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American Journal of Clinical Nutrition, Vol. 87, No. 5, 1282-1289, May 2008
© 2008 American Society for Nutrition

ORIGINAL RESEARCH COMMUNICATION

Glutamine is an important precursor for de novo synthesis of arginine in humans1,2,3,4

Gerdien C Ligthart-Melis, Marcel CG van de Poll, Petra G Boelens, Cornelis HC Dejong, Nicolaas EP Deutz and Paul AM van Leeuwen

1 From the Department of Surgery, VU University Medical Center, Amsterdam, Netherlands (GCL-M, PGB, and PAMvL), and the Department of Surgery, University Hospital Maastricht and the Nutrition and Toxicology Research Institute Maastricht (NUTRIM), Maastricht, Netherlands (MCGvdP, CHCD, and NEPD)

2 GCL-M and MCGvdP contributed equally to this work and share first authorship.

3 Supported by grants no. 920-03-317 AGIKO (to MCGvdP), 920-03-185 AGIKO (to PGB), and 907-00-033 Clinical Fellowship (to CHCD) from The Netherlands Organization for Health Research and Development and by a grant from Fresenius-Kabi, Bad Homburg, Germany.

4 Reprints not available. Address correspondence to PAM van Leeuwen, Department of Surgery, Vrije University Medical Center, PO Box 7057, 1007 MB Amsterdam, Netherlands. E-mail: pam.vleeuwen{at}vumc.nl.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
Background: A metabolic relation exists between glutamine and arginine, 2 amino acids with properties that enhance the recovery of seriously ill patients. It is possible that glutamine exerts part of its beneficial effects by enhancing the availability of arginine.

Objectives: We aimed to quantify under postabsorptive conditions the metabolic pathway of plasma glutamine into arginine via the intermediate citrulline and to establish the contribution of the kidneys to the synthesis of arginine.

Design: The study was conducted in patients during surgery. The metabolism of glutamine, citrulline, and arginine was studied by using intravenous administration of stable isotope tracers of the amino acids. Results were interpreted by using established equations. Parametric tests were used to test and correlate results. P < 0.05 was regarded as significant.

Results: Mean (±SE) whole-body plasma turnover rates of glutamine, citrulline, and arginine were 240 ± 14, 6.2 ± 0.6, and 42 ± 2.9 µmol · kg–1 · h–1, respectively (P < 0.01). Plasma turnover of citrulline derived from glutamine was shown to be 5.1 ± 0.7 µmol · kg–1 · h–1, and arginine derived from citrulline was shown to be 4.9 ± 0.9 µmol · kg–1 · h–1 (P < 0.01). The contribution of plasma glutamine to plasma arginine derived from plasma citrulline was calculated to be 64%. The kidneys were observed to take up >50% of circulating plasma citrulline and to release equimolar amounts of arginine into plasma.

Conclusions: This study shows that glutamine is an important precursor for the synthesis of arginine in humans. It also provides a firm basis for future studies exploring the effect of a treatment dose and the route of administration (enteral or parenteral) of glutamine.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
The relation between glutamine and arginine has been a subject of research by our group since the early 1990s, when Houdijk et al (1) observed that a glutamine-enriched enteral diet enhanced plasma concentrations of citrulline and arginine and resulted in a higher renal uptake of citrulline and a greater release of arginine in rats. A few years later, our group established that supplemental glutamine provided by the enteral route normalized depressed concentrations of arginine in trauma patients (2) and that intravenous administration of the dipeptide alanyl-glutamine in preoperative patients resulted in a rise in plasma concentrations of arginine (3).

The importance of these observations is supported by the clinical relevance of both glutamine and arginine—substrates with a broad spectrum of properties—in enhancing recovery after surgery, trauma, and other conditions of serious illness (4–6). In view of the apparent relation between glutamine and arginine, it could be speculated that glutamine exerts its positive effects partly by enhancing the availability of arginine.

