J Appl Physiol 104: 944-951, 2008.
First published January 10, 2008; doi:10.1152/japplphysiol.00752.2007
8750-7587/08 $8.00
Measurement of gluconeogenesis using glucose fragments and mass spectrometry after ingestion of deuterium oxide
Shaji K. Chacko,1
Agneta L. Sunehag,1
Susan Sharma,1
Pieter J. J. Sauer,2 and
Morey W. Haymond1
1Department of Pediatrics, Baylor College of Medicine, Children's Nutrition Research Center, US Department of Agriculture Agricultural Research Center, Houston, Texas; and 2Department of Pediatrics, Beatrix Children's Hospital, University Medical Center Groningen, University of Groningen, Groningen, The Netherlands
Submitted 12 July 2007
; accepted in final form 4 January 2008
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ABSTRACT
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We report a new method to measure the fraction of glucose derived from gluconeogenesis using gas chromatography-mass spectrometry and positive chemical ionization. After ingestion of deuterium oxide by subjects, glucose derived from gluconeogenesis is labeled with deuterium. Our calculations of gluconeogenesis are based on measurements of the average enrichment of deuterium on carbon 1, 3, 4, 5, and 6 of glucose and the deuterium enrichment in body water. In a sample from an adult volunteer after ingestion of deuterium oxide, fractional gluconeogenesis using the "average deuterium enrichment method" was 48.3 ± 0.5% (mean ± SD) and that with the C-5 hexamethylenetetramine (HMT) method by Landau et al. (Landau BR, Wahren J, Chandramouli V, Schumann WC, Ekberg K, Kalhan SC; J Clin Invest 98: 378–385, 1996) was 46.9 ± 5.4%. The coefficient of variation of 10 replicate analyses using the new method was 1.0% compared with 11.5% for the C-5 HMT method. In samples derived from an infant receiving total parenteral nutrition, fractional gluconeogenesis was 13.3 ± 0.3% using the new method and 13.7 ± 0.8% using the C-5 HMT method. Fractional gluconeogenesis measured in six adult volunteers after 66 h of continuous fasting was 83.7 ± 2.3% using the new method and 84.2 ± 5.0% using the C-5 HMT method. In conclusion, the average deuterium enrichment method is simple, highly reproducible, and cost effective. Furthermore, it requires only small blood sample volumes. With the use of an additional tracer, glucose rate of appearance can also be measured during the same analysis. Thus the new method makes measurements of gluconeogenesis available and affordable to large numbers of investigators under conditions of low and high fractional gluconeogenesis (
10 to 90) in all subject populations.
glucose kinetics; gas chromatography-mass spectrometry; isotope ratio mass spectrometry; hexamethylenetetramine; infants; fasting adults
UNTIL RECENTLY, IT HAS BEEN difficult to obtain quantitative in vivo estimates of gluconeogenesis, and it still remains a challenging problem. Over the past decade, several methods to measure gluconeogenesis using compounds labeled with stable isotopes, e.g., [U-13C]glucose, [2-13C]glycerol, and deuterium oxide (2H2O), have been published (3–5, 9–12, 20, 21). Each of these methods has its own advantages and disadvantages. The 2H2O method (10) is based on the measurement of deuterium in glucose carbon 5 (C-5), which is purported to represent total gluconeogenesis. The degree of analytic difficulty and high costs of the isotopes have limited the wide use of these methods. The necessity of a simple yet accurate and reproducible method to measure gluconeogenesis is a prerequisite for conducting quality research under various physiological and pathological conditions. We have developed a simple, straightforward, and cost-effective method to estimate gluconeogenesis using the average deuterium enrichment on glucose carbons in a specific glucose fragment measured by gas chromatography-mass spectrometry (GC-MS), thus making the measurement of gluconeogenesis available to large numbers of investigators.
