Oxidation of glutamine by the splanchnic bed in humans

M. Haisch, N. K. Fukagawa, and D. E. Matthews

Departments of Medicine and Chemistry, University of Vermont, Burlington, Vermont 05405


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

[1,2-13C2]glutamine and [ring-2H5]phenylalanine were infused for 7 h into five postabsorptive healthy subjects on two occasions. On one occasion, the tracers were infused intravenously for 3.5 h and then by a nasogastric tube for 3.5 h. The order of infusion was reversed on the other occasion. From the plasma tracer enrichment measurements at plateau during the intravenous and nasogastric infusion periods, we determined that 27 ± 2% of the enterally delivered phenylalanine and 64 ± 2% of the glutamine were removed on the first pass by the splanchnic bed. Glutamine flux was 303 ± 8 µmol · kg-1 · h-1. Of the enterally delivered [13C]glutamine tracer, 73 ± 2% was recovered as exhaled CO2 compared with 58 ± 1% of the intravenously infused tracer. The fraction of the enterally delivered tracer that was oxidized specifically on the first pass by the splanchnic bed was 53 ± 2%, comprising 83% of the total tracer extracted. From the appearance of 13C in plasma glucose, we estimated that 7 and 10% of the intravenously and nasogastrically infused glutamine tracers, respectively, were converted to glucose. The results for glutamine flux and first-pass extraction were similar to our previously reported values when a [2-15N]glutamine tracer [Matthews DE, Morano MA, and Campbell RG, Am J Physiol Endocrinol Metab 264: E848-E854, 1993] was used. The results of [13C]glutamine tracer disposal demonstrate that the major fate of enteral glutamine extraction is for oxidation and that only a minor portion is used for gluconeogenesis.

glutamine kinetics; gut; liver; stable isotopes; glutamine metabolism; phenylalanine kinetics


    INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

GLUTAMINE is one of the most important amino acids in the body. It has the largest free pool and one of the highest fluxes through blood of all amino acids (11). It is synthesized in large quantities in muscle and is the major vehicle of transport of amino-nitrogen from muscle (1, 11). Glutamine is also considered an important fuel for the gut (24) and plays a prominent role in the nitrogen metabolism of the liver (1). Glutamine is formed as a mechanism of scavenging excess ammonia from portal blood (9). Ammonia can also be derived from the glutamine amide group and incorporated into urea via hepatic urea synthesis. Glutamine carbon enters the tricarboxylic acid (TCA) cycle as alpha -ketoglutarate and is used for energy or for formation of new glucose (16).

Several studies have been performed to investigate the fate of enteral glutamine. We previously showed that the splanchnic bed removes about one-half of enterally administered glutamine tracer directly on the first pass during absorption (13). When Hankard et al. (7) infused enterally a glutamine tracer alone, they found that almost three-quarters of the glutamine tracer was retained by the splanchnic bed. When the tracer was infused simultaneously with a large glutamine load, the relative amount of glutamine retained by the splanchnic bed was reduced to 53%. The absolute amount, however, was substantial. They also determined that the majority of the infused glutamine tracer was oxidized to CO2 (7). However, Hankard et al. were unable to define whether the oxidation of the enteral glutamine was directly in the splanchnic bed or occurred after the glutamine tracer had passed through systemic circulation. The present study was designed to extend the former two studies (7, 13) by investigating the specific fate of glutamine carbon in the splanchnic bed in humans.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. L-[1,2-13C2]glutamine, L-[phenyl-2H5]phenylalanine (98.3% 2H5, abbreviated d5-phenylalanine), and sodium [13C]bicarbonate were obtained from Tracer Technologies (Somerville, MA; presently Masstrace, Woburn, MA). Chemical and isotopic purities were confirmed by gas chromatography-mass spectrometry (GC-MS).

The [1,2-13C2]glutamine was determined to have an average 13C enrichment of 98.2% in the two positions (1 and 2) on the basis of the measured distribution of 13C in the tracer of 96.3% dilabeled 13C, 3.6% monolabeled 13C, and 0.1% unlabeled glutamine. In the calculations described below, 96.3% was used for the dilabeled 13C enrichment (E2), and 98.2% 13C was used for the total 13C enrichment (Et). The [1,2-13C2]glutamine tracer was also determined to contain a small amount (2.3%) of [13C2]glutamic acid.

Before every infusion study, sterile solutions of the tracers were prepared using aseptic technique. Accurately weighed amounts of the labeled compounds were dissolved in weighed volumes of sterile, pyrogen-free saline and filtered through a 0.22-µm syringe filter before use. An aliquot of the sterile solution was initially verified to be pyrogen free before administration to human subjects. Solutions were prepared no more than 24 h in advance of use and were kept at 4°C before administration.

Subjects. Five healthy adults of normal weight for their height (Table 1) were studied at the University of Vermont General Clinical Research Center (CRC). Medical history, physical examination, and biochemical laboratory screening tests were obtained to verify that each subject was free of chemically evident metabolic, gastrointestinal, cardiovascular, neurological, or infectious disorders. The subjects were instructed of the purpose, benefits, and risks of the study and gave their written consent in accordance with protocols approved by the Committee on Human Research at the University of Vermont and by the CRC Scientific Advisory Committee.

