Nemours Children's Clinic, Jacksonville, Florida 32207; Centre de Recherche en Nutrition Humaine, 44093 Nantes Cedex 1, France; and United States Department of Agriculture Children's Nutrition Research Center, Houston, Texas 77030-2600
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ABSTRACT |
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The present study was designed to determine
whether sodium phenylbutyrate (B) acutely induces a decrease in
plasma glutamine in healthy humans, and, if so, will decrease estimates
of whole body protein synthesis. In a first group of three
healthy subjects, graded doses (0, 0.18, and 0.36 g · kg
1 · day
1)
of
B were administered for 24 h before study: postabsorptive plasma
glutamine concentration declined in a dose-dependent manner, achieving
an
25% decline for a dose of 0.36 g
B · kg
1 · day
1.
A second group of six healthy adults received 5-h infusions of
L-[1-14C]leucine
and
L-[1-13C]glutamine
in the postabsorptive state on two separate days: 1) under baseline conditions and
2) after 24 h of oral treatment with
B (0.36 g · kg
1 · day
1)
in a randomized order. The 24-h phenylbutyrate treatment was associated
with 1) an
26% decline in plasma
glutamine concentration from 514 ± 24 to 380 ± 15 µM (means ± SE; P < 0.01 with paired t-test) with no change in glutamine
appearance rate or de novo synthesis;
2) no change in leucine appearance
rate (Ra), an index of protein
breakdown (123 ± 7 vs. 117 ± 5 µmol · kg
1 · h
1;
not significant); 3) an
22% rise
in leucine oxidation (Ox) from 23 ± 2 to 28 ± 2 µmol · kg
1 · h
1
(P < 0.01), resulting in an
11%
decline in nonoxidative leucine disposal (NOLD = Ra
Ox), an index of
protein synthesis, from 100 ± 6 to 89 ± 5 µmol · kg
1 · h
1
(P < 0.05). The data suggest that,
in healthy adults, 1) large doses of
oral phenylbutyrate can be used as a "glutamine trap" to create a
model of glutamine depletion; 2) a
moderate decline in plasma glutamine does not enhance rates of
endogenous glutamine production; and
3) a short-term depletion of plasma
glutamine decreases estimates of whole body protein synthesis.
protein metabolism; nutrition; stable isotopes; radioactive tracers
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INTRODUCTION |
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GLUTAMINE IS the most abundant amino acid in the body
and comprises two-thirds of the intracellular free amino acid pool (3, 17). In human plasma, the concentration of glutamine (typically 400-600 µM) exceeds that of all other amino acids and is four times that of leucine (3, 17). Even though glutamine can be synthesized
de novo and is therefore considered a nonessential amino acid,
glutamine concentration may participate in the regulation of protein
homeostasis. Indeed, a twofold rise in plasma glutamine concentration,
obtained through a 5-h enteral infusion of natural L-glutamine, acutely inhibited leucine oxidation and
increased nonoxidative leucine disposal (NOLD), an index of whole body
protein synthesis, in healthy humans in the postabsorptive state (10). Conversely, a precipitous drop in the muscle free glutamine pool is
observed in life-threatening conditions that are associated with
protein wasting (17, 30-32), and replenishment of muscle glutamine
stores is associated with improved nitrogen balance in such conditions
(15, 30, 32, 36). These data collectively suggest that the maintenance
of glutamine at its high, normal concentration in most biological
fluids may play a unique role to sustain protein synthesis. If so, then
an acute decrease in plasma glutamine concentration may cause protein
wasting in healthy humans. The purpose of this study was to test the
latter hypothesis.
Sodium phenylbutyrate and sodium phenylacetate are used in the clinical
management of children with urea cycle enzymatic defects as a
"glutamine trap" (4, 19). As shown by Brusilow (4), phenylbutyrate does not accumulate in plasma: within minutes after ingestion of large doses of phenylbutyrate (up to 0.6 g · kg1 · day
1),
the latter is converted to phenylacetate by
-oxidation
(phenylacetate is normally produced only in small quantities from
hepatic metabolism of phenylalanine). Phenylacetate in turn reacts with
glutamine in liver and kidney to yield phenylacetylglutamine (Fig.
1). The latter is quantitatively excreted as such in the
urine and seems to substitute for urea as a means to eliminate excess
ammonia. In children with inborn errors of urea cycle metabolism and
high glutamine and ammonia concentrations, administration of
phenylbutyrate indeed caused an abrupt decrease in plasma glutamine.
