Assay of the 13C and 2H mass isotopomer distribution of phosphoenolpyruvate by gas chromatography/mass spectrometry.

ABSTRACT

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 NaBH4, 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 2H, the enzymatic conversion of PEP to pyruvate results in the loss of 2H. 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(trimethylsilyl) derivative. The procedures were applied to liver PEP labeled using [U-13C3]lactate, [U-13C3]glycerol or 2H2O. 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 2H enrichment between the hydrogens on C-3 of liver PEP and the hydrogens of water in 2 day fasted rats.