AJP - GI Ad Instruments
HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Am J Physiol Gastrointest Liver Physiol 293: G1061-G1067, 2007. First published September 27, 2007; doi:10.1152/ajpgi.00289.2007
0193-1857/07 $8.00
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/5/G1061    most recent
00289.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (4)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rougé, C.
Right arrow Articles by Darmaun, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rougé, C.
Right arrow Articles by Darmaun, D.

MUCOSAL BIOLOGY

Manipulation of citrulline availability in humans

Carole Rougé,1 Clotilde Des Robert,1,2,4 Alexander Robins,1 Olivier Le Bacquer,1 Christelle Volteau,5 Marie-France De La Cochetière,3 and Dominique Darmaun1,4

1Unité mixte de recherche 1280, Physiologie des Adaptations Nutritionnelles, Institut National de la Recherche Agronomique and University of Nantes, Human Nutrition Research Center, Nantes; 2Néonatologie, Hôpital Mère-et-Enfant, Centre hospitalier universitaire (CHU) de Nantes; 3EA 3826, Thérapeutiques cliniques et expérimentales des maladies infectieuses, University of Nantes; 4Centre d'Investigation Clinique, Institut National de la Santé et de la Recherche Médicale and Centre hospitalier universitaire (CHU) de Nantes; and 5Biostatistiques, Pôle Information médicale, épidémiologie et santé publique, CHU de Nantes, Nantes, France

Submitted 25 June 2007 ; accepted in final form 20 September 2007


   ABSTRACT
TOP
 ABSTRACT
MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
To determine whether circulating citrulline can be manipulated in vivo in humans, and, if so, whether citrulline availability affects the levels of related amino acids, nitric oxide, urinary citrulline, and urea nitrogen, 10 healthy volunteers were studied on 3 separate days: 1) under baseline conditions; 2) after a 24-h treatment with phenylbutyrate (0.36 g·kg–1·day–1), a glutamine "trapping" agent; and 3) during oral L-citrulline supplementation (0.18 g·kg–1·day–1), in randomized order. Plasma, erythrocyte (RBC), and urinary citrulline concentrations were determined by gas chromatography-mass spectrometry at 3-h intervals between 1100 and 2000 on each study day. Regardless of treatment, RBC citrulline was lower than plasma citrulline, with an RBC-to-plasma ratio of 0.60 ± 0.04, and urinary citrulline excretion accounted for <1% of the citrulline load filtered by kidney. Phenylbutyrate induced an ~7% drop in plasma glutamine (P = 0.013), and 18 ± 14% (P < 0.0001) and 19 ± 17% (P < 0.01) declines in plasma and urine citrulline, respectively, with no alteration in RBC citrulline. Oral L-citrulline administration was associated with 1) a rise in plasma, urine, and RBC citrulline (39 ± 4 vs. 225 ± 44 µmol/l, 0.9 ± 0.3 vs. 6.2 ± 3.8 µmol/mmol creatinine, and 23 ± 1 vs. 52 ± 9 µmol/l, respectively); and 2) a doubling in plasma arginine level, without altering blood urea or urinary urea nitrogen excretion, and thus enhanced nitrogen balance. We conclude that 1) depletion of glutamine, the main precursor of citrulline, depletes plasma citrulline; 2) oral citrulline can be used to enhance systemic citrulline and arginine availability, because citrulline is bioavailable and very little citrulline is lost in urine; and 3) further studies are warranted to determine the mechanisms by which citrulline may enhance nitrogen balance in vivo in humans.

nutrition; gut; glutamine; protein metabolism

CITRULLINE IS A NONPROTEIN amino acid found as a free amino acid in most biological fluids such as plasma, urine, and cerebrospinal fluid (20, 24, 31, 38, 39, 41). Two main pathways account for citrulline production (9, 32, 38, 39; Rougé C, Des Robert C, Robins A, Le Bacquer O, De La Cochetière MF, Darmaun D, unpublished observations): 1) citrulline is synthesized from glutamine in enterocytes, by condensation of ornithine and carbamyl phosphate (a reaction catalyzed by ornithine carbamyl-transferase); and 2) the conversion of arginine to nitric oxide produces citrulline as well, in a reaction catalyzed by nitric oxide (NO) synthetase (NOS) in most NO-producing tissues. Using stable isotope-labeled arginine and citrulline, other workers have found the contribution of arginine to citrulline synthesis to account for ~10% of circulating citrulline flux, and nearly 90% arise from glutamine (6, 38, 39, 43). Citrulline produced by gut is released as such into the bloodstream. Approximately 83% of the circulating citrulline is taken up by kidney (38, 39, 43), where citrulline is converted to arginine by arginosuccinate synthase (ASS) and arginosuccinate lyase (ASL) in cells of the proximal tubules (22). The production of arginine from citrulline accounts for 60% of the overall rate of de novo arginine synthesis in the body, but only 5–15% of circulating arginine (6, 26). Released as such into the bloodstream, arginine is used by most tissues for protein synthesis and other purposes (11, 14).

Interest about citrulline has risen substantially in the last decade, because plasma citrulline has been proposed as a noninvasive index of intestinal function. Plasma citrulline level indeed correlated with residual bowel length after extensive small intestinal resection (7, 33) or with small intestinal mass in celiac disease (8), intestinal transplantation (30, 16), or radiation-induced enteritis (23). In addition, recent animal studies suggest that citrulline may have a protein anabolic effect under specific settings: a citrulline-enriched diet indeed enhanced nitrogen balance after extensive small intestinal resection (28) and muscle protein synthesis in old, undernourished rats (29).

