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.

In type 2 diabetic patients, gluconeogenesis primarily accounts for increased endogenous glucose production (EGP) and fasting plasma glucose concentrations (1). Previous methods for measuring gluconeogenesis yielded a wide range of estimates in humans, which may relate to biochemical limitations of those methods (25). Gluconeogenesis can be also calculated from the difference between the rates of EGP determined with [6-3H]glucose and of liver glycogen breakdown determined by in vivo 13C-nuclear magnetic resonance spectroscopy (MRS) (1,6). This method quantifies rates of gluconeogenesis but requires patients to lie for hours within a magnet (7). An alternative method is based on incorporation of 2H into glucose after 2H2O administration (8). In humans, gluconeogenesis can be calculated from 2H enrichments in hexamethylenetetramine (HMT) derived from blood glucose measured with gas chromatography–mass spectrometry (9,10). Briefly, 2H is incorporated into position 3 of phosphoenolpyruvate (PEP) during the equilibration of pyruvate with alanine and via conversion of the pyruvate to oxaloacetate to the extent it equilibrates with fumarate. The carbon at position 3 of PEP becomes carbon 6 of glucose. 2H is incorporated into position 2 of glyceraldehyde-3-P via its isomerization with dihydroxyacetone-3-P and via hydration of PEP to 2-phosphoglycerate. During glycerol conversion, 2H is introduced at position 2 of glyceraldehyde-3-P. Carbon two of glyceraldehyde-3-P becomes carbon 5 of glucose. Subsequently, position 2 of glucose is labeled with 2H during both gluconeogenesis and glycogenolysis. Thus, the ratio of 2H enrichment at position 5 to that at two of glucose represents the fractional contribution of gluconeogenesis to EGP (9,10). This method was shown to overcome limitations of previous methods in humans but requires extensive and time-consuming biochemical preparations.

Determination of 5:2 ratios by in vitro 2H-NMR spectroscopy using monoacetone glucose (1114) has been reported. Biochemical short-comings of previous approaches and invasive or time-consuming procedures can be avoided by this method. In the present study, 5:2 ratios were obtained by an alternative approach applying in vitro 2H-NMR on a glucopyranosyl bromide derivative. The estimates of gluconeogenesis were compared with both in vivo 13C-MRS and the HMT method.

This method was tested under both the usually applied conditions of continuously increasing gluconeogenesis (i.e., overnight and prolonged fasting) and the conditions of rapidly decreasing fractional contribution of gluconeogenesis to EGP (i.e., intravenous galactose infusion after prolonged fasting). In the prolonged fasted state, gluconeogenesis almost completely accounts for EGP, but since galactose in liver is converted to glucose via glucose-1-P and glucose-6-P (the same intermediates as in glycogenolysis), galactose administration is expected to cause a rapid fall in the contribution of gluconeogenesis to EGP.

Clinical protocols

Study on the evaluation of the method.

Nine healthy male volunteers (control subjects) (age range 22–28 years, BMI 22.6 ± 0.5 kg/m2, and HbA1c 5.1 + 0.1%) and seven well-controlled type 2 diabetic patients (age range 49–62 years, BMI 26.1 ± 0.9 kg/m2, and HbA1c 7.1 + 0.2%) were on an isocaloric diet (>50% carbohydrates) and refrained from physical exercise for ≥3 days. The patients discontinued oral hypoglycemic agents at least 3 days before the study. None of them suffered from diabetes-related complications. All participants gave informed written consent to the protocols, which were approved by the institutional ethics board, and all participants participated in two protocols in randomized order. They ingested a liquid meal (60% carbohydrate, 20% protein, and 20% fat) at 6:00 p.m. (15) and fasted overnight. On day 1, they drank 5 g 2H2O (99.9% 2H; Cambridge Isotope Laboratory, Andover, MA) per kilograms of body water divided into four doses spaced 30 min from 6:00 (12 h of fasting) to 7:30 a.m. (10,16). Blood was drawn at 6:00, 8:00 (14 h of fasting), and 10:00 (16 h of fasting) a.m. and at the end of the study at noon (18 h of fasting). The volume of blood samples was 30 ml per time point and was doubled in some experiments for determination of the precision of the method. No side effects of 2H2O were observed.

On another day, subjects first received an intravenous bolus, adjusted for plasma glucose concentration, and from 6:00 until noon (18 h of fasting) they received a continuous infusion of d-[6,6-2H2]glucose (99% enriched; Cambridge Isotope Laboratory). Simultaneously, liver glycogen concentration was monitored by in vivo 13C-MRS from 12.5–13.5 and 15–16 h and at the end of the study (17–18 h of fasting). Blood samples were chilled and centrifuged, and supernatants were stored at −20°C.

Study on the effect of galactose.

Three healthy male subjects (age range 24–25 years, BMI 23.7 ± 2.0 kg/m2, and HbA1c 5.0 + 0.1%) participated in this protocol, which was identical to the first study including ingestion of 2H2O until completion of day 1, but they were continued for a total of 44 h of fasting. Throughout the study, subjects had free access to water containing 0.5% 2H2O to maintain isotopic equilibrium in body water. At 40 h of fasting, galactose (9 g/h) was intravenously infused for 4 h. Subjects were also infused with d-[6,6-2H2]glucose (99% enriched; Cambridge Isotope Laboratory), and liver glycogen concentrations were measured from 37.5 until 44 h of fasting. Blood samples were obtained at 16, 18, 40, 42, and 44 h and treated as above.

