Assaying erythrocyte haem biosynthetic enzyme with activities by high-performance liquid chromatography with the advanced automated sample processor

Abstract The four cyclic haem biosynthetic enzymes in erythrocytes were assayed with the Varian advanced automated sample processor (AASP) for rapid sample concentration and clean-up with fast and effective high-performance liquid chromatography systems for separation and quantitation. In the assay for 5-aminolae-vulinic acid dehydrase, the porphobilinogen (PBG) formed was extracted on a C 18 AASP cartridge and separated by reversed-phase ion-pair chromatography with 32% methanol in 0.05 M sodium acetate buffer (pH 3.5), containing 5.4 m M of 1-heptane-sulphonic acid as eluent. PBG was the substrate for the simultaneous assay of hydroxymethylbilane synthase and uroporphyrinogen III synthase. The uroporphyrinogen I and III isomers formed were oxidised to porphyrins, concetrated on a C 2 or C 8 cartridge, and separated by reversed-phase chromatography with 13% acetonitrile in 1 M ammonium acetate buffer (pH 5.16) as eluent. Uroporphyrinogen decarboxylase was estimated with pentacarboxylic porphyrinogen III as substrate. The coproporphyrinogen formed was extracted on a C 2 or C 8 cartridge, oxidised to coproporphyrin and separted by reversed-phase chromatography with 30% acetonitrile in 1 M ammonium acetate buffer (pH 5.16) as mobile phase.