Determination of delta-aminolaevulinic acid synthase activity in human bone marrow using high performance liquid chromatography.
A high performance liquid chromatographic (HPLC) method is described for the rapid and specific determination of the activity of the enzyme delta-aminolaevulinic acid synthase (ALA-S) in mitochondria prepared by sonication of human bone marrow cells. After incubation with 14C-alpha-ketoglutarate the 14C-delta-aminolaevulinic acid (ALA) formed is converted to a pyrrole derivative, 2-methyl-3-carbethoxy-4-(3-propionic acid) pyrrole. This is isolated by reversed-phase ion-pair chromatography on a Hypersil-SAS column with methanol-water (45:155, v/v) in the presence of 0.005 mol/l 1-heptanesulphonic acid (PIC B-7) as the mobile phase. The radioactivity of the isolated pyrrole is determined by scintillation counting. The optimal substrate concentration and pH were 0.17 mmol/l alpha-ketoglutarate and pH 7.4, with an optimal period of sonication of 18s. Under these conditions ALA production was proportional to the concentrations of erythroblasts in the initial sample and was linear with time up to 60 min. The addition of pyridoxal phosphate (PLP) did not affect ALA-S activity in normal subjects. The mean ALA-S activity in 10 haematologically normal control subjects was found to be 318.8 pmol.10-6 erythroblasts.h-1 (S.D.  125.8, range 193-444.6).
D000624:5-Aminolevulinate Synthetase / Q000378:metabolism*; D001853:Bone Marrow / Q000201:enzymology*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007656:Ketoglutaric Acids / Q000378:metabolism; D008832:Microchemistry; D008928:Mitochondria / Q000201:enzymology; D012016:Reference Values
D007656:Ketoglutaric Acids; D000624:5-Aminolevulinate Synthetase