M. C. G. van de Poll and G. C. Ligthart-Melis contributed equally to this work. This paper has online supplemental material.
Corresponding author P. A. M. van Leeuwen: Department of surgery, VU University Medical Center, PO box 7057 1007 MB Amsterdam, the Netherlands. Email email@example.com
Glutamine plays an important role in nitrogen homeostasis and intestinal substrate supply. It has been suggested that glutamine is a precursor for arginine through an intestinal–renal pathway involving inter-organ transport of citrulline. The importance of intestinal glutamine metabolism for endogenous arginine synthesis in humans, however, has remained unaddressed. The aim of this study was to investigate the intestinal conversion of glutamine to citrulline and the effect of the liver on splanchnic citrulline metabolism in humans. Eight patients undergoing upper gastrointestinal surgery received a primed continuous intravenous infusion of [2-15N]glutamine and [ureido-13C–2H2]citrulline. Arterial, portal venous and hepatic venous blood were sampled and portal and hepatic blood flows were measured. Organ specific amino acid uptake (disposal), production and net balance, as well as whole body rates of plasma appearance were calculated according to established methods. The intestines consumed glutamine at a rate that was dependent on glutamine supply. Approximately 13% of glutamine taken up by the intestines was converted to citrulline. Quantitatively glutamine was the only important precursor for intestinal citrulline release. Both glutamine and citrulline were consumed and produced by the liver, but net hepatic flux of both amino acids was not significantly different from zero. Plasma glutamine was the precursor of 80% of plasma citrulline and plasma citrulline in turn was the precursor of 10% of plasma arginine. In conclusion, glutamine is an important precursor for the synthesis of arginine after intestinal conversion to citrulline in humans.
The amino acid glutamine is well known for its pivotal role in nitrogen homeostasis and intestinal energy supply. From animal experiments (Windmueller & Spaeth, 1974; Boelens et al. 2005) and some human studies (van der Hulst et al. 1997; Olde Damink et al. 2002) it has become clear that the gut is an important player in whole body glutamine metabolism. Glutamine is degraded by the intestines by means of the enzyme glutaminase, yielding glutamate and ammonia. Subsequently ammonia is released in the portal vein (Olde Damink et al. 2002), and the glutamate is subsequently metabolized by the intestine, mostly to α-ketoglutarate or alanine (Windmueller & Spaeth, 1974). In addition glutamate can be converted to ornithine and subsequently to citrulline. By the conversion of glutamine to citrulline, glutamine metabolized by the intestines can become a precursor for the synthesis of arginine elsewhere in the body (Castillo et al. 1996). Arginine is the precursor for the pluripotent signalling molecule nitric oxide and as such is important in the regulation of amongst others organ perfusion, immune function and wound healing.
Although it is generally conceived that the gut is the principal organ for citrulline synthesis, the contribution of the gut to whole body glutamine turnover and whole body citrulline synthesis has not yet been quantified in man. Moreover the potential role of the liver in modulating the release of intestinally derived citrulline to the systemic circulation is generally neglected, since early in vitro studies suggested that the liver is inert towards citrulline passing through its vascular bed (Windmueller & Spaeth, 1981). We showed recently, however, that the human liver is capable of taking up a substantial amount of intestinally derived citrulline (van de Poll et al. 2007).
The aim of the present study was to quantify the intestinal conversion of glutamine to citrulline and to study hepatic metabolism of citrulline in man. Studies were conducted in patients undergoing upper gastrointestinal surgery, which enabled access to intra-abdominal vessels. This allowed us to sample blood from the portal and hepatic veins and to measure intestinal and hepatic blood flows to quantify net amino acid flux. By simultaneous stable isotope infusion we were also able to study the absolute uptake (disposal) and absolute release (production) of glutamine and citrulline by the intestines and the liver as well as the whole body plasma rates of appearance of these amino acids.