                              
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Table 1.   Subject characteristics

Infusion protocol. Each subject was admitted to the CRC two evenings before each infusion study. The dinner the first night was ad libitum. The next morning, the subjects consumed a weight-maintaining liquid-formula diet (Ensure-Plus, Ross Laboratories, Columbus OH) set at 1.5 times each subject's basal metabolic rate, given evenly as three meals: breakfast, lunch, and dinner. After 8 PM that day, subjects drank only water until completion of the infusion study the following day at 3:30 PM. An 8-Fr 109-cm weighted nasogastric tube (Corpak, Wheeling, IL) was placed by 9 PM on the evening before each infusion study. All subjects slept in the CRC during the night before each infusion study. At 6:30 AM subjects were awakened and allowed to void; two intravenous catheters were placed, one in a forearm vein for infusion of the tracers and one inserted retrograde into a superficial, dorsal vein of the hand of the contralateral arm for blood sampling. Before each blood sample was obtained, the hand was warmed in a heated-air box (55°C air temperature) to produce "arterialized"-venous blood samples. The catheters were kept patent with a slow infusion of sterile saline.

Two infusion protocols were used. In study 1, [1,2-13C2]glutamine and d5-phenylalanine were infused by the intravenous route for the 1st 3.5 h and then via the nasogastric tube for the 2nd 3.5 h. In study 2, the [1,2-13C2]glutamine and d5-phenylalanine were infused for the 1st 3.5 h by the nasogastric route and then for the last 3.5 h by the intravenous route. All subjects participated in both studies. Each subject completed both infusions within 2 wk, and the order of the infusions was randomized among subjects.

Just before the start of each infusion (7:30 AM), priming doses of d5-phenylalanine, [1,2-13C2]glutamine, and [13C]bicarbonate (0.9, 6.3, and 15.6 µmol/kg, respectively) were administered as an intravenous bolus dose. In study 2, the [13C]bicarbonate priming dose was increased by one-third to 20.3 µmol/kg. Immediately thereafter, in study 1 an intravenous infusion of 6.3 µmol · kg-1 · h-1 of [1,2-13C2]glutamine and 1.0 µmol · kg-1 · h-1 of d5-phenylalanine was begun by use of a calibrated syringe pump (model 2001, Medfusion, Duluth, GA) set at 7.1 ml/h. At 3.5 h, the route of infusion was switched from intravenous to nasogastric. Study 2 started with a nasogastric infusion at the same infusion rate for the glutamine and phenylalanine tracers. After 3.5 h the route of infusion was switched to intravenous infusion in study 2. Water was infused into the nasogastric tube at 30 ml/h during the intravenous tracer periods and at 90 ml/h during the nasogastric tracer periods to keep flow through the enteral system.

In both studies, blood and breath samples were drawn just before the start and at 15-min intervals during the last 1.5 h of each of the two 3.5-h tracer infusion periods. At hourly intervals, each subject's carbon dioxide production was measured for 10-min periods with an indirect calorimeter with a flow-through canopy system (DeltaTrak by Datek, Sensormedics, Yorba Linda, CA). The carbon dioxide production rate was used to calculate the glutamine oxidation.

Analytical methods. Aliquots of blood were placed in heparinized tubes and stored on ice until the plasma was prepared by centrifugation at 4°C, frozen, and stored at -60°C for later analysis. Breath samples were placed into 20-ml evacuated tubes until measurement of 13CO2 in the expired air by isotope ratio mass spectrometry. For measuring plasma amino acid enrichments, 0.5-ml aliquots of plasma were acidified and added to a cation-exchange column to isolate the amino acid fraction. The column was washed with distilled water; then the amino acids were eluted using 3 M ammonium hydroxide. The ammonia eluant from the column was collected into screw-cap vials and evaporated to dryness. A 50-µl aliquot of a 1:1 solution of N-methyl-N-(t-butyldimethylsilyl)-trifluoroacetamide (Pierce Chemical, Rockford, IL) and acetonitrile was added, and the vials were capped and heated at 100°C for 30 min to form the t-butyldimethylsilyl (t-BDMS) amino acid derivatives. Injections of the t-BDMS samples were made into a GC-MS instrument (model 5971, Hewlett-Packard, Palo Alto, CA) using electron impact ionization. The tris-t-BDMS glutamine and the bis-t-BDMS phenylalanine were separated in the gas chromatograph from other amino acids isothermally at 245°C. The [M-57]+ ions at mass-to-charge ratios (m/z) 431 and 433 were monitored for unlabeled glutamine and [1,2-13C2]glutamine, respectively, and ions at m/z 336 and 341 were monitored for unlabeled phenylalanine and d5-phenylalanine, respectively. The peak area ratios of 433/431 and 341/336 were determined by selected ion monitoring as performed previously. From these ratios, the background corrected amino acid enrichments in mole % excess (MPE) were calculated as previously defined (3, 12). These GC-MS measurements reflect specifically the measurement of the [1,2-13C2]glutamine and d5-phenylalanine species.

For measuring plasma glucose 13C enrichments, 0.1-ml aliquots of plasma were added to tubes containing 0.3 ml of ice-cold (4°C) acetone for deproteinization. The tubes were allowed to stand for 10 min at 4°C and then were centrifuged at 4°C for 10 min. The supernatant was decanted and placed into screw-cap vials, and the samples were evaporated to dryness under a gentle stream of dry nitrogen at 40°C. After addition of 50 µl of 2% butyl boron dihydroxide (Sigma, St. Louis, MO) in pyridine, the samples were allowed to sit for 24 h at room temperature. Acetic anhydride (50 µl) was added just before measurement.

