Healthy subjects ingested 2 H 2 O and after 14, 22, and 42 h of fasting the enrichments of deuterium in the hydrogens bound to carbons 2, 5, and 6 of blood glucose and in body water were determined. The hydrogens bound to the carbons were isolated in formaldehyde which was converted to hexamethylenetetramine for assay. Enrichment of the deuterium bound to carbon 5 of glucose to that in water or to carbon 2 directly equals the fraction of glucose formed by gluconeogenesis. The contribution of gluconeogenesis to glucose production was 47 Ϯ 4% after 14 h, 67 Ϯ 4% after 22 h, and 93 Ϯ 2% after 42 h of fasting. Glycerol's conversion to glucose is included in estimates using the enrichment at carbon 5, but not carbon 6. Equilibrations with water of the hydrogens bound to carbon 3 of pyruvate that become those bound to carbon 6 of glucose and of the hydrogen at carbon 2 of glucose produced via glycogenolysis are estimated from the enrichments to be ‫ف‬ 80% complete. Thus, rates of gluconeogenesis can be determined without corrections required in other tracer methodologies. After an overnight fast gluconeogenesis accounts for ‫ف‬ 50% and after 42 h of fasting for almost all of glucose production in healthy subjects. ( J. Clin. Invest. 1996. 98:378-385.)

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A method is introduced for estimating the contribution of gluconeogenesis to glucose production. 2H20 is administered orally to achieve 0.5% deuterium enrichment in body water. Enrichments are determined in the hydrogens bound to carbons 2 and 6 of blood glucose and in urinary water. Enrichment at carbon 6 of glucose is assayed in hexamethylenetetramine, formed from formaldehyde produced by periodate oxidation of the glucose. Enrichment at carbon 2 is assayed in lactate formed by enzymatic transfer of the hydrogen from glucose via sorbitol to pyruvate. The fraction gluconeogenesis contributes to glucose production equals the ratio of the enrichment at carbon 6 to that at carbon 2 or in urinary water. Applying the method, the contribution of gluconeogenesis in healthy subjects was 23-42% after fasting 14 h, increasing to 59-84% after fasting 42 h. Enrichment at carbon 2 to that in urinary water was 1.12±0.13. Therefore, the assumption that hydrogen equilibrated during hexose-6-P isomerization was fulfilled. The 3H/14C ratio in glucose formed from [3-3H,3-'4C]lactate given to healthy subjects was 0.1 to 0.2 of that in the lactate. Therefore equilibration during gluconeogenesis of the hydrogen bound to carbon 6 with that in body water was 80-90% complete, so that gluconeogenesis is underestimated by 10-20%. Glycerol's contribution to gluconeogenesis is not included in these estimates. The method is applicable to studies in humans of gluconeogenesis at safe doses of 21H20. (J. Clin. Invest. 1995. 95:172-178.)