Clinical Nutrition (2008) 27, 328e339

ava i lab le a t www.sc iencedi rec t .com

ht tp : / / in t l . e l sev ie rhea l th .com/ j ourna l s /c lnu

REVIEW

Citrulline as a biomarker of intestinal failuredue to enterocyte mass reduction*

Pascal Crenn a,b,c,*, Bernard Messing d,e, Luc Cynober c,f

a Departement de Medecine, Hopital Raymond Poincare, Assistance Publique-Hopitaux de Paris,Universite de Versailles Saint-Quentin-en-Yvelines, Garches, Franceb Hepato-gastro-enterologie et Oncologie digestive, Hopital Ambroise Pare, Assistance Publique-Hopitaux de Paris,Universite de Versailles Saint-Quentin-en-Yvelines, Boulogne, Francec Laboratoire de Biologie de la Nutrition, EA 2498, Universite Paris Descartes, Paris, Franced Pole des Maladies de l’Appareil Digestif, Hopital Beaujon, Assistance Publique-Hopitaux de Paris,Universite Paris 7 Denis Diderot, Clichy, Francee Service de Gastroenterologie et d’Assistance Nutritive, Assistance Publique-Hopitaux de Paris,Universite Paris 7 Denis Diderot, Clichy, Francef Service de Biochimie, Hotel Dieu, Assistance Publique-Hopitaux de Paris, Paris, France

Received 6 August 2007; accepted 22 February 2008

KEYWORDSCitrulline;Enterocyte;Short bowel syndrome;Intestinal failure;Parenteral nutrition

Abbreviations: SBS, short bowel syndramine oxydase; HIV, human immunod

* This work was partly presented du* Corresponding author. Departmenþ33 1 4710 7758.

E-mail address: pascal.crenn@rpc.

0261-5614/$ - see front matter ª 200doi:10.1016/j.clnu.2008.02.005

Summary

Background & aims: In human, citrulline (plasma concentration about 40 mmol/L) is an aminoacid involved in intermediary metabolism and that is not incorporated in proteins. Circulatingcitrulline is mainly produced by enterocytes of the small bowel. For this reason plasma or se-rum citrulline concentration has been proposed as a biomarker of remnant small bowel massand function. This article reviews this concept and its metabolic basis.Methods: Conditions in which there is a significantly reduced small bowel enterocyte mass andfunction and a plasma or serum citrulline were measured in adults and children. These studiesincluded patients with a short bowel syndrome, villous atrophy states, Crohn’s disease, duringmonitoring of digestive toxicity of chemotherapy and radiotherapy or follow-up of patientsafter small bowel transplantation.Results: In all these situations, with more than 500 studied patients a decreased level ofplasma citrulline correlated with the reduced enterocyte mass independently of nutritionaland inflammatory status. A close correlation between small bowel remnant length and

ome; PN, parenteral nutrition; ASL, arginosuccinate lyase; ASS, argino succinate synthase; DAO, di-eficiency virus; HPLC, high pressure liquid chromatography.ring the 28th ESPEN Congress in Istanbul, October 2006.t of Medicine, Raymond Poincare Hospital, 104 Bd Raymond Poincare, 92380 Garches, France. Tel.:

aphp.fr (P. Crenn).

8 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.

Citrulline and enterocyte mass 329

citrullinemia was found. In addition, diagnosis of intestinal failure was assessed throughplasma citrulline levels in severe small bowel diseases in which there is a marked enterocytemass reduction.Discussion: The threshold for establishing a diagnosis of intestinal failure is lower in villousatrophy disease (10 mmol/L) than in short bowel syndrome (20 mmol/L). Compromised renalfunction is an important factor when considering plasma citrulline levels as a marker of intes-tinal failure as this potentially can increase circulating citrulline values.Conclusions: Reduced plasma citrulline levels are an innovative quantitative biomarker ofsignificantly reduced enterocyte mass and function in different disease states in humans.ª 2008 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rightsreserved.

Introduction

Intestinal architecture supports a complex absorptivefunction. This function interrelates with other complexgut functions, notably defensive (either specific by adap-tive immunity or non-specific by physical barrier and innateimmunity), endocrine and neuromotor. The intestine, andespecially the small bowel, exerts a metabolic activity forapoprotein synthesis and amino acid metabolism and, in thecolon, short chain fatty acid production and consumption.We hypothesize that the quantification of the activemetabolic mass of the intestine, predominantly the smallbowel, due to its high cellular and metabolic capacity,could correctly represent and ‘‘model’’ the overall absorp-tive capacity of the gut and intestine. Accordingly, welooked at citrulline, an amino acid not included in proteinsbut produced almost exclusively by enterocytes of thesmall bowel mucosa, as a candidate plasma/serum bio-marker for intestinal enterocyte function.1,2

