Biological synthesis of l-ascorbic acid in animal tissues: conversion of d-glucuronolactone and l-gulonolactone into l-ascorbic acid

Abstract

The enzyme system catalysing the conversion of D-glucuronolactone into L-ascorbic acid in the presence of KCN (50 m[image]) has been found to be located entirely in the microsomal fractions of the liver homogenates of the rat and goat and the kidney homogenate of the chick. This conversion requires no added cofactor. A low concentration of cyanide (5 m[image]) is ineffective with rat-liver and chick-kidney microsomes for the synthesis of L-ascorbic acid from D-glucoronolactone. But the synthesis is stimulated by the addition of: (a) reduced di- or tri-phosphopyridine nucleotide, glutathione, adenosine tri- or di-phosphate, or sodium citrate; (b) a chelating agent such as sodium pyrophosphate, aa-dipyridyl or 8-hydroxyquinoline; (c) the boiled supernatant obtained from the liver tissue of the rat, the goat or the guinea pig or the kidney tissue of the chick. In the absence of cyanide these agents are ineffective. On the other hand, they do not further enhance the synthesis stimulated by 50 m[image]_-KCN. KCN (5 m[image]) can bring about the conversion of D-glucuronoiactone into L-ascorbic acid by the goat-liver microsomes. The conversion is not enhanced by the addition of any of the aforesaid agents. The microsomes can convert D-glucuronolactone into L-ascorbic acid even in the absence of cyanide if L-gulonolactone is added. Potassium ferricyanide (m[image]), but not methyleone blue or oxidized glutathione, completely inhibits the conversion of D-glucuronolactone into L-ascorbic acid by rat-liver microsomes in the presence of 5 m[image]-KCN plus sodium pyrophosphate. The conversion of L-gulonolactone into L-ascorbic acid is not inhibited by potassium ferricyanide. The microsomes can act only on D-glucuronolactone and not on the corresponding free acid. Sodium glucuronate can be converted into L-ascorbic acid but to effect this synthesis the presence of the soluble supernatant and reduced triphosphopyridine nucleotide along with the microsomes is necessary. This conversion is inhibited by 5 m[image] or a higher concentration of cyanide. Soluble-enzyme preparations were obtained from the microsomal fractions. Oxidation of L-gulonolactone by the soluble-enzyme preparations is an aerobic process. Hydrogen peroxide is formed in the process. The immediate product of oxidation of L-gulonolactone has been identified as L-xylohexulonolac-tone.