The current concept is that the intestinal conversion of glutamine leads to a release from the gut of citrulline, which, after its uptake from the bloodstream, is converted by the kidneys into arginine (4, 6–8). Evidence has accumulated for the existence of this pathway in mice and rats (1, 9–12). Boelens et al (9, 10) were the first to confirm the existence of the complete pathway of glutamine into citrulline and arginine in mice with both enteral and intravenous administration of glutamine. Their experiments also showed that the quantitative importance of glutamine for arginine synthesis was affected by the route of administration and that the enteral route is more favorable.

In humans, previous investigations show that the intestines are the most important site of the release of citrulline (13, 14), and glutamine was recently established to be quantitatively the only important precursor for this intestinal release of citrulline (15). Furthermore, other stable isotope studies in humans support the quantitative importance of citrulline in the generation of arginine at the whole-body (WB) level (16, 17), and a correlation was shown between the uptake of citrulline and the release of arginine by the human kidneys (14, 18, 19). These observations together suggest that glutamine is a precursor for arginine synthesis in humans, and that intestinal and renal metabolism may be involved in this pathway.

However, in humans, the metabolic route of glutamine to citrulline and then to arginine remains to be confirmed, and the importance of glutamine as a precursor for the synthesis of arginine remains to be quantified. The aim of this study was to investigate this pathway at the WB and organ levels in humans under postabsorptive conditions in an effort to provide a firm basis for future studies involving the effect of a treatment dose of glutamine or the route of administration. Therefore, we applied a classic stable-isotope in vivo approach in 8 subjects undergoing major abdominal surgery, which facilitated access to the portal, hepatic, and renal veins. The stable isotope tracers L-[2-15N]glutamine, L-[13C]ureido[5,5-2H2]citrulline, and L-[guanidino-15N2]arginine were used to investigate the metabolic relations of glutamine, citrulline, and arginine at the WB plasma level and across the intestines, the liver, and the kidneys in these surgical patients.

Intestinal and hepatic results are described elsewhere (15). In this report, we show the quantitative importance of glutamine for the de novo synthesis of arginine in fasting humans, as well as the contribution of the kidneys to the release of arginine.


   PATIENTS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Patients
Eight patients undergoing gastrointestinal surgery (n = 6, 1, and 1 undergoing liver resection, pancreaticoduodenectomy, and duodenectomy, respectively) at the University Hospital Maastricht (UHM), because of liver metastasis due to previously resected colonic cancer (n = 6), cancer in the head of the pancreas (n = 1), or familial adenomatous polyposis (n = 1), were included. On the day of admission, routine blood tests were performed, a dietary questionnaire was obtained, and body composition was measured by using bioelectrical impedance analysis (Xitron 4200; Xitron Technologies, San Diego, CA). Patients with known parenchymal liver disease, inborn errors of metabolism, diabetes mellitus type 1, recent weight loss, clear cachexia, or other indications of metabolic disorders were excluded from the study. Patient characteristics are presented in Table 1Go. Oral intake except for water was stopped at 2000 on the day before surgery. All patients were transported to the operation theater at {approx}0730 the next day.


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  		TABLE 1 Patient characteristics1

 
All patients gave written informed consent. The Medical Ethics Committee of the UHM approved the study.

Study design
The metabolic study was conducted during surgery. Stable isotope tracers were administered intravenously to quantify the plasma turnover of the L-amino acids glutamine, citrulline, and arginine and the conversions of glutamine into citrulline and of citrulline into arginine at the WB plasma level in the fasted state. The surgical procedure also enabled us to quantify renal citrulline and arginine turnover with the provided tracers and their metabolic products.

Stable isotope tracers
The tracers L-[2-15N]glutamine, L-[13C]ureido[5,5-2H2]citrulline, and L-[guanidino-15N2]arginine (all: >98% mol percent enrichment) were purchased from Cambridge Isotope Laboratories (Woburn, MA). Sterile and pyrogen-free stock solutions of the tracers were prepared by the Department of Clinical Pharmacy at the UHM and were kept at –20 °C until the evening before surgery. The stock solutions were diluted with normal saline before the start of each tracer infusion.