Even though several workers have reported plasma and urinary citrulline levels in various age groups in humans (5, 18, 24, 27), the factors that may regulate citrulline availability have not been specifically investigated. Since glutamine is the main precursor of citrulline, several groups have shown that enteral glutamine supplementation resulted in a rise in plasma citrulline concentration: for instance, plasma citrulline rose ~33, 30, and 27% in healthy volunteers who received enteral glutamine at rates of 0.04 (12), 0.05 (25), and 0.09 g·kg–1·h–1 (35), respectively. Yet, the effects of glutamine depletion, such as observed in stress, on plasma citrulline have not been addressed. Moreover, little is known on the effect of exogenous citrulline administration on the circulating levels and excretion rates of citrulline, other related nitrogen substrates, or urinary urea in vivo in humans.

The aims of the present study therefore were 1) to determine whether circulating citrulline levels can be decreased in vivo in humans, by depleting glutamine, its main endogenous precursor, and raised by oral citrulline supplementation, and 2) if so, whether citrulline availability affects the levels of related amino acids, nitric oxide, urinary citrulline, and urea nitrogen.


   MATERIAL AND METHODS
TOP
 ABSTRACT
 MATERIAL AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Chemicals

All chemicals (propan-1-ol, acetylchloride, hydrogen chloride, ammonium hydroxide, potassium hydroxide, 5-sulfosalicylic acid, ethylacetate, and heptafluorobutyric anhydride), citrulline (L-2-amino-5-ureidovaleric acid), and ion exchange resins (Dowex 50WX8–200 and Dowex 1X8) were purchased from Sigma-Aldrich (Saint Quentin Fallavier, France). L-[2,3,3,4,4,5,5-2H7] citrulline (d7-citrulline) was obtained from CDN Isotopes (Cil Cluzeau Info Labo; SteFoy la Grande, France) and homocitrulline ({varepsilon}-carbamoyl lysine) from Advanced Asymetrics (Millstadt). Phenylbutyrate (Ammoniaps) was purchased from Orphan Europe (Paris-La Défense, France), and L-citrulline (Stimol) from Biocodex (Gentilly, France).

Subjects

Ten young, healthy adult male volunteers were enrolled in studies performed in the Clinical Investigation Center at the Hôtel-Dieu Hospital, Nantes, France after signing an informed consent form, according to protocols approved by the local Institutional Review Board (Comité de Protection des Personnes n°2 des Pays de la Loire). Only subjects who were free of any gastrointestinal, metabolic, renal, or hematological disease and were not taking any medication were recruited. Subjects were 24.2 ± 3.3 yr old (range 20–32) and had a mean body mass index (weight/height2) of 22.5 ± 2.3 kg/m2.

Experimental Design

Each subject underwent three separate studies with a 1-wk washout period in between study days. On each study day, subjects reported after an overnight fast to the Clinical Investigation Center at Hotel-Dieu Hospital, Nantes, France, where they stayed all day. They remained at rest and received a standard diet (35 kcal·kg–1·day–1, 16% protein, 30% fat, and 54% carbohydrate) supplied as three daily meals at 0830, 1230, and 1900. Each subject was studied on three occasions: 1) under baseline conditions; 2) during oral L-citrulline administration (0.18 g·kg–1·day–1) divided into five equal doses given at 3-h intervals between 0700 and 1900 to increase circulating citrulline; and 3) after 24 h of treatment with phenylbutyrate per os (0.36 g·kg–1·day–1) split in six equal doses given at 4-h intervals to decrease circulating citrulline.

The baseline study was always performed first, the order of the other two studies was randomized. The dose of phenylbutyrate chosen was shown to result in an ~25% decline in plasma glutamine concentration (10). The dose of citrulline was chosen so as to represent approximately four times plasma citrulline flux, based on studies by other workers (6, 38, 39).

On each study day, 5 ml of blood were obtained at 1100, 1400, 1700, and 2000 into sampling tubes containing EDTA, and urine was collected two times, between 0800 and 1400 and between 1400 and 2000. Blood cells and plasma were immediately separated by centrifugation at 3,000 g for 10 min at 4°C, and the resulting samples were stored at –80°C until analysis. Urine samples were aliquoted and stored immediately at –80°C until analysis.

Analytical Methods

GC-MS. Blood samples (5 ml) were drawn into sampling tubes containing EDTA. Blood cells and plasma were immediately separated by centrifugation at 3,000 g for 10 min at 4°C, and the resulting samples were stored at –80°C until analysis. The concentration of citrulline in plasma, erythrocytes (RBCs), and urine was measured by GC-MS as described (Rougé et al., unpublished observations).

Briefly, before analysis of plasma and RBC, a 10-nmol aliquot of 0.1 mol/l homocitrulline, an analog of citrulline, was added to 200 µl of sample, as an internal standard. Citrulline was extracted from deproteinized plasma and blood cells on Dowex 50 cation exchange resin. Urine samples were aliquoted soon after collection and stored immediately at –80°C until analysis. Before analysis of urine, a 10-nmol aliquot of d7-citrulline was added to 400 µl of urine, as an internal standard. Urine samples were first passed on a Dowex 1X8 anion exchange resin and then on Dowex 50WX8 cation exchange resin.

