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Am J Physiol Endocrinol Metab 291: E683-E690, 2006. First published May 9, 2006; doi:10.1152/ajpendo.00252.2005
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Route of administration (enteral or parenteral) affects the contribution of L-glutamine to de novo L-arginine synthesis in mice: a stable-isotope study

Petra G. Boelens,1,* Gerdien C. Melis,1,* Paul A. van Leeuwen,1 Gabrie A. ten Have,2 and Nicolaas E. Deutz2

1Department of Surgery, Vrije Universiteit Medical Center, Amsterdam; and 2Department of Surgery, University of Maastricht, Maastricht, The Netherlands

Submitted 7 June 2005 ; accepted in final form 1 May 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
A pathway from enteral L-glutamine as substrate for L-arginine synthesis is suggested by previous studies. L-Glutamine and L-glutamine dipeptides exhibit numerous beneficial effects in experimental and clinical studies. In trauma patients, enteral L-glutamine supply increased plasma L-arginine. The present study was designed to quantify the contribution of L-glutamine to the de novo L-citrulline and L-arginine synthesis in mice when L-glutamine is administered in a high dose of labeled L-glutamine or L-alanyl-L-glutamine by the enteral or parenteral route. For this purpose, male Swiss mice (n = 43) underwent a laparotomy, and catheters were inserted for sampling and infusion. A primed, constant, and continuous infusion of L-alanyl-L-[2-15N]glutamine (dipeptide groups) or L-[2-15N]glutamine (free L-glutamine groups), simultaneously with L-[ureido-13C,2H2]citrulline and L-[guanidino-15N2,2H2]arginine, was given (steady-state model). Mice received the L-glutamine tracers intravenously (jugular vein) or enterally (duodenum). Enrichments of metabolites were measured by LC-MS. Arterial L-glutamine concentrations were the highest in the intravenous dipeptide group. L-Glutamine was converted to L-citrulline and L-arginine when L-[2-15N]glutamine and L-alanyl-L-[2-15N]glutamine were given by enteral or parenteral route. The contribution of L-glutamine to the de novo synthesis of L-citrulline and L-arginine was higher in the enteral groups when compared with the intravenous groups (P < 0.005). Therefore, the route of administration (enteral or parenteral) affects the contribution of L-glutamine, provided as free molecule or dipeptide, to the de novo synthesis of L-arginine in mice.

L-glutamine; L-arginine; enteral nutrition; parenteral nutrition; surgery

L-GLUTAMINE IS AN IMPORTANT AMINO ACID during a metabolic stress such as surgery (7). During metabolic stress, L-glutamine concentrations in plasma and muscle fall sharply due to increased requirements (3, 4, 19, 28–31). These low plasma L-glutamine concentrations are associated with poor clinical outcome (27). Fortunately, many clinical studies show that exogenous L-glutamine, as in L-glutamine-enriched parenteral or enteral formulas, improve recovery of the patient by reducing the number of infectious complications and the "6-mo mortality" (14, 15, 21, 40). The exact mechanism behind the beneficial effects of L-glutamine remains to be clarified.

The suggested metabolic pathway from L-glutamine through L-citrulline into L-arginine indicates that L-glutamine could serve as a substrate for intestinal L-citrulline production and that L-citrulline could serve as a substrate for renal L-arginine production (11, 39). L-Arginine is known to be the physiological precursor for the synthesis of nitric oxide, which has been identified as the endothelium-dependent relaxing factor, a mediator of immune responses, a neurotransmitter, and a signaling molecule (9). The clinical relevance of L-arginine itself is also well established (25).

It is conceivable that the beneficial effects of L-glutamine are exerted partially by its contribution to L-arginine production. The relationship between L-glutamine and L-arginine was already suggested from the results of a double-blind, randomized trial performed by our group (21), providing free L-glutamine-enriched enteral nutrition or an isonitrogenous and isocaloric control nutrition to severely injured trauma patients. Plasma L-arginine concentrations increased in the patients who received L-glutamine-enriched nutrition compared with the control group (21). In the majority of studies investigating L-glutamine-enriched nutrition, parenteral nutrition was given, and no significant changes in plasma L-arginine were found. Because the gut plays a key role in L-glutamine conversion to L-citrulline, we hypothesized that the contribution of L-glutamine to L-citrulline synthesis would be enhanced when L-glutamine was offered intraluminally compared with parenterally delivered L-glutamine. This way, enterally provided L-glutamine could also contribute more to de novo L-arginine production from L-citrulline.