Surgical procedure and anesthesia
To standardize metabolic and surgical conditions, all patients underwent surgery at the same time of the day and were operated on by the same surgical team. Anesthesia was applied by using isoflurane and propofol. In all patients, a thoracic epidural catheter was inserted for perioperative administration of analgesia; indwelling catheters were inserted in a jugular vein and a radial artery to monitor arterial and central venous blood pressure, as well as pH, HCO3–, and glucose. No exogenous bicarbonate was supplied, and lactate-containing infusates were routinely avoided in patients undergoing liver surgery. Urine output was monitored by using transurethral catheterization. Body temperature was kept constant by using a Bair Hugger system (Arizant Healthcare Inc, Eden Prairie, MN).

Tracer infusion, blood sampling, and renal blood flow measurement
After the induction of anesthesia, a catheter was placed in an antecubital vein for isotope infusion. Blood was sampled from the radial artery catheter. After baseline sampling and shortly after incision, a primed, continuous intravenous infusion of the stable isotope tracers was started and continued for 2.5 h (Table 2Go). For the tracer infusion, a calibrated, volume-controlled pump (Graseby 3000; Graseby Medical Ltd, Watford, United Kingdom) was used.


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  		TABLE 2 Tracer dosages

 
During infusion, blood samples were drawn at 30, 60, 90, 120, and 150 min after the beginning of the infusion for the study of WB plasma amino acid turnover. After 1 h, when experience has shown that an isotopic steady state would be present, blood was drawn from the right renal vein by direct puncture, and arterial blood sampling was done simultaneously to study renal metabolism. The renal vein was sampled before organ transection in those patients undergoing liver resection. To quantify renal amino acid fluxes, blood flow was measured in the right renal vein by using a color Doppler ultrasound (Prosound SSD 5000; Aloka Co, Ltd, Tokyo, Japan) before samples were taken from the renal vein. Time-averaged mean velocities of the bloodstream and the cross-sectional area of the right renal vein were measured as described previously (19). Briefly, the renal vein was traced by using a sterile transducer (UST-579T-7.5, 5–10-MHz linear; Aloka Co Ltd). For accurate measurements, care was taken to keep the angle between the ultrasonic beam direction and the blood flow direction <60 °. The cross-sectional area of the vessel was calculated by drawing an area ellipse at the same point at which the velocity was measured. Blood flow in the renal vein was calculated by multiplying the time-averaged velocity of the bloodstream by the cross-sectional area of the vessel. Renal plasma flow was calculated from measured blood flow and hematocrit [plasma flow = blood flow x (1–hematocrit)] and multiplied by 2 for each patient separately (assuming that both kidneys contribute equally to total renal flow).

Processing of blood samples
Blood was collected in chilled heparinized vacuum tubes (Vacutainer; Becton-Dickinson, Franklin Lakes, NJ) and placed on ice. Within 1 h, blood was centrifuged (10 min, 4000 rpm, 4 °C), and 500 µL of plasma was added to 80 mg dry sulfosalicylic acid (Across Inc, Geel, Belgium) to precipitate plasma proteins. After vortex mixing, deproteinized plasma samples were snap-frozen in liquid nitrogen and stored at –80 °C until they were analyzed. The use of stable isotopes requires plasma sampling, because sampling should occur from a pool in which the tracer mixes freely (20). Before centrifugation, the hematocrit of each blood sample was measured by using a microcapillary centrifuge.