The drug caused a decline in plasma glutamine even in those among the
infants whose plasma glutamine levels were within normal limits (4). Even though other investigators (20, 28) have administered small doses
of phenylacetate to healthy adults with the aim of using
phenylacetylglutamine as a probe of intrahepatic tracer dilution in
tricarboxylic acid cycle intermediates, it is not known whether large
doses of phenylbutyrate alter plasma glutamine concentration in healthy
adults with an intact urea synthetic pathway.
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The present studies were therefore designed to determine 1) whether phenylbutyrate treatment affects plasma glutamine in healthy adults; 2) if so, whether this effect is dose dependent; and 3) whether a phenylbutyrate-induced decline in plasma glutamine concentration will decrease estimates of whole body protein synthesis.
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METHODS |
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Materials
Solutions of L-[1-14C]leucine (>55 mCi/mmol; New England Nuclear, Boston, MA), L-[1-13C]glutamine (99% 13C), L-[5,5,5-2H3]leucine (98% 2H3), and H13CO3Na (99% 13C; all three from Cambridge Isotope Laboratories, Woburn, MA) were mixed with 0.9% saline and tested for chemical, isotopic, and optical purity by HPLC and gas chromatography-mass spectrometry (GC-MS) for radioactive and stable isotope tracers, respectively, and were verified to be sterile (plate culture) and pyrogen free (Limulus lysate assay). Solutions were passed through a 0.22-µm Millipore filter and stored in sterile containers at 4°C for <24 h before use. Sodium phenylbutyrate (0.5-g tablets) was purchased from Triple Crown America (Perkasie, PA) after our application for its use as an investigational new drug (IND 46,010) was approved by the Food and Drug Administration.Subjects
This protocol was reviewed and approved by the Nemours Children's Clinic Research Committee and the Institutional Review Committee at Baptist Medical Center (Jacksonville, FL). Subjects were judged to be free of any chronic or acute illness based on detailed medical history, physical exam, and routine blood chemistry. Women had to have a negative pregnancy test within 48 h of study and could not be breastfeeding. A dietary history was obtained, and subjects were instructed to maintain their usual intake and their regular level of activity for 3 days before the initial study and between the two isotope infusions. A total of 11 healthy 29 ± 3-yr-old (4 female, 7 male) volunteers received detailed information on the purpose and potential risks of the study and were enrolled after signing a written consent form. Their mean ± SE height, weight, and body mass index were 169 ± 4 cm, 63 ± 6 kg, and 21.7 ± 1.0 kg/m2, respectively.Experimental Design
Two different protocols (A and B) were carried out. Protocol A consisted of preliminary studies designed to assess the plasma glutamine response to graded doses of sodium phenylbutyrate. Three subjects (2 male, 1 female) underwent isotope infusions in the postabsorptive state on three separate days, several days apart. Each received 0, 0.18, and 0.36 g · kgIsotope Infusion Protocol
For each protocol, the night before each infusion study day, each subject ate dinner at 1800 and then remained fasting (with the exception of water and calorie-free, caffein-free drinks) until completion of the infusion study at 1300 the following day. On the next morning at 0700, each subject was studied as an outpatient in the Baptist Medical Center/Wolfson Children's Hospital Clinical Investigation Unit. Two short catheters were inserted, one in a forearm vein for isotope infusion and the other one in a superficial vein of the contralateral hand; the hand was placed in a warming pad at ~60°C to obtain arterialized venous blood samples (5). Five-hour continuous infusions of tracers were administered in the postabsorptive state between 0800 and 1300: 1) unprimed infusions of L-[1-13C]glutamine (Analytical Methods
Plasma amino acid concentrations were determined by ion exchange chromatography using a Beckman 6300 amino acid analyzer (Beckman Instruments, Palo Alto, CA). Breath 13CO2 enrichments were measured by gas chromatography-isotope ratio mass spectrometry (Isochrom III). Plasma [13C]glutamine enrichments and [2H3]leucine and [2H3]KIC were determined by selected ion monitoring GC-MS (Hewlett-Packard MSD 5970), as described previously (10, 26, 27). Plasma leucine 14C specific activity (SA) was determined by HPLC (on a Spectraphysics SP8800 instrument equipped with a Pharmacia LKB Frac 200 fraction collector) as described previously (14). The 14C specific activity of leucine fractions and breath CO2 was measured on a Beckman LS6500 scintillation counter. Because of interference of phenylacetate, the main metabolite of phenylbutyrate, with the HPLC assay of KIC, we used the "primary pool" model to calculate leucine kinetics, i.e., based on plasma leucine specific activities rather than the preferred "reciprocal pool model" in which leucine kinetics are derived from KIC specific activities (13).Calculations
Leucine appearance rate. Leucine appearance rate into plasma (Ra Leu; µmol · kgLeucine oxidation. Leucine oxidation
(OxLeu;
µmol · kg1 · h
1)
was calculated based on the excretion of labeled
CO2 in expired air:
OxLeu = F14CO2/(SALeu × 0.7), where
F14CO2
is the rate of
14CO2
excretion in expired air
(µmol · kg
1 · h
1),
and 0.7 is the assumed fractional recovery of labeled carbon in expired
air in the postabsorptive state (12).