This new method required identifying a glucose derivative with a mass fragment that carries all of the hydrogens except that on glucose C-2. Deuterium labeling at glucose C-2 is reported to be due to the isomerization process and glycogenolysis (7, 11). The careful analysis of the mass spectrum of the pentaacetate derivative of a variety of deuterium-labeled glucose isotopes using positive chemical ionization has revealed a simple approach for measuring fractional gluconeogenesis. Among the various fragments of glucose observed, mass-to-charge ratio (m/z) 169 is of specific interest because of the presence of six covalently bonded deuterium/hydrogen to the carbon chain skeleton of glucose except that on C-2 (2).
The development of this method required 1) identification of the carbons and hydrogens in the m/z 169 GC-MS fragment of the pentaacetate derivative of glucose, 2) determination of the average deuterium enrichment on the glucose carbons in a specific fragment (m/z 170/169) of the glucose molecule, 3) evaluation of the reproducibility of the deuterium enrichment measured over a wide range of abundances using the m/z 169 fragment, and 4) comparison of fractional gluconeogenesis obtained by the new method with those of the C-5 hexamethylenetetramine (HMT) method (10) under different physiological conditions. We demonstrate that the average deuterium enrichment method will provide estimates of gluconeogenesis equal to those obtained with the C-5 HMT method (10) under conditions of overnight fast, 3-day fast, and total parenteral nutrition.
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METHODS
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Materials.
[1-2H]glucose (99 atom % 2H), [3-2H]glucose (98 atom % 2H), [6,6-2H2]glucose (99 atom % 2H), [1,2,3,4,5,6,6-2H7]glucose (99 atom % 2H), [U-13C]glucose, and 2H2O (99 atom % 2H) were obtained from Cambridge Isotope Laboratories (Andover, MA); [2-2H]glucose (97 atom % 2H) was obtained from Isotec (Miamisburg, OH), and [5-2H]glucose (98 atom % 2H) was from Omicron Biochemicals (South Bend, IN). Reagent-grade acetic anhydride, pyridine, and ethyl acetate were purchased from Sigma (St. Louis, MO).
Derivatization and GC-MS and GC-C-IRMS analyses.
Enrichments of glucose labeled with deuterium and 13C were measured by GC-MS and gas chromatography combustion isotope ratio mass spectrometry (GC-C-IRMS), respectively, using the pentaacetate derivative (1). A volume of 25 µl of each standard and deproteinized plasma samples (deproteinized with 100 µl of cold acetone) were aliquoted into 4-ml vials and taken to dryness under nitrogen at room temperature. Fifty microliters of acetic anhydride/pyridine (2:1) were added to each sample, after which the samples were heated to 60°C for 10 min or allowed to sit overnight at room temperature. The samples were then dried under nitrogen at room temperature, reconstituted in 50 µl of ethyl acetate, and transferred to autosampler vials. The derivatized standards and samples were analyzed by GC-MS (GC 6890, MS 5973N; Agilent Technologies, Wilmington, DE) with an RTX-1701 column (30 m x 0.25 mm ID x 0.5 µm film; Restek, Bellefonte, PA). The GC conditions were as follows: injector, 250°C (splitless injection); oven, initially 70°C for 1.0 min; and ramp, 30°C/min to 275°C for 7 min. The positive chemical ionization mode of GC-MS (source at 250°C, quadrupole at 106°C) used methane as the reagent gas. Deuterium enrichment in plasma water was measured by IRMS (Delta+XL IRMS Thermo Finnigan, Bremen, Germany) as an average of multiple measurements.
Determining the rate of gluconeogenesis requires the use of an additional glucose tracer (to measure glucose appearance rate), which could potentially interfere with the measurement of the deuterium enrichment of glucose. The most commonly used stable isotope glucose tracers to measure plasma glucose appearance rate are [1-13C]glucose and [6,6-2H2]glucose. The [1-13C]glucose enrichment can be measured by GC-C-IRMS as previously described (6). This technique provides a measure of enrichment specifically reflecting the 13C tracer. Alternatively with the use of [6,6-2H2]glucose, the M+2 enrichment is measured with either m/z 171/169 or m/z 333/331 in the positive chemical ionization mode during the same run used to measure deuterium labeling from ingested deuterated water or m/z 244/242 in the electron impact ionization mode of GC-MS (6, 15–19). The slopes of standard curves are always used to correct instrument deviations. We have evaluated whether these tracers would interfere with our approach for measuring gluconeogenesis using 2H2O (see Potential interference of [1-13C]glucose or [6,6-2H2]glucose during glucose appearance rate measurement). In the studies described here, we have used [6,6-2H2]glucose for the measurement of glucose rate of appearance.