Citrulline from plasma, RBC, or urine extracts was derivatized to its propyl heptafluorobutyryl derivative (Rougé et al., unpublished observations), and analysis was performed using an HP 5890 series II gas chromatograph coupled with a 5971 mass selective detector (Hewlett-Packard, Palo Alto, CA) using a DB-1 (30 m x 0.25 mm ID, 0.25 µm film thickness; J & W Scientific, Courtaboeuf, France) capillary column operated in the splitless mode. The mass spectrometer was operated in the electron impact ionization mode. The acquisition time was 14 min. Ions at m/z = 114, 118, and 128, representing natural citrulline, d7-citrulline, and homocitrulline, respectively, were selectively monitored.

Amino acid analyzer. Glutamine, ornithine, and arginine analyses were performed by using a Biotronik LC5001 automatic amino acid analyzer.

Nitrate and nitrite assay. Urinary nitrates and nitrites were quantified by a commercial kit (Roche-Diagnostic, Meylan, France), following the Griess method.

Total urinary nitrogen excretion was quantified by the Kjeldhal method.

Calculations

The renal clearance of creatinine, a measure of glomerular filtration rate (GFR; ml/min), was calculated as GFR = [creat]u x V/[creat]p, where [creat]u and [creat]p are the concentrations of creatinine in urine and plasma (nmol/ml), respectively, and V is urinary output (ml/min). Citrulline clearance (ml/min) was calculated as: V x [cit]u/[cit]p, where [cit]u is urinary citrulline concentration (nmol/ml), and [cit]u is the concentration of citrulline in urine (nmol/ml). The fraction (%) of citrulline filtered by glomerulus that was excreted into urine was calculated as (100 x V x [cit]u)/([cit]p x GFR). Nitrogen balance (N balance, mmol N/12 h) was estimated as N balance = Nin – Nout, where Nin is overall nitrogen intake (mmol nitrogen/12 h). Nitrogen intake was calculated as the sum of dietary protein nitrogen (based on food composition tables), plus nitrogen provided as oral citrulline, and Nout is total urinary nitrogen output, as quantified by the Kjeldhal method, between 0800 and 2000.

Statistical Analysis

Results are reported as means ± SD for amino acid quantification. Mixed linear models were used to test order of treatment and the effects of time, phenylbutyrate treatment, and oral citrulline on amino acids concentrations and to assess the relationships between plasma and RBC citrulline levels and between plasma and urine citrulline levels. The use of a mixed linear model provided correlations that did not suffer from pseudoreplication, since it is especially designed for repeated data points obtained at various time points (13), contrary to conventional linear models.


   RESULTS
TOP
 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
Order of Regimens

Because the order of regimens per se affected neither plasma nor RBC or urine citrulline (data not shown), the 10 subjects were considered as a single group studied under three separate regimens: control (baseline), phenylbutyrate treatment, and oral citrulline supplementation.

Diurnal Variation of Plasma and RBC Citrulline

On the control day, plasma citrulline tended to decline at 1400 and 2000, compared with the earlier samples (Fig. 1; Table 1). In contrast, RBC citrulline level did not vary significantly throughout the day.


Figure 1
View larger version (10K):
[in this window]
[in a new window]

  		Fig. 1. Composite time course of plasma citrulline ({circ}) and erythrocyte (RBC) citrulline (bullet) concentration under baseline conditions (left), after phenylbutyrate treatment (middle), and during oral citrulline supplementation (right) in 10 healthy adult subjects.

 

View this table:
[in this window]
[in a new window]

  		Table 1. Plasma citrulline concentration, and urinary excretion of citrulline and related nitrogen compounds under control conditions, and during treatment with phenylbutyrate or oral citrulline supplementation

 
Effect of Oral Phenylbutyrate

As expected, treatment with phenylbutyrate (0.36 g·kg–1·day–1) induced a significant, 7.3 ± 9.2% (P = 0.013) decline in plasma glutamine, from 501 ± 103 µmol/l to 464 ± 106 µmol/l (n = 10 subjects, 1 sampling time per subject at 1100). As a consequence, and as expected, phenylbutyrate treatment was associated with a significant, 18 ± 14% (P < 0.0001) decline in plasma citrulline (39 ± 4 µmol/l vs. 32 ± 3 µmol/l; n = 10 subjects, 4 sampling times per subject; baseline vs. phenylbutyrate treatment), and a 19 ± 17% (P < 0.01) decline in urinary citrulline (0.9 ± 0.3 µmol/mmol of creatinine vs. 0.7 ± 0.2 µmol/mmol of creatinine; baseline vs. phenylbutyrate treatment). RBC citrulline was not altered (P = 0.19), nor was plasma ornithine (P = 0.26), plasma arginine (P = 0.26), or urinary arginine (P = 0.14).

Phenylbutyrate treatment was associated with a significant reduction in urinary nitrate (15 ± 39%; P = 0.06), and urinary urea excretion (17 ± 15% P < 0.0001), without any alteration in plasma urea (P = 0.40).