Preparation of blood glucose.

Blood (∼30 ml) was diluted with equal volumes of water and deproteinized with ZnSO4/Ba(OH)2. After lyophilization, plasma was purified on anion exchange column (1.6 × 10 cm) filled with Bio-Rad resins 4.5 g AG1-X8 (acetate form) and 4.0 g AG50W-X8 (proton form). The glucose was eluted with water, and glucose-containing fractions (∼6 ml) were analyzed for glucose by glucose test sticks. In test-runs with pure glucose, a yield of close to 100% of the starting material was achieved.

α,β-d-pentaacetyl glucose.

Lyophilized glucose was dissolved in dry pyridine (6.7 μl pyridine/mg glucose) and incubated (0°C, 18 h) with acetic anhydride (4.6 μl/mg glucose). After dilution with five times the volume of water and lyophilization, a mixture of α- and β-d-pentaacetylglucose (PAG) was obtained.

2,3,4,6-tetra-O-acetyl-α-d-glucopyranosyl bromide.

The mixture of α- and β-PAG was dissolved in dry chloroform (10 μl/mg PAG), and hydrogen bromide (10 μl/mg PAG) dissolved in glacial acetic acid was added at 0°C. After 1.5 h incubation and the addition of the same volume of chloroform, the solution was washed five times with water. After lyophilization, 2,3,4,6-tetra-O-acetyl-α-d-glucopyranosyl bromide was obtained as a yellowish oil and dissolved in natural abundance benzene. 1H spectra of the product were recorded in some samples and revealed a purity of the derivative of >95%.

1,2–5,6-di-O-isopropylidene-α-d-glucofuranose.

Glucose was dissolved in dry acetone (0.35 ml/mg glucose), and anhydrous ZnCl2 (5 mg/mg glucose) and concentrated phosphoric acid (0.5 μl/mg glucose) were added. After 18 h, Na2CO3 solution was added to precipitate zinc ions. 1,2–5,6-di-O-isopropylidene-α-d-glucofuranose was obtained by lyophilization of the supernatant and in natural abundance acetone.

In vitro2H-NMR.

2H enrichments at positions 1, 2, 3, 4, 5, 6, and 6‘ were obtained from spectra that were acquired on a Varian Unity INOVA spectrometer (9.4 T, 400 MHz; 5-mm broad band probe) (Fig. 1). For determination of optimal acquisition times, various 2,3,4,6-tetra-O-acetyl-α-d-glucopyranosyl bromide derivatives were prepared from d-[5-2H]glucose (98% enriched in 2H; Omicron Biochemicals, South Bend, IN), d-[2-2H]glucose (98% enriched in 2H; Cambridge Isotope Laboratory), and perdeuterated glucose (98% enriched in 2H; Sigma Chemical, Perth, Australia). Signal intensities were not different for acquisition times between 1.5 and 3 s, which were used in all further experiments. To reduce line-broadening in unlocked mode occurring during acquisition of spectra for 5–20 h, the transmitter frequency was adjusted by the shift of the solvent resonance after each set of 2,048 transients. This yielded a resolution of 1.5 Hz, even after 36 h. All spectra were multiplied with an exponential window function of 1.5-Hz line broadening before Fourier transformation. Spectra were least-square fitted with Lorentzian lines using the Varian Vnmr deconvolution routine (17). The natural line widths of the glucose resonances were 7–8 Hz, whereas that of the solvent, benzene, was 3.6 Hz. Spectra were acquired at a temperature of 303 K. The percent contribution of gluconeogenesis to EGP was set to 100 times the ratio of 2H enrichments at position 5 to that at carbon 2 (5:2) of blood glucose (10).

In vivo 13C MRS.

Subjects were lying within a magnet (3-T Medspec 30/80-DBX system; Bruker Medical, Ettlingen, Germany) using a 10-cm double-tuned 1H/13C circular coil (15,18). Spectra were acquired using a modified 1d inversion-based sequence (6) without 1H decoupling (19). Glycogen concentrations were measured by integration of the carbon 1 glycogen doublet at 100.5 ppm using the same frequency bandwidth (±300 Hz) for all spectra. Absolute quantification was performed by comparing the peak integral with that of a glycogen standard (6). Corrections for loading and sensitive volume of the coil were done. Liver volumes were measured in a 1.5-T Vision imager (Siemens, Germany) using a body array coil and in-phase and postphase multislice FLASH imaging sequences (15,18).

Gas chromatography-mass spectrometry.

2H enrichments at positions 2 and 5 of blood glucose were determined by isolating the hydrogens in formaldehyde derivatized to HMT (9,10,20). Briefly, deproteinized blood samples were deionized by ion exchange, and the glucose was purified by high-performance liquid chromatography. Aliquots of glucose were converted to arabitol-5-P and ribitol-5-P and to xylose, which were oxidized with periodate to yield formaldehyde containing carbons 2 and 5 of glucose, respectively. The HMTs were assayed for 2H enrichments by mass spectrometry using mass 141 for the enrichments at positions 2 and 5.

To assess EGP, 2H enrichments in carbon 6 of glucose pentaacetate were assayed on a Hewlett-Packard 5,890 gas chromatograph interfaced to a Hewlett-Packard 5971A mass selective detector (15,18). M + 2 enrichments were measured from the mass-to-charge ratio of 202:200 of the fragment ion consisting of carbons 2–6. Rates of EGP were calculated by using the Steele equations for non–steady-state conditions (21).

