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Plasma citrulline as a quantitative biomarker of HIV-associated villous atrophy in a tropical enteropathy population
Clinical Nutrition 29 (2010) 795e800
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Clinical Nutrition journal homepage: http://www.elsevier.com/locate/clnu
Original Article
Plasma citrulline as a quantitative biomarker of HIV-associated villous atrophy in a tropical enteropathy population Cinzia Papadia a, b, Paul Kelly c, d, Saverio Caini e, Gino Roberto Corazza f, Tamara Shawa c, d, Angelo Franzè b, Alastair Forbes a, *, Antonio Di Sabatino f a
Department of Gastroenterology, University College Hospital, London, United Kingdom Department of Gastroenterology, University of Parma, Parma, Italy Institute of Cell and Molecular Science, Barts & The London School of Medicine, Queen Mary University of London, United Kingdom d Tropical Gastroenterology and Nutrition Group, University of Zambia School of Medicine, Lusaka, Zambia e Molecular and Nutritional Epidemiology Unit, Istituto per lo Studio e la Prevenzione Oncologica, Florence, Italy f First Department of Medicine, S. Matteo Hospital, University of Pavia, Pavia, Italy b c
a r t i c l e i n f o
s u m m a r y
Article history: Received 8 December 2009 Accepted 13 April 2010
Background & aims: Studies have shown that the circulating citrulline concentration is decreased in patients with proximal small bowel villous atrophy from coeliac disease and more so in patients with extensive damage to the intestinal mucosa, but there have been few data on HIV enteritis and tropical enteropathy (TE). Our primary aim was to correlate serum citrulline with the degree of reduction of the enterocyte mass in HIV-infected patients with TE. Methods: Postabsorptive fasting serum citrulline was measured in 150 TE pts, 44 of whom had HIV infection, using reverse phase, high performance liquid chromatography. Absorptive capacity and permeability were measured after intrajejunal instillation of 4 sugars (5 g lactulose, 1 g L-rhamnose, 0.5 g D-xylose, 0.2 g 3-O methyl Dglucose) with assay by thin-layer chromatography. Morphometric analysis was carried out on jejunal biopsies. Results: In HIV positive patients, the median serum citrulline was significantly lower (median 19, interquartile range (IQR) 17e24 mmol/L) than in HIV negative patients (median 27, IQR 23e33 mmol/L; p < 0.001). There were statistically significant correlations (p < 0.005) between citrulline and: crypt depth; villous height/crypt depth ratio; Shenk-Klipstein score; and xylose absoption, only in the HIV positive. Conclusions: Serum citrulline concentration appears to be a quantitative biomarker of small bowel mass integrity in HIV positive enteropathy and desrves assessment as a surrogate for monitoring anti-retroviral therapy. Ó 2010 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.
Keywords: Citrulline HIV Tropical enteropathy Sprue Celiac Biomarker
1. Introduction Tropical enteropathy (TE) is a population shift in intestinal mucosal morphology and function which resembles an asymptomatic villous atrophy of the small bowel. It is characterised by subclinical malabsorption and increased permeability, but its clinical significance is unknown. It is not even clear whether it is truly an abnormality or an adaptive response to environmental factors. It is most prevalent in the developing world within 30 of either side * Corresponding author at: Department of Gastroenterology and Clinical Nutrition, Maple House, University College Hospital, 235 Euston Road, London, NW1 2BU, United Kingdom. Tel.: þ44 (0) 845 155 5000x9011; fax: þ44 (0) 20 7380 9699. E-mail address: [email protected] (A. Forbes).