The C-5 HMT method of Landau et al. (10), which was used for method comparison, involves HPLC purification of glucose from other plasma components, conversion of glucose to xylose, HPLC purification of xylose, distillation of formaldehyde, and subsequent derivatization to HMT. The resulting product (HMT) is then analyzed by GC-MS using the fragment m/z 141/140 to measure deuterium incorporation at C-5 of glucose.
Determination of sites of labeling on glucose fragments and measurement of enrichments on the carbons of specific fragments of glucose.
For determination of the deuterium or hydrogen labeling sites on the m/z 169 GC-MS fragment of glucose pentaacetate, specific deuterium-labeled glucose compounds were derivatized and analyzed in the positive chemical ionization scan mode of GC-MS (Fig. 1) with subsequent comparison of the mass spectral data (2). Selective ion monitoring of m/z 170/169 was performed to determine the M+1 enrichment of deuterium in the circulating glucose carbons (C-1,3,4,5,6,6). To measure accurately the deuterium labeling in glucose from 2H2O, the enrichment of M+1 resulting from the natural abundance was subtracted. The average enrichment of deuterium on a gluconeogenic glucose carbon was then calculated from this M+1 data. Measurement of the enrichment at M+1 gives the deuterium incorporation resulting from the exchange of deuterium from 2H2O with covalently bonded hydrogens on glucose carbons during the gluconeogenic pathway when no other tracers are infused or ingested.
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Fig. 1. Mass spectra of various deuterium- and carbon-labeled glucose compounds (as pentaacetate derivatives) (A–H) obtained in the positive chemical ionization mode of GC-MS for the determination of the deuterium and hydrogen labeling sites on different GC-MS fragments. m/z, Mass-to-charge ratio.
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Calculation.
As evident from the mass spectrum of glucose (Fig. 1), the fragment m/z 169 carries hydrogens at C-1,3,4,5,6,6 (see details in RESULTS).
1) With the assumption that the 2H labeling of exchangeable hydrogens is identical on all glucose carbons except that on C-2, which is in complete exchange with body water, the average enrichment of 2H on each glucose carbon is calculated with the following equation:
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where (M+1)d(m/z 169) is the M+1 enrichment of deuterium of glucose measured using m/z 170/169 and "6" is the number of 2H labeling sites on the m/z 169 fragment of glucose.
2) Because body water is the precursor pool for deuterium or hydrogen, the extent of deuterium labeling of glucose during the gluconeogenic process when 2H2O is ingested or infused is a measure of fractional gluconeogenesis. This is based on the assumption that the deuterium enrichment measurement is performed under near steady-state conditions. Therefore, with the average deuterium enrichment in m/z 170/169 for calculating fractional gluconeogenesis, the equation is
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where EH2O is the deuterium enrichment in body water.
Experiments with human plasma after ingestion of 2H2O.
Samples were utilized from studies intended to measure gluconeogenesis. The studies were approved by the Institutional Review Board for human research at Baylor College of Medicine and were performed after written consent had been obtained. We have compared gluconeogenic measurements with our method based on average deuterium enrichment with those of the C-5 HMT method (10) under conditions when fractional gluconeogenesis is low, intermediate, and high.
In an adult volunteer, after overnight fasting, five doses of 99.8% 2H2O were ingested at 2-h intervals (a total of 5 g/kg). Plasma samples were obtained before isotope administration and 6 h after the last dose, representing approximately steady state (17, 18). Pentaacetate derivatization of plasma glucose was performed, and the deuterium enrichment in plasma water was measured as described above.