The butylboronate glucose derivatives were measured for 13C content by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS) by use of a DELTA-Plus instrument with a GCC-III unit (Finnigan, Bremen, Germany). The gas chromatograph was equipped with an RTX-1 30-m, 0.25-mm ID, 0.25-µm film thickness fused silica column (Restek, Bellefonte, PA) and operated isothermally at a temperature of 230°C and a flow rate of 0.4 ml/min. One-microliter aliquots of the samples were injected into the GC by use of a split ratio of 1:20. The temperature of the combustion oven was set at 960°C. The butylboronate peak was chromatographically separated from all other compounds. Aliquots of CO2 of known 13C/12C content were injected into the IRMS during the chromatographic runs before and after the elution of the glucose butylboronate peak to provide the reference ratio of 13C to 12C (13C/12C) peaks. All samples were measured for the ratio of 13CO2 to 12CO2 (13CO2/12CO2) with the Faraday cup collectors for measurement of the M+ ion (including isotopes) of CO2 at m/z 44, 45, and 46. The m/z 45/44 isotope ratio data were transformed using standard software for IRMS into 13C/12C for the samples using the known 13C/12C content of the reference CO2. A standard curve of known [1-13C]glucose was prepared, derivatized, and measured with the plasma glucose samples to confirm that the GC-C-IRMS produced a 1:1 linear response with respect to 13C measurement.

The 13C/12C ratio data (R13C) from the GC-C-IRMS for each peak were transformed into mole fraction abundance of 13C: F13C = R13C/(R13C + 1). Enrichments of 13C for a derivatized sample taken at time i were calculated as the difference between the F13C measured for plasma glucose taken before administration of tracer (F13C,0) sample and the F13C of the sample: E13C,i = F13C,i - F13C,0. The specific enrichment of the 13C in the glucose was assumed to occur at a single position. This enrichment is equal to E13C,i multiplied by the number of total carbons in the derivatized glucose (i.e., 16).

Calculations. The subscripts iv and ng refer to the enrichments and tracer infusion rates during the intravenous and nasogastric infusion periods. The appearance rates of glutamine and phenylalanine into plasma (synonymous with "flux" or "turnover" in the steady state) were calculated for the intravenous infusion period from the standard relationship
R<SUB>a</SUB> = I(E<SUB>i</SUB>/E<SUB>p,iv</SUB> − 1) (1)
where Ra is the rate of amino acid appearance into plasma (µmol · kg-1 · h-1), Ei and Ep,iv are the amino acid tracer enrichments (MPE) in the infusate and in plasma at steady state during the intravenous tracer infusion period, and I denotes the tracer infusion rate (µmol · kg-1 · h-1). For glutamine, the dilabeled 13C enrichment, E2, was used for the tracer enrichment Ei.

As discussed previously (3, 13, 14), the fraction of nasogastric tracer not extracted on the first pass by the splanchnic bed was determined by comparing the enrichment of the nasogastrically administered tracer in plasma (Ep,ng) to the enrichment of the intravenously infused tracer in plasma (Ep,iv), normalized for tracer infusion rates. The fraction of nasogastric tracer extracted on the first pass by the splanchnic bed (f) is
f = 1 − (E<SUB>p,ng</SUB>/i<SUB>ng</SUB>)/(E<SUB>p,iv</SUB>/i<SUB>iv</SUB>) (2)
where ing and iiv are the rates of nasogastric and intravenous tracer infusions in micromoles per kilogram per hour of enriched species per se. In this case, the rate of tracer infusion was the same for the intravenous and nasogastric periods, and the equation reduces to f = 1 - Ep,ng/Ep,iv. A necessary assumption for this calculation is that all tracer delivered via the nasogastric tube is absorbed. The validity of this assumption was demonstrated for glutamine by Déchelotte et al. (6).

The rate of oxidation of the [13C]glutamine to 13CO2 (F13C) is the product of the rate of CO2 production and the breath 13CO2 enrichment. This rate was increased by 1/0.81, with the assumption that only 81% of the metabolic CO2 that is produced is released as exhaled CO2 (2). The fraction of infused tracer that was oxidized to CO2 was calculated by dividing F13C by the rate of [13C]glutamine infusion. The rate of [13C]glutamine infusion (µmol 13C · kg-1 · h-1) was the rate of total glutamine tracer infusion (I) times the average 13C enrichment in positions 1 and 2 (Et): it = I · 2 · Et. For the intravenously infused tracer, the fraction of tracer oxidized to CO2 is F13C,iv/it.

The nasogastrically infused 13C tracer may be oxidized either on the first pass, during absorption by the splanchnic bed, or later, after passing into the systemic circulation. As previously defined (14), the fraction of nasogastric tracer oxidized directly on the first pass (fox) is the fraction of nasogastric 13C tracer oxidized, F13C,ng/it, minus that fraction of tracer which escapes first-pass extraction, (1 - f), and is subsequently oxidized (F13C,iv/it)
f<SUB>ox</SUB> = F<SUB>13C,ng</SUB>/i<SUB>t</SUB> − (1 − f) ⋅ F<SUB>13C,iv</SUB>/i<SUB>t</SUB> (3)

