Double stable isotope ultra performance liquid chromatographic tandem mass spectrometric quantification of tissue content and activity of phenylethanolamine N-methyltransferase the crucial enzyme responsible for synthesis of epinephrine

摘要

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 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 5000 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 ng 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.