Several problems limit quantification of gluconeogenesis. We applied in vitro 2 H-nuclear magnetic resonance (NMR) spectroscopy to simultaneously measure 2 H in all glucose carbons for direct assessment of gluconeogenesis. This method was compared with 2 H measurement in carbons 5 and 2 using gas chromatography-mass spectrometry (hexamethylenetetramine [HMT]) and with in vivo 13 C magnetic resonance spectroscopy (MRS). After 14 h of fasting, and following 2 H 2 O ingestion, blood was obtained from nine healthy and seven type 2 diabetic subjects. Glucose was purified, acetylated, and analyzed for 2 H in carbons 1-6 with 2 H-NMR. Using 5:2 ratios, gluconeogenesis increased (P < 0.05) over time and mean gluconeogenesis was lower in control subjects than in type 2 diabetic patients (63 ؎ 3 vs. 75 ؎ 2%, P < 0.01).13 C-MRS revealed higher hepatic glycogenolysis in control subjects (3.9 ؎ 0.4 vs. 2.3 ؎ 0.2 mol ⅐ kg ؊1 ⅐ min ؊1 ) yielding mean contribution of gluconeogenesis of 65 ؎ 3 and 77 ؎ 2% (P < 0.005). Measurement of gluconeogenesis by 2 H-NMR correlated linearly with 13 C-MRS (r ‫؍‬ 0.758, P ‫؍‬ 0.0007) and HMT (r ‫؍‬ 0.759, P ‫؍‬ 0.0007). In an additional protocol, 2 H enrichments demonstrated a fast decline of gluconeogenesis from ϳ100 to ϳ68% (P < 0.02) within 4 h of galactose infusion after 40 -44 h of fasting. Thus, in vitro 2 H-NMR offers an alternative approach to determine fractional gluconeogenesis in good agreement with standard methods and allows monitoring of rapid metabolic alterations.

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