Abstract
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 13CO2 by a double-enzymatic-step reaction is reported. The 13CO2 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 13CO2 is performed with the formaldehyde (0.2 unit) and the formate (0.2 unit) dehydrogenase NAD-dependent enzymes. The recovery of 13CO2 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 [13C2]aminopyrine. This method is useful for the rapid determination of N-demethylase activity of human liver microsomes from methyl-13C-substrates.
Published Version
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