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1 From the Departments of Respiratory Medicine (EPAR, EFMW, and AMWJS) and Surgery (MPKJE and NEPD), University of Maastricht, Maastricht, the Netherlands.
2 Supported by grant no. 3.2.0034 from the Netherlands Asthma Foundation. 3 Reprints not available. Address correspondence to EPA Rutten, Department of Respiratory Medicine, University of Maastricht, PO Box 5800, 6202 AZ Maastricht, Netherlands. E-mail: e.rutten{at}pul.unimaas.nl.
ABSTRACT
Background: Because low plasma glutamate and glutamine concentrations are often seen in chronic obstructive pulmonary disease (COPD), glutamine or glutamate supplementation may be a good option for preventing further metabolic disturbances in COPD patients. However, the metabolic effects of glutamate supplementation have never been compared with those of glutamine supplementation.
Objective: We compared the metabolic effects of repeated ingestion of glutamine and glutamate in COPD patients and in age-matched healthy control subjects.
Design: On 3 d separated by intervals of 2 d, a protocol of primed constant and continuous infusion of [2H5]phenylalanine and [2H2]tyrosine was performed for 3 h in 8 stable male COPD patients and 8 healthy control subjects. After a 90-min tracer infusion, all subjects ingested a glutamine or glutamate drink or the same amount of water every 20 min for 80 min. Blood samples were taken at the end of the postabsorptive and ingestion periods to test for effects on plasma amino acid and substrate concentrations and whole-body protein turnover.
Results: Glutamate but not glutamine ingestion resulted in higher plasma ornithine concentrations than did water ingestion (P < 0.01). The change in plasma arginine, citrulline, and urea concentrations was significantly (P < 0.01) higher after glutamine ingestion than after water or glutamate ingestion. Whole-body protein turnover decreased overall, independent of the drink consumed.
Conclusions: Repeated ingestion of glutamine and glutamate resulted in different effects on the plasma amino acid concentration. In both groups, ingestion of glutamine but not of glutamate increased the plasma concentrations of citrulline and arginine, substrates produced in the intestine and the liver.
Key Words: Glutamine • glutamate • chronic obstructive pulmonary disease • supplementation • amino acids • protein turnover
INTRODUCTION
Glutamine is a widely investigated amino acid that is known to play an important role in many metabolic routes in various organs, such as the splanchnic bed and skeletal muscle. Supplementation with glutamine in enteral or parenteral feeding is often suggested to improve disturbed metabolic processes, eg, a low muscle glutathione status or a negative nitrogen balance during illness (1, 2). Glutamine solutions are, however, unstable (3) and thus not practical for use as a food supplement. In addition, disturbances in glutamate status but not in glutamine status were found in several diseases (4, 5). Glutamate is stable in water, and research by Walker and Lupien (6) has shown that, in contrast to what was reported in the past (7), there is no evidence that the intake of glutamate via glutamate-containing food products or dishes prepared with (monosodium) glutamate is responsible for inducing symptoms of Chinese restaurant syndrome (6). In this view, supplementation with glutamate may be a good alternative to that with glutamine to improve substrate metabolism during illness.
Glutamine and glutamate are closely linked because they can easily be converted to each other by the enzymes glutamine synthetase (glutamate + NH3 glutamine) and glutaminase (glutamine glutamate + NH3) (8). Although glutamate plays a key role in the transamination reactions, a small number of studies evaluated the effect of glutamine or glutamate supplementation on plasma amino acid concentrations. In general, these studies showed that the concentration of only few plasma amino acids was modified after the ingestion of glutamine (9) or glutamate (10). Moreover, in a catabolic state such as surgery, glutamine supplementation has been shown to increase protein synthesis (11, 12). A correlation between a low skeletal muscle glutamine concentration in a catabolic state and low protein synthesis is noticed (12), although the mechanism is not yet completely clear. It is interesting that the addition of glutamate to enteral nutrition also increased mucosal protein synthesis (13), which suggests that both glutamine and glutamate can modify protein metabolism.
In patients with chronic obstructive pulmonary disease (COPD), the plasma glutamine and glutamate and skeletal muscle glutamate concentrations were low (14, 15). Moreover, low muscle glutamate status was associated with metabolic disturbances such as a low skeletal muscle glutathione concentration (5). Supplementation with glutamine or glutamate may be an option to prevent further metabolic disturbances in COPD patients.
The aim of the current study was to compare the effects of oral ingestion of the closely linked amino acids glutamine and glutamate on plasma amino acid concentrations and whole-body (WB) protein turnover. The study was performed in stable COPD patients and age-matched healthy control subjects to allow simultaneous evaluation of potential disease-specific effects in response to glutamine and glutamate ingestion.
