Observations on the use of Escherichia coli for the reduction and estimation of dehydroascorbic acid

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Abstract
The reduction of dehydroascorbic acid by E. coli was reinvestigated. Complete recovery of dehydroascorbic acid as ascorbic acid was not obtained owing to small concurrent loss of dehydroascorbic acid by conversion to diketogulonic acid. Loss due to this cause can, however, be calculated and allowed for and dehydroascorbic acid may accordingly be estimated quantitatively by this method. The difficulty associated with this method of estimating dehydroascorbic acid in vegetable extracts, reported by earlier workers, was shown to be due to the reduction of nitrate to nitrite by E. coli under anaerobic conditions. On acidification, the nitrous acid formed rapidly oxidizes ascorbic acid. Interference in the estimation of ascorbic acid by nitrous acid may be overcome by the use of amidosulfonic acid, which reacts rapidly with nitrous acid and so protects ascorbic acid from oxidation. The nitratase enzyme of E. coli may be completely inhibited by KCN or NaN3, without affecting the enzyme systems associated with the reduction of dehydroascorbic acid. The presence of 0.001 [image] KCN accelerates the conversion of dehydroascorbic acid to diketogulonic acid, but NaN3 in a similar concn. has no effect. NaN3 may, therefore, be used to prevent the formation of nitrite by E. coli without any disadvantageous effect on the reduction of dehydroascorbic acid to ascorbic acid. The oxidized forms of other enediols including D-isoascorbic acid, hydroxytetronic acid, reductic acid, reductone (enol of tartronaldehyde) and the reductants present in a glucoreductone solution with the exception of reductone were reduced, but at a slower rate than dehydroascorbic acid.

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