Laboratory analysis
Amino acid concentrations in deproteinized plasma samples and infusates were measured by using HPLC as described elsewhere (21). Glutamine, citrulline, and arginine enrichments were measured by using liquid chromatography–mass spectrometry (22) and were expressed as a tracer-to-tracee ratio [(TTR) tracer = labeled substrate; tracee = unlabeled substrate]: TTR x 100 (TTR%). The mean (±SD) of the measured standards for tracer enrichments were: 6.8 ± 0.29 TTR% for [15N]glutamine, 8.4 ± 0.49 TTR% for [15N]citrulline, 1.4 ± 0.37 TTR% for [13C-2H2]citrulline, 8.4 ± 0.25 TTR% for [15N]arginine, and 0.8 ± 0.21 TTR% for [15N2]arginine and [13C-2H2]arginine.

Calculations
Isotopic enrichment was calculated by taking into account the contribution of overlapping isotopomer distributions of the tracee and tracers with lower masses to the measured TTR as described by Vogt et al (23). Metabolic fluxes and conversions were calculated by using established formulas (24, 25).

Steady state curve-fitting
For the estimation of individual steady state values, arterial enrichment curves at each mass of each amino acid under study were fitted for each patient with the use of PRISM for WINDOWS software (version 4.03; GraphPad Software Inc, San Diego, CA). For calculations of renal metabolism, the enrichment of the arterial sample taken simultaneously with the sample from the right renal vein was used for the calculations.

Whole-body plasma turnover of the amino acids studied
Whole-body plasma rate of appearance of glutamine, citrulline, and arginine
The WB plasma rate of appearance (WB Ra; µmol · kg–1 · h–1) of glutamine, citrulline, and arginine was calculated from the arterial TTR values (TTR-A) of [15N]glutamine, [13C-2H2]-citrulline, and [15N2]arginine, respectively, and the known infusion rate of these tracers by using the following equation (24):

Formula 1(1)
where I is the know infusion rate of the tracers.

Whole-body de novo synthesis of citrulline from glutamine
Calculation of the rate of WB plasma turnover (Q; µmol ·kg–1 · h–1) of glutamine into citrulline was performed by using the following equation from Castillo et al (17):

Formula 2(2)
where WB RaCit is the plasma WB Ra of citrulline, calculated from the TTR of the infused [13C-2H2]citrulline tracer by using equation 1, and CitM(mass)+1 is [15N]citrulline coming from [15N]glutamine (Gln M+1).

Rate of whole-body de novo synthesis of arginine from citrulline
Calculation of the WB plasma turnover of citrulline into arginine (de novo synthesis of arginine) was performed as described by Castillo et al (17) by using the following equation:

Formula 3(3)
where WB RaArg is the WB RA of arginine, calculated from the TTR of the [15N2]arginine tracer by using equation 1, and Arg M+3 is [13C-2H2]arginine coming from [13C-2H2]citrulline (Cit M+3).

Renal metabolism of the amino acids studied
Renal net balance, disposal, and production of the amino acids studied
The renal net balance (NB) of glutamine, citrulline, and arginine was calculated by subtracting renal output [renal venous (RV) amino acid concentration (in µmol/L) x plasma flow (F) (in L · kg–1 · h–1)] from renal input [arterial (A) amino acid concentration (in µmol/L) x F], according to the following equation:

Formula 4(4)
The tracer net balance (tnb) of the glutamine, citrulline, and arginine tracers was calculated in the same way as the renal NB. Plasma tracer concentrations were calculated by multiplying the plasma concentration of an amino acid by the concomitant TTR of this amino acid, according to the following equation:

Formula 5(5)
where TTR-A represents arterial tracer enrichment, and TTR-RV represents renal venous tracer enrichment.

