Application of isotopic ratio mass spectrometry for the in vitro determination of demethylation activity in human liver microsomes using N-methyl-13C-labeled substrates.
The reaction of demethylation mediated by cytochrome P450 (CYP) leads to the equimolar production of demethylated metabolite and formaldehyde. From a 13C-substrate labeled on a carbon of the methyl moiety, [13C]formaldehyde (H13CHO) is liberated. A highly sensitive and specific assay involving the oxidation of H13CHO to 13CO(2) by a double-enzymatic-step reaction is reported. The 13CO(2) was quantified by the method of reverse isotopic dilution based on gas chromatography-isotope ratio mass spectrometry analysis. The method first involves the limiting step of the CYP-dependent reaction, which is stopped with a mixture of zinc sulfate 5 mM and trichloroacetic acid 100 mM. Then, the transformation of H13CHO to 13CO(2) is performed with the formaldehyde (0.2 unit) and the formate (0.2 unit) dehydrogenase NAD-dependent enzymes. The recovery of 13CO(2) from the incubation mixture was equal to 91.4 +/- 3.0%. The accuracy and the precision of the present method were within 12 and 10%, respectively. The limit of quantification was set to 25 pmol. The performance of the assay was validated on human liver microsomes with five probes: [13C]erythromycin, [1-13C]caffeine, [3-13C]caffeine, [7-13C]caffeine, and [13C(2)]aminopyrine. This method is useful for the rapid determination of N-demethylase activity of human liver microsomes from methyl-13C-substrates.
Grand
Florence
F
Fédération de Biochimie, Laboratoire de Biochimie C, Hôpital E. Herriot, 69437 Lyon Cedex 03, France.
Kilinc
Izzet
I
Sarkis
Albert
A
Guitton
Jérôme
J
D000632:Aminopyrine / Q000378:metabolism; D002110:Caffeine / Q000378:metabolism; D002247:Carbon Isotopes / Q000378:metabolism; D003577:Cytochrome P-450 Enzyme System / Q000378:metabolism; D004917:Erythromycin / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D013058:Mass Spectrometry; D008745:Methylation; D008862:Microsomes, Liver / Q000201:enzymology / Q000378:metabolism*; D010089:Oxidoreductases, N-Demethylating / Q000378:metabolism*
D002247:Carbon Isotopes; D000632:Aminopyrine; D002110:Caffeine; D004917:Erythromycin; D003577:Cytochrome P-450 Enzyme System; D010089:Oxidoreductases, N-Demethylating