Kinetic measurement of bicarbonate in serum by thiocyanate inhibition of wheat germ phosphoenolpyruvate carboxylase

Abstract

We describe a kinetic enzymic method for serum bicarbonate analysis, using wheat germ phosphoenolpyruvate carboxylase (EC 4.1.1.31) coupled through oxaloacetate reduction with NADH in the presence of malate dehydrogenase (EC 1.1.1.37). Inhibition with potassium thiocyanate yielded first-order kinetics with respect to bicarbonate over the concentration range of 0-45 mmol/L. The inhibitor was chosen by evaluating reaction data in the presence of different anions, with use of a monoexponential model. Criteria for first-order kinetics included a constant reaction half-life over the concentration range and SDest for the model comparable with the magnitude of spectrophotometric noise. We compared our kinetic method (y) with an automated ion-selective electrode method (x), obtaining the regression relationship y = 0.97x + 1.2 mmol/L (r = 0.991; n = 77; mean = 25.5 mmol/L; y = 25.3 mmol/L). Within-run precision from duplicates was 3.1% (mean = 25.2 mmol/L; n = 72). Total analytical precision (n = 12) was 9.4% (mean = 15 mmol/L) for the low control and 4.3% (mean = 32 mmol/L) for the high control. We conclude that the kinetic assay allows use of large serum-to-reagent ratios (1:100) and smaller amounts of NADH than an equilibrium assay. The assay is suitable for automated kinetic analysis.