The tnb of arginine was corrected for [2H2]arginine as described by Hallemeesch et al (26). The rationale for this correction is that the [13C-2H2]arginine coming from [13C-2H2]citrulline may in turn be catabolized by arginase, which would lead to [2H2]ornithine, which in turn will give rise to [2H2]citrulline and [2H2]arginine. This correction assumes that [2H2]citrulline (Cit M+2) is converted into [2H2]arginine (Arg M+2) in the same way that [13C-2H2]citrulline (Cit M+3) is converted into [13C-2H2]arginine (Arg M+3), as described in the following equation:

Formula 6(6)
The fractional extraction (FE) of the amino acids studied was calculated by dividing tnb by input of the concomitantly provided tracer according to the following equation:

Formula 7(7)
Renal disposal, representing the unidirectional absolute renal uptake of glutamine, citrulline, or arginine, was calculated from the tnb and the TTR-RV of the respective tracer, which is known to most accurately represent intracellular enrichment (27). The calculation was done according to the following equation:

Formula 8(8)
Renal production, representing the unidirectional absolute release of glutamine, citrulline, and arginine was calculated by subtracting the renal NB from the renal disposal, according to the following equation:

Formula 9(9)

Renal de novo synthesis of arginine from citrulline
Renal de novo synthesis of arginine from citrulline (Q Cit->Arg) was calculated by using the TTR-A of [13C-2H2]citrulline (Cit M+3) and the TTR-RV of [13C-2H2]arginine (Arg M+3). First, the renal output of Arg M+3 was corrected for the fraction of bypassing arginine, which was calculated with the help of the renal FE of arginine, as in the following equation:

Formula 10(10)
Subsequently, Q Cit->Arg was calculated by multiplying the ratio of the corrected renal output of Arg M+3 to the renal input of Cit M+3 by the renal input of unlabeled citrulline, according to the following equation:

Formula 11(11)

Statistical analysis
Results are presented as means ± SEMs. With respect to renal metabolism, disposal (only in case of arginine), production, and conversion rates < 0 were considered absent and therefore equal to 0. This adaptation did not affect the direction of the results.

The one-factor analysis of variance for repeated measurements was used to test whether arterial enrichments were in steady state. The one-sample t test was used to test whether arterial and venous enrichments; WB turnover; renal NB, disposal, and production; and the renal conversion of citrulline into arginine differed from zero. Correlations were studied by using the Pearson test. We used EXCEL for WINDOWS software (version 2003; Microsoft Corp, Redmond, WA) to perform calculations, and SPSS for WINDOWS software (version 14.0.1; SPSS Inc, Chicago, IL) to perform statistical tests. P < 0.05 was considered to indicate significance.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Flow measured in the right renal vein was multiplied by 2 for each patient. Subsequently, the mean plasma flow in all patients was used in calculations, because results did not differ significantly from individual flow results, but variation was larger.

Arterial plasma enrichment (expressed as TTR) was observed to be in steady state for all infused tracers: 6.66 ± 0.35% for [15N]glutamine, 2.58 ± 0.44% for [13C-2H2]citrulline, and 6.7 ± 0.67% for [15N2]arginine (Figure 1Go). Moreover, the TTRs of the metabolic products of [15N]glutamine metabolism—[15N]citrulline and [15N]arginine—were observed to be significantly different from zero (5.52 ± 0.44% and 1.43 ± 0.12%, respectively; P < 0.001), which shows that the 15N label of glutamine found its way to citrulline and arginine.


Figure 1
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  		FIGURE 1. The arterial enrichments of the given stable isotope tracers L-[2-15N]glutamine, L-[13C]ureido[5,5-2H2]citrulline, and L-[guanidino-15N2]arginine. TTR%, tracer-to-tracee ratio (in %). No significant change in the slope of arterial enrichment was observed between 30 and 120 min (one-factor repeated-measures analysis of variance) for any of the given tracers. Therefore, the enrichment of all given tracers was considered to be in steady state. Furthermore, arterial enrichment curves were fitted for each patient to estimate individual steady state values. In 4 patients, the last measurement (at 150 min) was omitted from the analysis because the infusion of the tracers was terminated before the last sample was taken.