Eight patients undergoing gastrointestinal surgery (6 liver resections, 1 pancreaticoduodenectomy, 1 duodenectomy) at the University Hospital Maastricht were studied. All but one of the patients were operated on for malignant disease. On the day of admission routine blood tests were performed, a dietary questionnaire was obtained by a dietician and body composition was measured. Patients with known parenchymal liver disease, inborn errors of metabolism, diabetes mellitus type I or cancer cachexia were excluded from the study. Patient characteristics are presented in Table 1. Oral intake except for water was ceased at 20.00 h on the day of admission and all patients were transported to the operation theatre at approximately 07.30 h the next day. The study was approved by the Medical Ethical committee of the University Hospital Maastricht, all patients gave written informed consent, and the study conformed to the Declaration of Helsinki.
Table 1. Patient characteristics
Surgical procedures used were 6 × hepatectomy, 1 × pancreaticoduodenectomy and 1 × duodenectomy. AST, aspartate amino transferase; APPT, activated partial thromboplastin time; INR, international normalized ratio.
Body mass index (BMI)
Fat free mass (kg)
AST (IU l−1)
Alkaline phosphatase (IU l−1)
Bilirubine (μmol l−1)
Urea (mmol l−1)
Creatinine (μmol l−1)
Albumin (g l−1)
C-reactive protein (mg l−1)
Glucose (mmol l−1)
Stable isotope study
Isotopes [2-15N]Glutamine was obtained from Cambridge Isotope Laboratories (Woburn, MA, USA) and [ureido-13C–2H2]citrulline from ARC laboratories (Apeldoorn, the Netherlands). Both tracers were dissolved in sterile water, and thereafter sodium chloride was added to create an isotonic solution. Sterility and non-pyrogenity were tested and confirmed by the hospital pharmacy. Before the experiment aliquots of the stock solution were diluted in normal saline to obtain the final infusion solution.
Study protocol Anaesthetic management was in accordance with institutional routines and included placement of two peripheral venous catheters, an epidural catheter for pre- and postoperative analgesia, an arterial line, and a central venous line. Anaesthesia was maintained using isoflurane and propofol. After induction of anaesthesia, an additional peripheral venous catheter was placed in an antecubital vein for tracer infusion. This catheter was kept patent with normal saline until the start of the tracer infusion. After laparotomy a baseline blood sample was drawn from the arterial line, followed by the start of a primed continuous intravenous tracer infusion (dosages in Table 2). Blood samples were drawn from the arterial line every 30 min for the following 2 h. After 1 h, when based upon prior experience an isotopic steady state was known to be present, blood was drawn from the portal vein and the middle hepatic vein by direct puncture, simultaneously with arterial blood sampling. Blood sampling occurred before organ transection to resemble the physiological situation as closely as possible. Blood was collected in prechilled heparinized vacuum tubes (BD Vacutainer, Franklin Lakes, NJ, USA) and placed on ice. Within 1 h, blood was centrifuged (10 min, 4000 g, 4°C) and 500 μl of plasma were added to 80 mg dry sulphosalicylic 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 analysis. Before centrifugation, haematocrit of each blood sample was determined using a micro capillary centrifuge.
Table 2. Tracer dosages (μmol kg−1)
Duplex flow measurement
Hepatic and intestinal blood flow were measured by means of colour Doppler ultrasound (Aloka Prosound SSD 5000, Aloka Co., Ltd, Tokyo, Japan) as described before (Siroen et al. 2005). Briefly, time-averaged mean velocities of the blood stream and cross-sectional area of the portal vein and hepatic artery were measured before their hilar bifurcations. Blood flow was calculated by multiplying the cross-sectional area of the vessel by the velocity of the blood stream. Plasma flow (PF) was calculated by correcting blood flow (BF) for haematocrit (Hct): PF = BF × (1 − Hct). Hepatic (and splanchnic) plasma flow was calculated by adding up plasma flows in the portal vein and hepatic artery. Mean blood flows were used to calculate organ fluxes.