However, enriching nutrition with L-glutamine is pharmacologically complicated because L-glutamine has a relative aqueous instability (20). A dipeptide, for example L-alanyl-L-glutamine, which is stable in a watery solution, was shown to be a good alternative (1, 2, 23). In human volunteers, an intravenous bolus of L-alanyl-L-glutamine had an elimination half-life from plasma of only 4 min. Moreover, the dipeptide disappearance was accompanied by a prompt equimolar increase in the concentrations of L-alanine and L-glutamine (1). However, most of the information about dipeptides is derived from studies involving intravenous treatment (13), whereas a minimum of information is available on enteral dipeptide intervention (32, 34). No evidence is available yet comparing the effect of L-glutamine or glutamine-dipeptides on L-citrulline and L-arginine synthesis in vivo in mice.

The aim of this study was to investigate whether and how much L-glutamine contributes to L-citrulline and L-arginine synthesis when L-glutamine is provided parenterally or enterally in a high dose of free L-glutamine or as a dipeptide with L-alanine. For this purpose, the pathway of L-glutamine to L-citrulline and L-arginine was quantified using stable isotope technology.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animals

Male Swiss mice (n = 43) were obtained from IFFA Credo Broekman (Someren, The Netherlands). The mice were fed standard laboratory chow before the start of the experiments (SMRA 2131; Hope Pharms, Woerden, The Netherlands) and were operated on in the postabsorptive state. The mice were subjected to standard 12:12-h light-dark cycle periods (7:30 AM to 7:30 PM). Room temperature was maintained at 25°C. Radio music was on to acclimate the animals to noise in the environment in order to reduce their stress toward noise induced by the caretakers/researchers. Experiments were performed in accordance with the recommendations of the Guide for the Care and Use of Laboratory Animals and approved by the Ethics Committee of Animal Research of the Maastricht University.

Experimental Design

Two series of mouse experiments were performed. Mouse characteristics are shown in Table 1. The mice received a primed, constant, and continuous infusion of L-alanyl-L-[2-15N]glutamine (dipeptide groups) in series 1 or L-[2-15N]glutamine (free L-glutamine groups) in series 2, simultaneously with L-[ureido-13C,2H2]citrulline and L-[guanidino-15N2,2H2]arginine in both series, for 1 h in a steady-state experiment. Mice received a large dose of labeled L-glutamine or L-alanyl-glutamine to bring about an effect. Braulio et al. (8), who also studied the turnover of L-glutamine in mice from the same trunk as ours, giving labeled L-glutamine in a tracer dose, established the whole body rate of appearance of L-glutamine to be ~1,470 µmol·kg–1·h–1. Assuming that this figure represents the real turnover of L-glutamine in mice, it is almost six times higher than the turnover of L-glutamine in humans, which is ~260 µmol·kg–1·h–1 (35). To be able to bring about an effect of the supplemented L-glutamine comparable to an intervention 150 µmol·kg–1·h–1 of L-glutamine in humans, mice in this study received approximately six times this dose.


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  		Table 1. Mouse group characteristics

 
When the unit of the infusion rate was translated to micromoles per kilogram per hour, mice in series 1 received on average 858 µmol·kg–1·h–1, and mice in series 2 received on average 665 µmol·kg–1·h–1 L-[2,15N]glutamine. Differences between series were due to differences in body weight.

Tracer Infusion Protocol

Tracer experiments were conducted under ketamine-medetomidine anesthesia and fluid management, as described in detail before (18). A primed, constant, and continuous infusion of stable isotopes (Cambridge Isotope Laboratories, Andover, MA; the dipeptide tracer was a kind gift of Prof. D. E. Matthews, University of Vermont, Burlington, VT) was given (Table 2). All tracer primes were given intravenously. Continuous infusion of L-[2-15N]glutamine or L-alanyl-L-[2-15N]glutamine was given in the jugular vein (intravenous groups) or directly in the duodenum (enteral groups). Simultaneously, in all experiments, L-[ureido-13C,2H2]citrulline and L-[guanidino-15N2,2H2]arginine were infused into the jugular vein. Blood was collected from the carotid artery 30 and 40 min after the start of the primed continuous infusion of stable isotopes. In pilot experiments, this model yielded a steady state in 30 min, which is illustrated in Fig. 1 for this experiment (8). Amino acid concentrations and tracer-to-tracee ratios (TTRs) were determined in deproteinized plasma, as previously described (37). Briefly, 65 µl of plasma were added to 4 mg of solid 5'-sulfosalicylic acid, vortexed, frozen in liquid nitrogen, and stored at –80°C. Plasma amino acid concentrations and enrichments, calculated as TTRs, were measured using a fully automated liquid chromatography-mass spectrometry (LC-MS) system, using precolumn derivatization with o-phthalaldehyde (36, 37). Detailed information on the accuracy and precision of this method was given before (37).


