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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
- Source :
- Analytical and bioanalytical chemistry. 405(5)
- Publication Year :
- 2012
-
Abstract
- 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.
- Subjects :
- endocrine system
S-Adenosylmethionine
Antineoplastic Agents, Hormonal
Epinephrine
Adrenal Gland Neoplasms
Pheochromocytoma
Mass spectrometry
Tandem mass spectrometry
Biochemistry
Sensitivity and Specificity
Dexamethasone
Article
Analytical Chemistry
chemistry.chemical_compound
Norepinephrine
Mice
Tandem Mass Spectrometry
Cell Line, Tumor
medicine
Animals
Humans
Chromatography, High Pressure Liquid
Enzyme Assays
chemistry.chemical_classification
Chromatography
biology
Phenylethanolamine N-Methyltransferase
Deuterium
Phenylethanolamine N-methyltransferase
Enzyme assay
Phenylethanolamine
Enzyme
chemistry
biology.protein
medicine.drug
Subjects
Details
- ISSN :
- 16182650
- Volume :
- 405
- Issue :
- 5
- Database :
- OpenAIRE
- Journal :
- Analytical and bioanalytical chemistry
- Accession number :
- edsair.doi.dedup.....392868d0554cfbd118e0e1c7887993b8