 
Whole-body plasma amino acid turnover
Whole-body rate of appearance of glutamine, citrulline, and arginine
WB plasma turnover rates for glutamine, citrulline, and arginine were observed to be 240 ± 14, 6.2 ± 0.6, and 42 ± 2.9 µmol · kg–1 · h–1, respectively (P < 0.01).

Whole-body conversions of glutamine into citrulline and arginine
WB plasma citrulline synthesis from plasma glutamine was observed to be 5.1 ± 0.7 µmol · kg–1 · h–1 (P < 0.01), representing 83 ± 5% of the total plasma turnover of citrulline. Plasma arginine synthesis from plasma citrulline was observed to be 4.9 ± 0.9 µmol · kg–1 · h–1 (P < 0.01), representing 11 ± 2% of the total plasma turnover of arginine and 76 ± 10% of the total plasma turnover of citrulline. Both conversions are shown together with the WB Ra of citrulline in Figure 2Go. The contribution of glutamine to the de novo synthesis of arginine from citrulline in plasma was calculated to be 64 ± 11%.


Figure 2
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  		FIGURE 2. The conversion of glutamine into citrulline and of citrulline into arginine, together with the whole-body (WB) plasma rate of appearance (Ra) of citrulline. From these results, the contribution of glutamine to citrulline, of citrulline to arginine, and of glutamine to arginine synthesis can be calculated. When expressed as a percentage of the plasma WBRa of citrulline, glutamine contributes 83 ± 5% to the plasma Ra of citrulline, and 76 ± 10% of plasma citrulline turnover is converted into plasma arginine, which represents the de novo synthesis of arginine. Thus, the contribution of glutamine to de novo synthesis of arginine was calculated by multiplying the contribution of glutamine to citrulline synthesis (as % of citrulline turnover) with the percentage of citrulline converted into arginine (as % of citrulline turnover); the result was 64 ± 11%. Gln, glutamine; Cit, citrulline; Arg, arginine. *Significant difference from 0 (P < 0.05, 1-sample t test).

 
Renal metabolism of citrulline and arginine
Arterial and RV enrichments (expressed as TTRs) of the isotopic tracers [15N]glutamine, [13C-2H2]citrulline, and [15N2]arginine and the labeled conversion products [15N]citrulline, [15N]arginine, and [13C-2H2]arginine from [15N]glutamine and [13C-2H2]citrulline are summarized in Table 3Go. These results were used to calculate the renal NB of glutamine, citrulline, and arginine and the unidirectional uptake and release of these amino acids by the kidneys.


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  		TABLE 3 Arterial and renal venous enrichments (in tracer-to-tracee ratio) of given isotopic tracers1

 
Renal net balance, disposal, and production of citrulline and arginine
Renal NB, disposal, and production of glutamine, citrulline, and arginine are summarized in Table 4Go. Significant renal unidirectional uptake of citrulline (7.2 ± 2.2 µmol · kg–1 · h–1; P = 0.01) and release of arginine (6.2 ± 1.6 µmol · kg–1 · h–1; P < 0.01) were observed in all patients, which illustrated the role of the kidney in the plasma release of arginine.


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  		TABLE 4 Renal net balance (NB), disposal, and production of glutamine, citrulline, and arginine1

 
Renal de novo synthesis of arginine from citrulline
Renal synthesis of plasma arginine from plasma citrulline was observed to be 1.5 ± 0.7 µmol · kg–1 · h–1 (P = 0.05). This value represents {approx}21 ± 9% of the total renal production of plasma arginine and 57 ± 25% of the WB plasma turnover of citrulline into arginine.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
For the first time, we show in humans the qualitative and quantitative importance of glutamine as a precursor for the synthesis of citrulline and arginine under postabsorptive conditions. Our data suggest that {approx}83% of circulating plasma citrulline comes from plasma glutamine and that {approx}11% of plasma arginine is derived from plasma citrulline. It is important that the present data indicate that 64% of the arginine obtained by de novo synthesis is generated from citrulline that comes from glutamine.