Previous proposed biomarkers ofgut/intestinal functions

Over the years several biomarkers have been proposed toaccess overall intestinal gut function, including diamineoxidase (DAO), apoprotein AIV,3 beta-carotene and perme-ability tests. The most widely studied marker was DAO. Thisenzyme is involved in the catabolism of polyamines andappears to be largely produced by tissues with high cellturnover, e.g. intestinal mucosa. Since a single blood deter-mination was judged insufficiently sensitive, a dynamicstudy and sequential assay after injection of heparin wasproposed: post-heparin diamine oxidase activity.4 However,technical difficulties and disappointing specificity andsensitivity soon led to the withdrawal of this test. The per-meability test might have offered an alternative, but theprocess studied does not correspond exactly to ‘‘gut func-tion’’ and in addition it requires the use of several ratios,such as xylose/3-OMG (O-methyl-D-glucose) that areassumed to reflect absorptive epithelial capacity, or rham-nose/3-OMG to assess cellular transport.5 Other parame-ters, which remain the gold standard of absorption, suchas steatorrhea, or the hard-to-assess net digestive balancesof macro- (and micro) nutrients, are time-consuming anddifficult to use repeatedly in a clinical setting. The assayof FABP-I (fatty acid binding protein intestinal) was pro-posed as an index of epithelial integrity with a release in

the general circulation in cases of severe ischaemic lesionof the bowel.6 Fecal calpotrectin, a sensitive biomarkerof intestinal inflammation as showed in Crohn’s disease,lack of specificity and did not evaluate mucosal function.7

For all the above reasons, a simple and reliable biomarkerof gut function, analogous to the ones used for the liveror kidney, have seemed unattainable because intestinalfunction was deemed ‘‘elusive’’.8

Metabolism of citrulline

Splanchnic territory, including gut and liver, has a long-standing recognized major role in macronutrient metabo-lism and in quantitative and qualitative metabolic exchangeof amino acids. In the seventies, Felig9 established withmulticatheterization studies in healthy humans, that thesplanchnic area consumed a large amount of glutamineand alanine in the post-absorptive state. On the contrary,of all amino acids only citrulline was significantly produced.

Splanchnic metabolism

IntestineWindmueller et al.10 clearly showed in rats that only the in-testine produced circulating citrulline, with glutamine asits main precursor. This finding was further confirmed inadult omnivorous mammals such as pigs11 and humans,12,13

but not in strictly carnivorous animals like cats. In these lat-ter species a supply of exogenous amino acids, especiallyarginine, is mandatory to protect against hyperammonemiaand coma,14,15 In sheep, a strictly herbivorous animal,there is no significant conversion of glutamine into citrul-line.16 The specific tissue enzyme activities were later es-tablished (Fig. 1 and Table 1). Intestinal citrullineproduction originates mainly from the proximal small bowel(Table 1), and probably from the middle and upper parts ofintestinal villi. The main precursor is glutamine, and de-rived amino acids, either circulating or exogenous. It hasbeen estimated in a rat model that 24% of glutamine nitro-gen and 10% of glutamine carbon is converted into citrullineby the small bowel mucosa.10 This was measured inhumans, representing 13% of glutamine taken up by theintestine.13 Arginine, and possibly other amino acids suchas proline or ornithine, also contribute to the intestinal pro-duction of citrulline.17 However, the exact quantitativecontribution in vivo of all these potential precursors forcitrulline production is currently not well known,18,19 but

ENTEROCYTES

KIDNEY (PCT)

ARG ARG LIVER

ARGase Urea

GLN GLU ORN

GLNase P5CS+OAT

OTC Ureagenesis

CIT

ASS

ARG ASL

Figure 1 Interorgan metabolism of citrulline and related amino acids. ARGase: arginase; ASS: arginosuccinate synthase; GLNase:glutaminase; ASL: arginosuccinate lyase; P5CS: pyrroline carboxylate synthase; OAT : ornithine aminotransferase; PCT: proximalconvoluted tubule. In enterocytes there is an incomplete urea cycle, mitochondrial but not cytosolic.

330 P. Crenn et al.

is estimated at 80% from glutamine using a stable isotope-based study.13 Cellular and tissular intestinal models haveestablished that it is the enterocytes, the small intestinalabsorptive epithelial cells, and no other cells thatproduce citrulline,11,20 with the exception of the localnitric oxide (NO) pathway in associated immune cells.21

One of the major metabolic features of citrulline is that itis a non-protein amino acid. Hence we can consider the cir-culating citrulline as dependent only on de novo synthesis

Table 1 Enzyme activities of P5CS (pyrroline 5-carboxyl-ate synthase), OAT (ornithine aminotransferase), CPS (car-bomoyl phosphate synthase) and OTC (ornithinetranscarbamylase) in different tissues in rats (adaptedfrom Wakabayashi Y. The glutamate crossway. In: CynoberL, editor. Boca Raton: CRC Press; 2004. p. 135e152)

