title: Assaying erythrocyte haem biosynthetic enzyme activities by high-performance liquid chromatography with the advanced automated sample processor.
abstract: 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.
mesh_terms: D000642:Ammonia-Lyases / Q000032:analysis*; D001322:Autoanalysis; D002851:Chromatography, High Pressure Liquid / Q000295:instrumentation; D004912:Erythrocytes / Q000201:enzymology*; D006418:Heme / Q000096:biosynthesis*; D006801:Humans; D011163:Hydroxymethylbilane Synthase / Q000032:analysis*; D000623:Porphobilinogen Synthase / Q000097:blood; D014575:Uroporphyrinogen Decarboxylase / Q000097:blood; D014576:Uroporphyrinogen III Synthetase / Q000097:blood
chemical_list: D006418:Heme; D011163:Hydroxymethylbilane Synthase; D014575:Uroporphyrinogen Decarboxylase; D000623:Porphobilinogen Synthase; D014576:Uroporphyrinogen III Synthetase; D000642:Ammonia-Lyases