To determine the validity of the new method in samples with low fractional gluconeogenesis, samples were used from an infant [birth weight = 880 g, gestational age = 25 wk, postnatal age = 5 days, blood glucose 227 mg/dl (12.6 mM)]. The infant received total parenteral nutrition consisting of glucose at 16 g·kg–1·day–1 (11.13 mg·kg–1·min–1), lipid (intralipid 20%) at 4 g·kg–1·day–1 (2.65 mg·kg–1·min–1), and protein (TrophAmine) at 3.1 g·kg–1·day–1 (2.14 mg·kg–1·min–1). The infant also received sterile 2H2O (4 g/kg iv over a period of 2 h) dissolved in isotonic saline via an umbilical venous catheter to measure gluconeogenesis. Plasma samples were obtained before the 2H2O administration and at 9.5 and 10 h ("steady state") after the completion of the 2H2O infusion.
For validation of the new method under conditions of high fractional gluconeogenesis, samples from six healthy adult male human volunteers (26 ± 3 yr, weight = 69 ± 5 kg, and body mass index = 23 ± 1 kg/m2) were used. The volunteers had consumed a normal diet (50% carbohydrate, 15% protein, and 35% fat) for 3 days, after which they were admitted to the Metabolic Research Unit at the Children's Nutrition Research Center. In the afternoon of day 1 of the study, a standard meal was served, after which they were fasted continuously for 66 h except for water ad libitum. On day 3, 46 h into the fast, a baseline plasma sample was obtained before five oral doses of 99.8% 2H2O (3 g/kg) were administered at 2-h intervals. Blood samples were collected on day 4 at 15-min intervals between 65 and 66 h of the fast. These samples had previously been analyzed using the C-5 HMT method (10) to measure fractional gluconeogenesis. Because of the higher volume requirement with the C-5 HMT method (10), not all samples were available for reanalysis with the average deuterium enrichment method (new method) for comparative analysis. All five samples were available in two subjects, four samples in one subject, two samples in one subject, and one sample in two subjects for analysis with the new method (see Table 4).
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Table 4. Fractional gluconeogenesis measured in 6 adult male human volunteers after 66 h of continuous fasting using the average deuterium enrichment method and the C-5 HMT method
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Potential interference of [1-13C]glucose or [6,6-2H2]glucose during glucose appearance rate measurement.
To determine the rate of gluconeogenesis, the glucose appearance rate must also be measured (17–19). Therefore, it is crucial that the infused glucose tracers do not interfere with the primary measure of gluconeogenesis. To assess the effect of potential glucose tracers on the measurements of fractional gluconeogenesis when 2H2O is used, the adult overnight fasting plasma sample was spiked with either [1-13C]glucose or [6,6-2H2]glucose to achieve enrichments of 0–5% (Fig. 2), which covers the range of enrichments used in human studies. To calculate the M+1 enrichment representing the deuterium label derived from 2H2O, the 13C enrichment (obtained by GC-C-IRMS) was subtracted from the total M+1 enrichment of glucose (the sum of the 13C and 2H labeling of circulating glucose obtained by GC-MS). Measurements of fractional gluconeogenesis were performed with the new method to the unspiked sample and the sample spiked with either [1-13C]glucose or [6,6-2H2]glucose.
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Fig. 2. Effect of added [1-13C] and [6,6-2H2]glucose tracers to a plasma sample on percent gluconeogenesis (GNG) measurement. A cluster of data points (n = 5) representing unspiked samples are in the 47–51% range.
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Statistics.
Coefficient of variation (CV) was calculated by dividing standard deviation (SD) by the mean. The data obtained by the new method were compared with those of the C-5 HMT method (10) using paired t-test (data obtained from 10 analyses of 1 adult sample, triplicate analyses of 2 infant samples, and data obtained in 6 long-term fasting adults). A P value <0.05 was used to define significance. In addition, the Bland-Altman test was applied to the 34 paired measurements obtained by the two methods.