Tissues P5CS pmol/mg min

OATmmol/g h

CPSmmol/g h

OTCmmol/g h

Duodenum 197 þJejunum 252 117 10 100Ileum 101 þColon 12 <2Pancreas 12 40 3 2Thymus 8 0 2Stomach 2 <2Brain 1 13 4 0Lung <1 15 0 2Liver 0 188 284 4110Muscle 0 13 0 0Kidney 0 166 8 5

and absorption from food.22 However, citrulline is not com-monly present in food e with the exception of watermelon(1 g of citrulline in 780 g), but plasma citrulline concentra-tion is not significantly increased after regular watermeloningestion for 3 weeks23 e or in currently available enteral orparenteral nutrition (PN) solutions. The only condition inwhich citrulline occurs in endogenous protein is as citrulli-nated peptides through post-translationally modified argi-nine residues in various autoimmune disorders such asrheumatoid arthritis.24

Thus the main source of circulating citrulline, endproduct of glutamine metabolism, is intestinal de novo syn-thesis. Whole-body citrulline flux was established at 4e6 g/d in humans.25,26 The enzymatic pathway of intestinal cit-rulline biosynthesis is a complex sequence of at least fiveenzymes in enterocytes mitochondria27e29 (Fig. 1 andTable 1). Key enzymes are OAT (ornithine aminotransfer-ase)30 and P5CS (pyrroline 5-carboxylate synthase),27,31

the latter being almost solely expressed in intestinal mu-cosa.29 After weaning, the intestine no longer expressesactivity of villous ASS (arginosuccinate synthase) and ASL(arginosuccinate lyase)32 and so does not produce arginine.

LiverIt has been established in an ex vivo rat model of isolatedsmall bowel loop10 that the liver does not play a significantrole in splanchnic interorgan citrulline metabolism. This isdue to liver use and production of citrulline partitioned inperiportal hepatocytes that exhibit a complete urea cyclewith presence of cytosolic ASS and ASL, which convert cit-rulline to arginine recycled into ornithine and urea: thereis no hepatic release of citrulline. Conversely, one studysuggested that the metastatic liver may consume 8% of

Citrulline and enterocyte mass 331

intestinally produced citrulline,33 but net hepatic flux ofcitrulline is not significantly different from zero.13

Kidney metabolism

As demonstrated in rats, approximately 80% of the citrullineproduced by the intestine is equimolarly converted intoarginine in proximal convoluted tubules of kidneys express-ing the two enzymes (i.e. ASS and ASL) mandatory forendogenous arginine biosynthesis from citrulline.34 It is es-timated that citrulline contributes 10% of the whole bodyarginine production.13

Physiological implications of the intestine-kidneyinterorgan citrulline-arginine pathway

It has been hypothesized that this interorgan pathwayprevents excessive arginine degradation by the liver.17 Itis known that the enzymes of this incomplete intestinalurea cycle (only in mitochondria but not cytosolic) areup-regulated in situations in which the body has to sparenitrogen, such as in the post-absorptive state or when pro-tein intake is low, as opposed to the postprandial state.35 Itis thus logical that circulating citrulline concentration canbe found slightly reduced in the postprandial situation.22

We can consider that circulating citrulline is in its owna form of arginine masked to avoid liver capture and exces-sive ureagenesis.21 It may be that in omnivorous mammalsthe activation of the citrulline pathway is an alternativeto activation of the arginine pathway, which itself controlsureagenesis, and so the metabolism of amino acids. Thusthis process allows a fast response to changes in proteinintake with the citrulline pathway preferred for low or nor-mal protein intake and the arginine pathway preferred forhigh protein intake.36

Blood citrulline assay: analytical methods andinterpretation

Citrulline can be sampled in tubes for serum or plasmacollection with no significant difference in results or in-terpretation. Citrulline assay is unsuited to routine analysisin a clinical chemistry laboratory because it necessitatesspecialized techniques. The first step in the analyticalprocess is the deproteinization of serum or plasma. Theseparation of amino acids requires liquid chromatographymethods such as ion exchange chromatography with post-column detection with ninhydrin, HPLC with pre-columnderivatization using e.g. orthophthaldehyde, or mass spec-trometry. It has been suggested37 that the dried blood spot,generally used to screen for inborn error of metabolism,gives good results, such as close correlation (r2 Z 0.87)with plasma citrulline concentration, but with large varia-tions compared with results obtained with plasma. Henceusing the dried blood spot can result in misclassificationof patients in the follow-up of intestinal transplantation.36

Ion exchange chromatography gives good reproducibility(between-run below 5% and 7%) and repeatability (below1%)38 and is considered as the reference method. The useof quality control, with for example European Standard ISO9000 (ERNDIN), is mandatory to ensure reliable and

interpretable data. The cost of the assay (price charged bythe laboratory) is about V160 in France for a completeplasma aminoacidogram. This price can be appreciablyreduced by measuring citrulline only (V35) or combinedwith arginine and (or) glutamine determination by meansof specific rapid elution programs (Neveux et al., unpub-lished results).