of the equator, but it is not limited to indigenous populations as it also affects European and American expatriates living in tropical regions. Mild reduction in villous height, broadening of the villi and increased crypt depth are the most common histological features described in TE.1 An increase in inflammatory cell infiltrate may occur, as well as mucosal T cell activation and crypt hyperplasia.2 Functional changes have been described, including subclinical malabsorption of carbohydrates,3 fat and vitamin B12,4 and increased mucosal permeability.5,6 TE is observed in asymptomatic individuals, which distinguishes it from tropical sprue, a severe but now rare clinical illness. The histopathological changes of TE resemble those occurring in the early stages of coeliac disease and in other immune mediated enteropathies. It is likely that mucosal immune activation, perhaps
0261-5614/$ e see front matter Ó 2010 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved. doi:10.1016/j.clnu.2010.04.008
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in response to repeated intestinal infections, results in the observed microscopic changes described in TE. We have previously observed that Citrobacter rodentium may play such a role.7 In many tropical countries, the high prevalence of HIV leads to HIV enteropathy being superimposed on TE. On the whole the morphological and functional changes become significant in advanced AIDS.7 There are surprisingly few data on the responsiveness of the enteropathy to treatment of intestinal infection and to treatment with anti-retroviral drugs, and a non-invasive marker of intestinal mucosal health would be very useful in analysing the response of the mucosa to treatment. A relationship between plasma citrulline and intestinal function has been proposed from studies examining rejection of small bowel allografts, in which plasma citrulline concentrations were found to be reduced in cases of mucosal damage, and with an inverse correlation to severity on biopsy.8,9 Lower plasma citrulline concentrations have also been recorded in patients with villous atrophy than in healthy subjects or patients with anorexia nervosa. A range of thresholds was suggested: <10 mmol/L representing diffuse total villous atrophy; through 10e30 mmol/L for nonextensive or partial villous atrophy; to normal subjects who have levels >30 mmol/L10 One of the most exciting potential applications is the use of the circulating citrulline concentration as an objective tool for determining the need for and route of nutritional support.10e12 Evidence from human and animal studies suggests that citrulline may prove useful as a more general marker of small bowel epithelial damage regardless of cause.13,14 There are few data on citrulline levels in humans. In our previous work we reported a quadratic (non-linear) correlation between fasting citrulline concentrations and remnant small bowel length, indicating that decreased citrulline concentrations predominantly occur when intestinal disease (or loss) is severe.15 This observation supported the earlier work from Paris and elsewhere.10,13,14. However, Luo et al.16 reported no relation between fasting plasma citrulline concentrations and intestinal absorptive capacity in short bowel syndrome, Peters et al.17 described poor diagnostic value of citrulline concentrations in intestinal energy absorption in enterocyte damage, and Miceli et al.18 were unable to confirm the value of plasma citrulline as a sensitive marker of intestinal atrophy. The position of citrulline as a marker of intestinal function cannot therefore be considered fully established. In the current study we hypothesize that serum citrulline concentration might be a reliable marker of early morphological and functional changes in HIV enteropathy. 2. Methods 2.1. Patients Endoscopic jejunal biopsy specimens and peripheral blood samples were collected from 150 adult patients with TE to investigate whether serum citrulline concentrations mirrored the degree of mucosal injury. TE was defined histologically from the presence of reduction in villous height, broadening of the villi and increased crypt depth in the absence of serological evidence for coeliac disease or documentable gastrointestinal infection TE.1 We included both HIV seropositive and HIV seronegative TE patients. A cohort study had already been set up in a poor sector of Misisi township in Lusaka, the capital of Zambia, and we have previously reported the morphometric and functional data to which reference is made here.7,19 The study was reviewed and approved by the Research Ethics Committees of both the University of Zambia and the London School of Hygiene and Tropical Medicine.7 Patient characteristics at entry to the study are listed in Table 1. Participants
