Use of 2H2O for estimating rates of gluconeogenesis. Application to the fasted state.

Abstract

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.)

Changes in hepatic glycogen cycling during a glucose load in healthy humans

“…At 08.00 hours subjects started to drink a total of 5 ml of 2 H 2 O (99.9% enriched; Cambridge Isotope Laboratories, Andover, MA, USA) per kg body water, divided into four equal doses spaced at intervals of 30 min each [10], in order to assess the contribution of the indirect pathway to glycogen formation. Body water was estimated at 60% of body weight [9]. Thereafter, subjects were asked to drink a minimum of one glass of 0.5% 2 H 2 Oenriched water per hour to maintain isotopic equilibrium in body water.…”

“…To measure 3 H glucose-specific activity, the blood samples were diluted with water and deproteinised by adding equal volumes of 0.3 N Ba(OH) 2 and 0.3 N ZnSO 4 [9,29]. The supernatant was deionised by passage through a column of AG1-X8 (Bio-Rad Laboratories, Hercules, CA, USA) in the formate form layered over AG50W-X8 (Bio-Rad) in the hydrogen form.…”

“…Glycogen cycling has limited the interpretation of estimates of gluconeogenesis [2,8], since its contribution cannot be excluded by current methods, e.g. using 2 H 2 O [9,10] and magnetic resonance spectroscopy (MRS) [11]. Fluxes through the hepatic gluconeogenic pathway could also proceed directly to glycogen rather than into plasma glucose [12,13].…”

Quantifying the contribution of gluconeogenesis to glucose production in fasted human subjects using stable isotopes

“…During a fast, 2 H 2 O is ingested to reach a body water enrichment of 0·25-0·5 % (Landau et al 1995c(Landau et al , 1996Chandramouli et al 1997;Landau, 1997). Ingestion of a single dose to achieve 0·5 % enrichment can cause vertigo, therefore the dose is given over 2-4 h. When the enrichment at C-5 in blood glucose becomes constant, within a few hours after normal subjects ingest 2 H 2 O, i.e.…”

“…2 H enrichment at C-2 is approximately equal to 2 H 2 O at steady-state. From the retention of 20 % 3 H in the conversion of [2-3 H]galactose to glucose (Wajngot et al 1989), it might be expected to be only 80 % complete (Landau et al 1995c), but the 20 % retention is attributable to an isotope effect resulting in the incomplete removal of 3 H of [2-3 H]G6P (Katz et al 1978). Direct release of glucose from hydrolysis of 1,6-glucosyl branches in glycogen would also result in an overestimate of GNG.…”