Tracing gluconeogenesis with deuterated water: Measurement of low deuterium enrichments on carbons 6 and 2 of glucose

The contribution of gluconeogenesis to glucose production in vivo can be measured by enriching body water with 0.5% (H2O)-H-2 and measuring the glucose labeling ratio C6/C2 (Landau et al., J. Clin. Invest. 95, 172-178, 1995), We present further refinements of the measurements of the H-2 enrichments on C6 and C2 of glucose, The transfer of H-2 from C6 of glucose to hexamethylenetetramine (HMT) and extraction in preparation for gas chromatography-mass spectrometry can be done in a single test tube, without distillation of the intermediate formaldehyde, In addition, extraction of small amounts of HMT is greatly improved by making a HMT-iodine adduct. For C2, glucose is reduced to sorbitol, and H-2 on C2 is transferred enzymatically to [U-C-13(3)]pyruvate, forming [U-C-13(3),2-H-2]lactate. The latter is assayed by negative chemical ionization gas chromatography-mass spectrometry of the pentafluorobenzyl derivative. The natural enrichment of the [U-C-13(3)]lactyl ion is only 0.4%, allowing measurements of H-2 enrichment down to 0.1%. These techniques were used in dogs infused with (H2O)-H-2 and in isolated rat livers perfused with buffer containing 1 to 5% (H2O)-H-2. Our data reveal a difference in the rate of labeling of C6 and C2 of glucose in vivo. Lastly, in cows infused with [6,6-H-2(2)]glucose, we show that the turnover of glucose can be economically measured by assaying low tracer enrichment (down to 0.1%) via hexamethylenetetramine. (C) 1997 Academic Press.