Here, we describe a novel method utilizing double stable
isotope ultra performance liquid chromatography-tandem mass spectrometry to measure tissue contents and activity of
phenylethanolamine N-methyltransferase (PNMT), the
enzyme responsible for synthesis of the stress
hormone,
epinephrine. The method is based on measurement of
deuterium-labeled
epinephrine produced from the reaction of
norepinephrine with
deuterium-labeled
S-adenosyl-L-methionine as the methyl donor. In addition to
enzyme activity, the method allows for determination of tissue contents of PNMT using human recombinant
enzyme for calibration. The calibration curve for
epinephrine was linear over the range of 0.1 to 5,000 pM, with 0.5 pM
epinephrine representing the lower limit of quantification. The calibration curve relating PNMT to production of
deuterium-labeled
epinephrine was also linear from 0.01 to 100 ng PNMT. Intra- and inter-assay coefficients of variation were respectively 12.8 % (n = 10) and 10.9 to 13.6 % (n = 10). We established utility of the method by showing induction of the
enzyme by
dexamethasone in mouse
pheochromocytoma cells and strong relationships to PNMT gene expression and tissue
epinephrine levels in human
pheochromocytomas. Development of this assay provides new possibilities for investigations focusing on regulation of PNMT, the crucial final
enzyme responsible for synthesis of
epinephrine, the primary fight-or-flight stress
hormone.