Whole-body rate of appearance of glutamine, citrulline, and arginine
In the current study, the WB plasma flux of glutamine was in the range typically reported in the literature (28–31). Glutamine metabolism was not affected by the presence of cancer, perhaps because patients with a history of weight loss, clear cachexia, or other indications of metabolic disorders were excluded from the study. The WB turnover of arginine observed was comparable to values reported by Castillo et al (16, 17, 32, 33) in fasted subjects, but the WB turnover of citrulline was observed to be lower. This difference may be related to the study design, such as the inclusion of a different citrulline tracer in the present study. If the citrulline tracer was somehow recycled, the citrulline turnover may have been underestimated.

Moreover, patients were studied during surgery. Previous investigations suggested that general anesthesia does not affect metabolism but indicated that surgery causes a depression of WB protein metabolism (34–38). However, WB fluxes of glutamine and arginine were not observed to be affected by this phenomenon in the present study.

The acid-base status, which has a distinct influence on glutamine metabolism, was closely monitored and was not observed to be disturbed by the surgical procedure. Moreover, metabolic steady state was maintained during the entire study, and venous blood was sampled before the liver transection. During surgery, patients received more fluids than they excreted via blood loss and urine production. However, no significant plasma dilution, which could have affected results at the organ level, occurred when the percentage hematocrit was observed over time (data not shown). Because no patient received supplemental blood during the protocol, hematocrit may have been affected only by intravascular volume changes. Other investigators have suggested that, besides disappearing into blood loss or urine production, intraoperative infused fluids evaporate from the wound, the airways, and the skin and accumulate in peripheral tissues (39, 40). These losses may explain why the infusion of a large amount of fluid to sustain cardiac output does not automatically result in a dilution of the plasma pool.

The patients in the present study had an average BMI of 30. Unfortunately, such a high BMI currently is representative of well-nourished Dutch (and other Western) people >40 y old. Therefore, whereas the inclusion of slightly obese subjects may have influenced the results, for the same reason, it also contributes to the internal and external validity of the present data. However, no correlation was observed between BMI and metabolic results.

Conversion of glutamine into citrulline and arginine at the whole-body level
It was suggested by previous studies that glutamine can be deamidated in the intestines by phosphate-dependent glutaminase into glutamate, which is subsequently converted into ornithine and citrulline (8). Citrulline appears to be an end-product of intestinal glutamine metabolism (11–15, 41), and {approx}80% of circulating plasma citrulline is now known to come from this intestinal glutamine metabolism (15). Results of the current study at the intestinal level, showing this observation, are summarized in Table 5Go. At the WB level, glutamine was observed to be the precursor for 83% of circulating plasma citrulline. Therefore, it can be stated that the intestines are the most important site, if not the only site, for the conversion of glutamine into citrulline.


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  		TABLE 5 Metabolism of glutamine and citrulline across the portally drained viscera (PDV)1

 
Arginine obtained by de novo synthesis from citrulline was observed to represent 76% of the WB plasma turnover of citrulline and 11% of the WB plasma flux of arginine, both of which values are lower than the values observed by Castillo et al (17). Those investigators established that 100% of circulating plasma citrulline is used for the synthesis of plasma arginine and that the de novo synthesis of plasma arginine represents 15% of the total plasma arginine release (17). It is also possible that we underestimated the turnover of citrulline in our study, because of the choice of tracer.

Combining the 2 observations of the conversion of glutamine into citrulline and of citrulline into arginine (Figure 2Go), we conclude that glutamine is the precursor for 64 ± 11% of arginine generated by de novo synthesis. It is important to outline that glutamine contributes to the synthesis of arginine along 2 pathways: the carbon moiety together with the amino group can end up in citrulline and subsequently in arginine, and glutamine can donate its amino nitrogen atom to the guanidino group by transamination processes, as described by van de Poll et al (15). Using [15N]glutamine to study the conversion of glutamine into citrulline (amino group) and [13C-2H2]citrulline to study the conversion of citrulline into arginine (carbon moiety) at the WB level enabled us to quantify the contribution of glutamine to arginine through citrulline without including the contribution of glutamine to arginine synthesis by transamination processes.