2H enrichments in plasma water were measured by Metabolic Solutions (Nashua, NH) using an isotopic ratio mass spectrometer.

Metabolites and hormones.

Plasma glucose was measured on a Glucose Analyzer II (Beckman, Fullerton, CA). Plasma free fatty acids (FFAs) (Wako Chemical, Neuss, Germany) (intra- and inter-assay coefficient of variation [CV] <6%), lactate (CV 3.3%), and β-hydroxy-butyrate (CV <9%) were quantified enzymatically. Plasma insulin, C-peptide, glucagon, and cortisol were measured by radioimmunoassay as described (15,16,22).

Data analysis.

Data are presented as means ± SE unless otherwise stated. Linear regression analysis was performed by least-square fitting of data. One-way ANOVA with Bartlett’s test for equal variances and paired Student’s t tests were used as appropriate.

Study on the evaluation of the method

Metabolites and hormones.

Plasma glucose was higher in type 2 diabetic patients (9.9 ± 1.1 mmol/l) than in control subjects (5.4 ± 0.1 mmol/l; P < 0.005). Type 2 diabetic patients also exhibited increased plasma concentrations of FFA (0.48 ± 0.03 vs. 0.35 ± 0.03 mmol/l; P < 0.001) and lactate (1.26 ± 0.12 vs. 0.50 ± 0.09 mmol/l; P < 0.01) but not β-hydroxy-butyrate (0.12 ± 0.01 vs. 0.11 ± 0.01 mmol/l). Mean plasma insulin (60 ± 12 vs. 39 ± 3 pmol/l) and C-peptide (749 ± 149 vs. 484 ± 44 pmol/l) were higher (P < 0.005) in type 2 diabetic patients than in control subjects. Mean plasma glucagon (81 ± 10 vs. 70 ± 5 pg/ml), cortisol (337 ± 36 vs. 375 ± 37 nmol/l), and growth hormone (1.8 ± 0.7 vs. 1.0 ± 0.4 μg/l) were comparable between type 2 diabetic patients and control subjects. All of these parameters were not different between both days.

Precision of in vitro 2H-NMR.

After 10 h of accumulation of spectra in a sample containing 91 mg 2,3,4,6-tetra-O-acetyl-a-d-glucopyranosyl bromide, the signal-to-noise (s/n) ratio of the smallest peak, i.e., resonance at carbon 6′, was ∼20 for acquisition times of 3 s (12,000 transients).

To assess the reproducibility, spectra were recorded under identical conditions yielding an SD <3% for 5:2 ratios. At lowest s/n ratio of ∼10 (for the resonance at carbon 6′), the SD of the 5:2 ratio increased to ≤6%.

To estimate the accuracy of the NMR measurements as a function of the s/n ratio, 2 mg 2,4,6-tetra-O-acetyl-α-d-[2-2H]glucopyranosyl bromide were dissolved in natural abundance benzene, and four sets of 2H spectra were recorded with increasing numbers of transients (4, 16, 32, 64, 128, 256), giving a total of 24 measurements. The s/n ratio of the glucose signal ranged between 8 and 64 (4 and 256 transients). The ratios of the integrated peak areas as determined by deconvolution of the spectra were plotted as a function of the s/n ratios of the 2H signal of benzene and yielded CVs within 2% if the s/n ratio exceeded a value of ∼20.

The precision of data processing was determined by integrating the 2H peaks three times in two different samples, yielding a variance <2%.

Absolute enrichments were checked with solutions containing d-[5-2H]glucose and d-[2-2H]glucose giving a theoretical 5:2 ratio of 49.9%. The measured 5:2 ratios were ∼51.3% (0.595/1.160) with in vitro 2H-NMR and ∼50.5% (0.489/0.969) with the HMT method.

Gluconeogenesis from in vitro 2H-NMR

2H enrichments in plasma water.

After 2H2O ingestion, 2H enrichments in plasma water were 0.49 ± 0.03% at 14 h, 0.47 ± 0.01% at 16 h, and 0.46 ± 0.01% at 18 h of fasting.

2H-NMR spectra.

2H spectra of 2,3,4,6-tetra-O-acetyl-α-d-glucopyranosyl bromide exhibited well-resolved 2H signals at positions 1, 2, 3, 4, and 6′, whereas those at 5 and 6 were superimposed (Fig. 1). 2H spectra of 3-O-acetyl-1,2–5,6-di-O-isopropylidene-α-d-glucofuranose allowed separation of signals at positions 6 and 6′, whereas those at 2 and 5 overlapped. 2H enrichments at positions 6 and 6′ were identical (ratio at 6:6′ 1.03 ± 0.08). Thus, for the glucopyranosyl bromide derivative, the integral of the resonance at carbon 5 was obtained by subtracting the 2H enrichments at position 6′ from the combined resonances at carbons 5 and 6. This allowed us to quantify 2H enrichments at each of the seven positions and to calculate 2H incorporation into glucose. Because the glucopyranosyl bromide derivative is obtained in a yield of 75–95% in a simple two-step reaction, we used it as the compound of choice.

