Tracing Gluconeogenesis with Deuterated Water: Measurement of Low Deuterium Enrichments on Carbons 6 and 2 of Glucose

Abstract

The contribution of gluconeogenesis to glucose productionin vivocan be measured by enriching body water with 0.5%2H2O and measuring the glucose labeling ratio C6/C2 (Landauet al., J. Clin. Invest.95, 172–178, 1995). We present further refinements of the measurements of the2H enrichments on C6 and C2 of glucose. The transfer of2H 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, and2H on C2 is transferred enzymatically to [U-13C3]pyruvate, forming [U-13C3,2-2H]lactate. The latter is assayed by negative chemical ionization gas chromatography–mass spectrometry of the pentafluorobenzyl derivative. The natural enrichment of the [U-13C3]lactyl ion is only 0.4%, allowing measurements of2H enrichment down to 0.1%. These techniques were used in dogs infused with2H2O and in isolated rat livers perfused with buffer containing 1 to 5%2H2O. Our data reveal a difference in the rate of labeling of C6 and C2 of glucosein vivo.Lastly, in cows infused with [6,6-2H2]glucose, we show that the turnover of glucose can be economically measured by assaying low tracer enrichment (down to 0.1%) via hexamethylenetetramine.