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Measurement of Fractional Whole-Body Gluconeogenesis in Humans From Blood Samples Using ²H Nuclear Magnetic Resonance Spectroscopy

O. Kunert¹, H. Stingl², E. Rosian¹, M. Krák², E. Bernroider², W. Seebacher¹, K. Zangger¹, P. Staehr³, V. Chandramouli⁴, B.R. Landau⁴, P. Nowotny², W. Waldhäusl², E. Haslinger¹, and M. Roden²

¹ Department of Pharmaceutical Chemistry, University of Graz, Graz, Austria
² Division of Endocrinology and Metabolism, Department of Internal Medicine III, University of Vienna, Vienna, Austria
³ Department of Medical Endocrinology, Institute of Clinical Research, University of Southern Denmark, Odense, Denmark
⁴ Department of Medicine and Biochemistry, Case Western Reserve University, Cleveland, Ohio

Several problems limit quantification of gluconeogenesis. We applied in vitro ²H-nuclear magnetic resonance (NMR) spectroscopy to simultaneously measure ²H in all glucose carbons for direct assessment of gluconeogenesis. This method was compared with ²H measurement in carbons 5 and 2 using gas chromatography–mass spectrometry (hexamethylenetetramine [HMT]) and with in vivo ¹³C magnetic resonance spectroscopy (MRS). After 14 h of fasting, and following ²H₂O ingestion, blood was obtained from nine healthy and seven type 2 diabetic subjects. Glucose was purified, acetylated, and analyzed for ²H in carbons 1–6 with ²H-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). ¹³C-MRS revealed higher hepatic glycogenolysis in control subjects (3.9 ± 0.4 vs. 2.3 ± 0.2 µmol · kg^-1 · min^-1) yielding mean contribution of gluconeogenesis of 65 ± 3 and 77 ± 2% (P < 0.005). Measurement of gluconeogenesis by ²H-NMR correlated linearly with ¹³C-MRS (r = 0.758, P = 0.0007) and HMT (r = 0.759, P = 0.0007). In an additional protocol, ²H enrichments demonstrated a fast decline of gluconeogenesis from 100 to 68% (P < 0.02) within 4 h of galactose infusion after 40–44 h of fasting. Thus, in vitro ²H-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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