CLINICAL-EVALUATION OF HIGH-PERFORMANCE AFFINITY-CHROMATOGRAPHY FOR THE SEPARATION OF BONE AND LIVER ALKALINE-PHOSPHATASE ISOENZYMES

The newly described high-performance (HPLC) affinity chromatography method for the separation of human bone and liver alkaline phosphatase (ALP. EC 3.1.3.1) isoenzymes was clinically evaluated. The improved resolution of bone from liver isoenzyme and lower detection limit was achieved by conjugation of wheat-germ lectin (WGL) to a diol-bonded silica gel column, stepwise elution with N-acetylglucosamine (NAG) and post column derivatization using para-nitrophenyl phosphate substrate. To establish a reference interval, we measured bone ALP in 86 healthy women, ages 33 to 95 years. The normal reference interval is described by a piecewise linear regression on age (R2 = 0.20, P < 0.01). For women less-than-or-equal-to 45 years, bone ALP, U/l = 8.495. For normal women between ages of 45 to 55 years, bone ALP, U/l = -12.765 + 0.472* age. If age greater-than-or-equal-to 55 years, then bone ALP. U/l 13.219. In all 10 patients with primary biliary cirrhosis, serum bone ALP levels were elevated. In addition, sera from 43 patients with diverse metabolic bone diseases were evaluated. As expected, the sera from all 6 patients with Paget's disease and 2 with osteolytic metastasis had bone ALP activity which was > 3 standard deviations (SD) from the mean. In all 10 patients with hypoparathyroidism, bone ALP levels were depressed. Only 1 of the 9 patients with glucocorticoid excess and 2 of the 7 patients with primary hyperparathyroidism had elevated bone ALP when compared to the 95% confidence interval for the normal range. Results from the HPLC affinity chromatography correlated significantly with bone ALP levels obtained on the same patients by solid phase immunoassay using monoclonal antibodies specific for ALP (r = 0.91, P < 0.01, n = 139). Discordant results between these two methods were obtained in the patients with primary biliary cirrhosis. Whereas, bone ALP activity was undetectable by solid phase immunoassay, a fraction indistinguishable from bone ALP was detected by HPLC chromatography.