Statistics. Data are presented as means ± SE. To define steady state with time for plasma enrichments and 13CO2 excretion, a linear regression was performed for each data set for each half of each subject's infusion study. The slopes of the regression line for each subject were then averaged together for each infusion period, and these values were then tested by a two-tailed t-test for a slope being significantly different from zero. Where data were compared for all four infusion periods (i.e., iv 1st, ng 2nd, ng 1st, and iv 2nd), a two-period crossover design repeated-measures ANOVA (RMANOVA; SAS, version 6.12, SAS Institute, Cary, NC) was used. We analyzed the significance of differences between 1) the intravenous and nasogastric infusion periods and 2) the studies (i.e., whether there was a difference between study 1, iv 1st, and study 2, iv 2nd, infusion orders) for plasma tracer enrichment data. The glutamine enrichment was also analyzed by RMANOVA using the phenylalanine enrichments as a time-dependent covariate. The remainder of the comparisons were for data between two conditions, with an unpaired t-test and a pooled error term (comparisons between studies 1 and 2) and with a paired t-test (comparisons of iv vs. ng data within studies).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The time course of the plasma enrichments of [1,2-13C2]glutamine and d5-phenylalanine is shown in Figs. 1 and 2 for both studies 1 and 2. Although the plateaus during nasogastric infusion of tracer have more variance than during intravenous infusion of tracer, the time course of tracer enrichments during the last 1.25 h of each infusion period was constant, and the tracer enrichments were in isotopic steady state. The mean plasma enrichments of [1,2-13C2]glutamine and d5-phenylalanine are summarized in Table 2. The plasma tracer enrichments during the nasogastric infusion were less (P < 0.0001) than the enrichments during the intravenous infusion for both tracers. There was a significant effect of duration of glutamine tracer infusion time on enrichment between periods, but the effect was too small to be seen as a significant rise during the 1.25-h plateau periods. When the phenylalanine enrichment was used as covariate for the glutamine enrichment, this effect disappeared, indicating that the effect of time was also common to phenylalanine.


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Fig. 1.   Time course of L-[1,2-13C2]glutamine enrichment in plasma. Study 1: ; study 2: open circle . mpe, mole % excess [1,2-13C2]glutamine. Route of tracer delivery was switched in each study at 3.5 h between intravenous (iv) and nasogastric (ng) infusion routes. Data are means ± SE of 5 subjects. Horizontal lines associated with each infusion period represent mean value tracer enrichments for each time period. There was no significant change with time in enrichments during the period defined by horizontal lines (last 1.25 h) for any of the 4 infusion periods.



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Fig. 2.   Time course of L-[phenyl-2H5]phenylalanine (d5-phenylalanine) enrichment in plasma. Study 1: ; study 2: open circle . mpe, mole % excess d5-phenylalanine. Route of tracer delivery was switched in each study at 3.5 h between iv and ng routes. Data are means ± SE of 5 subjects. Horizontal lines associated with each infusion period represent mean value tracer enrichments for each time period. There was no significant change with time in enrichments during the period defined by horizontal lines (last 1.25 h) for any of the 4 infusion periods.


                              
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Table 2.   Tracer enrichments during steady state in breath and plasma

Phenylalanine and glutamine rates of appearance were calculated from the mean plasma enrichments for each tracer during the intravenous infusion period of each study. These data are shown in Table 3 for individual subjects. Although the phenylalanine Ra values appear 9% lower when the tracer was infused by the intravenous route in the second half of the study (study 2) compared with infusion of tracer during the first half of the study (study 1), there was no significant difference in the appearance rates between study 1 and study 2 for either phenylalanine or glutamine Ra.

                              
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Table 3.   Amino acid kinetic parameters calculated from plasma amino acid enrichments

The first-pass extraction by the splanchnic bed (f) of enteral phenylalanine and glutamine tracers is shown for each subject in Table 3. Significantly more of the nasogastrically infused glutamine tracer was extracted on the first pass (63.7 ± 1.6%) than for phenylalanine (26.9 ± 1.6%). There was also an effect of infusion order on the amount of tracer extracted by the splanchnic bed. The fraction extracted by the splanchnic bed was lower for glutamine and phenylalanine when the calculation was performed for study 1 (when the tracers were infused first by the iv route and then by the ng route) compared with study 2 (when the tracer infusion order was the reverse). When the f of phenylalanine was used as a covariate for the f of glutamine, the effect of infusion order became nonsignificant, indicating that the effect of infusion order on f was common to both glutamine and phenylalanine. The effect of tracer infusion order has been observed previously (3, 14).

Because each subject received two infusions with the intravenous-nasogastric order reversed between studies, we can also calculate f by comparing the tracer enrichments only from period 1 of each infusion study. The same calculation of f also can be done using only enrichment data from period 2. The first-pass extraction of phenylalanine for the five subjects was 26.8 ± 4.9% when the intravenous and nasogastric enrichment data for the first half of the tracer infusion from studies 1 and 2 were used, and it was 26.5 ± 5.2% for the second half of the tracer infusion. There was no significant difference between these values. The mean of these values was f = 26.7 ± 1.5% for the five subjects. The first-pass extraction of glutamine for the five subjects was 67.1 ± 2.2% when the intravenous and nasogastric enrichment data for the first half of the tracer infusion from studies 1 and 2 were used, and it was 60.7 ± 2.7% for the second half of the tracer infusion. There was no significant difference between these values. The mean of these values was f = 63.9 ± 2.7% for the five subjects. The overall means computed for glutamine and phenylalanine in Table 2 for f from enrichments on the same day are not different from the overall means calculated from enrichments from the same period. Either approach negates the effect of infusion duration on f.