Renal contribution to de novo synthesis of arginine
The kidney is considered to be the most important organ for arginine synthesis. According to animal experiments, {approx}60% of de novo synthesis of arginine occurs in the kidneys. Argininosuccinate synthase and argininosuccinate lyase catalyze the transamination reaction by which arginine is synthesized from citrulline (6).

Our study showed that the kidneys are indeed an important site for the uptake of citrulline and the release of arginine. More than 50% of the citrulline appearing in plasma was observed to be taken up by the kidneys, and this process resulted in equimolar releases of arginine in plasma. Tracer results indicate a renal uptake of citrulline and a release of arginine even greater than the renal NBs of both. The calculated renal conversion of citrulline into arginine suggests that this conversion is responsible for {approx}60% of the de novo synthesis of plasma arginine from plasma citrulline at the WB level, a possibility that is in agreement with results reported in the literature.

However, the results at the renal level do raise a few questions. The renal production of arginine was observed to exceed the calculated renal conversion of citrulline into arginine. An explanation may be that other substrates were converted within the kidney into citrulline and subsequently converted into arginine. A likely candidate as one of these other substrates is plasma glutamine, which was shown to be taken up by the kidneys in large quantities and which is able to serve arginine synthesis in a manner independent of its conversion into plasma citrulline. Unfortunately, the current study design impedes the distinction between [15N]glutamine and [15N]citrulline as the source for renal synthesis of plasma [15N]arginine.

It is not known where the other 40% of arginine is synthesized, assuming that the synthesis does not occur in the kidneys. The kidney is the only organ known to both take up and release arginine in mammals. The fact that argininosuccinate synthase and lyase are also widely expressed in other cell types, such as hepatocytes, endothelial cells, and macrophages, may clarify this uncertainty. In these cells, arginine is being formed and broken down in intracellular cycles such as the urea cycle (liver) and the nitric oxide cycle (endothelial cells and macrophages). Although compartmentalization of metabolites within these cycles should prohibit the net release of newly formed arginine into the circulation (42), results from the present study and a study by Wu et al (6) suggest that the conversion of citrulline into arginine within one of these cycles may result in the net release of arginine into the circulation.

In conclusion, the present study showed that glutamine contributes 64% to the synthesis of arginine from citrulline in plasma under postabsorptive conditions. It also showed that the kidneys take up >50% of circulating citrulline, which results in the release of equimolar amounts of arginine. However, the challenge remains to investigate the ways in which a treatment dose of glutamine affects the synthesis of arginine in patients, especially critically ill patients, and to determine whether different routes of administration (enteral or parenteral) result in different quantitative outcomes. Recent studies by our group in mice and humans indicated that more glutamine is taken up and converted into citrulline when glutamine is provided by the enteral route than by the parenteral route (10, 43). The present study provides a firm basis for future explorations along these lines.


   ACKNOWLEDGMENTS
 
We thank LR Belliot (Department of Radiology, Vrije University Medical Centre) for his help with the flow measurements.

The authors' responsibilities were as follows—GCL-M and MCGvdP: performance of the study, interpretation of the results, and writing of the manuscript; PGB: wrote the protocol for the study; CHCD: contributed to the design of the study, developed the model for in vivo measurement in humans, collected the abdominal blood samples of the patients during surgery, and participated in the interpretation of the results and the writing of the manuscript; NEPD: contributed to the design of the study and participated in the interpretation of the results and the writing of the manuscript; and PAMvL: intellectual responsibility for the objective of the study and supervision of all aspects of the project. None of the authors had a personal or financial conflict of interest.


   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 PATIENTS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 

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Received for publication November 5, 2007. Accepted for publication December 20, 2007.





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