Amino acid concentrations in deproteinized samples and infusates were measured using high performance liquid chromatography as described elsewhere (van Eijk et al. 1993). Glutamine, citrulline and arginine enrichments were measured by liquid chromatography–mass spectrometry (van Eijk et al. 1999). Coefficients of variation were 2.7% for [15N]glutamine enrichments, 5.9% for [15N]citrulline enrichments, 25% for [13C–2H2]citrulline enrichments and < 2% for amino acid concentrations. Arterial pH and HCO3− were measured by an automated analyser (GEM Premier 3000, Instrumentation Laboratory, Breda, the Netherlands).
Isotopic enrichment was expressed as tracer-to-tracee ratio (TTR,%), 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. (1993). Metabolic fluxes and conversions were calculated using established formulas for radioactive and stable isotopic tracers as described in detail by Bruins et al. (2002). In summary, whole body plasma rates of appearance of the tracee (Q) were calculated from the infusion rate of the tracer (IR) and the corresponding arterial enrichment (TTRA):
The whole body conversion of plasma glutamine (gln) to plasma citrulline (cit) (Qgln-cit) was calculated from arterial [15N]glutamine enrichment (), arterial [15N]citrulline enrichment () and citrulline plasma rate of appearance (Qcit) calculated from [13C–2H2]citrulline infusion rate and TTRA using eqn (1):
Net amino acid fluxes across the portal drained viscera (PDV) and the splanchnic area were calculated from arterial [A] and venous [V] concentrations (PDV: portal vein, splanchnic area: hepatic vein) and from portal and splanchnic plasma flow (pf), respectively.
Accordingly, a negative flux indicates organ specific net uptake whereas a positive flux indicates net release. Unidirectional absolute organ specific amino acid uptake (disposal) by the PDV and the splanchnic area was calculated from arteriovenous (A-V) balances of the infused tracers (tracer net balance, tNB) and from venous enrichments (TTRV) of the infused tracers, which most accurately represents intracellular enrichment (Biolo et al. 1995), needed to convert tracer uptake (tNB) to unidirectional tracee uptake (disposal):
Since organ specific net balance is the resultant of disposal and organ specific production, organ specific production can be calculated from net flux (eqn (3)) and disposal (eqn (5)):
The intestinal conversion of glutamine to citrulline (Qgln–cit,PDV) was calculated from the PDV tracer net balance of [15N]citrulline () (eqn (4)) and the arterial enrichment of [15N]glutamine (), corrected for the extraction of 15N citrulline reaching the PDV through the circulation:
Hepatic amino acid metabolism was quantified by subtracting PDV net flux, disposal and production from their corresponding splanchnic values.
In depth analysis of raw data revealed perceptibly too low arterial enrichments of [13C–2H2]citrulline in two cases (arterial enrichment below venous enrichments and below adjacent arterial enrichments) and too low portal [13C–2H2]citrulline enrichments in two other cases (portal enrichment below hepatic venous enrichment). The perceptibly erroneous arterial values were replaced by the average enrichment of two adjacent arterial samples. For ethical reasons the portal vein was sampled only once and no substitute values could be deduced from repeated measures in the two latter cases. Substitute values for portal [13C–2H2]citrulline enrichment in these two cases were chosen so that [13C–2H2]citrulline balance across the PDV (eqn (4)) was zero, which equalled the average value in the other subjects. No obvious erroneous values were found in other samples and for other isotopic enrichments.
Arterial enrichment curves were fitted to calculate a mean steady state value per individual. Arteriovenous gradients were tested versus a theoretical mean of zero using a one sample t test. Correlations were tested using Pearson's test. Results are expressed as means ±s.e.m. All statistical calculations were performed using Prism 4.0 for Windows (GraphPad Software Inc., San Diego, CA, USA). A P value < 0.05 was considered to indicate statistical significance.
Isotopic enrichments of glutamine and citrulline
Within 30 min isotopic steady state was achieved for both administered tracers (Figs 1A and B), which is in keeping with previous data from our group. Based upon arterial enrichments at steady state, it was calculated that whole body glutamine plasma rate of appearance was 240 ± 15 μmol kg−1 h−1 and whole body citrulline plasma rate of appearance was 6.2 ± 0.7 μmol kg−1 h−1. During the experiment citrulline enrichment could be detected at mass + 1 (m + 1) (Fig. 1A), reflecting the conversion of [15N]glutamine to [15N]citrulline. Average citrulline enrichment enrichment at m + 1 was 5.7 ± 0.4%. The ratio between arterial [15N]citrulline and [15N]glutamine enrichments indicates that approximately 80% of plasma citrulline is derived from plasma glutamine. According to eqn (2) this comes down to a conversion rate of 5.1 ± 0.7 μmol kg−1 h−1.