were enrolled in a three year program of investigation and treatment of each episode of ill-health. They were then asked to attend once a year for investigation, blood tests, enteroscopy with jejunal biopsy, and sugar absorption/permeability tests, all performed in the course of a single day in the endoscopy unit. Patients also underwent monthly stool sample examination to document parasitic and bacterial infections. Venous blood sampling after an overnight fast was carried out. Standard serum biochemical analyses (liver function tests albumin, urea, creatinine, electrolytes, C-reactive protein), and a full blood count were performed. Serum was saved for citrulline assay (see below). Testing for HIV was conducted and CD4 counts were determined using a FACSCount analyzer (Becton Dickinson, Franklin Lakes, NJ) according to the manufacturer’s instructions. Pregnant or lactating patients were temporarily excluded from the study. Endoscopy was also deferred in participants who had experienced diarrhoea and had taken antibiotics or non-steroidal anti-inflammatory drugs until one month had elapsed. The incidence of diarrhoea was assessed by recall using a simple questionnaire administered every two weeks, and by active case-finding while offering investigation and treatment of each episode. Participants then submitted a stool sample that was analyzed for the presence of protozoa, bacteria, and helminths. At the time of recruitment highly active anti-retroviral therapy was not available in Zambia. 2.2. Reverse phase liquid chromatography with UV detection for serum citrulline The blood samples were centrifuged (2500 g, 10 min) at room temperature. Aliquots of serum were then stored at 80 C until batched derivatization and reverse-phase HPLC. A 1 mmol/L stock solution of citrulline (Sigma) was diluted to 1, 5, 10, 50, and 100 mmol/L concentrations, and used as standards for assay calibration. Phenyl-isothiocyanate was selected as the derivatization reagent as it combines high sensitivity with ready detection by its ultraviolet (UV) absorption at 254 nm. The Thermo Analytical P4000 quaternary pump, AS3000 autosampler, 5 mm guard column 5 mm i.d., C18 (ODS) analytical column 25 mm long 5 mm i.d., the Uv 2000 UV detector, and the CHROMQUEST computing integrator (Thermo, St. Albans, UK) were employed. The blood samples were deproteinized with 10% sulfosalicylic acid, and 750 mmol/L norleucine was used as an internal standard. Supernatants were diluted with acetonitrile and phenyl-isothiocyanate coupling reagent for 20 min at room temperature, evaporated to dryness on a rotary evaporator (Genevac EZ2, Dublin, Ireland), and then resuspended in the mobile phase. Gradient elution was achieved with sodium acetate/acetonitrile. The gradient profile was modified to enable adequate separation of citrulline from taurine. At baseline, there was 90% buffer 1: sodium acetate 0.01 M, and 10% buffer 2: acetonitrile/sodium acetate (60% v/v) 0.01 M. The injection volume was 180 mL and the column flow rate was 1.0 mL/min. At 40 min, i.e. after the citrulline peak and the internal standard had eluted from the column (at approximately 12.3 min and at approximately 39 min, respectively) the percentage of acetonitrile was rapidly increased to 100% with the expectation of eluting all remaining amino acids within 5 min. The complete run time was therefore 45 min. The solvent gradient was returned to its starting composition over the following 5 min, and allowed to re-equilibrate, thus permitting introduction of the next sample 25 min later. 2.3. Sugar testing of absorption and permeability The four sugar test solution instilled into the small intestine contained 0.5 g of xylose, 1 g of rhamnose, 5 g of lactulose, and 0.2 g of 3-O-methyl Dglucose (Sigma Chemicals, Poole, UK) in 100 mL of
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Table 1 Median values (IQR) of age, anthropometric measures and malabsorption indices.
Number of Patients Age (yrs) BMI (kg/m2) MUAC (cm) Citrulline (mmol/L) D-Xylose (%) L/R ratio Villous height (mm) Crypt depth (mm) VH/CD ratio
pa
All
HIV þ
HIV
145 35 (27e44) 20.5 (18.7e23.5) 26.5 (25.5e29) 25 (19e31) 19.7 (15e24) 0.07 (0.04e0.10) 258 (229e296) 153 (137e175) 1.71 (1.47e1.95)
44 28 (24e34) 20.8 (19.5e22.1) 26 (25.5e28.5) 19 (17e24) 18.3 (13.2e21.2) 0.07 (0.05e0.10) 238.5 (224e279) 162 (146.5e193) 1.53 (1.26e1.72)
101 39 (29e48) 20.2 (18.7e24.6) 27 (25.5e29) 27 (23e33) 20.3 (15.3e24.6) 0.06 (0.04e0.10) 264 (234e306) 146 (132e170) 1.81 (1.61e2.01)
<0.001 0.698 0.675 <0.001 0.177 0.434 0.018 0.001 <0.001
Men
Women
62 37 (28e49) 19.7 (18.0e20.9) 26 (25e28) 28 (22e34) 20.8 (15.3e25) 0.06 (0.04e0.10) 253.5 (228e286) 147.5 (131e175) 1.71 (1.50e1.95)
83 32 (26e41) 21.6 (19.9e25.2) 28 (26e29) 24 (18e29) 18.5 (13.9e23.6) 0.07 (0.05e0.10) 261 (231e315) 157 (138e175) 1.71 (1.44e1.95)
pa 0.025 <0.001 0.004 0.001 0.209 0.299 0.103 0.212 0.903
BMI, body mass index; MUAC, mid-upper arm circumference; L/R, lactulose-to-rhamnose ratio; VH/CD, villous height-to-crypt depth ratio. a p-values are for Wilcoxon rank-sum test comparing medians.
