Measurement of 5-aminolevulinic acid synthase activity in whole and fractionated human bone marrow: effect of myeloid cell lysis by monoclonal antibody

Abstract

A sensitive radiochemical assay for the measurement of bone marrow and erythroblast 5-aminolevulinic acid (ALA) synthase (EC 2.3.1.37) was developed and optimized with respect to sample preparation and reagent concentration. Succinylacetone (4,6-dioxoheptanoic acid) was used to prevent ALA utilization during the incubation period. Sample purification on a Sep-Pak cartridge (Waters Associates) followed by reverse-phase high-performance liquid chromatography (HPLC) allowed rapid isolation of pure ALA-pyrrole, free from radioactive succinate and other contaminants. ALA synthase activity was measured in unfractionated bone marrow and in samples from which myeloid cells had been removed by monoclonal antibody-mediated cell lysis. Myeloid-derived ALA synthase was calculated and found to contribute approximately half of the total unfractionated marrow enzyme activity. This suggests that results from previous studies using unfractionated bone marrow which have assumed that myeloid cells are an insignificant source of ALA synthase require reappraisal.