REFERENCE-STANDARDS FOR QUANTIFICATION OF SKELETAL ALKALINE-PHOSPHATASE ACTIVITY IN SERUM BY HEAT INACTIVATION AND LECTIN PRECIPITATION

Putative standards of skeletal alkaline phosphatase (ALP) (from bone, bone cells, osteosarcoma cells, and Pagetic serum) and hepatic ALP (fr om cholestatic serum and bile) were used to compare three methods for quantifying skeletal ALP activity in serum: heat inactivation, precipi tation with wheat germ agglutinin (WGA), and precipitation with concan avalin A (Con A). All the skeletal ALP standards were similarly sensit ive to heat inactivation, as were the hepatic ALP standards. Heat inac tivation separated skeletal from hepatic ALP by a 50% difference in re maining ALP activities (e.g., 23% and 74% remaining skeletal and hepat ic ALP activities after 30 min at-52-degrees-C). Differential precipit ations with WGA and with Con A were less efficient at separating skele tal from hepatic ALP (maximum differences of <30% remaining ALP activi ty). Although both types of hepatic ALP standard (cholestatic serum an d bile) were precipitated with similar efficiencies by WGA and Con A, the skeletal ALP standards were not (e.g., at 2.7 g/L, WGA precipitate d 78-86% of the ALP activity in Pagetic serum, but only 49% of the ALP activity in extracts of human bone). These data suggest that heat ina ctivation is preferable to precipitation with WGA or Con A for quantif ying skeletal ALP activity in serum: it better separates skeletal from hepatic ALP activity and is not sensitive to glycosyl heterogeneity.