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  		Table 2. Tracer prime and infusion rates

 

Figure 1
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  		Fig. 1. Tracer steady state in plasma. {Delta}Time between t = 30 and t = 40 min of arterial tracer-tracee ratios (TTRs) of L-[2-15N]glutamine. Data represent means ± SE. Statistics with ANOVA. No significant change in time was observed. IV, intravenous; EN, enteral; L-ALA-L-GLN, L-alanyl-L-glutamine.

 
Calculations

Tracer-enrichments are shown for L-[2-15N]glutamine, L-[2-15N]citrulline, and L-[2-15N]arginine to illustrate the pathway. The pathway of L-glutamine to L-citrulline and L-arginine was quantified using the L-[2-15N]glutamine, L-[ureido-13C,2H2]citrulline, and L-[guanidino-15N2,2H2]arginine tracers, for tracer methodological reasons, to avoid inaccuracies.

Whole body rate of appearance. Tracer-to-tracee ratio (TTR) is an equivalent of specific activity. Therefore, formulas were derived from metabolic studies using radioactive tracers. Correction for background enrichment and when necessary for the contribution of lower isotopomers was performed as described by Wolfe (41). Plasma glutamine, citrulline, and arginine whole body rate of appearance (WbRa) were calculated from the arterial isotope TTR values of, respectively, L-[2-15N]glutamine [Gln mass (M) + 1], L-[ureido-13C,2H2]citrulline (Cit M + 3), and L-[guanidino-2H2,15N2]arginine (Arg M + 4) and the known infusion rate (I), using the steady-state isotope dilution equation (42)

Formula
Splanchnic extraction (SE) per series was calculated for glutamine as follows:

Formula

Whole body de novo L-citrulline production from L-glutamine. Calculation of the plasma whole body L-glutamine-to-L-citrulline flux (de novo L-citrulline production derived from L-glutamine) was made in the same fashion as the calculation described by Castillo et al. (10):

Formula
where WbRa Cit is the plasma whole body citrulline rate of appearance (nmol·10 g–1·min–1), estimated from the primed constant infusion of the L-[ureido-13C,2H2]citrulline tracer and TTR Cit M + 1 and TTR Gln M + 1, which are the respective TTRs of L-[2-15N]citrulline and L-[2-15N]glutamine.

Furthermore, the above-mentioned calculation according to Castillo et al. (10) was adapted to be able to include the contribution of the conversion of L-[2-15N]glutamine to L-[2-15N]citrulline in the total whole body conversion of L-glutamine to L-citrulline. This adaptation is necessary because the endogenously synthesized L-[2-15N]citrulline (I Cit M + 1), which might be substantial in this study providing L-[2-15N]glutamine or L-alanyl-L-[2-15N]glutamine in a therapeutic dose, is not part of the WbRa of L-citrulline. The calculation was adapted as follows:

Formula

Formula
I Cit M + 1 is the unknown factor. However, it is known how much L-glutamine contributes to L-citrulline turnover, calculated as a percentage of total glutamine turnover:

Formula
Because no difference is to be expected in metabolic handling of unlabeled L-glutamine or Gln M + 1, the I Cit M + 1 can be calculated:

Formula
Therefore the complete equation is

Formula

Whole body de novo L-arginine production rate from L-citrulline. Calculation of the plasma citrulline-to-arginine flux (whole body total de novo arginine production derived from citrulline) was made in a similar fashion:

Formula
where WbRa Arg is the plasma arginine flux (nmol·10 g–1·min–1), estimated from the primed constant infusions of the L-[guanidino-15N2,2H2]arginine tracer and TTR Arg M + 3 and TTR Cit M + 3, which are the respective TTRs of L-[ureido-13C,2H2]arginine and L-[ureido-13C,2H2]citrulline.