distilled water. The same solution was given orally in those not undergoing endoscopy. Urine samples were collected prior to endoscopy and for exactly 5 h afterwards and preserved with merthiolate (final concentration of approximately 0.04%). Urine samples were frozen at 80 C for transport and analyzed by thinlayer chromatography.
villi, and grade 4 is flat. For cellular infiltration: grade 0 is normal, grade 1 shows a mild increase, grade 2 a moderate increase, and grade 3 a marked increase. To ensure that the morphometric technique was reproducible, measurements were repeated in triplicate from the same areas of 8 jejunal biopsies. The inter-assay variation was 13% indicating that variation seen between individuals is unlikely to be explained by effects of sampling.
2.4. Morphometric analysis 2.5. Statistical analysis Morphometric analysis was carried out as previously described.7,19 Slides were evaluated by a senior pathologist before morphometry was performed by a single observer. The following variables were then measured: 1) cross-sectional area of the villous compartment, which is representative of villous compartment volume (VCV); 2) total length of epithelium, which is used as a measure of epithelial surface area (ESA); 3) villous height (VH), which is measured from villus tip to crypt-villus junction as judged by eye (care was taken to include every recognizable villous unit of well orientated mucosa); 4) crypt depth (CD), which was measured in the same way, one for one with each villus; and 5) villous width (VW), which was measured automatically as the maximum width of the villus along a line perpendicular to VH in each unit of the villous compartment. The criteria suggested by Schenk and Klipstein20 were used to grade histological sections for villous abnormalities and cellular infiltrations. For villous appearance: grade 0 is normal, grades 1e3 show progressive broadening and flattening of
Patients were studied according to the following criteria: gender, HIV status, degree of small bowel mucosa atrophy. To compare medians and interquartile ranges (IQR) of continuous variables, the Wilcoxon rank-sum test and KruskaleWallis test were used, with a p-value less than 0.05 considered to confer statistical significance. Permeability was expressed as lactuloseto-L-rhamnose ratio (where normal subjects have a ratio of <0.06%) and absorption determined by percentage urinary retrieval of D-xylose at 5 h (which is conventionally considered to be normal if greater than 20%). Correlations between citrulline and the other measured variables including permeability, absorption, villus height-to-crypt depth ratio, and CD4 counts, were also studied. A non-parametric correlation method (Spearman’s rank test) was applied to analyze these relationships. A Bonferroni correction test for multiple comparisons was then carried out. Sensitivity and specificity were calculated using
Table 2 Median values of citrulline according to Klipstein index and HIV status.
HIV negative (N ¼ 65) HIV positive (N ¼ 32) ALL (N ¼ 97)
a
N median citrulline (mmol/l) N median citrulline (mmol/l) N median citrulline (mmol/l)
Normal mucosa
Shortened villi
Partial atrophy
Subtotal atrophy
Flat mucosa
pa
0-1-2
3e4
pa
0
1
2
3
4
e e
6 25.0
50 29.0
9 31.0
e e
0.316
56 28.0
9 31.0
0.954
e e
1 25.0
16 21.0
14 18.0
1 18.0
0.784
17 22.0
15 18.0
0.496
e e
7 25.0
66 27.0
23 21.0
1 18.0
0.110
73 26.0
24 21.0
0.027
p-values are for KruskaleWallis test and Wilcoxon rank-sum test comparing medians.