Qualitative relationships between glutamine, citrulline and arginine
During the study significant enrichments of arginine were found in arterial plasma at m + 1 (Fig. 1A) and m + 3 (Fig. 1B), reflecting the conversion of [15N]glutamine to [15N]citrulline and [15N]arginine and the conversion of [13C–2H2]citrulline to [13C–2H2]arginine, respectively. The ratio between arginine m + 3 and citrulline m + 3 (0.10 ± 0.02) indicates that approximately 10% of arginine released in the circulation is newly formed from plasma citrulline.
Duplex flow measurements yielded mean portal venous and hepatic arterial blood flows of 836 ± 143 ml min−1 and 442 ± 85 ml min−1, respectively. Mean blood flows were in agreement with data in the literature (Siroen et al. 2005; Schindl et al. 2006).
Glutamine and citrulline metabolism across the portal drained viscera
Net amino acid flux across an organ (arteriovenous concentration gradient times plasma flow) is the resultant of absolute unidirectional uptake (disposal) and absolute unidirectional release (production). Disposal can be measured by stable isotope techniques since appropriately chosen isotopic tracers cannot be produced within the body. From the organ specific disposal calculated from tracer data and the organ specific net flux, absolute production rates can be quantified for organs that simultaneously consume and produce a single amino acid. As expected, net glutamine uptake by the PDV was observed (Fig. 2). Absolute PDV glutamine uptake (disposal) calculated from [15N]glutamine data was 45 ± 9 μmol kg−1 h−1 and could account for 19% of plasma glutamine turnover. In addition to their substantial glutamine uptake, the PDV tended to produce glutamine and release it to the portal vein (17 ± 8 μmol kg−1 h−1). This value, however, was not statistically different from zero (P= 0.07). Tracer data further revealed that glutamine disposal by the PDV was significantly correlated with glutamine supply with a fractional extraction of 0.15 ± 0.03. No such correlation was found between glutamine supply and net glutamine flux (Fig. 3). Thirteen per cent of glutamine taken up by the PDV was converted to citrulline (Fig. 2). The balance of [13C–2H2]citrulline across the PDV and hence PDV citrulline disposal (extraction) was not significantly different from zero (Fig. 4). This indicates that net PDV citrulline flux equals total citrulline production (Fig. 4). The production of citrulline derived from glutamine equalled the total production of citrulline, showing that glutamine is quantitatively the only important precursor for citrulline produced by the PDV (Fig. 4). There was an excellent agreement between total PDV citrulline production (calculated from [13C–2H2]citrulline, eqn (6)) and citrulline released from the PDV, derived from glutamine (calculated from [15N]citrulline and [15N]glutamine data, eqn (8)). No release of [13C–2H2] or [15N)arginine by the PDV was found.
Splanchnic and hepatic glutamine and citrulline metabolism
Arterial pH during sampling of hepatic venous blood was 7.4 ± 0.03. Net splanchnic glutamine flux (Fig. 5A) was similar to net PDV glutamine flux, indicating that net hepatic glutamine flux was not significantly different from zero (Fig. 5B). Tracer data, however, revealed that this zero net balance was the resultant of very active glutamine uptake and glutamine release by the liver (Fig. 5B). Hepatic glutamine release (73 ± 22 μmol kg−1 h−1) could account for about 30% of total plasma glutamine appearance. Net splanchnic citrulline flux (Fig. 6A) was dominated by intestinal citrulline production, resulting in a hepatic citrulline net flux that was not significantly different from zero. Tracer data, however, show that the liver is capable of taking up citrulline (hepatic citrulline disposal; P= 0.046 versus zero). This suggests that the net balance of citrulline across the liver, like that of glutamine, is determined by the opposing effects of active hepatic citrulline uptake and similarly active hepatic release, although hepatic citrulline release failed to reach statistical significance (P= 0.138) (Fig. 6B). No hepatic release of [15N]citrulline, [13C–2H2]arginine or [15N]arginine was found.