To calculate the contribution of the conversion of L-[2-15N]citrulline to L-[2-15N]arginine to the total whole body conversion of L-citrulline to L-arginine, this calculation was adapted in the same way as the equation calculating the conversion from L-glutamine to L-citrulline:

Formula

Percentage of L-citrulline and L-arginine derived from L-glutamine. For this calculation we used the conversions from L-glutamine to L-citrulline and from L-citrulline to L-arginine, QGln->Cit and QCit->Arg (without the contribution of the M + 1 tracers), which were divided by the WbRa of L-citrulline or L-arginine.

The contribution of L-glutamine to the de novo synthesis of L-arginine was calculated by multiplying QGln->Cit as a percentage of the WbRa of L-citrulline with QCit->Arg as a percentage of the WbRa of L-citrulline:

Formula
The contribution of L-glutamine to the total WbRa of L-arginine was calculated in the same fashion, but QCit->Arg was expressed as a percentage of the WbRa of L-arginine:

Formula
The contribution of L-glutamine to de novo synthesis of L-arginine or total turnover of L-arginine was solely described as a percentage of the de novo synthesis or total turnover of L-arginine, as the absolute figures involve more assumptions and are therefore less accurate.

Statistical Analysis

Results are presented as means ± SE. The significance of enrichment with L-[2-15N]glutamine, L-[2-15N]citrulline, and L-[2-15N]arginine was tested per group by use of the one-sample t-test. With respect to the quantitative results, comparisons were made between intravenous and enteral groups per series by use of a Student's t-test when normally distributed or a Mann-Whitney U-test when not normally distributed. (SPSS for Windows release 11.0.1 2001; SPSS, Chicago, IL). Significance was defined as P < 0.05.


   RESULTS
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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
The tracer enrichment with L-[2-15N]glutamine, L-[2-15N]citrulline, and L-[2-15N]arginine was significant (P < 0.001), which illustrates the existence of the metabolic pathway from L-glutamine to L-citrulline and subsequently to L-arginine (Fig. 2).


Figure 2
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  		Fig. 2. Tracer enrichment of L-[2-15N]glutamine, L-[2-15N]citrulline, and L-[2-15N]arginine. Data represent tracer enrichments of L-[2-15N]glutamine (GLN M+1), L-[2-15N]citrulline (CIT M+1), and L-[2-15N]arginine (ARG M+1), expressed in TTR% and corrected for background enrichment. All enrichments were significantly different from zero (1-sample t-test; P < 0.001).

 
Arterial Amino Acid Concentrations

Arterial plasma concentrations of L-glutamine were significantly higher when the dipeptide was given parenterally compared with enteral administration (P < 0.05; Table 3). L-Glutamate concentrations were higher after enteral administration of both glutamine tracers compared with the intravenous administration (P < 0.05). Other arterial amino acid concentrations were not different between the groups with respect to the route of administration.


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  		Table 3. Arterial amino acid concentrations

 
WbRa L-Glutamine, L-Citrulline, and L-Arginine

The WbRa of L-glutamine was higher after administration of both L-glutamine tracers in the enteral groups than in the parenteral groups, most likely due to extraction of the tracer by the splanchnic viscera (P < 0.05; Table 4). Splanchnic extraction was 53% in the enteral dipeptide group and 39% in the enteral free L-[2-15N]glutamine group.


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  		Table 4. WbRa L-glutamine, L-citrulline, and L-arginine, whole body substrate conversions, and %WbRa

 
The WbRa of L-citrulline and L-arginine were not different among the groups with regard to the route of administration. The total turnovers of L-citrulline and L-arginine were only marginally affected by the calculated contribution of the endogenously synthesized L-[2-15N]citrulline and L-[2-15N]arginine (within the margin of the SE). Therefore, adjusted figures are not shown.

Conversion from L-Glutamine to L-Citrulline (QGln->Cit)

Whole body de novo L-citrulline production derived from L-glutamine (QGln->Cit) resulted in a higher L-citrulline production when isotopes were given enterally (P < 0.05) compared with the parenteral route. The lowest production of de novo L-citrulline from L-glutamine was seen in the intravenous dipeptide group when quantitatively compared with the other groups. The tracer conversion of L-[2-15N]glutamine to L-[2-15N]citrulline was found to represent between 10 and 20% of the total de novo L-citrulline synthesis from L-glutamine (Table 4).