Effect of Oral Citrulline Supplementation

As expected, oral L-citrulline administration induced a dramatic, 488 ± 92% rise in plasma citrulline (from 39 ± 4 to 225 ± 44 µmol/l; P < 0.0001). Urinary citrulline excretion rose accordingly (+571 ± 266%; from 0.9 ± 0.3 to 6.2 ± 3.8 µmol/mmol of creatinine; P < 0.001), whereas RBC citrulline only rose 139 ± 52% (from 23 ± 1 to 52 ± 9 µmol/l; P < 0.0001). Plasma glutamine, ornithine, and arginine rose 24 ± 21% (from 501 ± 103 to 614 ± 110 µmol/l; P = 0.0007), 73 ± 42% (from 90 ± 29 to 150 ± 46 µmol/l; P = 0.0003), and 92 ± 57% (from 134 ± 33 to 247 ± 62 µmol/l; P = 0.00005), respectively. Oral citrulline supplementation did not alter urinary arginine (P = 0.15), nor plasma urea (P = 0.23), urinary urea (P = 0.56), or urinary nitrate excretion (P = 0.55).

Because GFR was assessed by using creatinine clearance, the load of citrulline filtered by glomerulus could be calculated (see Calculations), as well as the fraction of this load that is eventually excreted into urine. During exogenous citrulline supplementation, 0.2 ± 0.1% of the citrulline load filtered by glomerulus, and 0.4 ± 0.2% of the arginine filtered, were excreted in urine. The ratio of urinary urea nitrogen to total urinary nitrogen was 77 ± 11% on the control day, and 85 ± 8% on the citrulline supplementation day (P = 0.15; not significant). Oral citrulline supplementation was associated with an appreciable rise in nitrogen balance (+57%, from 683 ± 246 to 970 ± 187 mmol nitrogen/12 h; P = 0.0053) (Fig. 2).


Figure 2
View larger version (13K):
[in this window]
[in a new window]

  		Fig. 2. Nitrogen balance under baseline conditions and during oral citrulline supplementation in 8 healthy volunteers.

 
RBC vs. Plasma Citrulline Concentrations

On each of the 3 study days, RBC citrulline was significantly lower than plasma citrulline (Table 1). Even though the average ratio of RBC-to-plasma citrulline concentration (RBC/P) was 0.60 ± 0.04, this ratio varied widely between subjects, ranging between 0.26 and 1.09. Five subjects indeed had RBC/P ratios between 0.74 and 1.09 (not different from 1.0), i.e., they had no significant citrulline concentration gradient across red cell membrane, whereas five other subjects had RBC/P ratios that were below 1.0 (0.26–0.40). The RBC/P ratio was unaltered (0.71 ± 0.07; P = 0.126) after phenylbutyrate treatment but declined to 0.23 ± 0.03 (P = 0.001) during oral citrulline supplementation.

Relationship Between Changes in Red Cell and Plasma Citrulline

There was a significant linear relationship between RBC citrulline and plasma citrulline levels under baseline conditions (P = 0.024), and during oral citrulline supplementation (P < 0.0001) (Fig. 3). The slope of the regression line describing the correlation was, however, weak, and no significant linear relation was observed between plasma and RBC citrulline during phenylbutyrate treatment (P = 0.49).


Figure 3
View larger version (9K):
[in this window]
[in a new window]

  		Fig. 3. Relationship between citrulline concentrations in plasma (P) and RBCs under baseline conditions (left), after phenylbutyrate treatment (middle), and during oral citrulline supplementation (right) in 10 healthy adult subjects. bullet, Subjects in whom baseline RBC/P citrulline concentration ratio did not significantly differ from 1 (0.74–1.09); {circ}, subjects in whom baseline RBC/P ratio was below 1 (0.26–0.40).

 
Relationship Between Changes in Urine and Plasma Citrulline

Even though urinary citrulline excretion and plasma citrulline concentration changed in the same direction, and even though the rise with oral citrulline supplementation and the decline during phenylbutyrate treatment were of the same magnitude in urine and plasma, no significant linear relationship was observed between urine citrulline and plasma citrulline levels under any of the regimens.


   DISCUSSION
TOP
 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
The findings of the present study suggest plasma citrulline levels can be lowered by glutamine depletion and raised by oral citrulline administration. The data further suggest incomplete equilibration of citrulline between plasma and RBCs and extensive reabsorption of citrulline by renal tubule, because <1% of an oral dose of citrulline is excreted in urine. Finally, to the best of our knowledge, the present study is first to demonstrate that oral citrulline can be used to enhance the availability of citrulline and arginine in systemic bloodstream, whereas it does not enhance urea excretion and may thus improve nitrogen balance in vivo in humans.

Even though the plasma and urine citrulline levels found in our subjects are consistent with earlier reports (7, 15, 17, 21, 24, 27), we observed fluctuations in plasma citrulline through the day under baseline conditions. The exact mechanism for the fluctuations in plasma citrulline on the control day is unclear: although protein does not contain any citrulline, meal protein supplies amino acids that are potential precursors for citrulline synthesis, such as glutamate and glutamine, which could potentially increase plasma citrulline. However, we observed a decline in plasma citrulline: this suggests that either the amino acids derived from meal protein are predominantly used for purposes other than citrulline synthesis (e.g., protein synthesis) or citrulline utilization is increased after meals. Accordingly, Rabier and Kamoun (32) showed that plasma citrulline declined after a high-protein meal and returned to normal value 4 h later. This finding implies that if plasma citrulline is used as a noninvasive marker to monitor intestinal function attention should be paid to the time of sampling.