NOLD. NOLD
(µmol · kg1 · h
1)
was calculated as NOLD = Ra Leu
OxLeu. Because leucine is assumed
to be either oxidized or incorporated into protein, NOLD is an index of
whole body protein synthesis (13, 22).
Glutamine appearance into the plasma
compartment. Glutamine appearance into the
plasma compartment
(Ra Gln;
µmol · kg1 · h
1)
was calculated as Ra Gln = iGln × [(EiGln/EpGln)
1], where iGln is
the rate of
[13C]glutamine
infusion, and EiGln and
EpGln are the
[13C]glutamine
enrichments (mole %excess) in the infused tracer solution and plasma
at steady state, respectively.
Ra Gln is an index of interorgan glutamine exchange between tissues (7).
Glutamine release from protein
breakdown. Because glutamine is a nonessential amino
acid, both glutamine release from protein breakdown
(BGln) and glutamine de novo
synthesis (DGln) contribute to
glutamine Ra. Although body
protein is known to contain 13.9 g of glutamine plus glutamate per 100 g protein, the exact contribution of glutamine per se to that total
amount is not precisely known (10, 21). As in recent studies,
BGln was estimated as 0.78 × Ra Leu. This approach
assumes that 1) the release of an
amino acid from proteolysis is proportional to its abundance in body protein; 2) 100 g of body protein
contain 8,000 mg leucine, i.e., 61.1 mmol leucine (as 1 mmol leucine = 131 mg; and 8,000/131 = 61.1); and
3) glutamine contributes one-half
the total glutamine plus glutamate content of body protein, i.e.,
7,000 mg glutamine (= 47.9 mmol glutamine, since 1 mmol glutamine = 146 mg, and 7,000/146 = 47.9), or 0.78 mol glutamine/mol leucine
(47.9/61.1 = 0.78).
DGln.
The fraction of glutamine Ra that
cannot be accounted for by release of glutamine from protein breakdown
was attributed to DGln.
DGln was therefore estimated
by DGln = Ra Gln BGln (10).
Statistical Analysis
Data are presented as means ± SE. During the tracer infusions, steady state for plasma amino acid levels and enrichments were defined by the absence of a significant correlation of the measured parameter vs. time over the considered period. Data were compared using paired t-tests. Significance was established at P < 0.05. ![]() |
RESULTS |
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Response of Plasma Glutamine to Phenylbutyrate Treatment
Plasma glutamine concentrations were near steady state during the 5 h of each isotope infusion study; the average coefficients of variation (= 100 × SD/mean) of plasma glutamine concentration were 7.1, 12.9, and 11.6% with 0, 0.18, and 0.36 g · kg
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Effect of Phenylbutyrate on Leucine Kinetics
Based on the preliminary experiments described above, we elected to use the dose of 0.36 g · kg
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Because determination of leucine oxidation is based on excretion of 14CO2 during the infusion of labeled leucine, any factor affecting the recovery of labeled CO2 might affect the measured rates of leucine oxidation. In additional experiments performed in two subjects (subjects 7 and 8), we verified that the recovery of labeled CO2 was not significantly affected by phenylbutyrate treatment: upon primed, continuous infusion of H13CO3Na, the recovery of labeled 13C in expired air averaged 81% (76 and 87%) vs. 80% (77 and 83%) for baseline vs. phenylbutyrate treatment (subjects 7 and 8, respectively).
