Aldose reductase does catalyse the reduction of glyceraldehyde through a stoichiometric oxidation of NADPH

In order to define the ability of bovine lens aldose reductase (ALR2) to ge nerate polyols from aldoses, the quantitative determination of glycerol in the presence of glyceraldehyde was performed by gas chromatography after de rivatization with trifluoroacetic anhydride. The proposed method appears to be useful in quantifying low amounts of glycerol in the presence of relati vely high concentrations of glyceraldehyde and in following glycerol format ion in enzyme assay conditions. The generation of one equivalent of glycero l in the presence of ALR2, is paralleled by the oxidation of one equivalent of NADPH. A similar result was obtained when S-glutathionyl-modified ALR2 was used, instead of the native enzyme, as a catalyst of glyceraldehyde red uction. Sorbinil, a classical ALR2 inhibitor, present in the enzyme assay m ixture, inhibits to the same extent both NADPH oxidation and glycerol forma tion. The demonstration of the stoichiometric ratio of 1:1 occurring in the presence of bovine lens ALR2 between the synthesis of glycerol from D,L-gl yceraldehyde and the oxidation of NADPH, rules out doubts concerning the ab ility of the enzyme to catalyse the reduction of aldoses to the correspondi ng polyalcohols. Possible autooxidation processes of glyceraldehyde, in the enzyme assay conditions, appear to be irrelevant with respect to the enzym e-catalysed reduction of the aldose. This would indicate that the spectroph otometric monitoring of NADPH oxidation at 340 nm, in the presence of ALR2, is a reliable method to assay the enzyme activity. (C) 2000 Academic Press .