Physiologically, a concentration of a given amino acidcan be difficult to interpret.39 The pool of plasma aminoacids is low compared with the cellular pool. In comparisonto most non-essential and essential amino acids, theamount of free citrulline in cells such as muscle(170 mmol/L intracellular water) is low.40 Also, citrullinehas the great advantage of not being incorporated into pro-tein. Theorically, decreased citrulline concentration mayhave two causes: diminished production or increased utili-zation. The first corresponds to a significant reduction ofenterocyte mass. To our knowledge the second situationhas never been described. For increased citrulline concen-tration there are also two possible causes: increased pro-duction or decreased utilization. The first situation couldarise from an increase in intestinal production in responseto low protein diet (see above); the second correspondsto a decrease in clearance, i.e. renal failure (see below).

Clinical factors of variation of citrullinemia tobe taken into account for data interpretation

The most important points to check in a practical approachto patient management in a clinical setting will beconsidered here. In Western countries 97.5% of healthysubjects and patients with normal intestinal mucosa func-tion and no renal function impairment have post-absorptiveplasma citrulline concentrations between 20 and 60 mmol/L, with a mean of 40 mmol/L.41 Most of the conditions,either pathological or not, in which citrulline has beenstudied in humans are depicted on Table 2.

Postprandial or post-absorptive state andnutritional status

As for usual biochemical assays, it is preferable to avoida postprandial assay and to prefer the strict post-absorptivestate. In the postprandial state, the circulating concentra-tion of citrulline is stable or slightly depressed (10e20%),22

possibly owing to the diminution of key enzyme activities inintestinal mucosa,25 and return to pre-prandial value occurs2e4 h later. After prolonged starvation citrulline is loweredby 30%.42 During the postprandial period, unlike the post-absorptive period or fasting where it is important to sparearginine, the amount of exogenous amino acid supply maybe sufficient to allow arginine ‘‘wasting’’. Clinical nutri-tional status and level of albuminemia have no independenteffect on blood citrulline level (see below).41,43

Age and ethnic variations

In the first month of life, before breast weaning, citrullineconcentration is significantly decreased.22 Plasma citrullinelevel then progressively and significantly increases with re-pression of enterocyte ASS and ASL activities (see above)

Table 2 Plasma citrulline interpretation in different clinical situations

Situations Citrulline values Cut off forparenteralnutritionindication

Caveats Author(for additional see text)

Normal adult caucasiansubjects

40 � 10 mmol/L Fasting Rabier et al.22

Intestinal diseases/intestinalfailureShort bowel syndrome Decreased 20 mmol/L Normal hydration Crenn et al.41

Chronic villous atrophy Usually decreased 10 mmol/L Crenn et al.43

Intestinal toxicity(antineoplastic treatment.)

Decreased ND Reversible Lutgens et al.84

Small bowel transplantation Normal untilcomplication

ND Non-specific biomarkerfor rejection

Pappas et al.87

Isolated malnutrition(without kwashiorkor)

Normal Crenn et al.43

Severe metabolic stress Can be decreased ND Jeevanandam et al.50

Hepatocellular failure Normal Weber et al.56

Renal failure Increased If creatinine clearancebelow 50 ml/min

Ceballos et al.49

Urea cycle disorders Usually low Rare: OTC deficiencymost common (1/14 000)

Zschocke J,Hoffman GF. Vademecummetabolicum,2nd Ed. Shattauer,Friedrichsdorf,Germany 2004

Ageing Can be slightlyincreased after 70

Check renal function Pitkanen et al.46

ND: not determined.

332 P. Crenn et al.

and with adaptation to food diversification, as established inthe intestinal mucosa of young pigs.44,45 In the elderly (age>70 years) the frequent impairment of renal function mayexplain an increase in blood citrulline above the mean plustwo SD46 with a mean 20% increase compared with subjectsbelow age 50 years.47 A study reported lower mean valuesin healthy Chinese subjects (i.e. 17 mmol/L) than in Cauca-sians, possibly due, in addition to analytical differences(HPLC vs. ion exchange chromatography), to genetic differ-ences or differences in long-term dietary habits.48

Renal function

The main factor for clinical interpretation of plasmacitrulline concentration is renal function. Citrulline ismetabolized into arginine in proximal kidney tubules andincreases significantly in patients with compromised creat-inine clearance. A study49 has established that citrullinecan increase in patients with moderate renal impairmentevidenced by a creatinine clearance below 50 ml/min: atthis threshold there is a correlation between citrullineelevation and decrease in renal function assessed by creat-inine clearance. Consequently, all samples for citrullineassay must come from patients with a normal hydrationstatus, if necessary after saline intravenous infusion, and

with a concomitant determination of creatinine clearance,at least using a Cockroft formula.41

