Thin-layer Chromatographic Determination of L-Ascorbic, L-Dehydroascorbic and 2.3-Diketo-L-gulonic Acids in Animal Tissues, Blood and Urine

Abstract

A new method for the determination of ascorbic, dehydroascorbic and 2.3-diketogulonic acids in animal tissues, blood and urine is described. The material for analysis is homogenized in the presence of stannous chloride in trichloracetic acid and the extract is divided into three aliquots: in one part the ascorbic acid is converted into dehydroascorbic acid by oxidation with bromine water, in the second part the original content of dehydroascorbic and diketogulonic acids is stabilized by the addition of thiourea and in the third part dehydroascorbic acid is reduced to ascorbic acid by thioglycolic acid. Reaction with 2.4-dinitrophenylhydrazine produces a mixture of osazones, from which the bis-hydrazone of diketogulonic acid is isolated by thin-layer chromatography on Silicagel (solvent, ethyl aeetate-chloroform-. acetic acid 50:50:5 v/v). The amount of bis-hydrazone is determined photometrically at 502 nm after dissolution in ethyl-acetate-toluene(1:3 v/v). The content of ascorbic, dehydroascorbic and diketogulonic acids is calculated by comparison of the results from the three aliquots of the extract. The described method is more specific and more sensitive than the former colorimetric methods and is suitable for the isolation and evaluation of small amounts of ascorbic, dehydroascorbic and diketogulonic acids labelled with carbon 14 C. The most commonly used method for the simultaneous content of dehydroascorbic and diketogulonic acids in estimation of the content of ascorbic acid and its meta-the animal tissues and blood, for which the colorimetric bolites in animal material is based on the reaction of the method is not sensitive enough. oxidized form of ascorbic acid and diketogulonic acid The Chromatographie isolation of the osazones of dehywith 2.4-dinitrophenylhydrazine and the photometric droascorbic and diketogulonic acids on thin layers or evaluation of the resulting osazone dissolved in mineral on paper, which have already been used successfully on acid (1). In the simultaneous estimation of all of these plant and animal material (3, 4), increases the specificity substances advantage is taken of the reduction of the of the method and at the same time enables the treatdehydroascorbic acid to ascorbic acid, which does not ment of greater volumes of extracts with a low conreact with the phenylhydrazine reagent. In the difleren-centration of the studied substances than in the method tial method according to ROE and coworkers (2), before of direct colorimetry. Our method of independent the addition of dinitrophenylhydrazine two aliquots of estimation of ascorbic acid and its metabolits is based on the sample are subjected to the action of the oxidizing the method of ROE and coworkers (2) which we have or reducing agent, changing the ascorbic acid to its modified and linked to the isolation of the bis-2.4-oxidized form, and the dehydroascorbic acid to ascorbic dinitrophenylhydrazone of diketogulonic acid on a thin acid. To the third part of the sample the reagent is layer of Silicagel. Its advantage is a gr...

Platelet ascorbic acid levels in normal subjects and in disease

“…The mean platelet ascorbic acid content was 3 5 ± 1-4 ,tg/109 cells. This can be compared with 2-91 ,ug/109 cells (Barkhan and Howard, 1958) and 6-28 tzg/109 cells (Sahud and Aggeler, 1970); but these authors used the dinitrophenylhydrazine procedure which has been shown to overestimate the ascorbic acid content of tissues (Lloyd et al, 1969;Zloch and Ginter, 1970).…”