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RESULTS
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Glucose fragments and calculation of deuterium enrichment at glucose carbon 5.
Figure 1 depicts the mass spectra of natural glucose, [2H7]glucose, [1-2H]glucose, [2-2H]glucose, [3-2H]glucose, [5-2H]glucose, [6,6-2H2]glucose, and [U-13C]glucose obtained in the positive chemical ionization scan mode. The mass spectrum of glucose pentaacetate provides three important fragments at m/z 109, m/z 169, and m/z 331. The hydrogen or deuterium labeling sites in these ion fragments of different deuterium-labeled glucose pentaacetate compounds are depicted in Table 1. The m/z 169 fragment carries the hydrogen at C-1, C-3, C-4, C-5, and C-6,6 of glucose but not at C-2. The m/z 331 fragment carries all of the hydrogen at C-1, C-2, C-3, C-4, C-5, and C-6,6, whereas the m/z 109 fragment has the hydrogen at C-1, C-3, C-4, and C-6,6 of glucose. The presence of deuterium label at C-4 in the m/z 169 and m/z 109 fragments was confirmed by comparing mass spectrum obtained from [2H7]glucose with the mass spectra of all of the other deuterium-labeled glucose compounds. Comparisons of mass spectra (Fig. 1) and mass shifts in m/z 169 of various deuterium-labeled glucose compounds confirm that the m/z 169 fragment carries all of the covalently bonded hydrogens in the glucose carbon chain except that on C-2. Therefore, we selected fragment m/z 169 to calculate the average deuterium enrichment on a glucose carbon.
Measurement of average enrichment (analysis of reproducibility).
Our approach is based on the average deuterium enrichment in the glucose carbons of the m/z 170/169 fragment. The CV of M+1/M in a [1-2H]glucose standard at various levels of enrichment from 0 to 3 mole percent enrichment (MPE) (10 replicates each) was 0.39%. M+1/M of the adult human baseline plasma sample was 9.048 ± 0.006 (mean ± SD) (10 replicate analyses; CV = 0.1%) (Table 2). M+1/M of the adult human plasma sample (6 h) obtained after an overnight fast (after 2H2O ingestion) was 10.424 ± 0.010 (mean ± SD) (10 replicate analyses), with CV of 0.1%. The average MPE of deuterium was 1.376 ± 0.012 (mean ± SD), with CV of 0.9%. M+1/M of the 9.5- and 10-h infant plasma samples after 2H2O ingestion measured with GC-MS (3 replicate analysis each) were 9.641 ± 0.003 (mean ± SD) and 9.639 ± 0.010 (see Table 3). The average MPE of deuterium measured for the 9.5-h sample was 0.314 ± 0.005 (mean ± SD) (CV = 1.6%) and that for the 10-h sample was 0.311 ± 0.006 (mean ± SD) (CV = 1.9%). Mean M+1/M of all of the baseline samples from six adult male subjects was 9.239 ± 0.042 (mean ± SD) (3 replicate analysis each), and mean M+1/M for all samples between 65 and 66 h was 11.373 ± 0.081 (mean ± SD), with CV of 0.45% and 0.71%, respectively (3 replicates each). The average MPE of deuterium was 2.134 ± 0.099 (mean ± SD), with CV of 4.6%. The precision of the average enrichment method is evident from the statistics of the above GC-MS measurements. Because of the lower number of manipulations involved, the propagation of errors is less in measurements of fractional gluconeogenesis using the average enrichment method compared with the C-5 HMT method (10).
