A radioenzymatic isotope-dilution assay for oxalate in serum or plasma.

This assay for oxalate in serum or plasma, by a radioenzymatic isotope dilution technique, is sensitive, specific, and accurate. The oxalate in 5 mL of serum or plasma ultrafiltrate is precipitated with Ca2, dissolved in acid, extracted with ether, and the extract evaporated under nitrogen and redissolved in citrate buffer. The oxalate in this solution is decarboxylated with oxalate decarboxylase (EC 4.1.1.2) in the presence of [14C]oxalate to evolve labeled and unlabeled CO2. An equation derived by Newsholme and Taylor [Biochim. Biophys. Acta 158, 11(1968)] described quantitatively the simultaneous effects of variation of non-radioactive substrate on isotope dilution and enzyme velocity, resulting in a linear standard curve. We found the Km for this enzyme to be 31.0 zmoi/L, with an assay sensitivity of 0.5 tg and a linear assay range to 10 pg. The sensitivity of this result was consistent with the value of 0.1 Km predicted from the equations of Newsholme and Taylor. The K1values for phosphate and sulfate, two commonly occurring inhibitors of the enzyme that affect assay sensitivity by the relationship Km (1 + l/K), were 12 and 7 mmol/L, respectively. Analytical recovery of 0.5-2.0 mg of unlabeled oxalate added to serum per liter was 95.4 ± 5.7% (SE). Interand intra-assay precision was excellent (coefficients of variation of 6.0 and 7.7%, respectively). Dilution ofserum or plasma citrate buffer extracts over an eightfold range yielded plots parallel to the standard curve, indicating the absence of enzyme inhibitors or activators in the citrate buffer extracts. The mean oxalate concentrations in serum and plasmameasured in nine and six male volunteers, respectively-were 0.83 ± 0.24 mg/L (SD) and 0.96 ± 0.24 mg/L (SD).