The CO2 production rate measured by indirect calorimetry over the course of the 7-h infusion period did not change significantly with time and was not different between the two infusion studies (Table 2). The oxidation of [1,2-13C2]glutamine to 13CO2 was determined from the product of the CO2 production rate and the breath 13CO2 enrichment. The time course of 13CO2 excretion is shown in Fig. 3 for both studies. The 13CO2 excretion reached a steady state in both studies; there was no significant change in 13CO2 excretion with respect to time during the last 1.25 h during any of the four infusion periods. The mean rates of 13CO2 excretion are presented in Table 2 for each infusion study and period.


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Fig. 3.   Time course of rate of 13CO2 excretion in breath. Study 1: ; study 2: open circle . Route of tracer delivery was switched in each study at 3.5 h between iv and ng routes. Data are means ± SE of 5 subjects. Horizontal lines associated with each infusion period represent mean value excretion rate for each time period. There was no significant change with time in 13CO2 excretion during the period defined by horizontal lines (last 1.25 h) for any of the 4 infusion periods.

The fraction of the infused [13C]glutamine tracer oxidized to CO2 was determined by dividing the rate of 13CO2 excretion by the rate of [13C]glutamine infusion. These data are presented in Table 4. When the [13C]glutamine tracer was infused by the intravenous route, 57.5 ± 1.2% of the infused 13C tracer was recovered in breath CO2 (Table 4). A higher fraction of oxidation was determined for the nasogastrically infused tracer: 73.4 ± 1.9% of it was recovered as 13CO2. There was an effect of infusion order (duration of infusion time on the rate of excretion of 13CO2, P < 0.01; Table 4). The fractional oxidation data were used to calculate for each subject the amount of nasogastric tracer infused that was oxidized directly on the first pass by the splanchnic bed (fox). An average of 52.9 ± 2.4% of the [13C]glutamine tracer was extracted and oxidized directly on the first pass by the splanchnic bed (Table 4). There was no effect of infusion order in calculating first-pass oxidation, because all parameters in the calculation of fox are affected similarly by changes with time.

                              
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Table 4.   Glutamine kinetic parameters measured from exhaled 13CO2

The transfer of [13C]glutamine to glucose was measured from the increase in 13C in glucose with time during the study. The 13C enrichment in glucose was too small to be measured by GC-MS and therefore was measured by GC-C-IRMS. The GC-C-IRMS method measures 13C in the entire derivatized glucose molecule with great sensitivity but cannot distinguish where in the glucose is the 13C. For the purposes of the calculations in this paper, we have assumed on the basis of the low enrichments of 13C found in glucose that the 13C occurs no more frequently than one atom per molecule of glucose. The time course of the measured plasma glucose 13C enrichment is shown in Fig. 4. The enrichments rose during the first 3.5 h but tended toward plateau by the end of the period. The [13C]glucose enrichment was not significantly different during the last 1.25 h of the first half of study 2 (ng infusion) but did rise slightly and significantly (P < 0.05) during the last 1.25 h of the first half of study 1. Although glucose 13C enrichments rose or fell initially in the second half of the two infusion studies, the enrichments reached plateaus during the last 1.25 h. The mean glucose 13C enrichment values are presented in Table 2. The glucose 13C enrichments were significantly higher (P < 0.001) when [13C]glutamine was infused by the nasogastric vs. the intravenous route. There was also a significant (P < 0.001) rise in glucose 13C enrichment over time (comparison of the 1st period vs. the 2nd period) for studies 1 and 2.


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Fig. 4.   Time course of [13C]glucose enrichment in plasma. Study 1: ; study 2: open circle . Route of tracer delivery was switched in each study at 3.5 h between iv and ng routes. Data are means ± SE of 5 subjects. Enrichments are expressed as atom % excess 13C (ape) in one single position in the glucose carbon. Enrichment for all 6 carbons would be a factor of 6 less. Horizontal lines associated with each infusion period represent mean value excretion rate for each time period. There was a significant change with time (P < 0.05) in period defined by horizontal lines (last 1.25 h) in [13C]glucose enrichment only during the iv period of study 1.

Although we did not directly measure the rate of glucose production in individual subjects, we have assumed a glucose production rate (10.9 µmol · kg-1 · min-1) taken from a study that used a design and subjects similar to those reported here (8). Multiplying this glucose production rate by the plasma [13C1]glucose enrichment gives the rate of plasma glucose 13C formation from glutamine (Ra,13CGlc). These data are presented for individual subjects in Table 5 and mirror the plasma [13C]glucose enrichments in terms of changes: the rate of 13C appearance in glucose was significantly (P < 0.001) greater during nasogastric infusion of the [13C]glutamine tracer compared with the intravenous infusion. We can then divide Ra,13CGlc by the rate of [13C]glutamine tracer infusion to define the fraction of infused glutamine converted to glucose. However, because the first carbon of glutamine is released immediately when alpha -ketoglutarate enters the TCA cycle and is decarboxylated to form succinate, it cannot be transferred. The fraction of infused glutamine converted to glucose was then estimated as Ra,13CGlc/0.5 it), where the 0.5 indicates that only one-half of the infused 13C label could appear in glucose. Approximately 6.7 ± 0.4% of the glutamine infused by the intravenous route was converted to glucose. Significantly more infused glutamine (10.7 ± 0.7%) was converted to glucose when the tracer was infused by the nasogastric route.