SUBJECTS AND METHODS
Study population
Eight healthy male control subjects and 8 stable COPD patients, all age- and sex-matched, were studied. The COPD patients were characterized by significantly lower body weight (P < 0.05), fat-free mass (FFM) (P < 0.01), body mass index (in kg/m2) (P < 0.05), and FFM index (FFM/height2) (P < 0.01) (Table 1). Exclusion criteria for both groups were malignancy, cardiac failure, distal arteriopathy, recent surgery, and severe endocrine, hepatic, or renal disorder. In addition, patients who were using systemic corticosteroids 3 mo before the study were excluded because it has been shown that systemic corticosteroids may affect muscle amino acid metabolism (16). The number of current smokers was 2 in the control group and 3 in the COPD group. The COPD group was characterized by slight but significantly (P < 0.05) higher plasma concentrations of C-reactive protein.
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TABLE 1. General characteristics of the study population1
Written informed consent was obtained from all subjects. The study was approved by the medical ethics committee of the University Hospital Maastricht.
Pulmonary function tests
All subjects underwent spirometry for measurement of forced expiratory volume in 1 s, as a marker of disease severity, and the highest value from 3 technically acceptable maneuvers was used. The diffusion capacity of the lung for carbon monoxide, as a marker of emphysema, was measured by using the single-breath method (Masterlab; Jaeger, Wurzburg, Germany). All values obtained were related to a reference value and expressed as percentages of the predicted value (17). The COPD patients had significantly lower values of forced expiratory volume in 1 s and diffusions capacity of the lung for carbon dioxide than did the control group (P < 0.01 for both; Table 1).
Study design
The test drinks
On 3 test days separated by 2 d, subjects were invited to the metabolic ward of the University Hospital Maastricht after an overnight fast. On the different test days, the test drink contained glutamate, glutamine or only water in a randomized order. The test drinks consisted of a 2.4% solution to deliver 30.0 mg glutamate · kg body wt–1 · 20 min–1 or an isomolar amount of glutamine (29.8 mg glutamine · kg body wt–1 · 20 min–1). The water drink contained the equal amount of only water (1.25 mL water · kg body wt–1 · 20 min–1). The drinks were served at a temperature of 55 °C to ensure complete solution.
Study protocol
All subjects were in the supine position for 3 h (Figure 1). A catheter was placed in an antecubital vein of the arm for tracer infusion (85 mL/h) according to a primed constant and continuous infusion protocol. A venous blood sample was collected to measure baseline enrichment of phenylalanine (Phe) and tyrosine (Tyr). The stable isotopes L-[ring-2H5]-Phe and L-[ring-2H2]-Tyr were used to measure WB protein turnover. The following priming doses and infusion rates were used: for L-[ring-2H5]-Phe, 2.19 µmol/kg FFM and 2.26 µmol · kg FFM–1 · h–1; for L-[ring-2H2]-Tyr, 0.95 µmol/kg FFM and 0.77 µmol · kg FFM–1 · h–1, respectively. Moreover, a bolus dose of L-[ring-2H4]-Tyr was given to prime the phenylalanine-derived plasma tyrosine pool (priming dose: 0.31 µmol/kg FFM). The tracers were obtained from Cambridge Isotopic Laboratories (Woburn, MA). The tracer infusion was begun after intravenous administration of the priming dose, and the infusion continued to the end of the test day. A second catheter for blood sampling was placed in a superficial dorsal vein of the hand of the contralateral arm, which was placed in a thermostatically controlled hot-box (internal temperature: 60 °C) 20 min before the first blood sampling. The hot-box technique is used to mimic direct arterial sampling (18). Ninety minutes after the start of the tracer infusion, all subjects began to ingest 1 of the 3 test drinks every 20 min for 80 min. Triple arterialized venous blood samples were taken between 80 and 90 min after the start of the tracer infusion and between 70 and 80 min after the start of the ingestion.
FIGURE 1.. Overview of the study protocol. On 3 test days, intravenous infusion of the stable isotopes was given for 3 h. Ingestion involved 1.25 mL water/kg body wt, 29.8 mg glutamine/kg body wt, or 30 mg glutamate/kg body wt every 20 min. Arteriovenous blood samples were taken between 80 and 90 min after the start of the infusion and between 70 and 80 min after the start of the ingestion.
