Assaying erythrocyte haem biosynthetic enzyme activities by high-performance liquid chromatography with the advanced...

The four cytosolic 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-aminolaevulinic acid dehydrase, the porphobilinogen (PBG) formed was extracted on a C18 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 mM 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, concentrated on a C2 or C8 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 C2 or C8 cartridge, oxidised to coproporphyrin and separated by reversed-phase chromatography with 30% acetonitrile in 1 M ammonium acetate buffer (pH 5.16) as mobile phase.