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Table 2. Comparison of fractional gluconeogenesis of adult human plasma (overnight fasting study), applying the new method using average deuterium enrichment of glucose and the C-5 HMT method
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Table 3. Gluconeogenesis as a fraction of total plasma glucose appearance rate based on two "steady-state" plasma samples from an infant who received total parenteral nutrition and comparison of the new method using average deuterium enrichment of glucose and the C-5 HMT method
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As anticipated, no significant increase in the enrichment of M+2 glucose in the plasma sample was observed at the low 2H enrichment in body water used in human studies. With the singly labeled glucose standards, the enrichments measured with the m/z 170/169 fragment was extremely stable and reproducible over a wide abundance range (2–30 million area counts) (Fig. 3), as demonstrated by the slope of 0.0024. This observation further demonstrates the robust nature of this method using fragment m/z 170/169 to measure deuterium enrichment of gluconeogenic glucose molecules.
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Fig. 3. Stability of deuterium enrichment measured over a wide-abundance range using a 2.1% [5-2H]glucose standard.
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Measurement of fractional gluconeogenesis.
The measurements of fractional gluconeogenesis using 10 aliquots of the same adult human plasma sample (overnight fasting) applying the new method and C-5 HMT method (10), respectively, are provided in Table 2. Fractional gluconeogenesis measured with the average enrichment in the adult plasma sample was 48.3% ± 0.5 (mean ± SD) and that using the C-5 HMT method (10) was 46.9% ± 5.4 (mean ± SD). These measures were not significantly different (P = 0.35). Thus the CV of 10 replicate analyses for the new method was 1.0%, whereas that of the C-5 HMT method (10) was 11.5%. The deuterium enrichment of body water in the adult plasma was 0.4746 ± 0.0021 MPE (mean ± SD; average of 10 measurements). Thus measurements of gluconeogenesis using the new method compare extremely well with those obtained by the C-5 HMT method (10), supporting the validity of this new method. Ten independent measures of fractional gluconeogenesis were calculated from 10 replicate analyses of the same sample using 4 different instruments with the new method. Fractional gluconeogenesis was 48.3 ± 0.5% (mean ± SD) for instrument A, 48.6 ± 0.3% for instrument B, 46.5 ± 1.5% for instrument C, and for 49.4 ± 1.5% instrument D, with intra-assay CVs of 1.0, 0.6, 3.2, and 3.0%, respectively. The average fractional gluconeogenesis was 48.2 ± 1.2%. With these four values, the interassay CV was 2.5%.
Fractional gluconeogenesis based on three replicate analyses of two steady-state samples (9.5 and 10 h) from the infant study was 13.3 ± 0.3% (mean ± SD) for the new method and 13.7 ± 0.8% (mean ± SD) (Table 3) with the C-5 HMT method (10). These measures were not significantly different (P = 0.46). The CV of 3 replicate fractional gluconeogenesis measurements of the 9.5-h sample was 1.5% and that of the 10-h sample was 2.2% using the new method and 2.3% and 8.4%, respectively, with the C-5 HMT method (10). The deuterium enrichment of body water in the infant study was calculated from the means of two separate measurements for each steady-state sample (9.5 h: 0.3978, 10 h: 0.3877).
The fractional gluconeogenesis measurement, which was performed in six adult male subjects, was 83.7 ± 2.3% (mean ± SD) via the new method and 84.2 ± 5.0% (mean ± SD) via the C-5 HMT method (10) (see Table 4). These data were not significantly different (P = 0.78). In addition, the new method was compared with the C-5 HMT method (10) using the Bland-Altman test (Fig. 4) including all fractional gluconeogenic measurements (n = 34 pair). The results indicate that the new method, on average, overestimated fractional gluconeogenesis by 0.5% (mean difference between the methods). The two methods provide similar measurements of fractional gluconeogenesis (P = 0.59). The mean differences between the methods for infant, overnight fasting, and 66-h fasting conditions were 0.4%, –1.7%, and 1.8% respectively. All data points except for one (97%) were within ±2 SD, and 24 of 34 data points (71%) were within ±1 SD.
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Fig. 4. Plot of the differences between the %fractional GNG measured by the new method and the %fractional GNG measured by the C-5 hexamethylenetetramine (HMT) method vs. the average of %fractional GNG by the 2 methods. Results were obtained by the Bland-Altman procedure. The solid line represents the bias between the methods (0.5%). The dotted lines represent the upper and lower limits of agreement (11.7 and –10.7 respectively), calculated as bias ± (2 x the SD of the differences).