The present data suggest plasma citrulline can be depleted by treatment with phenylbutyrate, a glutamine-trapping agent. The ~18% decline in plasma citrulline observed in the present study during phenylbutyrate treatment is likely secondary to the 7% drop in plasma glutamine, the main precursor of citrulline. The lesser degree of glutamine depletion observed in this study, compared with our previous study (~7% vs. ~25%; P = 0.01) despite the same dose of phenylbutyrate (0.36 g·kg–1·day–1) (10), likely reflects differences in study design, since subjects were fed in the present study, rather than fasted as in earlier reports (10). The ~17% decline in plasma and urinary urea during treatment with phenylbutyrate is consistent with the fact that glutamine is the main precursor of urea. The fact that plasma citrulline depends on prevailing glutamine levels suggests that caution should be exercised when using plasma citrulline as an index of intestinal function in clinical situations known to produce glutamine depletion, such as acute illness and stress (40).

We are not aware of earlier studies on RBC citrulline in humans. The concomitant assessment of RBC and plasma citrulline concentration revealed a wide range of variation in the RBC/P citrulline concentration ratio. Under baseline conditions, citrulline concentration was consistently lower in RBC than plasma; yet, whereas RBC/P ratio was close to 1.0 in five subjects, it was below 1.0 (0.26–0.40) in the remaining five. When the entire data set is considered as a whole, the correlation between plasma and RBC citrulline levels is weak (Fig. 3). This may be due to the heterogeneity of the population studied. When the population was divided in two subgroups, based on the RBC/P citrulline concentrations under baseline conditions, a different picture emerged. In subjects who had a low baseline RBC/P ratio (<1.0), changes in RBC citrulline did not correlate with changes in plasma citrulline. In contrast, in the five other subjects in whom baseline RBC/P ratio was close to 1.0, the changes in RBC citrulline induced by phenylbutyrate and oral citrulline tended to correlate with changes in plasma.

The observation that two groups were identified with regard to plasma-to-RBC citrulline ratio is intriguing. This heterogeneity may reflect differences either in citrulline metabolism or in citrulline transport across the membrane of RBC. Citrulline uptake involves an active, Na+-dependent transport in rat small intestine (37) and in Caco-2 cells, a model of human enterocytes (3). Although citrulline transport has been characterized in arterial smooth muscle (42) and neurons as well (36), we are not aware of studies of citrulline transport across the RBC membrane. Studies using incubation of fresh RBCs in the presence of labeled citrulline would be required to address this issue. On the other hand, differences in the expression of enzymes involved in RBC citrulline metabolism among subjects could explain the results. In a population of healthy volunteers, Apostol and Tayek (2) observed two different patterns in the response of plasma citrulline to arginine supplementation: some increased plasma citrulline, whereas others did not. They speculate that NO synthase activity differed widely between these groups (2). The differences we observed in RBC citrulline could result from differences in NOS activities; Kang et al. (19), however, reported that NOS is not active in RBCs, so this mechanism cannot account for our findings. In contrast, arginase is abundant in RBC. Differences in arginase activities in RBC have been reported in humans, since patients with sickle cell disease had a 60% elevation in RBC arginase activity, compared with controls (26). As arginine is both a product and a precursor of citrulline via ornithine, differences in arginase activity could play a role as well. This is, however, unlikely, because differences in arginase activity were reported in African Americans who suffer from sickle cell disease, and our subjects were Caucasian and had normal blood counts. Further studies would be warranted to determine the precise mechanisms responsible for the apparent compartmentation of citrulline between plasma and RBCs observed in a substantial fraction of the healthy volunteers.

Little is known about the handling and potential effects of an exogenous load of citrulline in humans. In the present study, plasma citrulline rose nearly fivefold (~490%) in volunteers supplemented with oral citrulline, implying that exogenous citrulline is efficiently absorbed in the gut and may escape uptake in splanchnic territory without any significant conversion to urea, as plasma urea remained unaltered (Table 1). Even though low levels of citrulline have been reported in urine from subjects of various age groups, the fraction of citrulline reabsorbed by kidney is not known. Because urine was collected throughout the study, and because creatinine clearance reflects glomerular filtration rate in healthy subjects, we were able to calculate that <1% of the amount of citrulline filtered by glomerulus is excreted as such; the rest, >99%, is therefore either reabsorbed or converted to arginine. Accordingly, the fivefold rise in plasma citrulline resulted in a near doubling in plasma arginine. Regardless of the specific mechanisms of citrulline reabsorption and/or metabolic conversion in kidney, the present data therefore suggest that orally administered citrulline is highly bioavailable, since systemic citrulline rose dramatically, whereas urinary citrulline loss was minimal. This finding implies that the oral route could be used effectively to increase citrulline and arginine availability in specific clinical situations.