The steady-state plasma [2H3]KIC-to-[2H3]leucine enrichment ratio was not affected by phenylbutyrate treatment in the two subjects who received an L-[5,5,5-2H3]leucine infusion (1.03 ± 0.04 vs. 0.99 ± 0.04 in subject 7 and 0.72 ± 0.02 vs. 0.67 ± 0.02 in subject 8, control vs. phenylbutyrate treatment days, respectively).
Effect of phenylbutyrate on glutamine
kinetics. The rate of appearance of glutamine into
plasma, glutamine oxidation rate, and the estimated release from
protein breakdown and de novo synthesis remained unaltered after
phenylbutyrate treatment compared with baseline in the six subjects who
received infusions of
L-[1-13C]glutamine
(Table 2). Because plasma glutamine was
25% lower, whereas glutamine
Ra remained unaltered, glutamine
metabolic clearance rate (=
Ra Gln/[Gln],
where [Gln] is glutamine concentration) was higher on the
phenylbutyrate treatment day than at baseline (776 ± 43 vs. 589 ± 46 ml · kg
1 · h
1;
P < 0.05).
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DISCUSSION |
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The results of the current study demonstrate that a short course of treatment with the drug phenylbutyrate is able to induce acute glutamine depletion even in healthy adult humans with an intact urea synthetic pathway and normal baseline glutamine levels. They further show that, upon treatment with phenylbutyrate, 1) rates of glutamine de novo synthesis fail to increase, and 2) leucine oxidation increases, whereas NOLD, an index of protein synthesis, declines. This study suggests that 1) short-term treatment with phenylbutyrate can be used as an experimental model to create glutamine depletion in healthy humans and 2) an acute depletion of plasma glutamine may decrease estimates of whole body protein synthesis.
Although the "trapping effect" of phenylbutyrate only occurs in
intracellular space in selected tissues (presumably liver and kidney),
a highly significant, dose-dependent decline in glutamine concentration
was detected in systemic plasma (Fig. 2). The main repository of
glutamine in the body is, however, skeletal muscle rather than plasma
(3, 17). Assuming 1) a muscle mass
of 28 kg in a 70-kg subject, 2)
an intramuscular glutamine concentration of
19.5 mmol/kg of
intracellular water (3, 17), and 3)
an intracellular water content of
77% in muscle, baseline
intramuscular glutamine content can be estimated at
420 mmol. Based
on the stoichiometry of phenylacetylglutamine synthesis (Fig. 1), 0.36 g · kg
1 · day
1
phenylbutyrate should "trap" an equimolar amount of glutamine, i.e., 1.935 mmol
glutamine · kg
1 · day
1,
or 135.5 mmol/day in a 70-kg subject. Thus the amount of glutamine presumably "trapped" by phenylbutyrate over a 24-h regimen is equivalent to
32% of the estimated glutamine content of skeletal muscle. As muscle is the main source of plasma glutamine, a loss of
this magnitude (wherever it occurred within the body) is unlikely to
have spared the intramuscular glutamine pool, especially since the loss
was not compensated by any appreciable rise in rates of glutamine de
novo synthesis and/or release from protein breakdown (Table 2).
We therefore speculate that phenylbutyrate induced depletion of the
muscle free glutamine pool.
In vitro studies suggest that glutamine itself regulates the activity of glutamine synthetase: for instance, increasing the glutamine concentration of the culture medium inhibits glutamine synthetase in cultured muscle cells (29). Accordingly, we observed in an earlier study that rates of glutamine de novo synthesis were suppressed in vivo when exogenous glutamine was infused to raise plasma glutamine concentration in healthy volunteers (10). In contrast, in the current study, the acute drop in plasma glutamine concentration was not associated with a rise in estimated rates of whole body glutamine de novo synthesis. The lack of a stimulation of glutamine de novo synthesis ultimately accounts for the fact that the subjects did not compensate for the continuous "trapping" of glutamine by phenylacetate and were therefore unable to maintain plasma glutamine concentrations at their baseline level. The current data suggest that, in this experimental model, the body's capacity to synthesize glutamine may have been limited, or, in other words, that glutamine may have become an essential amino acid. In that regard, short-term phenylbutyrate treatment may therefore be used as an experimental model for other states of glutamine depletion in which increased glutamine utilization by splanchnic tissues exceeds the endogenous capacity of glutamine synthesis, as is the case during severe illness.