Metabolic stress and inflammation

A severe metabolic stress is associated with a decrease incitrulline concentration,50,51 such as for example in acutepancreatitis.52,53 It is likely, though unproven, that thereis a diminution of intestinal citrulline production in this sit-uation. Nevertheless, this does not always imply an anatom-ical or even functional measurable intestinal abnormality:for instance, a low citrulline concentration may be due inpart to diminished availability of the main precursor gluta-mine, or to a decrease in intestinal glutaminase activity, asis well known in sepsis.54 Inflammation, either systemic orintestinal, has no significant effect on citrulline level.55

Liver function

Hepatocellular function, for example in severe cirrhosiswith decompensation, does not significantly influencecirculating citrulline concentration.56,57 Liver transplanta-tion does not change hypocitrullinemia in inborn errors ofmetabolism such as OTC (ornithine transcarbamylase) andCPS (carbamol phosphate synthase) deficiency,58,59 further

Citrulline and enterocyte mass 333

indicating that circulating citrullinemia does not originatefrom the liver.

Minimal biologically significant difference

The magnitude of the minimal biologically significantdifference between two assays that reflects a physiologicalor a clinical fate is important to assess, but data are lacking.In our experience we estimate that for citrulline concen-tration, with our current biochemical technology, thismagnitude will be probably greater than or equal to 15e20%.

Blood citrulline assay in intestinal diseases

Short bowel syndrome

Historically, after animal models, the short bowel syndrome(SBS) was the first to be studied in a clinical setting1,2,41 ow-ing to the near-‘‘experimental’’ situation created by the re-moval of a large amount of the anatomical and functionalmass of the intestine with a well-documented and measur-able reduction of the enterocyte mass. Early studies hadshown that interorgan glutamine flux was reduced by 20%in these patients, whether adults60 or children.61 In stableSBS patients, amino acids with a prevailing intestinal siteof metabolism have been found to be significantly higherfor plasma glutamine (10%) and lower for plasma citrulline(50%) than in controls,41,62 whereas argininemia was normalor lowered by 30%.62 There are now at least nine additionalpublished studies in adults SBS,48,55,62e68 with a total ofmore than 220 patients, and four studies in childrenSBS68e71 that clearly and constantly confirm that citrullineconcentration is a reliable biological marker of remnantsmall bowel (see for example Fig. 2). Orotic aciduria, indi-cating arginine deficiency, can be found in some shortbowel syndrome patients,63 but in most cases adaptationin arginine metabolism is observed when an adequate

Figure 2 Correlation between plasma citrulline concentra-tion and remnant small bowel length in 57 short bowel pa-tients. C patients with permanent intestinal failure; B

patients with transient intestinal failure [reproduced from ref-erence [41] with permission of Gastroenterology, 2000,Elsevier].

supply of arginine is provided.62 All studies in humanshave found a strong significant positive correlation, withthe r coefficient ranging from 0.47 to 0.90, between plasmaor serum post-absorptive citrulline concentration and rem-nant small bowel length measured peroperatively or withradiological techniques, including estimation of smallbowel surface area.41 It is suggested that citrulline concen-tration reflects the entire small bowel including the smallbowel excluded from the digestive circuit (n Z 11) in 23 pa-tients with enterostomy.67 There are fewer results concern-ing the relation between citrulline and absorptive function.Our original study in 57 SBS patients41 showed a correlationbetween citrullinemia and fat (r Z 0.53) and nitrogen(r Z 0.47) absorption in univariate analysis and for fatonly in multivariate analysis. Another study in children sug-gested that citrulline determination provided relevant in-formation on intestinal macronutrient absorption: in 24children with SBS, plasma citrulline concentration was cor-related (r Z 0.85) with the percent of enteral calories thata short gut can tolerate, i.e. without diarrhea defined bymore than eight stools per day.69 Citrulline concentrationwas closely correlated to D-xylose absorption (P < 0.0001)in SBS patients,48,55,71 Crohn’s disease, celiac patients andhealthy subjects.55 However, in another study in adult SBS(n Z 24) there was no significant correlation between cit-rulline and macronutrients (nitrogen, fat, carbohydrateand calories), fluids and electrolytes (sodium, potassium,phosphorus and magnesium) absorption expressed in per-cent of amount ingested.66 One of the problems with thisstudy is that the assay of citrulline was performed only1 h after PN was discontinued, raising the question of argi-nine, contained in PN, recycling into citrulline.18 Absorp-tion is nevertheless a complex integrated function relatedto small bowel mucosa, biliopancreatic secretions, gut lu-men and colonic absorption. Absorption is also, accordingto various nutrients, very different in capacity and location.Hence it is likely that plasma citrulline concentration can-not monitor precisely specific nutrient absorption and in-testinal energy absorption as recently suggested in 30adult patients with short bowel syndrome or celiac dis-ease.72 Citrulline explores the integrity of the intestinal ep-ithelium at the enterocyte level, with a predominant site ofproduction in the proximal small bowel. We therefore pos-tulate that citrulline is a true indicator of the remnant en-terocyte metabolic mass function.