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permeability (p ¼ 0.01) in both HIV positive and HIV negative patients, however this significance was not confirmed after Bonferroni’s correction test for multiple correlations (Table 3). A significant correlation between citrulline concentrations and the xylose absorption test was however confirmed with the Bonferroni method (Table 3). Although the specificity was 69%, the sensitivity and predictive values were disappointingly low (<50%) 4. Discussion
Fig. 1. Correlation between D-xylose urinary test and serum citrulline concentration in 101 HIV negative and 44 HIV-positive patients with tropical enteropathy.
a citrulline threshold of 20 mmol/l below which intestinal insufficiency was to be diagnosed. All analyses were performed using Stata 9.0 SE. The clinical study was registered as a trial with clinicaltrials.gov/ ct2/show/results/NCT00816842.
3. Results Our data set included 150 subjects (64 men and 86 women) affected by TE. HIV status was available for 145 subjects and only these individuals are considered further. Some 30.3% of the total sample were HIV seropositive (44 of 145; 21% in men:13 of 62; 37.3% in women: 31 of 83). One quarter (n ¼ 11) of the HIV seropositive patients had a CD4 count below 200. Citrulline concentrations were very similar in all HIV seronegative subjects (Table 1). The median villus-height-to-crypt depth ratio was significantly lower in HIV positive (1.53 mm IQR 1.26e1.72 mm) than in HIV negative patients (1.81 mm; IQR 1.61e2.01 mm). Citrulline concentrations were significantly lower (p < 0.001) in HIV positive patients (median 19 mmol/L; IQR 17e2 mmol/L) than in HIV negative patients (median 27 mm; IQR 23e33 mm). The percentage of patients with citrulline concentrations lower than 20 mmol/L was 14.9% and 52.3% respectively in HIV negative and HIV positive patients. Serum citrulline concentration showed a statistically significant negative correlation with the Schenk and Klipstein score in HIV positive patients (p < 0.004). Moreover the percentage of patients with a citrulline concentration below 20 mmol/L was inversely correlated with the Schenk and Klipstein score (Table 2). There was a sensitivity of 60%, a specificity of 59%, and positive and negative predictive values of 56% and 63% respectively in the HIV positive. Serum citrulline concentration also showed a statistically significant (p < 0.001) positive correlation with xylose absorption (Fig. 1) in the HIV positive patients, but the sensitivity, specificity and predictive values were too low to be of clinical value. Citrulline levels correlated significantly with mid-upper arm circumference (p ¼ 0.04) in the whole group, and were significantly reduced in all those with diarrhoea (p ¼ 0.003). A statistically significant positive correlation was found between citrulline concentrations and
Citrulline contained in food is absorbed by the intestine. Its presence in the lumen, leads to citrulline accumulation in enterocytes, especially in the middle and lower ileum.21 A broad set of transporters seems to be able to transport citrulline from the lumen to the cell.22 Most of the circulating citrulline however comes from glutamine conversion in enterocytes.23 Citrulline is excreted by the kidneys after conversion to arginine. In severe villous atrophy or in massive intestinal resection the main site of net citrulline production is greatly reduced. Probably as a consequence of the diminished circulating citrulline, decreased arginine is also observed,24,25 and it is suggested that arginine becomes an essential amino acid after massive intestinal resection.25 In this metabolic context the citrulline pathway might be thought to be a more important alternative route for arginine metabolism. Citrulline might therefore better express small bowel enterocyte functioning mass in the presence of poor nitrogen balance, since in this condition it becomes the most important amino acid substrate for protein synthesis in enterocytes. Osowska et al. tested this possibility in animals with strongly supportive results.26 However, while these concepts are becoming well established in intestinal failure and short bowel syndrome, their application to functional intestinal failure (ie intestinal failure in the absence of massive resection) has been more controversial and was one of the drivers for the present study. The pioneering study by Crenn et al.10 showed that in chronic villous atrophy (e.g. coeliac disease, tropical enteropathy and various infectious enteritides) citrulline is decreased (<20 mmol/L) in patients with destructive lesions of the proximal small intestinal villous architecture. It is more severely decreased in patients with extensive (proximal and distal) impairment of intestinal mucosa, and in these patients, a citrulline value below 10 mmol/L was highly predictive of the need for parenteral nutrition. Similar results were recently reported in patients with HIV enteropathy or severe intestinal infectious disease.11 In our study serum citrulline concentrations showed a significant inverse correlation with the Schenk and Klipstein score only in HIV positive patients. Our data are in agreement with previous studies10,11 where citrulline levels correlated with severity and extent of villous atrophy according to the modified Marsh classification in coeliac disease patients and to the degree of villous enteropathy in HIV positive patients. However our data show an important distinction between the behaviour of those with and without HIV infection, with a definite reduction of serum citrulline in those with infection compared to those without. This corresponded to worse evaluations on Schenk and Klipstein scoring and villus height-to-crypt depth ratio. Citrulline concentrations significantly correlated with xylose absorption test results but at a level too low for clinical application.