Biochemical analyses
Venous and arterialized venous blood was divided into a tube containing heparin and a coagulation tube. The heparin-containing tube was immediately put on ice and centrifuged at 3120 x g and 4 °C for 10 min to obtain plasma. Subsequently, 250 µL plasma was deproteinized with 20 mg dry sulfosalicylic acid for analysis of plasma amino acid concentrations and enrichment. Another 900 µL plasma was deproteinized with 90 µL trichloroacetic acid for measurement of plasma glucose, ammonia, and urea concentrations. All samples were frozen in liquid nitrogen and stored at –80 °C until they were analyzed. The amino acid concentrations were analyzed by using an HPLC system (19). Phenylalanine and tyrosine enrichment (tracer-to-tracee ratio) was analyzed by using liquid chromatography–mass spectrometry (LC-MS; Thermoquest, Veenendaal, Netherlands) (20). The concentrations of substrate (ie, glucose, ammonia, and urea) were measured by using the COBAS Mira S (Roche Diagnostica, Hoffman-La Roche, Basel, Switzerland) (21). C-reactive protein was measured in plasma by using the Synchron LX 20 system (Beckman Coulter, Mijdrecht, Netherlands). Blood collected in the cloth tube was stored uncooled for 20 min and subsequently centrifuged at 3120 x g and at room temperature for 10 min to obtain serum. The serum was stored at –80 °C until it was analyzed with a radioimmunoassay kit to measure insulin concentrations.
Calculations
The amino acids glutamate, asparagine, serine, glutamine, histidine, glycine, threonine, alanine, taurine, tyrosine, methionine, phenylalanine, tryptophan, lysine, valine, isoleucine, leucine, ornithine, citrulline, and arginine were analyzed. The sum of the amino acids represents the sum of all analyzed amino acids. The sum of essential amino acids represents the sum of threonine, phenylalanine, trpyptophan, methionine, lysine, isoleucine, valine, and leucine. The branched-chain amino acids (BCAA) represent the sum of leucine, isoleucine, and valine.
All metabolic data were determined under steady state conditions. Therefore, whole-body rate of appearance of phenylalanine represents whole-body protein breakdown and is calculated as follows:
RESULTS
Plasma amino acid concentration
At baseline, plasma ornithine concentration was significantly (P < 0.05) higher in the COPD group than in the control group (Table 2). The change in glutamate and alanine concentrations after glutamine and glutamate ingestion was significantly higher than the change after water ingestion, but the increase in both groups was highest after glutamate ingestion (plasma glutamate, P < 0.01; plasma alanine, P < 0.05; Figure 2). Moreover, the increase in plasma glutamate concentrations after repeated glutamate ingestion was significantly (P < 0.05) lower in the COPD group than in the control group. Because there was no group effect for the remaining amino acids, statistical effects on the plasma amino acid concentration are presented in the total study group. Glutamine ingestion resulted in a significantly (P < 0.01) greater change in plasma glutamine concentrations than did glutamate and water ingestion (Figure 2). Glutamate ingestion resulted in a greater change in taurine and methionine concentrations than did water and glutamine ingestion. The changes in valine, leucine, and isoleucine concentrations and the sum of BCAA after glutamine and glutamate ingestion were significantly (P < 0.01) greater than those after water ingestion. Glutamine ingestion resulted in a significantly (P < 0.01) greater change in citrulline and arginine concentrations than did water and glutamate ingestion, whereas plasma ornithine concentrations did not change significantly after glutamine ingestion. Glutamate ingestion, on the other hand, resulted in a significantly (P < 0.01) greater change in ornithine concentrations than did water and glutamine ingestion, whereas citrulline concentrations decreased after glutamate ingestion and were significantly (P < 0.01) lower than after water and glutamine ingestion. Plasma arginine concentrations did not change after glutamate ingestion.
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TABLE 2. Plasma amino acid concentrations at baseline (t0) and after 80 min of repeated ingestion (t80) of water, glutamine, or glutamate in control subjects and subjects with chronic obstructive pulmonary disease (COPD)1
FIGURE 2.. Mean (±SEM) plasma concentrations of glutamate, glutamine, and alanine at baseline (t0) and after 80 min of ingestion (t80) of water (WA), glutamine (Gln), or glutamate (Glu) in the control (; n = 8) and COPD (; n = 8) groups. Univariate ANOVA and the Bonferroni tests were used to test drink and group effects on the change from t0 to t80. There was no significant difference between the groups at t0. The group x drink interaction for plasma glutamate concentration after glutamate ingestion in both groups was significant (P < 0.01). Drink effects were significant for plasma concentrations of glutamate (between glutamine and water ingestion and between glutamine and glutamate ingestion; both: P < 0.01), glutamine (between glutamine and water ingestion and between glutamine and glutamate ingestion; both: P < 0.01), and alanine (between glutamine and water ingestion, P < 0.01, and between glutamine and glutamate ingestion, P < 0.05).
