STUDIES ON OXALATE IN UROLITHIASIS
I. A Radioenzymatic Assay for Oxalate in Urine
1981 Volume 72 Issue 6 Pages 694-700
Details
1981 Volume 72 Issue 6 Pages 694-700
The present assay for oxalate in urine is a modification of the radioenzymatic isotope dilution technique developed by D. J. Bennett. This assay has become sensitive and reproducible by the use of a new CO2 trapping apparatus. The oxalate in 2ml of urine is precipitated with Ca++, dissolved in a citrate buffer. The oxalate is then decarboxylated with oxalate decarboxylase in the presence of approximately 405ng of [14C] oxalic acid (18.0nCi) to evolve labeled and unlabeled CO2. An equation derived by Newsholme and Taylor describes quantitatively the simultaneous effects of the variation in the non-radioactive substrate on the isotope dilution and the enzyme velocity, resulting in a linear standard curve. With the extraction procedure 70% to 90% of the [14C] oxalic acid internal standard added to urine was recovered and the recoveries of added unlabeled oxalate averaged 103.5±2.1% (SE). The Km for this enzyme was found to be 35.6μM. The KI for phosphate and sulfate, which are competitive inhibitors of the enzyme, was found to be 0.9mM and 1.3mM respectively. The sensitivity of this method was 0.5μg, which is close to the theoretical sensitivity of 0.1Km, The coefficient of variation (CV) for triplicate determinations of triplicate precipitations of urine was 9.3%. The 24-hr urine excretion of oxalate from 20 patients who had normal kidney function without urinary stones or metabolic deseases, was 24.4±6.9mg (Mean±SD).
