A spectrophotometric method for the determination of glycolate in urine and plasma with glycolate oxidase

An enzymatic assay was developed for the spectrophotometric determination o f glycolate in urine and plasma. Glycolate was first converted to glyoxylat e with glycolate oxidase, and the glyoxylate formed was condensed with phen ylhydrazine, The glyoxylate phenylhydrazone formed was then oxidized with K 3Fe(CN)(6) in the presence of excess phenylhydrazine, and A(515) of the res ulting 1,5-diphenylformazan was measured. Since glycolate oxidase also acts on glyoxylate and L-lactate, the incubation of samples with glycolate oxid ase was carried out in 120-170 mM Tris-HCl (pH 8.3) to obtain glyoxylate as its adduct with Tris, The pyruvate formed from lactate was removed by subs equent brief incubation with alanine aminotransferase in the presence of L- glutamate, and alpha-ketoglutarate formed was converted back to L-glutamate by glutamate dehydrogenase and an NADPH generating system. Thus the specif icity of the assay relies principally on the substrate specificity of glyco late oxidase, and high sensitivity is provided by the high absorbance of 1, 5-diphenylformazan at 515-520 nm, Plasma was deproteinized with perchloric acid, and then neutralized with MOH, Plasma and urine samples were then inc ubated with similar to 5 mM phenylhydrazine, and then treated with stearate -deactivated activated charcoal to remove endogenous keto and aldehyde acid s as their phenylhydrazones. The normal plasma glycolate and urinary glycol ate/creatinine ratio for adults determined by this method are similar to 8 mu M and similar to 0.036, respectively.