This study was set up to address the role of the intestines and the liver in the metabolism of glutamine and citrulline in humans. Net fluxes of glutamine and citrulline across these organs are the resultant of both uptake and production, and therefore assessment of net fluxes may not give reliable information about the absolute unidirectional uptake (disposal) or absolute unidirectional release (production). We used stable isotope techniques to unravel the contribution of both unidirectional disposal and unidirectional release to net organ flux. Whole body rate of plasma appearance of glutamine (240 μmol kg−1 h−1) was in the range typically reported in the literature (Van Acker et al. 1998; van Acker et al. 2000; Bourreille et al. 2004; Raj et al. 2005). Also in agreement with data in the literature (Windmueller & Spaeth, 1974; van der Hulst et al. 1997; Olde Damink et al. 2002; Boelens et al. 2005), we show that the PDV take up glutamine from the circulation. Interestingly, tracer data revealed that absolute unidirectional glutamine disposal by the portal drained viscera is related to glutamine supply, a phenomenon that could not be detected before by measurement of net fluxes (van der Hulst et al. 1997; Olde Damink et al. 2002). This observation is of interest because it indicates that intestinal glutamine metabolism is regulated by glutamine supply in non-depleted humans.
The liver simultaneously disposed of and released glutamine, ultimately leading to a zero net balance. This high bidirectional flux enables the liver to respond rapidly to changes in nitrogen metabolism and acid–base balance. Mechanisms of hepatic glutamine uptake and release have been disentangled in depth. Both processes occur in different parts of the lobule by zonation of glutamine metabolizing enzymes (Haussinger et al. 1985). Glutaminase is primarily located in periportal hepatocytes and serves the provision of nitrogen and carbon for ureagenesis and gluconeogenesis, respectively. Glutamine synthetase is expressed in perivenous hepatocytes and is suggested to function as an ammonia overflow trap that serves hepatic ammonia detoxification under conditions of diminished hepatic ureagenesis, such as during acidosis (van de Poll et al. 2004). Arterial pH in our patients was normal and also ureagenesis is not affected by this type of surgery (Dejong et al. 2001). This shows that also under physiological conditions the liver is a key organ in glutamine homeostasis that accounts for approximately 30% of whole body plasma glutamine release in humans. However, we cannot rule out that these numbers may be somewhat different in human subjects without malignant disease. Unfortunately this subject category is virtually inaccessible with the currently applied methods.
Glutamine is a known precursor of citrulline. We found a rate of appearance of citrulline in plasma of 6.2 μmol kg−1 h−1. That is approximately 35% lower than values reported by Castillo et al. (1995, 1996), which to our knowledge are so far the only published human data. Unpublished data from our own group have yielded whole body rates of plasma appearance of citrulline varying between 13.4 and 22.0 μmol kg−1 h−1 (Luiking & Deutz, 2003). Against this background it should be noted that splanchnic unidirectional citrulline production exceeded the calculated whole body plasma rate of appearance of citrulline. Hence the value for whole body citrulline flux obtained in the present study may be an underestimate of the true plasma rate of appearance of citrulline. Further studies are needed to elucidate this. The conversion of glutamine to citrulline has not been quantified before in vivo in man. From the ratio between [15N]glutamine and [15N]citrulline plasma enrichments, however, it can be concluded that approximately 80% of plasma citrulline is immediately derived from plasma glutamine. Although measurement of [13C–2H2]citrulline enrichment yielded slightly more variable data than measurement of [15N]glutamine and [15N]citrulline enrichment, the validity of the resulting data was confirmed by the excellent agreement between PDV citrulline production calculated from [13C–2H2]citrulline data and from [15N]citrulline data.