Plasma substrate concentration
At baseline, plasma glucose, ammonia, insulin and urea concentrations did not differ significantly between the COPD group and the control group (Table 3). The group x drink interaction for the change in ammonia concentration was significant, which indicated that the drink effect differed significantly between the groups. In the control group, glutamine ingestion resulted in a significantly (P < 0.01) greater change in ammonia concentration than did water and glutamate ingestion, and this effect differed significantly (P < 0.05) between the groups. Glutamate ingestion resulted in a significantly (P < 0.01) greater change in insulin concentrations than did water ingestion. The change in urea concentration was significantly greater after glutamine ingestion than after glutamate (P < 0.05) or water (P < 0.01) ingestion.
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TABLE 3. Plasma glucose, ammonia, insulin, and urea concentrations at baseline (t0) and after 80 min of repeated ingestion (t80) of water, glutamine, or glutamate in control subjects and subjects with chronic obstructive pulmonary disease (COPD)1
Whole-body protein turnover
Baseline values for WB protein breakdown, synthesis, and net balance did not differ significantly between the control group and the COPD group (Table 4). There was no drink-specific effect on WB protein turnover in either group. However, the overall change in WB protein breakdown and synthesis was significantly different from zero (P < 0.01), independent of the drink consumed.
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TABLE 4. Measures of whole-body (WB) protein metabolism at baseline (t0) and after 80 min of repeated ingestion (t80) of water, glutamine, or glutamate in control subjects and subjects with chronic obstructive pulmonary disease (COPD)1
DISCUSSION
Supplementatin with glutamine and glutamate had different effects on plasma amino acid and urea concentrations. The effects were similar in healthy control subjects and in COPD patients except for the effect of glutamate ingestion on plasma glutamate concentrations. Glutamine ingestion resulted in higher plasma citrulline and arginine concentrations, whereas glutamate ingestion reduced citrulline concentrations, did not increase plasma arginine concentrations, but did increase ornithine concentrations. We could not detect a drink-specific effect of glutamine and glutamate supplementation on WB protein turnover compared with water ingestion. Thus, we concluded that supplementation with glutamine and glutamate has different effects on plasma amino acid concentrations. We hypothesized that these changes reflect intestinal metabolism.
Plasma amino acid and substrate concentration
The only amino acid that differed significantly between COPD patients and control subjects was ornithine, a finding that is in line with previous findings (15). It is suggested that plasma ornithine concentrations are elevated during inflammation, as a result of enhanced arginase activity (25). Because our patient group was characterized by high concentrations of C-reactive protein, a marker for systemic inflammatory response, the high plasma ornithine concentrations may well be related to the low-grade clinical inflammation in these patients.
Except for the glutamate concentration, supplementation with glutamine and glutamate resulted in the same effect on plasma amino acids concentrations and WB protein turnover in both groups. The underlying cause of the smaller increase in plasma glutamate concentrations after glutamate ingestion in the COPD group is speculative. That smaller increase may be due to an enhanced extraction of glutamate in the intestine or to a greater consumption of glutamate elsewhere.
Repeated ingestion of glutamine and glutamate (separately) induced a significant increase in plasma glutamate concentrations, although the increase was significantly lower after the intake of glutamine than after that of glutamate (50% and 500%, respectively). This finding suggests that the intestinal capacity to oxidize glutamate has reached its maximum and that an increase in the plasma glutamate concentrations follows. Furthermore, it is known that the splanchnic bed produces small amounts of glutamate from glutamine (glutamine glutamate + NH3), which results in an increase in plasma glutamate concentrations after glutamine ingestion (26). The glutamate-specific increase in plasma taurine concentration that we found in the current study is consistent with earlier findings after ingestion of monosodium glutamate (27). Glutamate is linked with taurine via 2 different routes. In the presence of the enzyme taurine:-keto-glutarate aminotransferase in skeletal muscle, glutamate can form taurine (glutamate + sulfoacetaldehyde taurine + -keto-glutarate) (28). More often, however, glutamate has been associated with taurine because the glutamate receptor agonists N-methyl D-aspartate and -amino-3-hydroxy-5-methyl-4- isoxazole propionic acid receptors evoke the release of taurine from the brain into the extracellular tissue (29). The functional importance of the glutamate-induced taurine increase should be investigated further.