                              
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Table 5.   Transfer of [13C]glutamine to plasma glucose


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

In the first study from this laboratory of splanchnic uptake of glutamine in humans (13), we used a [2-15N]glutamine tracer and determined that the splanchnic bed extracted directly on the first pass 54 ± 4% of enteral glutamine tracer. Enteral glutamate extraction was also determined by use of a [15N]glutamate tracer to be 88 ± 2% (13). As pointed out in a subsequent paper (3), in which we used a [1,2-13C2]glutamate tracer, the studies measure the metabolism of the tracers used. Thus different tracers may follow and measure different metabolic fates. In the case of glutamate, which is rapidly transaminated to/from alpha -ketoglutarate, the 15N label may be rapidly lost during reversible transamination of the glutamate in passage through the splanchnic bed. Such a result would give an artificially high measurement of splanchnic uptake of glutamate. The [13C]glutamate tracer gave us an alternative measure of splanchnic glutamate metabolism and also gave us the opportunity to follow the irreversible oxidation of the glutamate tracer to 13CO2 by the splanchnic bed. The results of the [13C]glutamate tracer study were similar to what was found previously for [15N]glutamate: 96 ± 1% of the [13C]glutamate tracer was extracted on the first pass by the splanchnic bed. Furthermore, we found that 78% of the glutamate tracer that was sequestered was oxidized. The results of these studies (3, 13) demonstrated that 1) the splanchnic bed extracts significantly more glutamate than glutamine and 2) the primary fate of the extracted glutamate is oxidation.

These results are surprising if glutamine is considered to be the most important fuel for the gut (18, 19). The assumption that glutamine is a key fuel for the gut arises from the studies of Windmueller and Spaeth (20, 21, 23). However, these authors also noted that the fractional extraction by the rat intestine of glutamate was considerably greater than that for glutamine and that glutamate was also readily oxidized to CO2 (21, 22).

The only other study of first-pass uptake of glutamine tracers by the splanchnic bed in humans is the study of Hankard et al. (7). They infused [1-13C]glutamine by the nasogastric route and [3,4-3H]glutamine by the intravenous route and determined a splanchnic uptake of 74 ± 4%. This amount of uptake is significantly greater than the 54% we determined using a [2-15N]glutamine tracer (13). Hankard et al. also determined that the fraction of nasogastrically infused [13C]glutamine tracer oxidized was 69 ± 2% (7). These results imply that almost all of the glutamine extracted by the splanchnic bed was oxidized. However, the 13C tracer was infused only by the nasogastric route, so that Hankard et al. were unable to distinguish between glutamine oxidation by the splanchnic bed on the first pass and subsequent systemic oxidation.

We performed the present study to extend our earlier work with the [2-15N]glutamine tracer and the study of Hankard et al. (7). We also infused the 13C-labeled glutamine tracer both by intravenous and nasogastric routes to determine the fraction of glutamine that is oxidized directly on the first pass during splanchnic uptake. Because the fate of the 1st carbon of glutamine in the TCA cycle is immediate release as CO2 when alpha -ketoglutarate is converted to succinate, we used a [1,2-13C2]glutamine tracer to follow the carbon fate through subsequent passes through the TCA cycle. The recovery of the 2nd carbon as CO2 better reflects oxidation of the entire glutamine molecule per se. The use of the [1,2-13C2]glutamine tracer also allowed us to compare our glutamine results obtained in this study with our former glutamate results using [1,2-13C2]glutamate (3).

The glutamine appearance rate determined during the intravenous infusion periods of [1,2-13C2]glutamine was 303 ± 8 µmol · kg-1 · h-1 in our postabsorptive subjects. This value was not different from what we determined previously for glutamine appearance using a [2-15N]glutamine tracer (295 ± 13 µmol · kg-1 · h-1) (13). These results suggest that the metabolic fate is similar for both tracers. This conclusion is in contrast to the report of Kreider et al. (10) that showed different glutamine fluxes for different tracers. When Kreider et al. infused simultaneously [2-15N]-, [U-14C]-, and [3,4-3H]glutamine tracers, they determined postabsorptive glutamine Ra values of 280 ± 23, 343 ± 32, and 368 ± 32 µmol · kg-1 · h-1, respectively. Hankard et al. (7) infused simultaneously [1-13C]-, [1-14C]-, and [3,4-3H]glutamine tracers and measured postabsorptive Ra values of 355 ± 24, 373 ± 19, and 393 ± 24 µmol · kg-1 · h-1, respectively. However, Hankard et al. infused another group of subjects under the same conditions with [3,4-3H]glutamine and measured a glutamine appearance rate of 258 ± 20 µmol · kg-1 · h-1. Thus the range of reported glutamine fluxes is just as great for a single tracer as it is when different tracers are compared. In our hands, the flux of glutamine was not significantly different when 13C and 15N tracers were compared. Much of the reported difference may arise as differences between radioactive and stable isotope tracer methods.

The fraction of nasogastrically infused [1,2-13C2]glutamine sequestered directly on the first pass in this study was 64 ± 2%. However, we noted an effect of infusion duration on the observed plasma glutamine enrichments (Table 2) and the calculated f derived from these values as the mean of studies 1 and 2 for each individual (Table 4). In our previous study (13) using [2-15N]glutamine, the experimental design was limited to the single design of study 1. We found that 54 ± 4% of the [2-15N]glutamine tracer was sequestered on the first pass in that study. The value of f for study 1, here, when [1,2-13C2]glutamine was used, was 58 ± 2%. Thus there would appear to be no difference between the metabolic fate in terms of uptake of the amino-15N vs. the [13C]glutamine tracers by the splanchnic bed. The conclusion from the present and former studies is that the uptake of the glutamine tracer is not an artifact of the label used.