In the present study, no significant net flux of citrulline across the liver was observed, although there was a trend towards net hepatic uptake of citrulline. Absolute unidirectional hepatic citrulline uptake, measured by tracer data, however, was significant and coincided with a trend towards hepatic citrulline release. Studies in animals (Remesy et al. 1978) and in humans with liver cirrhosis (Olde Damink et al. 2002) have previously demonstrated the capability of the liver to take up citrulline. Recent data on humans with a normal non-cirrhotic liver are in keeping with this (van de Poll et al. 2007). Since unidirectional hepatic citrulline uptake has not been recognized, mechanisms regulating hepatic citrulline uptake and release are unknown. This observation requires further study and is subject to ongoing investigation in our group.
Since citrulline is the precursor for arginine synthesis it can be conceived that arginine is an ultimate product of glutamine metabolism. This is supported by the observation of increased arginine levels during glutamine supplementation (Houdijk et al. 1994; Melis et al. 2005). The conversion of glutamine to arginine is thought to be effected in an inter-organ pathway involving intestinal glutamine to citrulline and renal citrulline to arginine conversion (Windmueller & Spaeth, 1981; Cynober, 1994; Mistry et al. 2002; van de Poll et al. 2004), but the existence of this pathway and its potential significance has not been quantified before in vivo in humans. We now show that the human gut indeed converts glutamine to citrulline. In addition, significant enrichments of arginine at m + 1 and m + 3 were found in arterial plasma. This reflects the conversion of plasma citrulline to plasma arginine and confirms that glutamine is a precursor for arginine de novo synthesis. The ratio between citrulline m + 3 and arginine m + 3 (0.10) indicates that approximately 10% of plasma arginine is derived from citrulline via de novo synthesis (probably in the kidney) (van de Poll et al. 2007), which is in agreement with data in the literature (Castillo et al. 1996), the remainder probably being derived from protein breakdown (Luiking & Deutz, 2003). Interestingly, the ratio between arginine m + 1 and glutamine m + 1 (0.19) exceeded the ratio between citrulline m + 1 and arginine m + 1 by a factor of 2. This probably indicates that the interorgan transport of citrulline is not the only way to transfer the amino nitrogen atom of glutamine to arginine. Most likely these data are explained by a pathway involving the conversion of [15N]glutamine to [15N]glutamate and subsequent transamination of [15N]glutamate. Subsequen transamination reactions ultimately can lead to the formation of [15N]aspartate, which can become the precursor for [15N]argininosuccinate and guanido-[15N]arginine (in contrast to amino-[15N]arginine, derived from the glutamine–citrulline–arginine pathway). It has been suggested that the availability of nitrogen donors for argininosuccinate synthesis limits the endogenous synthesis rate of arginine (Dhanakoti et al. 1990).
In conclusion, this is the first human study that quantifies the role of the intestines and the liver in the conversion of glutamine to citrulline and arginine. Plasma glutamine can account for approximately 80% of plasma citrulline production. The intestines account for the major part of this conversion although splanchnic citrulline release to the systemic circulation may be limited by hepatic citrulline extraction. Plasma citrulline accounts for 10% of plasma arginine release. Intestinal glutamine metabolism is crucial for endogenous arginine synthesis in humans.
M.C.G.vdP. participated in study design, data collection, data analysis and writing of the manuscript; G.C.L.M. participated in study design, data collection, data analysis and writing of the manuscript; M.C.G.vdP. and G.C.L.M. contributed equally to this manuscript and share first authorship; P.G.B. participated in study design and critically reviewed the manuscript; N.E.P.D. participated in study design, advised on data interpretation and critically reviewed the manuscript; P.A.M.vL. participated in study design and critically reviewed the manuscript; C.H.C.D. participated in study design operated on the patients and sampled blood, advised on data interpretation and critically reviewed and revised the manuscript. None of the authors has a potential financial or other personal interest from the presented results. This work was supported by grants from the Netherlands Organization for Health Research and Development to M.C.G.vdP. (920-03-317 AGIKO), P.G.B. (920-03-185 AGIKO) and C.H.C.D. (907-00-033 Clinical Fellowship) and from Fresenius-Kabi, Bad Homburg, Germany.