A sensitive capillary GC assay for the determination of sparteine oxidation products in microsomal fractions of human liver.
A sensitive method for the assay of sparteine 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 N2-FID detection. The limit of detection of the two metabolites, 2- and 5-dehydrosparteine, was 10 pmol (2.3 ng) per sample. Sparteine oxidase activity was linear with microsomal protein concentration ranging from 25 to 200 ug 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. 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 Vmax = 136 +/- 53 pmol/min/mg and Km = 44 +/- 12 microM for 2-dehydrosparteine formation. For 5-dehydrosparteine formation the following values were obtained: Vmax = 57 +/- 18 pmol/min/mg and Km = 42 +/- 26 microM. 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 microM) was noted, while Vmax was very similar to those obtained for 2-dehydrosparteine formation in EM subjects (Vmax PM = 147 pmol min/mg).
B A
BA
Osikowska-Evers
M
M
Eichelbaum
D002849:Chromatography, Gas / Q000379:methods; D019389:Cytochrome P-450 CYP2D6; D006801:Humans; D008862:Microsomes, Liver / Q000032:analysis* / Q000201:enzymology; D006899:Mixed Function Oxygenases / Q000032:analysis / Q000378:metabolism; D010088:Oxidoreductases / Q000032:analysis; D013034:Sparteine / Q000031:analogs & derivatives / Q000032:analysis* / Q000378:metabolism; D013997:Time Factors
D013034:Sparteine; D006899:Mixed Function Oxygenases; D010088:Oxidoreductases; D019389:Cytochrome P-450 CYP2D6