A sensitive capillary GC assay for the determination of sparteine oxidation products in microsomal fractions of human liver

Abstract

1. A sensitive method for the assay of spartein oxidase activity in vitro by microsomal fractions of human liver is described. The activity of sparteine oxidase was assessed by the formation of 2- and 5-dehydrosparteines, which were estimated by capillary gas chromatography with N 2-FID detection. The limit of detection of the two metabolites, 2- and 5-dehydrosparteine, was 10 pmol (2.3 ng) per sample. 2. Sparteine oxidase activity was linear with microsomal protein concentration ranging from 25 to 200 μg and with incubation times between 5 and 60 minutes. Omission of NADPH on incubation under an atmosphere of carbon monoxide inhibited formation of both metabolites, thus indicating that aforementioned metabolites arise in reaction catalyzed by cytochrome P-450. 3. In three liver samples from humans classified as extensive (EM) metabolizers the formation of 2- and 5-dehydrosparteines was observed, 2-dehydrosparteine being the major metabolite. In these samples sparteine oxidase activity was characterised by V max = 136 ± 53 pmol/min/mg and Km = 44 ± 12 μM for 2-dehydrosparteine formation. For 5-dehydrosparteine formation the following values were obtained: V max = 57 ± 18 pmol/min/mg and Km = 42 ± 26 μM. In a liver sample from a poor metabolizer (PM) only the formation of 2-dehydrosparteine was detected with the method of analysis used. In this sample a great increase in Km (Km Pm = 3033 μM) was noted, while V max was very similar to those obtained for 2-dehydrosparteine formation in EM subjects (V max PM = 147 pmol min/mg).