Hankard et al. (7) infused simultaneously a [3,4-3H]glutamine tracer intravenously and the [1-13C]glutamine tracer nasogastrically to determine f. They found that 74 ± 4% of the glutamine tracer was sequestered on the first pass. This value is greater than what we determined using [1,2-13C2]glutamine (64 ± 2%). The higher value for f reported by Hankard et al. can be explained by the intravenous glutamine kinetics with the [3H]glutamine tracer. During their validation of the 3H tracer against [13C]- and [14C]glutamine tracers, they determined a glutamine Ra of 393 ± 24 µmol · kg-1 · h-1 with the 3H tracer, but during the infusions when they determined f, their [3H]glutamine measured Ra was only 258 ± 20 µmol · kg-1 · h-1, a value 35% lower. If a glutamine Ra of 393 µmol · kg-1 · h-1 were used to determine their f, then their first-pass extraction for glutamine would be 61 ± 4%. In fact, one of their five subjects had a glutamine Ra of 337 µmol · kg-1 · h-1; that subject also had an f of 58%. We believe that differences in glutamine Ra measured using the [3H]glutamine tracer in the study by Hankard et al. explain differences between their results and ours.

We measured the fraction of infused [13C]glutamine tracer oxidized both during intravenous infusion and nasogastric infusion. During the intravenous infusion periods, we recovered 58 ± 2% of the [13C]glutamine tracer as 13CO2. Nurjhan et al. (15) and Perriello et al. (16) both infused [U-14C]glutamine for 5 h into healthy subjects. They recovered 44 and 59% of the 14C tracer as 14CO2, respectively. Hankard et al. (7) simultaneously infused [1-13C]- and [U-14C]glutamine tracers intravenously and recovered 51 ± 5 and 32 ± 3%, respectively, of the labels as CO2. Another study reported recovery of 34 ± 2% of intravenously infused [3,4-13C2]glutamine as CO2 (8). Finally, Darmaun et al. (5) recovered 64% of intravenous infused [1-13C]glutamine as CO2. The differences among studies do not appear to be protocol related, because similar infusion durations and similarly fasted subjects were used. The initial suggestion would be that different tracers have different recoveries as CO2.

We would expect highest recovery of the carbon labeled in the first position of glutamine, because it is released immediately when alpha -ketoglutarate enters the TCA cycle and is decarboxylated to form succinate. Additional passes through the TCA cycle are required to liberate the remaining carbons. There should be little difference in the recovery of label in the other positions, although slightly more retention in the TCA cycle may occur for the 3rd and 4th carbons. However, the variability of measurement of recovery among studies is high, even for use of the same tracer [e.g., 14CO2 recoveries from [U-14C]glutamine ranging from 59% (16) to 32% (7)]. The fractional recovery of intravenously infused carbon-labeled glutamine tracer ranges from 32 to 64% in the literature, and our results are intermediate.

We also infused the [1,2-13C2]glutamine tracer by the nasogastric route and determined that 73 ± 2% of the tracer was recovered as 13CO2, a significantly higher fraction of the tracer being oxidized when infused by the nasogastric route compared with the intravenous route. Only Hankard et al. (7) infused a carbon-labeled glutamine by the nasogastric route. They recovered 69 ± 2% of the [1-13C]glutamine as exhaled 13CO2 when it was infused by the nasogastric route. Their value for recovery of nasogastrically infused tracer is not different from what we report here.

To our knowledge, this report is the first in which the same subjects have been infused on the same day by both the intravenous and nasogastric routes with a carbon-labeled glutamine tracer. Using the intravenously infused tracer to define the systemic kinetics for oxidation of glutamine to CO2, we calculated the fraction of nasogastrically infused [13C]glutamine tracer sequestered on the first pass by the splanchnic bed and oxidized directly to be 53 ± 2% (Table 4). Thus, of the 64% of tracer glutamine sequestered by the splanchnic bed, 53/64, or 83%, was oxidized and only 17% was retained for other uses. Other uses would include incorporation of the glutamine into new protein and conversion to other forms of carbon, such as glucose. What is clear is that only a small amount of the glutamine tracer sequestered by the splanchnic bed had a fate other than oxidation via the TCA cycle.

We previously used the same protocol to measure the kinetics of [1,2-13C2]glutamate in the whole body and in the splanchnic bed (3). In that study we found that 96 ± 1% of the nasogastrically infused glutamate tracer was extracted on the first pass by the splanchnic bed and that 78 ± 3% of the nasogastrically infused tracer was oxidized directly on the first pass. Thus, when the glutamine and glutamate were presented enterally in tracer amounts to postabsorptive subjects, significantly more of the glutamate than the glutamine was sequestered by the splanchnic bed (96 vs. 64%, respectively). However, the fate of the extracted glutamine and glutamate tracers was similar: 53/64 = 83% of the glutamine sequestered by the splanchnic bed was oxidized, whereas 78/96 = 81% of the sequestered glutamate was oxidized. Thus delivery of glutamine and glutamate enterally at similar rates to the splanchnic bed gave similar results: oxidation.