During exogenous citrulline supplementation, the bulk of the citrulline administered was thus presumably converted to arginine. Arginine can be utilized by three main pathways: urea synthesis, NO production, and incorporation into protein synthesis (11, 14). In the present study, neither plasma urea nor urea excretion rose during oral citrulline administration, suggesting that little arginine underwent conversion to urea. Similarly, the excretion of nitrate and nitrite remained unaltered. We therefore speculate that the incorporation of arginine into protein synthesis may have risen. Even though rates of protein synthesis were not assessed in the present study, calculated nitrogen balance rose appreciably (+57%, from 683 to 970 mmol of nitrogen/12 h; P = 0.0053) during citrulline administration. This improved nitrogen balance is consistent with a putative rise in the rate of whole body protein synthesis and agrees with the enhanced rate of nitrogen accretion observed upon citrulline supplementation in animals by Osowska et al. (28, 29).

Taken together, the findings of the present study lead us to speculate oral citrulline could be used, either as a means to deliver arginine to the systemic circulation or as a putative protein anabolic agent, in specific clinical situations. Arginine indeed may become a conditionally essential amino acid in conditions associated with intestinal damage, such as radiation-induced enteritis (23), celiac disease (8), short bowel syndrome (7, 33), or premature birth (4, 44). For instance, after intestinal resection, the main site of citrulline production is essentially removed, and consequently plasma arginine level is reduced. Accordingly, citrulline supplementation increased arginine pools and restored nitrogen balance in rodents who had undergone extensive intestinal resection (28). The authors argue that citrulline may be a better candidate than arginine for supplementation, because extensive uptake and metabolism of arginine by the liver may cause excessive ureagenesis (9). In preterm infants, the limited expression of pyrroline-5-carboxylase, ASS, and ASL results in low rates of citrulline and arginine synthesis (4, 44). Because arginine is a precursor of NO, arginine deficiency results in decreased NO production, which could lead to intestinal dysfunction or necrotizing enterocolitis. Accordingly, in a clinical trial, arginine supplementation prevented necrotizing enterocolitis in a group of very-low-birth-weight infants (1). ASS and ASL activities can be enhanced by corticosteroid treatment (44). To our knowledge, the effects of combined corticosteroids and citrulline to raise arginine availability and potentially prevent necrotizing enterocolitis have yet to be examined in the latter population of patients.

In summary, the present study demonstrates that plasma citrulline can be depleted by depletion of glutamine and raised by oral citrulline supplementation, implying that citrulline is efficiently absorbed in the gut and reabsorbed by the kidney. In addition, to the best of our knowledge, this study is the first to suggest that oral citrulline can be used to raise arginine availability without affecting urea excretion and may enhance nitrogen balance in healthy humans. Further studies are warranted to determine whether citrulline has a true protein anabolic effect in vivo in humans and, if so, determine what mechanism(s) could mediate this putative effect.


   GRANTS
TOP
 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
C. Rougé was the recipient of a doctoral research fellowship from Nestlé (Vers-chez-les-Blanc, Switzerland). O. Le Bacquer was supported, in part, by a Nutricia postdoctoral fellowship. Part of this work was supported by grants from the Programme Hospitalier de Recherche Clinique (PHRC 2003) from the French Ministry of Health; the Délégation à la Recherche Clinique (DRC 2003), CHU de Nantes; and the Agence Nationale de la Recherche (ANR PRNA-2005).


   ACKNOWLEDGMENTS
 
We thank Y. Colleaux (Institut National de Recherche Agronomique, Systèmes d'Elevage et Nutrition Animale et Humaine, St Gilles, France) and J. Le Garzic (Laboratoire de Biologie médicale, Centre Régional de Gériatrie, Chantepie, France) for technical assistance and gratefully acknowledge the volunteers who took part in the study.


