Assay of Vitamin

Because it is a potent reducing agent, vitamin C is commonly determined by titrimetric orpotentiometric redoxmethods. Such methods 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, after 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 dehydroascorbate 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). 

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 

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Because of the time and expense involved, biological assays are used primarily for research purposes. The first chemical method for assaying L-ascorbic acid was the titration with 2,6-dichlorophenolindophenol solution (76). This method is not appHcable in the presence of a variety of interfering substances, eg, reduced metal ions, sulfites, tannins, or colored dyes. This 2,6-dichlorophenolindophenol method and other chemical and physiochemical methods are based on the reducing character of L-ascorbic acid (77). Colorimetric reactions with metal ions as weU as other redox systems, eg, potassium hexacyanoferrate(III), methylene blue, chloramine, etc, have been used for the assay, but they are unspecific because of interferences from a large number of reducing substances contained in foods and natural products (78). These methods have been used extensively in fish research (79). A specific photometric method for the assay of vitamin C in biological samples is based on the oxidation of ascorbic acid to dehydroascorbic acid with 2,4-dinitrophenylhydrazine (80). In the microfluorometric method, ascorbic acid is oxidized to dehydroascorbic acid in the presence of charcoal. The oxidized form is reacted with o-phenylenediamine to produce a fluorescent compound that is detected with an excitation maximum of ca 350 nm and an emission maximum of ca 430 nm (81). 

The Editors have striven, as in previous years, to include in the present volume reviews on greatly diversified subjects, all of timely importance. The article on mellituria in Volume 4 has been supplemented by a survey of galactosemia, and we expect to follow in future volumes with reviews of other inborn errors of metabolism or, in modern parlance, of molecular diseases. Likewise, the article on peptiduria supplements that on aminoaciduria in Volume 2 and that on microbiological assay of vitamins extends previous summaries on the nucleogenic vitamins. The haptoglobins lie on the borderline of hematology. 

F2. Ford, J. E., Microbiological assay of vitamin B12. The specificity of the requirement of Ochromonas malhamensis for cyanocobalamin. Brit. J. Nutrition 7, 299-306 (1953). 

Sobotka, H., Microbiological assays of vitamins in clinical chemistry, Clin. Chem. 4, 93-106 (1958). 

It is well recognized that in biological assays of vitamin A with rats, high variability in response is continually encountered. For this reason as many as 10 to 15 carefully selected animals, generally males, are used for each testing level, and the responses averaged. Certain stocks of animals then yield concordant results, whereas other stocks cannot be used. Through the courtesy of H. J. Deuel, Jr., the writer has been furnished the raw data on which several satisfactory vitamin A assays were based.34 When the level of intake was such that all the... 

JA Keverling Buisman, KH Hanewald, FJ Mulder, JR Roborgh, KJ Keuning. Evaluation of the effect of isomerization on the chemical and biological assay of vitamin D. J Pharm Sci 57 13 26-1329,1968. 

JW DeVries. Chromatographic assay of vitamins. In J Augustin, BP Klein, D Becker, PB Venugopal, eds. Methods of Vitamin Assay. 4th ed. New York Wiley, 1985, pp 65-94. 

T Okano, A Takeuchi, T Kobayashi. Simplified assay of vitamin D2 in fortified dried milk by using two steps of high-performance liquid chromatography. J Nutr Sci Vitaminol 27 539-550,1981. 

DeVries et al. (67) reported the summary of studies by a number of collaborating laboraties for the HPLC assay of vitamin D in multivitamin preparations. Saponification and a reverse-phase Merck LiChrosorb RP-8 column were used for sample cleanup. The analytical column was a Partisil , 5 nm column (Whatman, Clifton, N.J.) with hexane-amyl alcohol (99.65 0.35%) as the mobile phase. The cleanup procedure although a deparature from the usual analytical methods, was incorporated to ensure predictable, interference-free vitamin D assays (D2 and D3 co-elute). 

J3. Jacobs, W. L. W., and Zondag, H. A., Radioisotope assay of vitamin B12 in human blood serum. Clin. Chim. Acta 24, 93-103 (1969). 

Later in the Second World War, Bradford worked at the Charterhouse Rheumatism Clinic, London, from where she coauthored papers on winter sources of vitamin C and on the determination of riboflavin in blood. After the war, she was employed by J. C. Lyons Co. Ltd., from where she authored four papers for The Analyst two on riboflavin in tea, one on the microbiological assay of vitamins, and one on the use of a single tap source to simultaneously run three different applications. Later in life, Bradford married Mr. Bentley and became a Consultant. She died on 20 August 1981, aged 79 years. 

Numerous mass spectra of tocopherols and tocotrienols have been published and many are available in the NIST/EPA/NIH Mass Spectral Library (2011) and MassBank (2011). In addition to the above chromatographic and nonchromatographic methods for tocol analysis, kits are also commercially available for the ELISA (Enzyme-linked im-munoabsorbent assay) of vitamin E. 

There is also increased erythrocyte hemolysis, which responds to synthetic antioxidants or selenium. The sensitivity of erythrocytes to chemically induced hemolysis can be used both as a biological assay of vitamin E in... 

The assay of vitamins A, D, E, and K in food, feed-stuffs, pharmaceutical preparations, organs, blood, and vegetable matter is described in Refs. 1-4. The application of TLC techniques, together with a densit-ometric scanning apparatus, now permit precise and sensitive quantification of vitamins A, D, E, and K compounds on TLC plates. 

Commercial kits are available for the CPB assays of vitamin The vitamin B]2 binder used is often nonhuman IF, usually obtained from hog stomach. If the IF is not highly purified, it may contain R proteins, which bind not only vitamin B but also related metaboUcally inactive compounds, giving higher values. IF must therefore be either highly purified or have cobinamide (a vitainm B analogue) added to the IF to saturate all binding sites on the R proteins. Cobinamide is not bound by IF. 

Reynolds RD. Nationwide assay of vitamin B6 in human plasma by different methods. Fed Proc 1983 42 665. 

Vuilleumier JP, Keck E. Flourimetric assay of vitamin C in biological materials, using a centrifugal analyser with fluorescence attachment. J Micronutr Anal 1989 5 25-34. 

Jones G. Assay of vitamins D and D3, and 25-hydroxyvitamins D2 and D3 in human plasma by high-performance liquid chromatography. CUn Chem 1978 24 287-98. 

The editors concept of clinical chemistry, as stated in previous volumes of this serial publication, encompasses a wide field of subjects— from the mechanism of blood coagulation to the microbiological assay of vitamins, and from the significance of trace metals in health and disease to the therapeutic application of increased gas pressure. The natural history of clinical conditions and their comparison with normal controls share one common feature, namely, the central position of some species of molecules, be they small or large, all of them the natural subject of chemical research. Hence, practical methods develop, either manual or mechanized, the results of which become increasingly more useful for the clinician in diagnosis, therapy, and prognosis. 

Boxer, G.E. and Rickards, J.C., Chemical determination of vitamin B12. IV. Assay of vitamin B12 in multivitamin preparations and biological materials. Arch. Biochem., 30, 392-401, 1951. 

Diaoetyl-L-BScorbic acid was equally as potent, and 2,3,5,6-tetracetyl-L-ascorbic acid was one-tenth as potent as L-ascorbic acid in the odontoblast assay of vitamin C activity (W. Feldheim and M. Czemy, Preparation of acetyl derivatives of L-ascorbic acid and their properties. Biochem. Z. 831, 150-154, 1959). 

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