From the 5:2 ratios of 2H enrichments, gluconeogenesis increased from ∼62 and ∼51% at 14 h to ∼87 (P < 0.001) and ∼73% (P = 0.005) at 18 h of fasting in type 2 diabetic patients and control subjects, respectively (Table 1, Fig. 2). Type 2 diabetic patients presented with higher gluconeogenesis than control subjects (P < 0.01). Mean gluconeogenesis over the whole study period (14–18 h fasting) was ∼75% in type 2 diabetic patients and ∼63% in control subjects. In both type 2 diabetic patients and control subjects, the 3:2 and 6:2 ratios were lower (P < 0.001), whereas the 1:2 and 4:2 ratios were higher (P < 0.001) than the 5:2 ratios (Table 1). Using the HMT method, the 5:2 ratios gave an estimate of gluconeogenesis of 60 ± 3 and 50 ± 3% at 16 h and 67 ± 3% as well as 51 ± 3% at 18 h in type 2 diabetic patients and control subjects (P < 0.001), respectively (mean 63 ± 3 and 51 ± 3%, P < 0.005 vs. NMR method) (Table 2).

Gluconeogenesis from in vivo 13C MRS.

Hepatic glycogen concentrations were lower (P < 0.0001) in type 2 diabetic patients at 12.5–13.5 h (210 ± 8 vs. 244 ± 6 mmol/l liver) (Fig. 2). In all individuals of both groups, glycogen declined linearly (type 2 diabetic patients r2 = 0.73 ± 0.07, P < 0.01; control subjects r2 = 0.85 ± 0.04, P < 0.01) and was also lower in type 2 diabetic patients at 17–18 h of fasting (175 ± 7 vs. 197 ± 6 mmol/l liver, P < 0.0001). Mean rates of glycogen breakdown were also lower in type 2 diabetic patients than in control subjects (2.3 ± 0.2 vs. 3.9 ± 0.4 μmol · kg body wt−1 · min−1P < 0.005) (Table 2). Rates of EGP were higher in type 2 diabetic patients (16.3 ± 1.2 vs. 11.2 ± 0.4 μmol · kg−1 · min−1, P < 0.001) (Table 2). The difference between EGP and glycogenolysis gives the absolute rate of gluconeogenesis (7.9 ± 0.6 and 7.2 ± 0.5 μmol · kg body wt−1 · min−1), which accounted for 85 ± 2 and 65 ± 3% of EGP, respectively (P < 0.0005 type 2 diabetic patients vs. control subjects) (Table 2). Individual data of body weight (type 2 diabetic patients 85 ± 5 kg and control subjects 73 ± 2 kg) and liver volume (1.62 ± 0.04 and 1.59 ± 0.04 l, respectively) were used to calculate rates of gluconeogenesis and glycogenolysis.

Estimates of the contributions of gluconeogenesis to EGP from in vitro 2H-NMR correlated linearly with that from in vivo 13C-MRS (r = 0.757, P = 0.0007) (Fig. 3). In vitro 2H-NMR also linearly correlated with HMT measurements (r = 0.759, P = 0.0007). Finally, in vivo 13C-MRS and HMT measurements were linearly related (r = 0.713, P = 0.002).

Study on the effect of galactose

Metabolites and hormones.

Plasma glucose declined (P < 0.05) from 5.1 ± 0.1 mmol/l at 12 h to 4.1 ± 0.1 mmol/l at 40 h. Thereafter, plasma glucose rose by ∼24% (P < 0.01) during galactose infusion (Fig. 4). Plasma insulin concentrations were 17 ± 1 pmol/l at 40 h and 27 ± 5 pmol/l at 44 h. During galactose infusion, plasma C-peptide increased from 245 ± 35 to 428 ± 59 pmol/l (P < 0.05). Plasma FFAs were 0.70 ± 0.12 mmol/l at 12 h, 0.86 ± 0.06 mmol/l at 40 h, and 0.73 ± 0.08 mmol/l at 44 h.

Glycogen metabolism from in vivo 13C MRS.

Liver glycogen concentrations were 86 ± 4 mmol/l liver at the 37- to 38-h fast and declined (P < 0.005) to 77 ± 3 mmol/l at 39–40 h (Fig. 4), giving rates of hepatic glycogenolysis of 59 ± 12 μmol · l liver−1 · min−1. EGP was 9.2 ± 0.6 μmol · kg−1 · min−1 at 40 h so that gluconeogenesis measured by in vivo 13C-MRS was 89 ± 2% at ∼37–40 h of fasting. During galactose infusion, liver glycogen concentrations rose from 78 ± 9 mmol/l (∼40–41 h) to 104 ± 11 (∼41.5–42.5 h, P < 0.0005) and 109 ± 10 mmol/l (∼43–44 h, P < 0.0001), giving rates of hepatic glycogen synthesis of 3.1 ± 0.6 μmol · kg−1 · min−1. EGP remained constant during galactose infusion (mean 8.8 ± 0.2 μmol · kg−1 · min−1).

Gluconeogenesis from 2H enrichments.

Using the HMT method, gluconeogenesis contributed to EGP by 94 ± 5% at 40 h (P < 0.005 vs. 16 h 61 ± 5%) and declined to 72 ± 2% (42 h, P < 0.05) and 69 ± 6% (44 h, P < 0.005) during galactose infusion. Using in vitro 2H-NMR, gluconeogenesis contributed to EGP by 99 ± 6% at 40 h and declined to 79 ± 2 and 70 ± 3% (44 h, P < 0.02) during galactose infusion (Fig. 4). Estimates for gluconeogenesis were not different between both methods.