   FOOTNOTES
 

Address for reprint requests and other correspondence: D. Darmaun, UMR 1280, Physiologie des Adaptations Nutritionnelles, Centre de Recherche en Nutrition Humaine, Hotel-Dieu Hospital, 2èmeétage aile nord, 44093 Nantes cedex 1, France (e-mail: ddarmaun{at}chu-nantes.fr)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIAL AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Amin HJ, Zamora SA, McMillan DD, Fick GH, Butzner JD, Parsons HG, Scott RB. Arginine supplementation prevents necrotizing enterocolitis in the premature infant. J Pediatr 140: 389–391, 2002.[CrossRef][Web of Science][Medline]
  2. Apostol AT, Tayek JA. A decrease in glucose production is associated with an increase in plasma citrulline response to oral arginine in normal volunteers. Metabolism 52: 1512–1516, 2003.[CrossRef][Web of Science][Medline]
  3. Bahri S, Curis E, Aussel C, Chaumeil J, Cynober L, Zerrouk N. Characterization of L-citrulline transport in human intestinal Caco-2 cells. Nutr Clin Métab 20, Suppl 2: S111–S112, 2006.
  4. Becker RM, Wu G, Galanko JA, Chen W, Maynor A, Bose CL, Rhoads JM. Reduced serum amino acid concentrations in infants with necrotizing enterocolitis. J Pediatr 137: 785–793, 2000.[CrossRef][Web of Science][Medline]
  5. Bremer HJ, Duran M, Kamerling JP, Przyrembel H, Wadman SK. Disturbances of Amino Acid Metabolism: Clinical Chemistry and Diagnosis. Baltimore, MD: Urban and Schwarzenberg, p. 203–227, 1981.
  6. Castillo L, Beaumier L, Ajami AM, Young VR. Whole body nitric oxide synthesis in healthy men determined from [15N]arginine-to-[15N]citrulline labeling. Proc Natl Acad Sci USA 93: 11460–11465, 1996.[Abstract/Free Full Text]
  7. Crenn P, Coudray-Lucas C, Thuillier F, Cynober L, Messing M. Postabsorptive plasma citrulline concentration is a marker of absorptive enterocyte mass and intestinal failure in humans. Gastroenterology 119: 1496–1505, 2000.[CrossRef][Web of Science][Medline]
  8. Crenn P, Vahedi K, Lavergne-Slove A, Cynober L, Matuchansky C, Messing M. Plasma citrulline: a marker of enterocyte mass in villous atrophy-associated small bowel disease. Gastroenterology 124: 1210–1219, 2003.[CrossRef][Web of Science][Medline]
  9. Curis E, Nicolis I, Moinard C, Osowska S, Zerrouk N, Benazeth S, Cynober L. Almost all about citrulline in mammals. Amino Acids 29: 177–205, 2005.[CrossRef][Web of Science][Medline]
  10. Dhanakoti SN, Brosnan JT, Herzberg GR, Brosnan ME. Renal arginine synthesis: studies in vitro and in vivo. Am J Physiol Endocrinol Metab 259: E437–E442, 1990.[Abstract/Free Full Text]
  11. Darmaun D, Welch S, Rini A, Sager BK, Altomare A, Haymond MW. Phenylbutyrate-induced glutamine depletion in humans: effect on leucine metabolism. Am J Physiol Endocrinol Metab 274: E801–E807, 1998.[Abstract/Free Full Text]
  12. Déchelotte P, Darmaun D, Rongier M, Hecketsweiler B, Rigal O, Desjeux JF. Absorption and metabolic effects of enterally administered glutamine in humans. Am J Physiol Gastrointest Liver Physiol 260: G677–G682, 1991.[Abstract/Free Full Text]
  13. Diggle PJ, Heagerty P, Liang KY, Zeger SL. Analysis of Longitudinal Data. Oxford, UK: Oxford University Press, 1994.
  14. Featherston WR, Rogers QR, Freedland RA. Relative importance of kidney and liver in synthesis of arginine by the rat. Am J Physiol 224: 127–129, 1973.[Free Full Text]
  15. Fekkes D, van Dalen A, Edelman M, Voskuilen A. Validation of the determination of amino acids in plasma by high-performance liquid chromatography using automated pre-column derivatization with o-phthaldialdehyde. J Chromatogr B 669: 177–186, 1995.[CrossRef][Web of Science][Medline]
  16. Gondolesi G, Fishbein T, Chehade M, Tschernia A, Magid M, Kaufman S, Raymond K, Sansaricq C, LeLeiko N. Serum citrulline is a potential marker for rejection of intestinal allografts. Transplant Proc 34: 918–920, 2002.[CrossRef][Web of Science][Medline]
  17. Kamoun P, Parvy P, Cathelineau L. A rapid and simple method for citrulline determination in plasma. Clin Chem 29: 398–400, 1983.[Free Full Text]
  18. Kamoun P, Parvy PH, Rabier D. Indications et interpretation de la chromatographie des acides amines pour le diagnostic des maladies métaboliques. In: Maladies métaboliques, edited by Saudubray JM. Paris, France: Doin, p. 1–13, 1991.
  19. Kang ES, Ford K, Grokulsky G, Wang YB, Chiang TM, Acchiardo SR. Normal circulating adult human red blood cells contain inactive NOS proteins. J Lab Clin Med 135: 444–451, 2000.[CrossRef][Web of Science][Medline]
  20. Lau T, Owen W, Yu YM, Noviski N, Lyons J, Zurakowski D, Tsay R, Ajami A, Young VR, Castillo L. Arginine, citrulline, and nitric oxide metabolism in end-stage renal disease patients. J Clin Invest 105: 1217–1225, 2000.[Web of Science][Medline]
  21. Le Boucher J, Charret C, Coudray-Lucas C, Giboudeau J, Cynober L. Amino acid determination in biological fluids by automated ion exchange chromatography: performance of Hitachi L-8500A. Clin Chem 43: 1421–1428, 1997.[Abstract/Free Full Text]
  22. Levillain O, Parvy P, Hassler C. Amino acid handling in uremic rats: citrulline, a reliable marker of renal insufficiency and proximal tubular dysfunction. Metabolism 46: 611–618, 1997.[CrossRef][Web of Science][Medline]
  23. Lutgens LC, Blijlevens NM, Deutz NE, Donnellu JP, Lambin P, de Pauw BE. Monitoring myeloalative therapy-induced small bowel toxicity by serum citrulline concentration: a comparison with sugar permeability test. Cancer 103: 191–199, 2005.[CrossRef][Web of Science][Medline]