Although glutamine is the most abundant amino acid in the body and can be synthesized de novo, a precipitous fall in muscle glutamine concentration indeed is a hallmark of multiple clinical situations associated with protein wasting such as burns, surgical stress, or bone marrow transplantation (17, 30-32). With stress, a large increase in glutamine synthesis occurs concomitantly with the depletion in the muscle glutamine pool (1, 2). The precise mechanisms by which both glutamine production and glutamine utilization are enhanced with stress are, however, still debated. Because glutamine is predominantly released by skeletal muscle and taken up in splanchnic tissues, the depletion of the muscle glutamine pool observed during stress could result from an initial, dramatic rise in glutamine release from muscle; increased glutamine release would, in turn, enhance glutamine uptake by the gut and liver. Alternatively, the primary event may be a dramatic surge in splanchnic glutamine uptake, which could lead to a secondary rise in glutamine outflow from muscle. Furthermore, it is not known whether glutamine depletion is simply a meaningless epiphenomenon, reflecting the severity of stress, or, alternatively, a major disturbance that has severe consequences by itself. The glutamine depletion induced by phenylbutyrate differs from stress-induced protein wasting inasmuch as it specifically depletes glutamine pools, without altering glutamine production (Table 2); it therefore creates a much simpler model of an isolated increase in glutamine "clearance." Although the "model" of glutamine depletion created by phenylbutyrate treatment may not give any clue as to the mechanisms leading to glutamine depletion during stress, the "phenylbutyrate model" may prove useful to examine the consequences of an isolated increase in glutamine depletion per se on various functions in the body and thus help delineate some of the complex consequences of stress-induced protein wasting.
The rise in leucine oxidation observed during phenylbutyrate treatment is remarkable as it occurred at a time when plasma leucine concentration was lowered. Indeed, in most circumstances, rates of leucine oxidation are a direct function of the prevailing plasma leucine concentration (9, 23). Because neither bicarbonate retention nor the ratio of plasma [2H3]KIC to [2H3]leucine enrichment was affected by phenylbutyrate (see RESULTS), the change in measured rates of leucine oxidation must result from an actual change rather than from a change in the leucine-to-KIC isotopic ratio.
It should be pointed out that, in theory, the observed increased rate of leucine oxidation could be a direct effect of sodium phenylbutyrate through stimulation of branched-chain ketoacid dehydrogenase or other mechanisms, rather than a direct effect of glutamine depletion per se. Further experiments with the simultaneous administration of phenylbutyrate and glutamine supplementation are underway to resolve this issue.
Animal studies revealed a striking correlation between the size of the intracellular glutamine pool and the rates of muscle protein synthesis in rats submitted to dietary restriction or endotoxin injection (16, 18). Improvement in nitrogen balance was observed upon replenishment of the muscle glutamine pool with intravenous glutamine supplementation in several clinical studies (30, 32, 36). Yet conflicting data have appeared more recently, as several groups failed to observe a correlation between muscle protein fractional synthetic rate and muscle glutamine concentration in various models of stress in rats (8, 25, 34, 35) and humans (15). It therefore remains to be established whether the decline in muscle free glutamine concentration observed in stress is merely a reflection of a protein catabolic state or a causative factor. Indeed, the multiple metabolic and hormonal disturbances associated with the disease process or stress per se (33) might concomitantly affect protein synthesis and glutamine concentration without implying a causal relationship between the two events. In the current study, we observed that a moderate decline in plasma glutamine concentration, in otherwise healthy adult humans, was associated with a reduction in estimates of whole body protein synthesis (Fig. 3). In an earlier study, we had found that a doubling in plasma glutamine concentration was associated with an increase in estimates of whole body protein synthesis in healthy volunteers (11). Taken together, these data collectively support a physiological role of glutamine in the regulation of whole body protein synthesis.
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ACKNOWLEDGEMENTS |
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We are indebted to Bernice Rutledge and her nursing team at Wolfson Children's Hospital for the care of our patients. We acknowledge the superb technical assistance of Lynda Everline and Ed Jones. We thank Dr. Saul Brusilow and Dr. Pamela Arn for assistance in the use of phenylbutyrate.
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FOOTNOTES |
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This work was supported in part by National Institute of Diabetes and Digestive and Kidney Diseases Grant R01 DK-51477 and by a grant from the Nemours Foundation, Jacksonville, FL.
Address for reprint requests: D. Darmaun, Centre de Recherche en Nutrition Humaine, Hotel-Dieu, 44093 Nantes Cedex 1, France.
Received 1 October 1997; accepted in final form 28 January 1998.
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