Departments of Medicine and Chemistry, University of Vermont, Burlington, Vermont 05405
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ABSTRACT |
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[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 · kg1 · 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
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INTRODUCTION |
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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 -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.
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METHODS |
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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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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 · kgAnalytical 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.
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
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(1) |
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(2) |
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(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).
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RESULTS |
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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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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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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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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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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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Although we did not directly measure the rate of glucose production in
individual subjects, we have assumed a glucose production rate
(10.9 µmol · kg1 · 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
-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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DISCUSSION |
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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
-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 -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 · kg1 · 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 · kg1 · 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
-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 · kg1 · 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
-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.
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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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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.
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