We postulated previously that glutamate is an excellent fuel for the gut and that the gut will extract and oxidize glutamate preferentially to glutamine. These data are supported by the perfused rat intestine results of Windmueller and Spaeth (21, 22) and in the gut of the piglet (17). However, the results of this present study and our former study (3) show that, when both glutamine and glutamate are delivered in tracer amounts, they are both oxidized equally and nearly completely in the splanchnic bed. The primary difference is that considerably less glutamine than glutamate is extracted on the first pass. In addition, several multiples of increase more glutamine than glutamate are delivered from the arterial side to the gut. Thus the gut needs to remove a smaller fraction of the enteral glutamine tracer because of the large glutamine supply coming in from the arterial side compared with arterial glutamate delivery.

We also infused d5-phenylalanine along with the [13C]glutamine tracer in this study to provide a measure of essential amino acid kinetics and uptake by the splanchnic bed. We have used the d5-phenylalanine tracer in previous studies of splanchnic bed metabolism (3, 14). The phenylalanine Ra measured here (35.7 ± 0.7 µmol · kg-1 · h-1) was not significantly different from the Ra value determined previously in the former studies of identical format (3, 14). Likewise, the fraction of phenylalanine extracted on the first pass in this study (27 ± 2%) was not significantly different from the first-pass extraction determined previously. These results for phenylalanine emphasize that nonessential amino acids, such as glutamine, are extracted by the splanchnic bed in significantly greater amounts than essential amino acids. The agreement between phenylalanine kinetics in this study and the former study (3) supports the comparison of the glutamine and glutamate kinetic results between this study and the former study in terms of concordance of infusion protocol and subjects between the two studies. Finally, the phenylalanine results may be used to track changes with infusion duration that are independent of glutamine metabolism.

In our former report (14), we observed that enrichments rose slowly with duration of infusion and that the effect was more prominent during the nasogastric tracer infusion. We concluded that this increase was due to equilibration of rapidly turning over proteins, probably of splanchnic origin, with time. A similar effect was seen previously (3) and in the present study (Table 2). These changes in time result in a difference in f calculated during the first or the second one-half of the study. The same effect is noted in the glutamine 13C plasma enrichments. We assume that the effect of increasing enrichment with duration of infusion is related to glutamine incorporation and equilibration in fast-turnover proteins. When the glutamine 13C enrichments are tested for differences between infusion periods with the d5-phenylalanine enrichments used as a covariate, we find that the relationship with infusion duration disappears. Thus the glutamine and phenylalanine tracers are affected similarly. This problem was addressed by performing two sets of infusions per subject by use of different starting infusion orders (iv vs. ng 1st).

Although the initial purpose of this study was to determine the amount of glutamine carbon extracted by the splanchnic bed for oxidation, we were able to define the utilization of glutamine for gluconeogenesis from the measurement of the transfer of [13C]glutamine to glucose. These measurements were made for both intravenous and nasogastric infusion of the [13C]glutamine tracer. Although there were changes during the early portion of each 3.5-h infusion period, the 13C enrichment in plasma glucose settled toward a steady state by the end of each infusion period (Fig. 4). From these enrichments and an assumed glucose Ra, we estimated the rate of glutamine tracer 13C conversion to glucose. We then calculated the fraction of infused [13C]glutamine converted to glucose. These calculations showed that 50% more of the nasogastrically infused glutamine tracer was converted to glucose compared with intravenously infused glucose (10.7 vs. 6.7%, respectively; Table 5). This calculation assumes that the first 13C in the [1,2-13C2]glutamine tracer is removed as CO2 upon entry into the TCA cycle and is not incorporated into glucose. The primary point of these calculations is that the rate of 13C transfer to glucose increases when the glutamine tracer is infused by the enteral route, indicating that most of the systemic conversion of glutamine to glucose is through the splanchnic bed, not through other organs such as the kidney.

Glutamine conversion to glucose accounts for some of the remaining 17% of the tracer that is sequestered on the first pass by the splanchnic bed and not oxidized. Presumably the remaining small portion of tracer enters newly synthesized splanchnic protein. Again, the situation of uptake of nonessential amino acids, such as glutamine, glutamate (3), and alanine (4), is considerably different from the splanchnic fate for essential amino acids, such as leucine and phenylalanine (14). For leucine, we have previously estimated that as much as one-half of the sequestered leucine is sequestered for new protein synthesis (14). The fate of the key gluconeogenic nonessential amino acids that have been studied to date is sequestration by the splanchnic bed and oxidation. The direct measurement of the appearance of [13C]glutamine into glucose verifies that only a small portion of the sequestered tracer is directly converted to glucose. We assume that the gut is the key organ in the process of sequestration of glutamine and glutamate for the purpose of oxidation.

In conclusion, the splanchnic bed sequesters 64 ± 2% of enteral glutamine tracer on the first pass in humans. The glutamine uptake is not due to simple loss of tracer during transamination or alpha -ketoglutarate exchange in the TCA cycle. Most of the extracted glutamine is oxidized directly in the splanchnic bed. Only a small fraction of the carbon is converted to other compounds, such as glucose. The gut extracts considerably less glutamine than glutamate, but the primary fate of extraction of both fates is identical: oxidation.


    ACKNOWLEDGEMENTS

This work was supported in part by National Institutes of Health Grants DK-38429 and RR-00109. M. Haisch was supported by the Deutsche Forschungsgemeinschaft.


    FOOTNOTES

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. §1734 solely to indicate this fact.

Address for reprint requests and other correspondence: D. E. Matthews, Univ. of Vermont, Clinical Pharmacology, Given Bldg. B217, Burlington, VT 05405 (E-mail: dmatthew{at}zoo.uvm.edu).

Received 7 July 1999; accepted in final form 25 October 1999.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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