De novo L-citrulline derived from L-glutamine, as a percentage of WbRa of L-citrulline, was the lowest in the intravenous dipeptide group, with 15%. In the enteral dipeptide group this accounted for 32%. In the enteral free L-glutamine group, citrulline production derived from L-glutamine accounted for 36% of the total body L-citrulline turnover, whereas intravenous free L-glutamine accounted for 24%.

Conversion of Citrulline to Arginine (QCit->Arg) and the Contribution of Glutamine

No significant difference was observed within series between the enteral and the intravenous groups with respect to the absolute de novo synthesis of L-arginine from L-citrulline. When the tracer conversion of L-[2-15N]citrulline to L-[2-15N]arginine was included in the equation, the contribution of this route was found to represent only a marginal part of the total de novo L-arginine synthesis from L-citrulline.

The relative contribution of L-citrulline to the de novo synthesis of L-arginine was significantly lower in the enteral free L-glutamine group compared with the intravenous free L-glutamine group. This was the other way around for the dipeptide groups, although this difference was not significant. On the other hand, the conversion from L-citrulline to L-arginine contributed significantly more to total L-arginine production in the enteral dipeptide group compared with the intravenous dipeptide group, which was the other way around in the free L-glutamine groups (not significant).

The de novo synthesis of L-arginine from L-citrulline at whole body level, expressed as a percentage of the WbRa of L-citrulline, tended to be higher in the free L-[2-15N]glutamine groups (IV 65%; EN 55%) compared with the dipeptide groups (IV 43%; EN 47%).

The contribution of L-glutamine to the de novo synthesis of L-arginine was significantly higher in the enteral groups (dipeptide 16%; free L-[2-15N]glutamine 20%) compared with the intravenous groups (dipeptide 6%; free L-[2-15N]glutamine 16%).

When the contribution of L-glutamine to the novo synthesis of L-arginine was expressed as a percentage of the WbRa of L-arginine, the same differences were observed.


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Endogenous generated and exogenous delivered L-glutamine, unbound or derived from L-alanyl-L-glutamine, serves as a substrate for L-citrulline and L-arginine de novo synthesis at the whole body level in mice. Enterally given L-glutamine contributed more to the de novo synhthesis of L-arginine than parenterally supplied L-glutamine.

Validity of the Model

In this study, we used stable isotope tracers to quantify the metabolic interrelationships of L-glutamine, L-citrulline, and L-arginine in vivo in mice. The model assumes that when a steady state of tracer infusion is reached the amount of isotope entering and leaving per unit of time is equal (41). Additionally, assuming that the isotopes move freely among the pools, the calculated whole body substrate turnover will thus represent the rate of appearance and disappearance of the unlabeled substrate from or into the free plasma pool (41).

The metabolic pathway in our model was quantified with the help of the the isotopic transfer of L-[2-15N]glutamine (from the free L-[2-15N]glutamine or indirectly derived from the L-alanyl-L-[2-15N]glutamine infusion) to L-[2-15N]citrulline and of L-[ureido-13C,2H2]citrulline to L-[guanidino-13C,2H2]arginine. The quantification of the conversions of L-glutamine to L-citrulline and L-citruline to L-arginine were both necessary to calculate the relative contribution of L-glutamine to de novo synthesis of L-arginine.

Furthermore, a large dose of L-[2-15N]glutamine or L-alanyl-L-[2-15N]glutamine was given to bring about an effect. To include the contribution of the conversions of L-[2-15N]glutamine to L-[2-15N]citrulline and L-[2-15N]citrulline to L-[2-15N]arginine in the total conversions of L-glutamine to L-citrulline and L-citrulline to L-arginine, equations were slightly adapted.