Citrulline assay provides a practical clinical decision-generating index of the global function, and so thenutritional prognosis, of a compromised intestine. In 57patients with SBS, studied after 2 years following intestinalresection, i.e. beyond the period in which most of the ad-aptation occurs, a cut-off of 20 mmol/L, i.e. 50% of meancontrol level, was highly predictive (92% sensitivity, 90%specificity, 95% positive predictive value, 86% negativepredictive value) in distinguishing transient (n Z 20) frompermanent (n Z 37) chronic intestinal failure.41 The samecut-off was confirmed in children.69 Another study in adultsindicates that this threshold can be used as a preoperativemarker of PN indication before digestive circuit reestablish-ment.67 Surprisingly, in this last study citrullinemia did notchange significantly before and after re-establishment ofdigestive continuity. Therefore a prognosis indication ofchronic intestinal failure, i.e. PN dependency, can be

334 P. Crenn et al.

addressed with an analytically and clinically valid plasmacitrulline assay. One major point to be underlined is thatcitrulline levels are independent of nutritional status.41 Inanother study, in nine SBS patients, we demonstrated thatcitrulline concentration was a reliable surrogate marker ofits production by means of a protein kinetic study with citrul-line labeled with a stable isotope: citrulline fluxes werelowered by 68% compared with reference values and signif-icantly correlated to citrulline concentration (r Z 0.83,P < 0.01) and small bowel length (r Z 0.89, P Z 0.001).73

This confirms in vivo that citrulline is a marker of remnantenterocyte mass in SBS patients. We note that hyperphagicSBS patients had higher citrulline levels than non-hyper-phagic patients, with a mean increase of 7 mmol/L,41 espe-cially in SBS patients with the longest remnant.55 In ouropinion, this last observation suggests that citrulline isa true marker of the remnant absorptive function in shortgut patients.

Figure 3 Plasma citrulline concentrations in 51 healthy con-trols, 10 patients with anorexia nervosa, and 52 patients withsmall bowel disease according to severity and extent of villousatrophy. Statistical results are to group-to-group comparisonsby ANOVA: *P < 0.05 for group 3 compared with group 0,yP < 0.05 for group 4 compared with groups 0, 1, 2, and 3;zP < 0.05 for group 5 compared with groups 0, 1, 2, 3, and 4.Differences between groups 0, 1, and 2 were not statisticallysignificant. Patients with celiac disease are indicated by opencircles and patients with non-celiac disease villous atrophy-as-sociated small bowel disease by solid squares. [Reproducedfrom reference [43] with permission of Gastroenterology,2003, Elsevier]. Non-destructive mucosal lesions: partial villousatrophy. Destructive mucosal lesions: total or subtotal villousatrophy.

Villous atrophy diseases

Villous atrophy is a condition of which celiac disease is themain etiology in Western countries and ‘‘tropical’’ sprueand various kinds of infectious enteritis in the developingworld.74 Until recently grade and extension of villous atro-phy could be estimated only with multiple biopsies underenteroscopy. In 52 HIV-negative patients, citrulline waslowered by less than 20 mmol/L in patients with proximal-only total or subtotal villous atrophy, and by less than10 mmol in patients with extensive (proximal and distal)villous atrophy, irrespective of the underlying etiology43

(Fig. 3). In these patients, a citrulline value below10 mmol/L was highly predictive of need for PN reflectingintestinal failure. We obtained the same results in 21 HIV-positive patients with severe enteropathy, either specificor associated with intestinal infectious diseases,75 ofwhom eight needed PN including the seven patients withcitrulline levels below 10 mmol/L. Patients with only mildenterocyte involvement, i.e., partial proximal villous atro-phy, showed a normal or moderately lowered citrulline con-centration (Fig. 3).43,55,76 In this situation a ‘‘citrullinestimulation test’’, following, for example, an oral bolus ofglutamine, might give more sensitive results than a basalassay only.76 Adults43,77e79 or children74 with celiac diseasewho respond clinically and histologically to a gluten-freediet or some with a ‘‘refractory’’ sprue42,73 may experi-ence a net increase with (sub)normalization of citrullinemiaafter one year.

