. at least, begin their lives in freshwater. Vitamin A exhibiLs chemical, physical, and biological properties very similar to those of vitamin A. It has the structural formula depicted above (see page 869). Vitamin A has a biological potency nf 1.3 million USP U/g. which is approximately 40% of the activity of crystalline vitamin A acetate. 

C 83.86%, H 10.56%. O 5.59%. A naturally occurring isomer of vitamin A. Isolated in cryst form from soup-fin shark liver oil. Also present in cod, dogfish, halibut, and California jewfish liver oils to the extent of approx 357° of the total vitamin A content. Synthetic vitamin A concentrates also contain neovitamin A in the same proportion. The biological potencies of neovitamin A and vitamin A, as measured by the U.S.P. growth method in cats, proved to be identical. Isoln Robeson, Baxter. J- Am. Chem. Soc. 69, 136 (1947) Robeson, U.S. pat. 2,552,908 (1951 to Eastman Kodak). Discussion of stereoisomerism Zechmeister. 

Morton, R. A., Stubbs A.L., (1946). Photoelectric Spectrophotometry Applied to the Analysis of Mixtures and Vitamin A Oils, Vol.Tl, pp. 348-350 Morton, R.A., Stubbs, A.L. (1947). A Re-examination of Halibut-liver Oil. Relation Between Biological Potency and Ultraviolet Absorption Due to Vitamin A, Biochem. /., Vol.41, pp. 525-529... 

Estimation of true vitamin E in foods requires quantitative determination of all its components since they vary in their biological potency. This vitamin consists of four tocopherols (a, jS, y, and 6) and four tocotrienols (a, jS, y, and d), but the three major constituents responsible for vitamin E activity are the a-, jS-, and y-tocopherols. While these compounds are fluorescent, their esters must be reduced to free alcohols for total tocopherol assays. Total vitamin E can be directly obtained through fluorimetry, but the determination of individual components is carried out using LC with fluorimetric detection. This procedure has been used to determine the composition of vitamin E in seed oils from maize, olives, soya beans, sesame, safflower, and sunflower by measuring the content of all the four tocopherols plus a-tocotrienol. The simultaneous determination of tocopherols, carotenes, and retinol in cheese has been carried out using LC with two programmable detectors coimected in series, a spectrophotometer and a fluorimeter. Carotenes have been determined photometrically, and fluorimetric measurements have been obtained for tocopherol and retinol. 

From a nutritional viewpoint, the carotenoids are classified as provitamins and inactive carotenoids. To have vitamin A activity, the carotenoid molecule must incorporate a molecule of retinol, i.e., an unsubstituted /3-ionone ring with an 11-carbon polyene chain. /3-carotene (C40H56, MW = 536.88), the most ubiquitous provitamin A carotenoid, is composed of two molecules of retinol joined tail to tail thus the compound possesses maximal (100%) vitamin A activity. The structures of all other provitamin A carotenoids incorporate one molecule of retinol and hence theoretically contribute 50% of the biological activity of /3-carotene. Among the 600 or so carotenoids that exist in nature, only about 50 possess vitamin A activity in varying degrees of potency. 

In the analysis of foods that contain significant amounts of both naturally occurring toco-pherols and supplemental a-tocopheryl acetate, saponification, by hydrolyzing the esterified vitamin E, allows the total a-tocopherol content to be measured as a single peak by HPLC. It should be noted that if totally synthetic all-rac-a-tocopheryl acetate is the supplemental form used, its hydrolysis product, all-rac-a-tocopherol, is less biologically active than is naturally occurring RRR-a-tocopherol, making it impossible to calculate a potency value for the total vitamin E. This problem does not arise if the supplement used is / / / -a-tocopheryl acetate. 

Other studies have shown biological actions from these kinds of structure and indicate interrelationships between the chemicals in terms of their potencies. Tomita [229] found that the substances, vitamin A, vitamin K, vitamin E, /1-carotene, ubiquinone (15), phytol and squalene (16), from green-yellow vegetables could suppress the growth of tumour cells and enhance T-cell cytotoxicity, but /1-carotene, which does have both ends of the chain substituted with a bulky / -ionone ring on each end-group did not. Hydrophobic chain... 

FIGURE 66.1 Vitamins D3 and D2 are produced by ultraviolet irradiation of animal skin and plants, respectively. The precursor of vitamin D3 in skin is 7-dehydrocholesterol, or provitamin D. In humans, the storage, transport, metabolism, and potency of vitamins D2 and D3 are identical, and the net biologic activity of vitamin D in vivo results from the combined effects of the hydroxylated derivatives of vitamins D2 and D3. 

A better method is to first add an equal volume of dimethylsulfoxide (DMSO) or dimethylformamide (DMF) to the aqueous sample. This breaks both biological and encapsulation membranes and pulls polar and nonpolar material into solution. The second step is to dilute the sample with 10 volumes of water. At this point, nonpolars can be removed by solvent extraction or with a Cig SFE. Charged molecules can be recovered with pH-controlled extraction or with ion pairing reagents. The DMSO or DMF stays with the water layer. Customers have told me they can achieve almost complete recovery of both fat-soluble and water-soluble vitamins from polymer-encapsulated mixtures. Vitamins are encapsulated to protect potency from air-oxidation. Water-soluble vitamins have nonpolar encapsulation fat-soluble vitamins have polar encapsulation. Either vitamin can be extracted by themselves, but they are difficult to extract under the same condition unless DMSO or DMF are used to break both capsules. 

The predominant form of vitamin E in food is a-tocopherol. This form of the vitamin is also the most biologically potent form (100%), as determined by the rat fertility test. Other forms (and their relative potencies) are P-tocopherol (40%), y-tocopherol (10%), 6-tocopherol (1%), and a-tocotrienol (25%). The rat fertility test is performed as follows. Female rats are fed diets deficient in vitamin E, sufficient in a-tocopherol, or containing a known amount of the test compoimd. The rats are then mated with male rats. The number of living fetuses in the uterus of the female rat is then used to assess the potency of the test compoimd, relative to a-tocopherol. The deficient state results in dead fetuses, spontaneous abortions, and fetal resorptions. 

Retinoic acid is involved in only two of the known functions of Vitamin A, namely growth and cellular differentiation. Retinoic acid is a potent inducer of cellular differentiation, in vitro (17-191. and may be necessary for normal gene expression (17.271. Several nuclear transcription factors for retinoic acid have been identified (28.291 and the associated genes products may be necessary to prevent cellular transformation. Therefore, retinoic acid may be necessary for homeostasis. However, because of its potency, it exists in low concentrations in biological systems. 

Ergosterol is the provitamin of vitamin D2, which differs from 7-dehy-drocholesterol and vitamin D3, respectively, only by having a double bond between C22 and C23 and a methyl group at C24. Vitamin D2 is the constituent in many commercial vitamin preparations and in irradiated milk and bread. The antirachitic potencies of D2 and D3 in humans are equal, but both must be converted to 25-(OH)-cholecalciferol and eventually to the active form calcitriol (1,25-(0H)2D3) for biologic activity. 

The relative potencies of the different tocopherols (Harris et al., 1944 Joffe and Harris, 1943) in various biological tests have recently been summarized (Bunyan et al., 1961). Comparison of the data shows that the dimethyl- or monomethyltocols have a much lower vitamin E activity than a-tocopherol. 

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