Table 3 Spearman’s ranks correlation coefficients (p-value) for citrulline versus xylose test, lactulose-to-rhamnose ratio, and villous height-to-crypt depth ratio according to Bonferroni’s method in the group of HIV positive patients.
Citrulline Xylose test Lactulose-to-rhamnose ratio Villous height-to-crypt depth ratio
Citrulline
Xylose test
Lactulose-to-rhamnose ratio
Villous height-to-crypt depth ratio
1000 0.431 (0.05) 0.076 (1.00) 0.038 (1.00)
1000 0.073 (1.00) 0.064 (1.00)
1000 0.192 (1.00)
1000
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In patients with short bowel syndrome, there is a positive relationship between citrulline and absorptive enterocyte mass, but our previous data indicated that this relationship was less pronounced at higher citrulline values and in patients with borderline status.14 This might help to explain why citrulline concentrations correlate relatively weakly with xylose absorption, but the notoriously poor reproducibility of the xylose test may also be partly to blame. This concept might be extended to the potential use of serum or plasma citrulline concentration as an objective tool for the indication for and route of nutritional support as has been previously suggested in HIV-associated intestinal disease.11 The eight of Crenn’s patients who needed parenteral nutrition included all six patients with citrulline levels below 10 mmol/L, and citrulline normalized with recovery from enteropathy through specific treatment.11 However, another study17 using energy absorptiometry to assess the severity of enterocyte damage reported contradictory results in showing that plasma citrulline concentrations yielded poor diagnostic accuracy for the detection of decreased intestinal absorption capacity. In interpreting and integrating these results it should be taken into account that citrulline concentration cannot alone reflect overall digestive function. Citrulline appears however to be a reliable marker of enterocyte absorptive mass, especially in severe malabsorption, while small bowel energy absorption incorporates (inter alia) other mucosal functions, bilio-pancreatic secretions, and digestive motility. The gold-standard in this context might be considered to come from balance studies, but these are time consuming and highly dependent on unusual levels of patient compliance (especially in an outpatient setting). Against this, citrulline compares well, with the important reservation that it is more reliable for severe malabsorption than for milder disease.15 Citrulline was significantly reduced in our adults who had recently experienced diarrhoea. However, diarrhoea is partially independent of intestinal function and small bowel mucosal architectural changes in HIV related malabsorption.27e30 Ullrich et al.28 have also demonstrated a reduction in villus length and surface area in HIV positive patients compared to unmatched, presumably HIV negative controls, but with no relationship to intestinal infection or CD4 count. The crypt length was increased only in patients with intestinal infection However, enteropathy is largely a feature of late stage disease, and the characteristics of early pathological events, and their timing during HIV infection, remain largely unknown.31e33 In our study the citrulline concentration correlated significantly with crypt depth in all subjects affected by tropical enteropathy. However there was a statistically significant correlation between citrulline and crypt depth, villous height/crypt depth ratio and Klipstein score only in the HIV positive. We have previously concluded that increased crypt depth is the earliest significant change in the small bowel mucosa in HIV-infected adults,7 and can now - in the light of the present data - suggest that circulating citrulline is a reliable marker of early morphological and functional changes in HIV-infected small bowel mucosa. In our study HIV-infected patients without known gastrointestinal pathogens had shortened villi compared to age- and sexmatched controls, as well as having abnormal D-xylose absorption tests. These phenomena were accounted for mainly by those with low CD counts. Pathogen-negative HIV positive enteropathy thus reflects malabsorption resulting from small bowel architectural and functional abnormalities, but extrapolation to a causal link with the HIV itself, in keeping with the concept of “AIDS enteropathy” of chronic pathogen-negative diarrhoea and associated histological and functional changes would be inappropriate.34,35 Explants of small intestine from aborted 14- to 21- week human fetuses survive in culture for up to 14 days and can be used to study