In vitro 2H-NMR has recently been used to measure 2H incorporation from 2H2O in glucose from various natural sources. In one study (23), glucose, isolated from plants, was converted to 3,6-anhydro-1.2-O-isopropylidene-α-d-glucofuranose. That derivative exhibited all seven separated 2H resonances at 61 MHz, but multiple preparation steps prevent its efficient application on a micro scale. In another study (12), glucose, isolated from rat plasma, was oxidized to its gluconate. 2H at carbon 1 is lost during the conversion, and resolved signals are only obtained for carbons 2 and 3. The 3:2 ratio was then used to estimate gluconeogenesis. The same group reported fully resolved 2H resonances employing monoacetone glucose (11) using body water enrichments of 2H between 2.5–5% in rats. More recently, this method was used to estimate gluconeogenesis in humans at body water enrichments of 0.5% (13). We found our derivative to be easier to prepare and provide a higher overall yield. However, the resolution of monoacetone glucose allows the baseline separation of all 2H resonances at 10 T, and the line width of NMR signals was slightly superior in our hands.

The present study also used 2H enrichments of 0.5% in body water as reported previously in other studies in humans (9,10,13,16). These procedures make determination of 2H enrichments at all carbons of glucose feasible at a safe dose of 2H2O in humans. Although the 2H resonances at carbons 5 and 6 of glucose overlapped, identical 2H enrichments at positions 6 and 6′—which is in agreement with studies in rats (11)—allowed us to calculate 5:2 ratios. Our estimates of gluconeogenesis are similar to those reported at 15 and 18.5 h of fasting (24) but somewhat higher than in other studies (2,9) using the HMT method. Measurements of gluconeogenesis by the HMT method and in vitro 2H-NMR were linearly correlated, although the latter method gave higher estimates of 5:2 ratios. As both methods measure 5:2 ratios in blood glucose, no differences are to be expected. One potential explanation for the higher estimates from in vitro 2H-NMR could reside in the calculation of 5:2 ratios, which requires subtracting the peak area at position 6′ from that at 5 and 6 in order to obtain the peak area at position 5. Although the peak areas at positions 6′ and 6 of glucopyranosyl bromide were found to be identical, small variations in these peaks could affect the 5:2 ratios. Thus, the HMT method could be quantitatively more correct, whereas this 2H-NMR method would be most useful for evaluating relative changes in the fractional enrichments. Nevertheless, both estimates are in the range of 50–65% observed after overnight fasting (2,6,16,18,24).

Interestingly, the 3:2 ratios gave lower (P = 0.029) estimates of gluconeogenesis than the 5:2 ratios, which is similar to findings in fasted rats (11). The differences between the 6:2 and 5:2 ratios were even greater (19%, P < 0.001) and similar to that reported for the HMT method (16–22% at 14–22 h of fasting) (9,10). The 6:2 ratio has been used for measuring gluconeogenesis (9) but gives an underestimation because 1) binding of 2H at position 6 depends on the exchange of the hydrogens of pyruvate and alanine and of oxaloacetate and fumarate with those of water in their equilibrations before oxaloacetate’s conversion to glucose, and 2) the conversion of glycerol to glucose does not result in binding of 2H at position 6 (25). Assuming complete exchange of hydrogens in body water at the level of pyruvate (pyruvate alanine and oxalaceatate fumarate), the 6:2 ratios reflect gluconeogenesis from the level of PEP, and the difference of 5:2 and 6:2 ratios reflects gluconeogenesis from glycerol. Using the individual data at each time point, gluconeogenesis from glycerol contributes to EGP by 27 ± 4 and 19 ± 4% in type 2 diabetic patients and control subjects (P = 0.05), respectively. The estimates of the contribution of glycerol are somewhat higher than in previous reports (13,26). Nevertheless, in type 2 diabetic patients, the lower contribution of glycogen to EGP could be due to augmented gluconeogenesis from glycerol rather than from PEP, which was similar in type 2 diabetic patients and control subjects, as demonstrated by comparable 6:2 ratios (Table 1).

In contrast, the 1:2 ratio would have overestimated the contribution of gluconeogenesis to EGP, which is in agreement with a previous study (25). That is presumably explained by a contribution of glycogenolysis to the 2H at position 1 during short-term fasting because of equilibration of mannose-6-P with glucose-6-P via fructose-6-P (25).

To our knowledge, 4:2 ratios have not yet been compared with 5:2 ratios in humans in a similar experimental setup, i.e., after 2H2O ingestion. The 4:2 ratios were higher than the 5:2 ratios in both healthy and type 2 diabetic subjects, suggesting that this finding could be a constant phenomenon. With the equilibration at the triose-P level, one might expect that labeling at position 3 and 4 would approach that at position 5 but not exceed it.

Current limitations such as the large sample volume and long duration of spectra accumulation should be reduced by employing a custom-built NMR probe. With such equipment the costs of our method will be in the range of those of the HMT method but much less compared with in vivo 13C MRS.

Our study demonstrates that with all methods used, the contribution of gluconeogenesis to EGP is also higher in well-controlled type 2 diabetic patients as previously demonstrated for type 2 diabetic patients with moderate metabolic control by 13C-MRS (1,27) and the HMT method (24,28). Likewise, EGP was elevated in type 2 diabetic patients, which is in line with the majority of studies (29,30). As glycogenolysis was decreased in type 2 diabetic patients, probably due to lower hepatic glycogen concentrations, augmented gluconeogenesis mainly explains the increased EGP in these patients.

