From the Baylor College of Medicine, Childrens Nutrition Research Center, USDA/ARS, Department of Pediatrics-Nutrition, Houston, Texas
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ABSTRACT |
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INTRODUCTION |
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Use of isotopes of glucose to estimate the Ra and/or rate of production of glucose in humans is a fundamental research tool for the clinical investigator involved in studies of glucose homeostasis. Despite wide application of the isotope dilution techniques, the factors that affect the findings of such studies are incompletely understood. The purposes of the present study were to quantify the impact of the duration of infusion and choice of stable isotope tracer on tracer recycling. Using a dual isotope infusion approach, we assessed how these measures are affected by 1) potential recycling of labeled glucose molecules via gluconeogenesis and/or glycogenolysis and 2) the equilibration of the tracer with the tracee.
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RESEARCH DESIGN AND METHODS |
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Experimental design.
See Fig. 1 for graphic representation of protocols.
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At 0800 h, [6,6-2H2]glucose and [1-13C]glucose were given as primed constant rate infusions (prime dose 33 µmol/kg; infusion rate 0.554 ± 0.001 µmol · kg-1 · min-1 for both [1-13C]glucose and [6,6-2H2]glucose). Blood samples were obtained at 10-min intervals from 1000 to 1030 h. At 1030 h, the isotope infusions were discontinued and the catheter was immediately removed to guarantee the absence of any additional isotope being derived from the infusion catheter line. Samples were obtained at 15-min intervals during the next 3 h. At 3 h after discontinuing the isotopes, each subject received an intravenous bolus of 1 mg of glucagon and blood sampling continued at 10-min intervals for an additional 60 min.
Study protocol B.
Protocol B was identical to protocol A with the exception that the infusion of [1-13C]glucose was initiated at 2000 h on the day of admission (duration of infusion 14.5 h).
Study protocol C.
Protocol C was identical to protocol A with the exception that the infusion of [6,6-2H2]glucose was initiated at 2000 h on the day of admission (duration of infusion 14.5 h).
Study protocol D.
Protocol D was identical to protocol A with the exception that the infusion of [6,6-2H2]glucose was initiated at 0530 h (duration of infusion 5 h).
Isotopes.
[6,6 2H2]glucose (99% 2H) and [1-13C]glucose (99% 13C) were obtained from Cambridge Isotope Laboratories (Andover, MA). Isotope solutions were tested for sterility and pyrogenicity. The isotopes were dissolved in 0.45% saline, and the solution was filtered through a 0.2-µm Millipore filter into sterile syringes. All sterile isotopes were prepared <48 h before study and maintained at 4°C until used. Separate solutions were prepared for each of the two tracers.
Analyses.
Blood samples were placed on ice, and the plasma was separated and kept at -70°C until assayed. Plasma glucose concentrations were determined by a glucose oxidase method (YSI glucose analyzer; YSI, Yellow Springs, OH), and plasma glucagon concentrations were determined by a commercial radioimmunoassay (Linco, St. Charles, MO). The penta-acetate derivative of glucose was prepared as described previously (4,5). The isotopic enrichments of [6,6 2H2]glucose were measured by gas chromatography-mass spectrometry (GCMS) using a quadrupole instrument. The electron impact ionization fragments m/z 242244 do not contain the carbon-1 of glucose, thus ensuring no interference from the [1-13C]glucose in the determination of the enrichment of [6,6-2H2]glucose. The contribution of 13C randomized in other glucose carbons (C2-C6) contributed <1% to the M+2 enrichment after prolonged infusion of [1-13C]glucose. The isotopic enrichments of the [1-13C]glucose were measured by gas chromatography-combustion-isotope ratio mass spectrometry (Europa Scientific, ANCA-NT 2020, GC: HP 5890 with an HP 1701 column 30 m x 0.25 mm x 1 µm; Agilent Technologies, Wilmington, DE). Finally, the acetyl-pentafluorobenzyl derivative of lactate was prepared as described previously (6,7). The 13C enrichments in lactate were analyzed by negative chemical ionization GCMS as previously described (6,7).
Calculations.
Plasma glucose Ra at steady state was calculated using established equations for isotope dilution (8,9):
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where Ei is the isotopic enrichment of the infusate, Ep is the isotopic enrichment in plasma glucose, and I is the isotope infusion rate.
Regression analysis was used to calculate the rate constant (k) of the exponential curves representing enrichment decay of the [1-13C]glucose and [6,6-2H2]glucose tracers from the plasma pool during the 3 h after discontinuation of the isotope infusion. The enrichment values over time are described by the following equation:
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where E0 is the isotopic enrichment at steady state, and Et is isotopic enrichment at time t. The half-life (t1/2) of each of the two isotopes was then calculated from equation 1, as follows:
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The absolute amount of new tracer released from glycogen into the plasma pool (mmol/l) before and after administration of glucagon was calculated by multiplying the enrichment of the tracer (after subtracting the enrichment value obtained immediately before the glucagon bolus) by the glucose concentration.
