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Purpose: Enrichment of glucose position 5 (H5) from deuterated water ((2)H2O) is widely used for quantifying gluconeogenesis. Exchanges of hexose and triose phosphates mediated by transaldolase have been postulated to enrich H5 independently of gluconeogenesis, but to date this mechanism has not been proven. We determined the enrichment of glucose-6-phosphate (G6P), the immediate precursor of endogenously produced glucose, from (2)H2O in erythrocyte hemolysate preparations. Here, transaldolase exchange is active but gluconeogenesis is absent., Methods: Hemolysates were prepared from human erythrocytes and incubated with a buffer containing 5% [U-(13)C]G6P, unlabeled fructose 1,6-bisphosphate, and 10% (2)H2O. G6P (2)H-enrichment and (13)C-isotopomer distributions were analyzed by (2)H and (13)C NMR following derivatization to monoacetone glucose., Results: (2)H NMR analysis revealed high (2)H-enrichment of G6P hydrogens 2, 4, and 5; low enrichment of hydrogen 3, and residual enrichments of hydrogens 1, 6R, and 6S. (13)C NMR isotopomer analysis revealed that [U-(13)C]G6P was converted to [1,2,3-(13)C3]G6P, a predicted product of transaldolase-mediated exchange, as well as [1,2-(13)C2]G6P and [3-(13)C]G6P, predicted products of combined transaldolase and transketolase exchanges., Conclusion: Hydrogen 5 of G6P was enriched from (2)H2O through exchanges mediated by transaldolase. These studies prove that G6P can be enriched in hydrogen 5 by (2)H2O independently of gluconeogenesis., (© 2015 Wiley Periodicals, Inc.)

Incorporation of deuterium from deuterium oxide ((2) H2 O) into biological components is a commonly used approach in metabolic studies. Determining the dilution of deuterium in the body water (BW) pool can be used to estimate body composition. We describe three sensitive GC/MS/MS methods to measure water enrichment in BW. Samples were reacted with NaOH and U-(13) C3 -acetone in an autosampler vial to promote deuterium exchange with U-(13) C3 -acetone hydrogens. Headspace injections were made of U-(13) C3 -acetone-saturated air onto a 30-m DB-1MS column in electron impact-mode. Subjects ingested 30 ml (2) H2 O, and plasma samples were collected. BW was determined by standard equation. Dual-energy X-ray absorptiometry scans were performed to calculate body mass, body volume and bone mineral content. A four-compartmental model was used to estimate body composition (fat and fat free mass). Full-scan experiments generated an m/z 45 peak and to a lesser extent an m/z 61 peak. Product fragment ions further monitored included 45 and 46 using selected ion monitoring (Method1), the 61 > 45 and 62 > 46 transition using multiple reaction monitoring (MRM; Method2) and the neutral loss, 62 > 45, transition (Method3). MRM methods were optimized for collision energy (CE) and collision-induced dissociation (CID) argon gas pressure with 6 eV CE and 1.5 mTorr CID gas being optimal. Method2 was used for final determination of (2) H2 O enrichment of subjects because of lower natural background. We have developed a sensitive method to determine (2) H2 O enrichment in BW to enable measurement of FM and FFM., (Copyright © 2015 John Wiley & Sons, Ltd.)

The contribution of gluconeogenesis to hepatic glucose production (GP) was quantified after (2)H(2)O ingestion by Bayesian analysis of the position 2 and 5 (2)H-NMR signals (H2 and H5) of monoacetone glucose (MAG) derived from urinary acetaminophen glucuronide. Six controls and 10 kidney transplant (KTx) patients with cyclosporine A (CsA) immunosuppressant therapy were studied. Seven KTx patients were lean and euglycemic (BMI = 24.3 +/- 1.0 kg/m(2); fasting glucose = 4.7 +/- 0.1 mM) while three were obese and hyperglycemic (BMI = 30.5 +/- 0.7 kg/m(2); fasting glucose = 7.1 +/- 0.5 mM). For the 16 spectra analyzed, the mean coefficient of variation for the gluconeogenesis contribution was 10% +/- 5%. This uncertainty was associated with a mean signal-to-noise ratio (SNR) of 79:1 and 45:1 for the MAG H2 and H5 signals, respectively. For control subjects, gluconeogenesis contributed 54% +/- 7% of GP as determined by the mean and standard deviation (SD) of individual Bayesian analyses. For the lean/normoglycemic KTx subjects, the gluconeogenic contribution to GP was 62% +/- 7% (P = 0.06 vs. controls), while hyperglycemic/obese KTx patients had a gluconeogenic contribution of 68% +/- 3% (P < 0.005 vs. controls). These data suggest that in KTx patients, an increased gluconeogenic contribution to GP is strongly associated with obesity and hyperglycemia.

Lipogenesis and cholesterol synthesis can be studied by measuring the incorporation into fatty acids and cholesterol of deuterium from deuterated water. This has been previously achieved in human subjects using low levels of deuterium enrichment in plasma water, and thus in fatty acids and cholesterol. For the measurement of enrichment in lipids, this required the use of isotope ratio mass spectrometry, a tedious and time-consuming technique. It is shown that these measurements can be performed using the much simpler gas chromatography/mass spectrometry if higher, but always safe, deuterium enrichments in plasma water are obtained. Normal subjects ingested deuterated water in order to obtain stable enrichment in plasma water of 0.3% during a 60 h period. Enrichment in palmitate of plasma triglycerides (TG) plateaued (0.6-0.76%) whereas plasma cholesterol enrichment increased progressively [0.32 +/- 0.08% (12 h) to 0.78 +/- 0.18% (60 h)]. Endogenous synthesis was estimated to contribute, in post-absorptive subjects, 8-10% of the plasma TG pool and 3-5% of plasma free cholesterol pool. These data agree with results obtained previously using isotope ratio mass spectrometry. The present method will be useful for studies of normal and abnormal lipid metabolism in humans.

The exchange of individual protons of hepatic metabolites against the solvent deuterons has been investigated in perfused rat liver. Livers from starved rats were perfused for 20 min with a 10 mM solution of unlabeled or 3-13C-labeled L-alanine in Krebs Ringer bicarbonate buffer, with or without 50% deuterium oxide (2H2O). High resolution 13C NMR analysis of deuterium-induced isotopic shifts and of 2H-13C couplings revealed a differential 1H-2H exchange depending on the chemical nature of the metabolite and on the site of 13C labeling. [3-13C]Aspartate isotopomers showed similar 2H/1H ratios in the C3 and in the C2 carbons while [2-13C]aspartate isotopomers had much smaller 2H/1H ratios in the C2 than in the C3 carbons. Similarly, [2-13C]glutamate isotopomers had 2H/1H ratios significantly smaller in the C2 than in the C3 carbon. These results suggest that the hydration-dehydration reactions of the citric acid cycle, which result in exchange at the C3 carbons of aspartate and glutamate, approach equilibrium with the perfusate faster than the aminotransferases of aspartate and alanine, which induce exchange at the C2 carbons of these amino acids. Taken together, the results obtained are consistent with a heterogeneous solvent exchange environment in the perfused liver.