The conversion of galactose to glucose serves as a surrogate for glycogenolysis, since the same pathway is followed in both processes, i.e., the galactose-1-P formed is converted to glucose-1-P and hence to glucose-6-P. The decline in the percent of contribution of gluconeogenesis to glucose production was the same, ∼30% over the 4 h of galactose infusion, confirmed by either the HMT or 2H-NMR method. Of the approximate 11.4 μmol · kg−1 · min−1 infused, ∼29%, i.e., 3.1 μmol · kg−1 · min−1, appears to have been deposited in liver glycogen and ∼23% (fractional decrease in gluconeogenesis times EGP), i.e., 2.6 μmol · kg−1 · min−1, was converted to glucose that entered the circulation. While the use of 5:2 ratios gives a measure of the fractional contribution of gluconeogenesis to glucose production, the increase in glycogen content cannot be taken as a measure of the contribution of gluconeogenesis to glycogen formation under these conditions (31), since the conversion of galactose to glycogen does not require glucose 6-P as an intermediate, i.e., galactose-1-P is converted to UDP-glucose and hence glycogen. Also, galactose may not be considered a gluconeogenic substrate, since its conversion to glucose requires only epimerization of its hydroxyl at position, while its carbon skeleton remains intact.

In conclusion, in vitro 2H-NMR offers an alternative valid approach to directly quantify gluconeogenesis because this method 1) correlates linearly with established methods such as the HMT method and in vivo 13C MRS, 2) can be effectively applied in type 2 diabetic patients, and 3) allows monitoring of rapid changes in glycogenolysis and gluconeogenesis in humans. This could be of clinical relevance for studying glucose metabolism and new therapeutic strategies for diabetes treatment.

FIG. 1.

2H-NMR spectra of 2,3,4,6-tetra-O-acetyl-α-d-glucopyranosyl bromide obtained in a single blood sample of one healthy volunteer at 14, 16, and 18 h of fasting upon ingestion of 2H2O (5 g/kg body water).

FIG. 1.

2H-NMR spectra of 2,3,4,6-tetra-O-acetyl-α-d-glucopyranosyl bromide obtained in a single blood sample of one healthy volunteer at 14, 16, and 18 h of fasting upon ingestion of 2H2O (5 g/kg body water).

FIG. 2.

Liver glycogen concentrations (A) determined by in vivo 13C-MRS (*P < 0.0001 vs. basal, §P < 0.0001 vs. healthy subjects) and percent gluconeogenesis (B) determined by in vitro 2H-NMR (*P < 0.05 vs. basal, §P < 0.05 vs. healthy subjects) in healthy volunteers (n = 9; •) and well-controlled type 2 diabetic patients (n = 7; ▴) during 18 h fasting. Data are given as means ± SE.

FIG. 2.

Liver glycogen concentrations (A) determined by in vivo 13C-MRS (*P < 0.0001 vs. basal, §P < 0.0001 vs. healthy subjects) and percent gluconeogenesis (B) determined by in vitro 2H-NMR (*P < 0.05 vs. basal, §P < 0.05 vs. healthy subjects) in healthy volunteers (n = 9; •) and well-controlled type 2 diabetic patients (n = 7; ▴) during 18 h fasting. Data are given as means ± SE.

FIG. 3.

Correlation of estimates of percent gluconeogenesis obtained by in vitro 2H-NMR and in vivo 13C-MRS in healthy subjects (n = 9; •) and well-controlled type 2 diabetic patients (n = 7; ▴).

FIG. 3.

Correlation of estimates of percent gluconeogenesis obtained by in vitro 2H-NMR and in vivo 13C-MRS in healthy subjects (n = 9; •) and well-controlled type 2 diabetic patients (n = 7; ▴).

FIG. 4.

Plasma glucose concentrations (A) (*P < 0.05 vs. 40 h), liver glycogen concentrations (B) (*P < 0.01 vs. 40 h), and percent gluconeogenesis obtained from in vitro 2H-NMR (C) (*P = 0.02 vs. 40 h) during infusion of galactose (9 g/h) in healthy volunteers (n = 3).

FIG. 4.

Plasma glucose concentrations (A) (*P < 0.05 vs. 40 h), liver glycogen concentrations (B) (*P < 0.01 vs. 40 h), and percent gluconeogenesis obtained from in vitro 2H-NMR (C) (*P = 0.02 vs. 40 h) during infusion of galactose (9 g/h) in healthy volunteers (n = 3).

TABLE 1

2H enrichments (%) at carbons 1, 3, 4, 5, and 6 relative to those at carbon 2 in plasma glucose of healthy subjects (n = 9) and type 2 diabetic subjects (n = 7) between 14 and 18 h of fasting after ingestion of 5 g 2H2O per kg body water