Statistics.
All values were expressed as mean ± SE. ANOVA procedure for repeated measures (using Fishers least squares difference for multiple comparisons) was used to test for differences in the mean glucose Ra, glucose concentration, and the rate constant of isotope decay curve and t1/2 values in the various subgroups under study. Differences in the lactate enrichment at the end of 14.5- vs. 2.5-h infusion of [1-13C]glucose were tested using paired Students t test. P 0.05 was considered to indicate statistical significance. All statistical analyses were performed on a personal computer with the statistical program SPSS (version 8.0) for Windows.
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RESULTS |
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Plasma glucagon concentrations.
Plasma glucagon concentrations during the steady-state period were similar on all four study occasions (66 ± 11, 62 ± 5, 65 ± 5, and 59 ± 7 pg/ml for protocols A, B, C, and D, respectively; NS). After the intravenous administration of a glucagon bolus (1 mg), the plasma levels rose to >2,000 pg/ml in all four groups.
Glucose isotopic enrichments and appearance rates.
Isotopic enrichments during the steady-state period -0.5 to 0 h increased with the duration of isotope infusion, independent of tracer (Fig. 2, Table 1). The mean isotopic enrichments (moles % excess) at steady state and the rates of isotope infusions of 13C and 2H2 glucose in each of the four study protocols are provided in Table 1. The calculated glucose Ras are illustrated in Fig. 3.
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Isotope decay curves and t1/2 of [6,6-2H2]glucose and [1-13C]glucose.
The rate constants (k) of the isotopic enrichment decays after discontinuation of the isotope infusion and the t1/2 of the isotopes are provided in Table 2. Figure 4 depicts the isotopic enrichments after discontinuation of the infusion through the end of the study (expressed as percentage of the steady-state value), plasma glucose concentrations, and amount of tracer released into the plasma pool after glucagon administration. The decay (k = rate constant x 10-3) of the [1-13C]glucose tracer after a 2.5-h infusion (7.1 ± 0.3) was similar (NS) to that after a 14.5-h infusion (6.4 ± 0.2).
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Glucagon effect on glucose enrichment.
There was no detectable tracer released from glycogen into the plasma pool during the 1 h after administration of glucagon (Fig. 4).
Lactate enrichment.
The steady-state [13C]lactate enrichment was nearly twice as high (1.55 ± 0.1) during the 14.5-h vs. the 2.5-h [1-13C]glucose infusion (0.85 ± 0.05) in protocols B and C, respectively (P < 0.05).
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DISCUSSION |
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Although both the [1-13C]glucose and [6,6-2H2]glucose tracers could theoretically recycle through glycogen via the direct pathway, only the carbon-labeled tracer can recycle back to glucose through the Cori cycle (35,10). In the present study, neither the [1-13C]glucose nor the [6,6-2H2]glucose tracer was released into the plasma glucose pool after glucagon infusion, making any significant recycling of tracer from glycogen unlikely.
The [6,6-2H2]glucose tracer decay after 14.5 h or 5 h of infusion was slightly slower (10%) than that after the 2.5-h infusion. Because no detectable isotope decay curve change was observed after stimulation of glycogenolysis by glucagon infusion, it seems unlikely that this difference would be the result of release of tracer into the plasma space from glycogen. The decay of the [6,6-2H2]glucose tracer was faster than that of the [1-13C]glucose tracer independent of the duration of tracer infusion (Table 2). However, unlike the [6,6-2H2]glucose tracer, the decays of the [1-13C]glucose tracer after 2.5-h or 14.5-h of infusion were similar (NS). This suggests an ongoing contribution of 13C label from lactate to glucose during the decay period obscuring a small difference between the 2.5- and 14.5-h infusions as was observed with the [6,6-2H2]glucose tracer. Consistent with this speculation is that the higher lactate 13C enrichment was almost twice as high during the prolonged compared with the short [1-13C]glucose infusion. Comparing the isotopic enrichment after the 14.5-h infusion of [6,6-2H2]glucose and [13C]glucose indicates Cori cycle activities of
10%. This is less than that of Reichard et al. (11) of 16% in normal subjects but greater than that estimated by Tayek et al. (12) of 5%.
Having excluded significant recycling of tracer via glycogen or the Cori cycle, we hypothesized that the 30% difference in glucose Ra between the 2.5- and 14.5-h infusion was due to lack of complete equilibration of substrate and isotopes within the extracellular glucose space during the short (2.5 h) isotope infusion study. To test this hypothesis, we conducted protocol D using a 5-h infusion of [6,6-2H2]glucose. The glucose Ra obtained after 5 h of tracer infusion was higher (P < 0.05) than that obtained after 14.5 h and lower (P < 0.05) than that observed after the 2.5-h infusion. Notably, extending the isotope infusion from 2.5 to 5 h reduced the overestimation of glucose Ra by nearly 80%. A glucose tracer infused into the plasma pool needs to equilibrate with both the intravascular pool and the pool in the extracellular-extravascular space, which is twice that of the intravascular space. Thus, 2.5-h isotope infusion is not sufficient to achieve isotope and substrate equilibration in adults. In addition, this slowly equilibrating pool will complicate any attempt at non-steady-state modeling.