Arterial Plasma L-Glutamine, L-Citrulline, and L-Arginine Pool

First, we will discuss the arterial amino acid responses. Because all of our mice received L-glutamine or the glutamine-dipeptide in equimolar concentrations, we expected equimolar increases in the concentrations of plasma L-glutamine when they were given intravenously. In contrast, we found 50% higher plasma concentrations of L-glutamine in the intravenous dipeptide group compared with the intravenous free L-glutamine group. An explanation might be provided by the increase in the plasma L-alanine concentration with the administration of the dipeptide. L-Alanine and L-glutamine are related amino acids with the same property of interorgan nitrogen/carbon carrier for ureagenesis and gluconeogenesis (42). Battezzati et al. (5) investigated the amino acid kinetics during the anhepatic phase of liver transplantation and confirmed the importance of the liver in the clearance of both amino acids. Furthermore, it is known that in the postabsorptive state L-glutamine-derived L-glutamate is converted to L-alanine in the gut (33), and it was shown by Funovics et al. (12) that the postoperative infusion of L-alanine (0,9g·kg–1·h–1) significantly increased the plasma L-glutamine concentration. In the view of this evidence, it could be speculated that the increase in the plasma L-alanine concentration as a consequence of the administration of L-alanyl-L-[2-15N]glutamine might have affected the plasma L-glutamine concentration as well. The plasma L-glutamine concentration was highest in the group receiving L-alanyl-L-[2-15N]glutamine by the intravenous route. In the enteral group, the splanchnic extraction of L-alanyl-L-[2-15N]glutamine or its constituent metabolites L-alanine and L-[2-15N]glutamine might have blunted the effect observed in the group receiving L-alanyl-L-[2-15N] glutamine by the intravenous route. In future experiments, it would probably be wise to give free L-alanine next to free L-glutamine, when one is comparing the effects of L-glutamine as a free amino acid or bound in a dipeptide as L-alanyl-L-glutamine.

Intravenous supply of L-glutamine resulted in higher arterial L-glutamine concentrations than enteral L-glutamine administration in both series, which was expected because of the partial breakdown of L-glutamine in the intestinal wall. Plasma L-citrulline or L-arginine concentrations were not affected by the feeding route or the difference in molecular form of L-glutamine in our mice model. All groups had higher plasma arterial L-citrulline and L-arginine concentrations than the control mice described before in a separate experimental series [60 ± 6 µM and 105 ± 7 µM, respectively (18)]. The higher arterial values in our series might have caused a suppression of endogenous production of L-citrulline and L-arginine.

WbRa of L-Glutamine, L-Citrulline, and L-Arginine

The WbRa of L-glutamine was not different between the intravenous groups. Lower plasma L-[2-15N]glutamine enrichments were found after enteral administration of L-[2-15N]glutamine or L-alanyl-L-[2-15N]glutamine, presumably due to their first pass through the splanchnic region, resulting in a higher calculated WbRa of glutamine. Haisch et al. (16) found in humans, consistent with our findings, lower plasma L-glutamine enrichments after enteral administration of labeled L-glutamine compared with intravenous infusion and estimated human splanchnic extraction to be ~64%. In our mouse model, splanchnic L-glutamine extraction was estimated to be somewhat lower than in the human study. It has been shown in human intestines that L-glutamine dipeptides are predominantly absorbed as intact dipeptide rather than being hydrolyzed into L-glutamine (26). Our observations suggest that this might be different in mice, since the splanchnic extraction was calculated to be higher in the groups receiving the dipeptide, suggesting hydrolysis of the dipeptide in the intestine or the liver.

Interestingly, the WbRa of L-citrulline and L-arginine were not affected by the route of administration or the molecular form of L-glutamine. In a previous mouse study, when no L-glutamine or dipeptide of L-glutamine was administered the WbRa of L-citrulline was ~26% higher and the WbRa of L-arginine was 31% higher than in the present study (17). Because the arterial plasma L-citrulline and L-arginine values were, respectively, 50 and 30% lower in the previous study, the present study indicates that the exogenous L-glutamine dosage might have suppressed the metabolic turnover of L-citrulline and L-arginine. This finding is in agreement with the observation that the large dose of L-[2-15N]glutamine or L-alanyl-L-[2-15N]glutamine probably suppressed the turnover of L-glutamine when compared with the observed turnover of 245 nmol·10 g–1·min–1 L-glutamine by Braulio et al. (8).

Summarized and compared with the other experiments (8, 17), the results from this study might indicate that the plasma concentrations of L-glutamine, enhanced by the exogenous supply of labeled L-glutamine, L-arginine, and L-citrulline, regulate the metabolic turnover of the respective amino acids independently of precursor supply.

Furthermore, it should be taken into account that the WbRa of L-arginine reflects the entry of L-arginine into the plasma pool from mostly 1) whole body protein breakdown and less from 2) de novo L-arginine synthesis from L-citrulline (43).

L-Glutamine Conversion to L-Citrulline and L-Arginine

L-Glutamine contributed more to the de novo synthesis of L-citrulline and L-arginine when L-[2-15N]glutamine or L-alanyl-L-[2-15N]glutamine was provided by the enteral route. The contribution of L-glutamine to the conversion of L-citrulline to L-arginine was highest in the enteral groups and the free L-glutamine groups without the introduction of a difference in the absolute de novo synthesis of L-arginine.