1/23/24/25/26/2
Control subjects      
 14 h 73 ± 4 49 ± 8 75 ± 4 51 ± 5 43 ± 3 
 16 h 67 ± 5 42 ± 6 73 ± 7 65 ± 4 48 ± 6 
 18 h 81 ± 3 53 ± 6 94 ± 6 73 ± 5 46 ± 3 
 14–18 h 74 ± 3 49 ± 6 81 ± 4 63 ± 3 46 ± 3 
Type 2 diabetic subjects      
 14 h 81 ± 3 43 ± 3 73 ± 3 62 ± 2 44 ± 4 
 16 h 81 ± 5 42 ± 4 85 ± 6 75 ± 3 47 ± 3 
 18 h 84 ± 2 49 ± 2 88 ± 3 87 ± 2 51 ± 3 
 14–18 h 82 ± 3 45 ± 2 82 ± 3 75 ± 2 47 ± 3 
1/23/24/25/26/2
Control subjects      
 14 h 73 ± 4 49 ± 8 75 ± 4 51 ± 5 43 ± 3 
 16 h 67 ± 5 42 ± 6 73 ± 7 65 ± 4 48 ± 6 
 18 h 81 ± 3 53 ± 6 94 ± 6 73 ± 5 46 ± 3 
 14–18 h 74 ± 3 49 ± 6 81 ± 4 63 ± 3 46 ± 3 
Type 2 diabetic subjects      
 14 h 81 ± 3 43 ± 3 73 ± 3 62 ± 2 44 ± 4 
 16 h 81 ± 5 42 ± 4 85 ± 6 75 ± 3 47 ± 3 
 18 h 84 ± 2 49 ± 2 88 ± 3 87 ± 2 51 ± 3 
 14–18 h 82 ± 3 45 ± 2 82 ± 3 75 ± 2 47 ± 3 

Data are means ± SEM.

TABLE 2

Summary of EGP, hepatic glycogenolysis, and gluconeogenesis in 7 type 2 diabetic and 9 nondiabetic volunteers

Type 2 diabetic subjects
Control subjects
Absolute rate (μmol · kg−1 · min−1)Fraction of EGP (%)Absolute rate (μmol · kg−1 · min−1)Fraction of EGP (%)
EGP 16.3 ± 1.2* 100 11.2 ± 0.4 100 
Glycogenolysis 2.3 ± 0.2* 15 ± 2* 3.9 ± 0.4 35 ± 3 
GNG (13C-MRS) 14 ± 1.3* 85 ± 2* 7.3 ± 0.4 65 ± 3 
GNG (2H-NMR) — 75 ± 2* — 63 ± 3 
GNG (HMT) — 63 ± 3* — 51 ± 3 
Type 2 diabetic subjects
Control subjects
Absolute rate (μmol · kg−1 · min−1)Fraction of EGP (%)Absolute rate (μmol · kg−1 · min−1)Fraction of EGP (%)
EGP 16.3 ± 1.2* 100 11.2 ± 0.4 100 
Glycogenolysis 2.3 ± 0.2* 15 ± 2* 3.9 ± 0.4 35 ± 3 
GNG (13C-MRS) 14 ± 1.3* 85 ± 2* 7.3 ± 0.4 65 ± 3 
GNG (2H-NMR) — 75 ± 2* — 63 ± 3 
GNG (HMT) — 63 ± 3* — 51 ± 3 

Data are means ± SEM. Comparison of estimates of gluconeogenesis (GNG) as calculated from 5:2 ratios in plasma glucose measured with in vitro 2H-NMR spectroscopy, from HMT and from the difference between EGP and hepatic glycogenolysis measured with in vivo 13C-MRS.

*

P < 0.01 vs. control subjects

P < 0.005 vs. HMT.

O.K. and H.S. contributed equally to this work.

The studies were supported by the Austrian Science Foundation (P13213-MOB, P13722-MED to M.R., P13718-CHE to E.H.), Novo Nordisk (to W.W.), National Institutes of Health (RO1-DK-14507 to B.R.L.), Austrian National Bank (9127 to H.S. and M.R.), and the Austrian Academy of Sciences (427/1997 to H.S.).

We thank D. Weghuber and the lab of the Division of Endocrinology and Metabolism, University of Vienna, for technical assistance and E. Moser, PhD, and the Department of Radiology for support.

