Assay of Vitamin C

Because it is a potent reducing agent, vitamin C is commonly determined by titrimetric orpotentiometric redoxmethods. Suchmethods underestimate the amount of the vitamin present because dehydroascorbate - which has vitamin activity- is formed by atmospheric oxidation of ascorbate in the sample, especially under neutral conditions, and is not detected by redox assay methods.

Vitamin C can also be determined colorimetrically, alter oxidation to dehydroascorbate, by reaction with dinitrophenylhydrazine. Under appropriate conditions, neither ascorbic acid itself nor potentially interfering sugars react with dinitrophenylhydrazine. However, diketogulonate, which has no vitamin activity, also reacts with dinitrophenylhydrazine under the same conditions. Unless diketogulonate is determined separately after reduction of de-hydroascorbate to ascorbate, this method overestimates the vitamin.

These problems can be overcome by using more specific assay methods: either high-performance liquid chromatography or a fluorescence assay (Brubacher et al., 1985).

13.2 METABOLISM OF VITAMIN C

As shown in Figure 13.2, ascorbate is an intermediate in the gulonolactone pathway of glucuronic acid metabolism. In those species for which ascorbate is not a vitamin, this is a major pathway of glucuronic acid catabolism, and ascorbate is a metabolic intermediate whose rate of synthesis and turnover bear no relation to physiological requirements for ascorbate per se. In these species, rates of ascorbate synthesis and turnover range between 5 mg per kg of body weight per day (cats and dogs) and 30 to 40 mg per kg per day (goats, rats, and mice). Metabolic stress and the administration of xenobiotics

Figure 13.2. Biosynthesis of ascorbate. Glucuronate reductase, EC 1.1.1.19; glucono-lactone 3-lactonase, EC 3.1.1.17; gulonolactone oxidase, EC 1.1.3.8; NADPH-dependent dehydroascorbate reductase, EC 1.6.5.4; and glutathione-dependent dehydroascorbate reductase, EC 1.8.5.1.

(Section 13.3.8) can increase the rate of ascorbate turnover several-fold in species for which it is not a vitamin.

Species for which ascorbate is a vitamin lack gulonolactone oxidase, and metabolize gulonic acid by reduction and decarboxylation directly to xylulose. The loss of gulonolactone oxidase seems to be the result of nonexpression of the gene rather than a gene deletion (Sato and Udenfriend, 1978).

An autosomal recessive mutant strain of rat, which lacks gulonolactone oxidase and hence is unable to synthesize ascorbic acid, has been described (Mizushima et al., 1984). The animals have an osteogenic disorder akin to scurvy in human infants, and homozygotes are sterile. The addition of ascorbate to their diet restores normal growth and fertility, but because, like all species for which it is not normally a vitamin, they lack the intestinal active transport carrier for the ascorbate (Section 13.2.1), and they require relatively large amounts of the vitamin.

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