These conclusions in normal adult volunteers are supported by previous studies in patients with type 2 diabetes. In subjects with type 2 diabetes, hepatic glucose production was overestimated when the duration of a [3-3H]glucose infusion was insufficient for complete isotope equilibration, and the error was related to the degree of hyperglycemia (13). Chen et al. (14) demonstrated that at least 4 h of tracer administration was necessary to reach steady state and accurately measure glucose Ra in patients with type 2 diabetes but failed to observe this in normal subjects. They attributed this difference to the expanded plasma glucose pool size and the markedly reduced glucose uptake in the subjects with type 2 diabetes. Using a paired study design, we were able to demonstrate that this applies equally to normal individuals with normal plasma concentrations. Moreover, studies using prolonged isotope infusions in patients with type 2 diabetes could not confirm the increase in hepatic glucose production that was reported in some of the earlier studies using short (90120 min) infusion periods of [3H]glucose (15,16). These observations are in agreement with the findings of the present study.
In this study, we used a 60-min priming dose of labeled glucose. We and others have used a variety of priming doses ranging from 60 to 100 min (1719). It is of interest that despite a 90-min prime in the study by Hovorka et al. (3), the change in enrichment from a 120- to a 240-min time point was 15%, which is nearly identical to the difference that we observed after infusion of isotope for 120 vs. 270 min, i.e., 13% (using a 60-min prime). In addition, these results are consistent with those of Hother-Nielsen et al. (20). These investigators used 3H glucose and provided data from normal control and type 2 diabetic subjects using a fixed prime or a prime adjusted to glycemia. In the normal control subjects, using a 100-min prime, the specific activity increased by 21% between 2.5 and 5 h of the isotope infusion as compared with 13% in our study (using a 60-min prime). In subjects with type 2 diabetes, the corresponding changes were 50% using a fixed 100-min prime and 25% using a prime adjusted to glycemia. Collectively, these results demonstrate that increasing the prime from 60 to 100 min did not overcome the isotopic disequilibrium in normal control subjects during short periods of isotope infusion. In subjects with type 2 diabetes, the use of an adjusted prime was helpful but did not completely overcome isotopic disequilibrium during short periods of isotope infusion. Thus, priming the substrate pool may be useful in approaching an "approximate" steady state during short infusions of glucose tracers but may still result in incomplete equilibration and an underestimation of glucose Ra.
Erroneous measures of glucose Ra will also result in propagating errors of measures of gluconeogenesis and glycogenolysis. The most recently described methods to measure gluconeogenesis ([U-13C]glucose [21,22] and [2-13C]glycerol MIDA [23]) and deuterated water with measurement of incorporation of deuterium in glucose carbons 5 and 6 (24) express gluconeogenesis as a fraction of glucose Ra. Rates of gluconeogenesis are subsequently calculated as the product of fractional gluconeogenesis and glucose Ra and, therefore, directly affected by an erroneous measure of glucose Ra. Likewise, measures of glycogenolysis, which are calculated as the difference between glucose Ra and the rate of gluconeogenesis, will also be affected. Similar errors may occur in studies using nuclear magnetic resonance (NMR). NMR provides an objective measure of glycogenolysis, whereas gluconeogenesis is calculated by subtracting glycogenolysis from glucose Ra (measured by GCMS). In some reports using this NMR technique, short duration of tracer infusion was used to measure glucose Ra (24 h) (2527). In these studies (despite using a prime dose corresponding to 100-min infusion), rates of glucose Ra decreased from 12.6 µmol · kg-1 · min-1 · day-1 at 2 h of isotope infusion to 8.4 µmol · kg-1 · min-1 · day-1 after 4 h of infusion, i.e., 33%, which is in agreement with our results and reemphasizes the importance of using sufficient duration of isotope infusion.
These studies unequivocally demonstrate the importance of the duration of isotope infusion in achieving valid results. Thus, investigators who conduct studies on glucose turnover rates using tracer infusion periods of <45 h should be cognizant that their measurements of glucose Ra using steady-state equations may be overestimated, which has to be taken into account when drawing conclusions from their data.
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ACKNOWLEDGMENTS |
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We thank Shaji Chacko, Cindy Clarke, Dan Donaldson, Kathryn Louie, and Matt Moore for technical assistance and our nurse coordinator Andrea Dotting-Jones and the nursing staff of the Metabolic Research Unit at the Childrens Nutrition Research Center for assistance with the execution of these studies.
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FOOTNOTES |
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Received for publication 1 January 2002 and accepted in revised form 13 August 2002.
GCMS, gas chromatography-mass spectrometry; NMR, nuclear magnetic resonance; Ra, rate of appearance.
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REFERENCES |
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