In the current study, the ingestion of either glutamine and glutamate resulted in a decrease in plasma BCAA concentrations. In general, BCAA can be transaminated in skeletal muscle, where they act as the most important nitrogen donor in the synthesis of glutamine and alanine (30). It has been shown in catabolic illness that the infusion of glutamine or alanine reduces the release of BCAA from the liver (31), which may result in a lower plasma BCAA concentration. Moreover, glutamate acts as the precursor for BCAA in the liver via transamination reactions. Therefore, oral ingestion of glutamine and glutamate may have a BCAA-sparing effect that results in less BCAA production in the liver and, hence, in lower plasma BCAA concentrations.
We observed a significantly increased plasma citrulline and arginine concentration after glutamine ingestion. The intestine and the liver are the predominant sources for citrulline production. Glutamine is deaminated to glutamate and ammonia, and the latter stimulates the formation of carbamoyl phosphate (Figure 3). Citrulline is formed from the transamination reaction of carbamoyl phosphate and ornithine via the enzyme ornithine carbamoyltransferase. In the liver, citrulline is further metabolized to arginine to produce urea and ornithine in the hepatic ornithine cycle. Therefore, circulating citrulline originates from the intestine rather than from the liver. Because the enzymes that convert citrulline to arginine are not present in the intestine (32), citrulline is released to the circulation and taken up by the kidneys. In the presence of the enzymes argininosuccinate synthetase (ASS) and argininosuccinate lyase (ASC), citrulline is converted to arginine that is released in the circulation (33). In the current study, plasma citrulline concentrations decreased after glutamate ingestion, whereas plasma ornithine concentrations increased; the opposite results were seen after glutamate ingestion. This finding implies that the amide amino group of glutamine as source of NH3 could be the rate-limiting substrate for carbamoyl phosphate production in the intestine. In the absence of NH3, glutamate may, instead, rather convert to glutamate--semialdehyde and be transaminated to ornithine by the enzyme ornithine-oxo-acid aminotransferase. The decrease in plasma urea concentrations after glutamate ingestion but not after glutamine ingestion in the current study confirms the hypothesis that, in the intestine as well as in the liver, the production of carbamoyl phosphate is dependent on ammonia from glutamine. Bolus ingestion of monosodium glutamate (150 mg/kg body wt) did not significantly increase plasma ornithine concentrations in healthy young volunteers (27), although there was a tendency toward an increase of 33% after 60 min of ingestion, which is comparable to the 32% increased observed in the current study.
FIGURE 3.. Schematic overview of the ornithine-citrulline-arginine cascade in the intestine and the kidney.
Whole-body protein turnover
Baseline WB protein breakdown, synthesis, and net balance did not differ significantly between the control group and the COPD group. Engelen et al (34) observed significantly more WB protein synthesis and breakdown in COPD patients than in age-matched healthy control subjects. One factor that may contribute to the different findings in the 2 studies is the fact that the COPD patients had a different disease severity; that is, the patients in the study by Engelen et al had a greater degree of airflow obstruction than did the subjects in the current study. It can be posited that an increase in WB protein turnover is an adaptive process in the disturbed metabolism of patients with more severe COPD.
In time, WB protein breakdown and synthesis decreased overall, independent of the drink consumed. Supplementation with the amino acids glutamine and glutamate dissolved in water had no additional effect on WB protein turnover, although skeletal muscle glutamine concentration is often related to protein synthesis (12). It is often suggested that preventing tissue glutamine decrease during catabolism could have a sparing effect on muscular amino acid concentrations and hence could increase protein synthesis (35). Because hypo-osmolarity is known to have a protein-sparing effect (36), it can be suggested that a water load of 500 mL in 80 min, as in the current study, caused a decrease in protein turnover. Consequently, under the current circumstances, specific effects of glutamine and glutamate ingestion on WB protein turnover probably could not be detected. Future research is needed to study the effect of these amino acids when supplemented under different conditions—in the form of capsules, for example.
We can conclude that supplementation with glutamate results in a different response of several plasma amino acids than does that with glutamine, both in the healthy elderly and in COPD patients. Ingestion of glutamine but not of glutamate increased plasma concentrations of citrulline and arginine, substrates produced in the intestine and the liver. It may be possible that the amide amino group of glutamine as the source of NH3 is the rate-limiting substrate in this cascade of reactions. Moreover, except for taurine, the other amino acids and WB protein turnover responded similarly to glutamine and glutamate ingestion.
ACKNOWLEDGMENTS
EPAR participated in the study design, data collection, data analysis, and manuscript writing; MPKJE and NEPD participated in the study design, data analysis, and manuscript revision; EFMW and AMWJS participated in study design and manuscript revision. None of the authors had any personal or financial conflict of interest.
REFERENCES