Simultaneous determinations of liver- and bone-type alkaline phosphatase by curve-fitting of inhibition kinetic data. I. Development and evaluation of an absorbance-based method.

We describe an approach for the simultaneous determination of isoenzymes of alkaline phosphatase (EC 3.1.3.1) based on the kinetic behavior of inhibition reactions. Data for absorbance vs time, collected while enzymes are being inhibited, are fitted with suitable models to obtain results related to activities of the individual isoenzymes. The primary focus is on two-component mixtures of the bone and liver isoenzymes of alkaline phosphatase, but some results are reported for three- and four-component mixtures. Factors studied include choices of inhibitors, buffers, pH, ionic strength, substrate concentration, kinetic models, data ranges, data densities, and data-processing approaches and programs. Criteria used to select optimal conditions include measurement times, detection limits, useful range, and agreement between expected and computed results for mixtures of isoenzymes. For two-component mixtures, a linear least-squares fit of isoenzyme content computed with the curve-fitting method (y) v a comparison method (x) gave y = 0.96 (+/- 0.05)x + 3.8 (+/- 3)% with r = 0.97 and standard error of the estimate of 9.4% for a range from 15 to 300 U/L. The pooled relative standard deviation (CV) for results was about 5%. Results were degraded for three- and four-component samples.