  24. Martens-Lobenhoffer J, Bode-Böger SM. Simultaneous detection of arginine, asymmetric dimethylarginine, symmetric dimethylarginine and citrulline in human plasma and urine applying liquid chromatography-mass spectrometry with very straightforward sample preparation. J Chromatogr B 798: 231–239, 2003.[CrossRef]
  25. Melis GC, Boelens PG, van der Sijp JR, Popovici T, De Bandt JP, Cynober L, van Leeuwen PA. The feeding route (enteral or parenteral) affects the plasma response of the dipeptide Ala-Gln and the amino acids glutamine, citrulline and arginine, with the administration of Ala-Gln in preoperative patients. Br J Nutr 94: 19–26, 2005.[CrossRef][Web of Science][Medline]
  26. Morris SM Jr. Regulation of arginine availability and its impact on NO synthesis. In: Nitric Oxide: Biology and Pathobiology, edited by Ignarro LG. San Diego, CA: Academic, p. 187–197, 2000.
  27. Neveux N, David P, Cynober L. Measurement of amino acids concentrations in biological fluids and tissues using ion exchange chromatography. In: Metabolic and Therapeutic Aspects of Amino Acids in Clinical Nutrition (2nd ed.), edited by Cynober L. Boca Raton, FL: CRC, p. 17–28, 2004.
  28. Osowska S, Moinard C, Neveux N, Loi C, Cynober L. Citrulline increases arginine pools and restores nitrogen balance after massive intestinal resection. Gut 53: 1781–1786, 2004.[Abstract/Free Full Text]
  29. Osowska S, Duchemann T, Walrand S, Paillard A, Boirie Y, Cynober L, Moinard C. Citrulline modulates muscle protein metabolism in old malnourished rats. Am J Physiol Endocrinol Metab 291: E582–E586, 2006.[Abstract/Free Full Text]
  30. Pappas PA, Saudubray JM, Tzakis AG, Rabier D, Carreno MR, Gomez-Marin O, Huijing F, Gelman B, Levi DM, Nery JR, Kato T, Mittal N, Nishida S, Thompson JF, Ruiz P. Serum citrulline as a marker of acute cellular rejection for intestinal transplantation. Transplant Proc 34: 915–917, 2002.[CrossRef][Web of Science][Medline]
  31. Perry TL. Chemical and cellular architecture. In: Handbook of Neurochemistry, edited by Lajtha A. New York: Plenum, 1982, p. 151–180.
  32. Rabier D, Kamoun P. Metabolism of citrulline in man. Amino Acids 9: 299–316, 1995.[CrossRef][Web of Science]
  33. Rhoads JM, Plunkett E, Galanko J, Lichtman S, Taylor L, Maynor A, Weiner T, Freeman K, Guarisco JL, Wu GY. Serum citrulline levels correlate with enteral tolerance and bowel length in infants with short bowel syndrome. J Pediatr 146: 542–547, 2005.[CrossRef][Web of Science][Medline]
  34. Rutten EP, Engelen MP, Wouters EF, Schols AM, Deutz NE. Metabolic effects of glutamine and glutamate ingestion in healthy subjects and in persons with chronic obstructive pulmonary disease. Am J Clin Nutr 83: 115–123, 2006.[Abstract/Free Full Text]
  35. Schmidlin A, Fischer S, Wiesinger H. Transport of L-citrulline in neural cell cultures. Dev Neurosci 22: 393–398, 2000.[CrossRef][Web of Science][Medline]
  36. Vadgama JV, Evered DF. Characteristics of L-citrulline transport across rat small intestine in vitro. Pediatr Res 32: 472–478, 1992.[Web of Science][Medline]
  37. Van de Poll MC, Siroen MP, Van Leeuwen PA, Soeters PB, Melis GC, Boelens PG, Deutz NE, Dejong CH. Interorgan amino acid exchange in humans: consequences for arginine and citrulline metabolism. Am J Clin Nutr 85: 167–172, 2007.[Abstract/Free Full Text]
  38. Van de Poll MC, Ligthart-Melis GC, Boelens PG, Deutz NE, Van Leeuwenen PA, Dejong CH. Intestinal and hepatic metabolism of glutamine and citrulline in humans. J Physiol 581: 819–827, 2007.[Abstract/Free Full Text]
  39. Vinnars E, Bergström J, Fürst P. Influence of the postoperative state on the intracellular free amino acids in human muscle tissue. Ann Surg 182: 665–671, 1975.[Web of Science][Medline]
  40. Wiesinger H. Arginine metabolism and the synthesis of nitric oxide in the nervous system. Prog Neurobiol 64: 365–391, 2001.[CrossRef][Web of Science][Medline]
  41. Wileman SM, Mann GE, Pearson JD, Baydoun AR. Role of L-citrulline transport in nitric oxide synthesis in rat aortic smooth muscle cells activated with LPS and interferon-gamma. Br J Pharmacol 140: 179–185, 2003.[CrossRef][Web of Science][Medline]
  42. Windmueller HG, Spaeth AE. Source and fate of circulating citrulline. Am J Physiol Endocrinol Metab 241: E473–E480, 1981.[Abstract/Free Full Text]
  43. Wu G, Jaeger LA, Bazer FW, Rhoads JM. Arginine deficiency in preterm infants: biochemical mechanisms and nutritional implications. J Nutr Biochem 15: 447–451, 2004.



This article has been cited by other articles:


Home page
 Am. J. Clin. Nutr.Home page
Y. C Luiking, M. Poeze, G. Ramsay, and N. E. Deutz
Reduced citrulline production in sepsis is related to diminished de novo arginine and nitric oxide production
Am. J. Clinical Nutrition, January 1, 2009; 89(1): 142 - 152.
[Abstract] [Full Text] [PDF]

This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
293/5/G1061    most recent
00289.2007v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in Web of Science
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Web of Science (4)
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Rougé, C.
Right arrow Articles by Darmaun, D.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Rougé, C.
Right arrow Articles by Darmaun, D.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2007 by the American Physiological Society.