Criteria for establishing a standardized method for determining alkaline phosphatase activity in human serum.

We investigated factors influencing alkaline phosphatase activity in the course of developing criteria for the establishment of a standardized method for its determination in human serum at 30 degrees C. The effects of pH, phosphorylatable acceptor (2-amino-2-methyl-1-propanol and diethanolamine), 4-nitrophenyl phosphate, magnesium ion, zinc ion, temperature, volume fraction of specimen, and details of initiation of the reaction have been studied, with use of partly purified enzymes from bone, intestine, liver, and placenta, and sera from patients with a predominant characterized isoenzyme. The purity of the diethanolamine was examined and contaminant monoethanolamine was characterized as a competitive inhibitor. Two sets of recommended conditions are: 2-amino-2-methyl-1-propanol, 0.9 mol/liter; 4-nitrophenyl phosphate, 16 mmol/liter; magnesium ion, 1 mmol/liter; volume fraction of specimen 1/30, and pH30 degrees C 10.5; diethanolamine, 1.8 mol/liter; 4-nitrophenyl phosphate, 18 mmol/liter; magnesium ion, 1 mmol/liter; volume fraction of specimen 1/60, and pH30 degrees C 10.1. Serum is preincubated with all reagents but 4-nitrophenyl phosphate, which is used as the reaction-initiating substrate.