1.
Magnusson I, Rothman DL, Katz LD, Shulman RG, Shulman GI: Increased rate of gluconeogenesis in type II diabetes mellitus: A 13C nuclear magnetic resonance study.
J Clin Invest
90
:
1323
–1327,
1992
2.
Landau BR: Quantifying the contribution of gluconeogenesis to glucose production in fasted human subjects using stable isotopes.
Proc Nutr Soc
58
:
963
–972,
1999
3.
Radziuk J, Lee WP: Measurement of gluconeogenesis and mass isotopomer analysis based on [U-13C]glucose.
Am J Physiol
277
:
E199
–E207,
1999
4.
Inzucchi SE, Maggs DG, Spollett GR, Page SL, Rife FS, Walton V, Shulman GI: Efficacy and metabolic effects of metformin and troglitazone in type II diabetes mellitus.
N Engl J Med
338
:
867
–872,
1998
5.
Mao CS, Bassilian S, Lim SK, Lee WN: Underestimation of gluconeogenesis by the [U-13C6]glucose method: effect of lack of isotope equilibrium.
Am J Physiol Endocrinol Metab
282
:
E376
–E385,
2002
6.
Rothman DL, Magnusson I, Katz LD, Shulman RG, Shulman GI: Quantitation of hepatic glycogenolysis and gluconeogenesis in fasting humans with 13C NMR.
Science
254
:
573
–576,
1991
7.
Roden M, Shulman GI: Applications of NMR spectroscopy to study muscle glycogen metabolism in man.
Annu Rev Med
50
:
277
–290,
1999
8.
Guo ZK, Lee WN, Katz J, Bergner AE: Quantitation of positional isomers of deuterium-labeled glucose by gas chromatography/mass spectrometry.
Anal Biochem
204
:
273
–282,
1992
9.
Landau BR, Wahren J, Chandramouli V, Schumann WC, Ekberg K, Kalhan SC: Use of 2H2O for estimating rates of gluconeogenesis: application to the fasted state.
J Clin Invest
95
:
172
–178,
1995
10.
Landau BR, Wahren J, Chandramouli V, Schumann WC, Ekberg K, Kalhan SC: Contributions of gluconeogenesis to glucose production in the fasted state.
J Clin Invest
98
:
378
–385,
1996
11.
Jones JG, Sherry AD, Malloy CR: Analysis of 2H enrichment in all positions of plasma glucose by 2H-NMR spectroscopy following infusion of 2H2O.
Proc Intl Soc Mag Reson Med
8
:
876
,
2000
12.
Jones JG, Carvalho RA, Sherry AD, Malloy CR: Quantitation of gluconeogenesis by 2H nuclear magnetic resonance analysis of plasma glucose following ingestion of 2H2O.
Anal Biochem
277
:
121
–126,
2000
13.
Jones JG, Solomon MA, Cole SM, Sherry AD, Malloy CR: An integrated (2)H and (13)C NMR study of gluconeogenesis and TCA cycle flux in humans.
Am J Physiol Endocrinol Metab
281
:
E848
–E856,
2001
14.
Jones JG, Merritt M, Malloy C: Quantifying tracer levels of 2H2O enrichment from microliter amounts of plasma and urine by 2H-NMR.
Magn Reson Med
45
:
156
–158,
2001
15.
Bischof MG, Krssak M, Krebs M, Bernroider E, Stingl H, Waldhäusl W, Roden M: Effects of short-term improvement of insulin treatment and glycemia on hepatic glycogen metabolism in type 1 diabetes.
Diabetes
50
:
392
–398,
2001
16.
Roden M, Stingl H, Chandramouli V, Schumann WC, Hofer A, Landau BR, Nowotny P, Waldhausl W, Shulman GI: Effects of free fatty acid elevation on postabsorptive endogenous glucose production and gluconeogenesis in humans.
Diabetes
49
:
701
–707,
2000
17.
Martin YL: A global approach to accurate and automatic quantitative analysis of NMR spectra by complex least-squares curve fitting.
J Magn Reson
111
:
1
–10,
1994
18.
Stingl H, Krssak M, Krebs M, Bischof MG, Nowotny P, Furnsinn C, Shulman GI, Waldhausl W, Roden M: Lipid-dependent control of hepatic glycogen stores in healthy humans.
Diabetologia
44
:
48
–54,
2001
19.
Stingl H, Schnedl WJ, Krssak M, Bernroider E, Bischof MG, Lahousen T, Pacini G, Roden M: Reduction of hepatic glycogen synthesis and breakdown in patients with agenesis of the dorsal pancreas.
J Clin Endocrinol Metab
87
:
4678
–4685,
2002
20.
Schumann WC, Gastaldelli A, Chandramouli V, Previs SF, Pettiti M, Ferrannini E, Landau BR: Determination of the enrichment of the hydrogen bound to carbon 5 of glucose on 2H2O administration.
Anal Biochem
297
:
195
–197,
2001
21.
Steele R: Influences of glucose loading and of injected insulin on hepatic glucose output.
Ann N Y Acad Sci
82
:
420
–430,
1959
22.
Stingl H, Raffesberg W, Nowotny P, Waldhausl W, Roden M: Reduction of plasma leptin concentrations by arginine but not lipid infusion in humans.
Obes Res
10
:
1111
–1119,
2002
23.
Schleucher J, Vanderveer PJ, Markley JL, Sharkey TD: Intramolecular deuterium distribution reveal disequilibrium of chloroplast phosphoglucose isomerase.
Plant Cell Environ
22
:
525
–533,
1999
24.
Wajngot A, Chandramouli V, Schumann WC, Ekberg K, Jones PK, Efendic S, Landau BR: Quantitative contributions of gluconeogenesis to glucose production during fasting in type 2 diabetes mellitus.
Metabolism
50
:
47
–52,
2001
25.
Chandramouli V, Ekberg K, Schumann WC, Wahren J, Landau BR: Origins of the hydrogen bound to carbon 1 of glucose in fasting: significance in gluconeogenesis quantitation.
Am J Physiol
277
:
E717
–E723,
1999
26.
Nurjhan N, Campbell PJ, Kennedy FP, Miles JM, Gerich JE: Insulin dose-response characteristics for suppression of glycerol release and conversion to glucose in humans.
Diabetes
35
:
1326
–1331,
1986
27.
Hundal RS, Krssak M, Dufour S, Laurent D, Lebon V, Chandramouli V, Inzucchi SE, Schumann WC, Petersen KF, Landau BR, Shulman GI: Mechanism by which metformin reduces glucose production in type 2 diabetes.
Diabetes
49
:
2063
–2069,
2000
28.
Gastaldelli A, Baldi S, Pettiti M, Toschi E, Camastra S, Natali A, Landau BR, Ferrannini E: Influence of obesity and type 2 diabetes on gluconeogenesis and glucose output in humans: a quantitative study.
Diabetes
49
:
1367
–1373,
2000
29.
Boden G, Chen X, Stein TP: Gluconeogenesis in moderately and severely hyperglycemic patients with type 2 diabetes mellitus.
Am J Physiol Endocrinol Metab
280
:
E23
–E30,
2001
30.
Radziuk J, Pye S: Quantitation of basal endogenous glucose production in type II diabetes: importance of the volume of distribution.
Diabetologia
45
:
1053
–1084,
2002
31.
Landau BR: Methods for measuring glycogen cycling.
Am J Physiol Endocrinol Metab
281
:
E413
–419,
2001