Assay of the13C and2H Mass Isotopomer Distribution of Phosphoenolpyruvate by Gas Chromatography/Mass Spectrometry

✍ Scribed by Previs, Stephen F.; Rosiers, Christine Des; Beylot, Michel; David, France; Brunengraber, Henri

• Publisher: John Wiley and Sons
• Year: 1996
• Tongue: English
• Weight: 582 KB
• Volume: 31
• Category: Article
• ISSN: 1076-5174
• DOI: 10.1002/(sici)1096-9888(199606)31:6<643::aid-jms335>3.0.co;2-b

✦ Synopsis

The 13C mass isotopomer distribution of liver phosphoenolpyruvate (PEP) yields important information on the regulation of gluconeogenesis and the citric acid cycle. A convenient technique is presented for measuring the mass isotopomer distribution of PEP in tissue extracts. The procedure involves reduction of extant pyruvate to lactate with NaBH, , enzymatic conversion of PEP to pyruvate, extraction of pyruvate hydroxamate and gas chromatographic/mass spectrometric determination of pyruvate hydroxamate di-tert-butyldimethylsilyl derivative. When PEP is labeled with 'H, the enzymatic conversion of PEP to pyruvate results in the loss of 'H. Therefore, to assay the enrichment of [ 2H]PEP, the tissue extract is chromatographed on an anion-exchange column. The fraction containing PEP is treated to form PEP tri(trimethylsily1) derivative. The procedures were applied to liver PEP labeled using [ U-13C, jlactate, [ U-'"C3 1 glycerol or ' H' O. The results show the compatibility between the mass isotopomer distributions of PEP and glucose in rat livers perfused with [ U-13C3]lactate or [ U-13C3]glycerol. There is a 78% isotopic equilibration of 'H enrichment between the hydrogens on C-3 of liver PEP and the hydrogens of water in 2 day fasted rats.