Thus in villous atrophy diseases, citrulline can beconsidered as a biomarker of the extent of the villousatrophy and enterocyte mass and of repair under specifictreatments. In addition, citrulline threshold can serve as anindicator to guide the type of nutritional support: a citrul-line value below 10 mmol/L predicts a very high probability(near 100%) of PN dependency43,75 (i.e. intestinal failure),whereas citrulline higher than 10 mmol/L might be compat-ible with nutritional replenishment through the enteral(tube feeding) route.75 In villous atrophy diseases, theROC curves indicate that citrulline is more powerful thanalbumin level in predicting the degree of reduction of theenterocyte mass.43

Crohn’s disease

Crohn’s disease is an immunological disease of the gutlinked to abnormal bacterial antigen sampling and process-ing in intestinal mucosal cells. There is no extensiveenterocyte damage per se, except in the case of significantintestinal resection. The presence of non-extensive resid-ual small bowel abnormalities did not significantly influencecitrulline concentration in our SBS population.41 It wasshown55 that systemic inflammation, defined by CRP level>10 mg/L and a Crohn’s disease activity index (CDAI) ofmore than 150, did not significantly influence citrullinemiain 31 Crohn’s disease patients. In the whole group of 55patients, including Crohn’s disease, mesenteric infarction,celiac disease and controls, there was no correlation be-tween citrullinemia and small bowel permeability (urinarylactulose/rhamnose), but a correlation was found with

Citrulline and enterocyte mass 335

bowel length and D-xylose absorption.55 Thus the authorsconclude that plasma citrulline level is a simple surrogatebiomarker for small bowel absorptive capacity not influ-enced by intestinal inflammation.

Acute mucosal enteropathy and antineoplastictreatments

Acute mucosal enteropathy can cause a significant loss ofenterocytes, as suggested by secondary lactose intoler-ance, and can lead to acute intestinal failure. Citrulline islowered in these situations, for example in adenovirusenteritis and all infectious intestinal diseases with highcytopathic effects.80 The normalization of citrullinemia israpid in most situations, i.e. in 1e3 weeks. Chemotherapyin hemato-oncology (bone marrow allograft) induces de-creased citrulline levels.81 This effect is consistent withknown cytokinetic renewal of intestinal mucosa with anadir in citrulline levels (40% of pre-treatment level) 5e8 days after hematopoietic stem cell transplantation initi-ation in 10 patients82 (Fig. 4). Citrulline variations aremore sensitive, occur earlier (1e2 weeks) and are morespecific than various sugar-based permeability tests for de-tecting chemotherapy-induced gut damage in patientswith hematological malignancies. After bone marrowtransplantation, the decrease in citrulline plasma levelsappeared to be a risk factor for infections.81 This suggeststhat during antineoplastic therapies citrullinemia could beused to monitor the intestinal mucosa toxicity, just as neu-trophil polynuclears are used to monitor bone marrow tox-icity. Mucositis and epithelitis can be treated or preventedin part by KGF (keratinocyte growth factor or palifermineas DCI). In a mouse model, rhKGF, acting as an epithelialtrophic factor, allowed citrulline levels to be maintained

Figure 4 Time course of the plasma citrulline concentrationbetween hematopoietic stem cell transplantation (HSCT day�6 to þ21). [Reproduced from reference [82] with permissionof Cancer, 2005, Wiley Interscience]. Lines represent meancitrulline concentration and 95% confidence intervals.

at normal values during chemotherapy instead of the usualdecrease.83 The effect of radiotherapy on citrulline con-centrations was studied in a mouse model84 and later con-firmed in humans treated with abdominal or pelvic sitecancer.85 Acute radiation enteritis induced by total bodyirradiation or fractioned localized irradiation can be mon-itored by citrulline, which correlated in 23 patients withthe morphological endpoints of epithelial radiation dam-age, the dose received and the volume of small bowel inthe radiation field.85,86 The relation between citrullineconcentration and clinical symptoms such as diarrheawas less obvious.

Small bowel transplantation

Intestinal transplantation, radical treatment of severe,permanent and PN-related major complications in chronicintestinal failure is one situation in which the potentialusefulness of citrulline measurement was observed. Wecurrently lack a powerful indicator of acute dysfunction ofintestinal grafts,80 and so citrulline was proposed as a bio-marker of acute cellular epithelial rejection after smallbowel transplantation87 if a prompt result is obtained.From a practical point of view, the rapidity with whichthe result is obtained, which has to be the same day in allcases, is a crucial challenge. The duration of chromatogra-phy, including time of preanalytical preparation (centrifu-gation and deproteinization) must take no longer than1 h. Also, citrulline has to be interpreted in the light of mul-tiple interrelated parameters such as (a) time after surgerywith progressive elevation of citrulline (1e3 months),88 (b)renal function (dehydration, drugs, etc., with higher citrul-linemia than expected)65 and (c) graft pathology, for exam-ple, enteritidis80 and acute or chronic rejection whateverits cause80,89e92 with decline in citrullinemia. As shown ina pig model, the citrulline decline is only observed whendiffuse severe mucosal damage has occurred, i.e. moderateand severe acute cellular rejection, but not in cases ofindeterminate or mild acute cellular rejection.93

Miscellaneous

Situations without significant reduction of enterocyte massshould not show hypocitrullinemia. For example, (sub)-normal citrulline determination can be used for the purposeof excluding an extensive small bowel disease in a case ofa malabsorptive diarrhea. Also, citrulline plasma levels arein the normal range in patients with anorexia nervosa withsevere malnutrition and body mass index below 1343

(Fig. 3). Hypocitrullinemia, more specifically than hypoal-buminaemia, can be used as an index of the extent andresponse to treatment of villous atrophy diseases in bothHIV-negative43 and HIV-positive75 patients.