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Isotope interference from infused glucose tracers.
When [6,6-2H2]glucose was added to the plasma sample, no contamination of the M+1 fragment was observed; thus fractional gluconeogenesis measurements were unaffected by plasma enrichment up to 5% MPE of the [6,6-2H2]glucose (see Fig. 2). When [1-13C]glucose was added to the adult plasma sample to assess the interference of an additional tracer in the measurement of gluconeogenesis using 2H2O, fractional gluconeogenesis was slightly overestimated when the [1-13C]glucose enrichment exceeded 2.5% MPE (see Fig. 2). However, at an enrichment of 0.5–1.0% MPE of [1-13C]glucose, this error was essentially eliminated. Accurate measurement of 13C enrichment at these levels can be easily obtained by GC-C-IRMS. Although [6,6-2H2]glucose is the preferred tracer for this purpose because of the potential recycling of 13C glucose tracers (1), [1-13C]glucose at low enrichment can be used and might be the only alternative in complex studies requiring more than one glucose tracer.
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DISCUSSION
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After the ingestion or infusion of deuterium oxide and equilibration in the total body water pool, deuterium is incorporated into intermediary substrates along the glycolytic/gluconeogenic pathway. Hence, the degree of deuterium labeling in plasma glucose is a measure of gluconeogenesis. The isomerization of glyceraldehyde-3-phosphate to dihydroxyacetone phosphate by triose phosphate isomerase and a series of equilibration reactions between phosphoenolpyruvate and dihydroxyacetone phosphate is consistent with deuterium incorporation in C-1, C-3, C-4, C-5, and C-6 of glucose during gluconeogenesis. Our proposed method using average deuterium enrichment is based on the assumption that the labeling of 2H in glucose C-1,3,4,5,6,6 are all essentially equal. The 2H enrichment at C-2 is purported to be due to complete 2H exchange with body water during the extensive glucose-6-phosphate to fructose-6-phosphate isomerization. Therefore, the 2H enrichment in glucose C-2 represents body water enrichment and is not a reflection of the gluconeogenic process (11). This is supported by the studies of Rognstad et al. (13), who observed that the incorporation of tritium on C-2 of glucose was higher than in all of the other glucose carbons. Therefore, the ratio of the average enrichment of deuterium at the glucose carbons in fragment m/z 170/169 (which does not carry the hydrogen of glucose C-2) to that of deuterium in body water represents an estimate of fractional gluconeogenesis.
Rognstad et al. (13) reported that the incorporation of 3H on C-1 of glucose formed by hepatocytes incubated with 3H2O using different substrates is essentially half that on C-6 (two tritium on C-6), suggesting that the primary mechanism of labeling is similar between C-1 and C-6. The extensive tritium incorporation in C-3, C-4, and C-5 of glucose when incubated with various gluconeogenic substrates indicates that the labeling occurs throughout the gluconeogenic process (13). Thus, with the exception of C-2, these series of isomerization and equilibration reactions throughout the gluconeogenic pathway should result in equal deuterium labeling on each glucose carbon. We have provided data supporting that the average 2H enrichment (M+1) in C-1,3,4,5,6,6 of glucose provides measures of fractional gluconeogenesis equivalent to the C-5 HMT method (10).
We have observed that multideuterated tracers (d7-glucose) exhibited isotope discrimination when analyzed in the GC-MS-positive chemical ionization mode using fragment m/z 169 as reported by Guo et al. (2) during their studies of quantitation of positional isomers of deuterium-labeled glucose. Our studies have shown that such discrimination is below the level of detection when singly labeled deuterated glucose compounds are used (also reported by Guo et al.). The average enrichment method used in our study only measures singly labeled glucose because the probability of multiple deuteriums getting labeled on the same glucose molecule is negligible at a lower level of deuterium enrichment in body water. Unlike studies performed by Guo et al. (2) in rats, the deuterium enrichment in body water used in our human study to measure gluconeogenesis is substantially lower.