By the enteral route, L-glutamine is first passing the gut, which is the main "L-glutamine-consuming" and "L-citrulline-producing" organ (38, 39). In rats, it was shown that a normal rat chow containing L-glutamine stimulates glutaminase specific activity the most, followed by L-glutamine-enriched parenteral nutrition. Glutaminase specific activity was lowest in rats receiving conventional parenteral nutrition (24). Therefore, it could be hypothesized that luminal delivery of L-glutamine will enhance intestinal L-glutamine metabolism. Exact mechanisms behind the preference for luminally or intravenously offered precursors remain to be unravelled.

The conversion of glutamine into citrulline is 2–3% of the total appearance rate of L-glutamine, of which approximately one-half is converted into L-arginine. Thus glutamine is quantitatively an important source for de novo synthesis of L-arginine, but L-arginine is quantitatively an unimportant product of L-glutamine metabolism. The results from the adapted equation, revealing the direct contribution of the conversion of L-[2-15N]glutamine to L-[2-15N]citrulline to the total conversion of L-glutamine to L-citrulline, suggest that the effect of the provision of L-glutamine by the enteral route is an effect on total intestinal glutamine metabolism.

The relative contribution of the synthesis of L-arginine from L-citrulline to the de novo synthesis and total appearance of L-arginine was observed to be different for route between the dipeptide and the free L-glutamine group. This observation suggests an interaction between route and molecular form of L-glutamine. This finding deserves further attention in future research.

Free L-[2-15N]Glutamine or Dipeptide

More de novo L-arginine was synthesized from L-citrulline when free L-[2-15N]glutamine was given compared with the dipeptide. De novo L-arginine production was ~60% of the substrate pool (WbRa of L-citrulline) in the free L-glutamine groups and 45% in the dipeptide groups. This finding supports the hypothesis that the molecular form of L-glutamine also affects the contribution of L-glutamine to L-arginine. This possible effect deserves further exploration.

Summary of Main Findings

In conclusion, L-glutamine serves as substrate for de novo L-citrulline and L-arginine synthesis in mice. Also, quantities of the product were observed to be affected by the feeding route (enteral or intravenous) and the different molecular forms (free amino acid or dipeptide) of the provided exogenous L-glutamine. When L-alanyl-L-glutamine was admistered by the enteral route, L-glutamine contributed significantly more to the de novo L-citrulline and L-arginine synthesis in vivo in mice. The finding that feeding route and molecular structure of L-glutamine affect the contribution of L-glutamine to the de novo synthesis of L-arginine raises a number of questions for future research, most importantly: what is the contribution of L-glutamine to de novo synthesis of L-arginine in humans?

After the contribution of L-glutamine to de novo synthesis of L-arginine under postabsorptive circumstances in humans is established, it will be interesting to investigate the effect of an exogenous supplementation with L-glutamine, parenteral or enteral, free or as a dipeptide with L-alanine, on the de novo synthesis of L-arginine.

Furthermore, considering the observed effect of enterally provided L-alanyl-L-glutamine on de novo synthesis of L-citrulline and L-arginine, the question arises what will be the quantitative importance of the intestinal-renal axis in the de novo synthesis of L-arginine. This last question was recently answered in mice (6) but remains to be answered in humans.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
G. Boelens is a recipient of a fellowship (NWO 920-03-185) of the Council for Medical Research of the Netherlands Organization of Scientific Research.


   ACKNOWLEDGMENTS
 
With special gratitude we acknowledge the laboratory staff of Metabolic Research Center of the Maastricht University, Department of Surgery, in particular Hans van Eijk and Marieke G. van den Heuvel, who assisted in data management and the conduct of the second series of experiments. The dipeptide tracer was a kind gift from Prof. D. E. Matthews.

Part of this work was presented at the European Society of Parenteral and Enteral Nutrition 2004 and was awarded for being the highest scoring abstract.


   FOOTNOTES
 

Address for reprint requests and other correspondence: P. A. M. van Leeuwen, Dept. of Surgery, VU Univ. Medical Center, PO Box 7057, 1007 MB Amsterdam, The Netherlands (e-mail pam.vleeuwen{at}vumc.nl)

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. Section 1734 solely to indicate this fact.

* These two authors contributed equally to this paper. Back


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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