Perspectives and clinical outcome

Citrulline is at the present time the only biological tool usedin a clinical setting to quantitatively investigate intestinalepithelial integrity at the enterocyte level. Different sitesof reduction of the enterocyte mass (proximal in celiacvillous atrophy, distal in most patients with SBS) as well as

336 P. Crenn et al.

functional adaptive changes in SBS can give a partialexplanation of differences between these two models ofenterocyte mass reduction. For example it can be esti-mated with citrulline assay that duodenal enterocyte massrepresents 10e15% of the normal total enterocyte mass inhumans, but 25% of a remnant enterocyte mass compatiblewith non-PN dependence in SBS.41,94 This suggests a poten-tial role for citrulline as a biomarker to follow intestinaladaptation, both physiological,95 post-surgical and pharma-cological. In six SBS patients followed in the first year afterresection, one study suggested an ongoing adaptive mecha-nism by showing an increase in plasma citrulline (þ15%)correlated with protein absorption improvement and urineD-xylose excretion.48 This may be related to hyperphagia96

and ensuing increased enterocyte function.1,41 Growth hor-mone administration had a borderline effect on citrullineconcentration despite its non-specific action on the 15% im-provement of macronutrient absorption.66,97 There is nopublished data on the action of GLP-2, a specific enterotro-phic factor, on citrulline plasma level. Surgical proceduresfor experimental intestinal lengthening improved intestinalcapacity, absorption and serum citrulline in a porcine shortbowel model,98 data that are confirmed in 14 childrenSBS,71 either newborn and infants, where plasma citrullineincreased by 90% 1 year after enteroplasty. To our knowl-edge there are no reports of citrulline evolution after bari-atric surgery for severe obesity such as gastric bypass,a procedure that creates proximal malabsorption.

In situations with overlapping values between healthysubjects and patients with moderate intestinal involve-ment, such as partial villous atrophy or patchy lesion, i.e.where citrullinemia is between 20 and 30 mmol/L43

(Fig. 3), the citrulline stimulation test, such the oneproposed after oral ingestion of glutamine, may be useful.In situations such as proximal villous atrophy or shortbowel without intestinal failure, this dynamic test appearsto be more powerful than a simple assay with time-to-peak(but not peak per se) and area under the curve up to180 min after glutamine ingestion.99 These preliminarydata need to be confirmed. However, a dynamic test isless easy to perform and more costly than a single assaycarried out with clinical precautions and adequate analyt-ical methods. The respective utility and reliability of staticand dynamic tests need to be prospectively studied intargeted small bowel diseases.

In addition, the citrulline deficiency in SBS patients mayhave clinical consequence as suggested in a rat model ofextensive small bowel resection: citrulline supplementa-tion, either enterally100 or parenterally,101 promoted nitro-gen balance more than isonitrogeneous supplement ofarginine. In human, hyperammonemic encephalopathy canbe observed in SBS with hypocitrullinemia.102

Conclusions

Plasma citrulline concentration is a quantitative biomarkerof the remnant metabolically active enterocyte mass thatreflects the functional absorptive capacity of a remnantsmall bowel. This biomarker is not significantly influencedby nutritional or inflammatory status. A cut-off of 20 mmol/L serves as an objective measurement for quantifying the

degree of intestinal failure in SBS, whether transient orpermanent, and so helps to select and evaluate appropri-ate treatment. However, citrulline concentration doesnot provide an accurate indication of the overall or specificabsorptive function of the whole gut, especially in patientswho have not had a significantly reduced enterocyte mass.Decreased citrulline plasma concentration is not specific toa disease, but can be considered as an innovative tool fora significant epithelial small bowel loss. Citrulline thusseems to be a reliable marker of intestinal function analo-gously to that used for the liver, i.e. proaccelerin (factorV), or for renal function, i.e. creatinine. Citrulline, proac-celerin and creatinine are not representative of overall orspecific organ functions. The absorptive enterocyte masscan be assessed by blood citrulline levels, which cannotrepresent all the functions of the intestine. However, as-sessing enterocyte mass with citrulline is invalidated incases of renal insufficiency. Regarding clinical and analyti-cal variation factors, circulating citrulline assay for smallbowel assessment appears in most cases to be convenient,valid and reproducible.

Conflict of interest statement

None declared.

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