The 2H-labeled NMR data from Jin et al. (7) in rats that received 2H2O suggest relatively lower exchange of deuterium at C-3 of glucose. However, these studies were performed with high deuterium enrichment in body water, resulting in deuterium labeling at multiple positions of the same glucose molecule. This could potentially affect the accuracy of the deuterium enrichment measurements on different glucose carbons. Because of the toxicity of 2H2O when given at high doses, human studies need to be performed at severalfold lower levels of deuterium enrichment in body water. Jones et al. (8) reported in humans unequal deuterium labeling at various glucose carbons using NMR at 0.5% deuterium enrichment in body water. However, no precision or accuracy data were provided at this level of enrichment to determine whether these differences were significant.
Our method in which average deuterium enrichment was used to measure fractional gluconeogenesis is analytically easy to perform and highly reproducible compared with the C-5 HMT method (10). Furthermore, our method is robust in that the M+1 ratio measured with the m/z 170/169 fragment does not fluctuate over a wide range of abundances (Fig. 3). The low tracer cost and ease of analysis make this method affordable and accessible to a wide number of investigators. In addition, the small sample volume requirement allows this method to be used in studies with infants and children. Although the C-5 HMT method (10) is conceptually straightforward, it is a very tedious, time-consuming analysis (and thus expensive) that requires a high level of expertise. In addition, the C-5 HMT procedure requires a minimum of 0.5 ml of plasma per sample, limiting its use in infants and children. We have compared measurements obtained by our method based on average deuterium enrichment with those of the C-5 HMT method (10) under conditions when fractional gluconeogenesis is low, intermediate, and high (10 to 90), with results showing that the two methods provide nearly identical values. This supports the validity of the method at different levels of fractional gluconeogenesis. Because it is extremely accurate and reproducible even when fractional gluconeogenesis is low, it can be used in subjects receiving parenteral or enteral feedings and during insulin clamp studies.
In summary, our method based on the average deuterium enrichment has a number of advantages: it is simple and straightforward, it requires only 25 µl of plasma to accomplish the analysis, and it can be completed in a few hours. Furthermore, this method has high reproducibility and accuracy even when fractional gluconeogenesis is low. Therefore, the measurement of gluconeogenesis using the average deuterium labeling on a glucose carbon using GC-MS can be performed in most laboratories and can be applied to all populations, including in infants with very low birth weight. In addition, the high sample throughput will provide speedy results in studies that include large numbers of subjects.
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GRANTS
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These studies were supported by grants from the Children's Nutrition Research Center (USDA/ARS Cooperative Agreement 6250-51000), National Institute of Child Health and Human Development Grant 2RO1 HD-037957, MARS Inc., National Institute of Child Health and Human Development Grant RO1 HD-044609, National Institute of Diabetes and Digestive and Kidney Diseases Grant 5-RO1 DK-55478, and National Institutes of Health Grant M01 RR-00188 (General Clinical Research Center).
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ACKNOWLEDGMENTS
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This work is a publication of the US Department of Agriculture/Agricultural Research Service, Children's Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine (Houston, TX). The contents of this publication do not necessarily reflect the views or policies of the US Department of Agriculture, and mention of trade names, commercial products, or organizations does not imply endorsement from the US Government.
We thank Dr. E O'Brian Smith (biostatistician) for invaluable advice and Daniel Donaldson, Cindy Bryant, Pamela Gordon, Amy Pontius, and the staff of the Metabolic Research Unit at the Children's Nutrition Research Center for skillful assistance with the studies.
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FOOTNOTES
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Address for reprint requests and other correspondence: M. W. Haymond, USDA/ARS Children's Nutrition Research Center, 1100 Bates St., Houston, TX 77030-2600 (e-mail: mhaymond{at}bcm.tmc.edu)
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
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