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the gut mucosa and the effects of various stimuli on small bowel cell turnover and differentiation.36 Exposure of explants to HIV results in infection of lamina propria lymphocytes and induction of epithelial crypt cell proliferation,37 but the enterocytes themselves are not infected. Any enteropathic effect of HIV itself is therefore likely to be mediated by cytokine production from infected cells in the gut associated lymphoid tissue.32,37e39 This would explain the lack of correlation of severity of intestinal injury with the intensity of mucosal HIV infection. As crypt cell hyperplasia and villous atrophy are predominantly T cell dependent,40 it is unsurprising that these are affected in HIV infection of the gut mucosal T cells. It is possible that estimation of the circulating citrulline is acting as a close surrogate for the measurement of these parameters and the consequent functional changes in HIV positive patients. In conclusion serum citrulline concentration appears to be a reliable marker of the structural and functional integrity of the small intestine in those with HIV infection. As measuring citrulline is relatively easy, inexpensive and repeatable, it deserves attention as a potentially valuable tool in monitoring the effects of antiretroviral therapy on the gut. Its putative value is may indeed be increased by the frequent discordance of the presence of diarrhoea and features of malnutrition with the severity of HIV-related small bowel enteropathy. 5. What is current knowledge Circulating citrulline concentration correlates with reduced enterocyte mass independently of nutritional and inflammatory status. Citrulline concentration does not seem to be a sufficiently sensitive marker for rejection or viral enteritis as its values decline only in more severe intestinal mucosal damage. The time course of blood citrulline following radiation is in agreement with well known radiation effects on intestinal mucosa and clinical observations of acute intestinal injury.
6. What is new here The citrulline concentration appears to be a reliable marker of small bowel structural and functional integrity. It warrants attention as a possible surrogate for evaluation of HIV related enteropathy. Citrulline concentrations show a significant inverse correlation with the Schenk and Klipstein score in HIV positive patients Serum citrulline measurement is feasible and repeatable. It has the potential to be used as a marker of response to therapy in patients with HIV infection.
Statement of authorship All authors made a substantial contribution to the conception and design of the study, and to the interpretation of the results. All were involved in the drafting and/or revision of the manuscript, and all gave their approval for the submitted manuscript. Conflict of interest None of the authors has any conflict of interest to declare. Acknowledgements We gratefully acknowledge Roy Sherwood from King’s College Hospital who devised the citrulline assay method and carried out all the measurements. Many thanks to Simone Bartolini and Winnie
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Dhaliwal for their kind help. This study was supported in part by the Italian Government Research Fund. The support of the UK NIHR via the Comprehensive Biomedical Research Centre at UCL/UCLH is also gratefully acknowledged.
References 1. Cook GC, Segal I, Farthing MJ. ‘Tropical sprue’: some early investigators favoured an infective cause, but was a coccidian protozoan involved? Gut 1996;39:428e9. 2. Veitch AM, Kelly P, Zulu IS, et al. Tropical enteropathy: a T-cell mediated crypt hyperplastic enteropathy. Eur J Gastroenterol Hepatol 2001;13:1175e81. 3. Keusch GT. Subclinical malabsorption in Thailand. I. Intestinal absorption in Thai children. Am J Clin Nutr 1972;25:1062e6. 4. Lindenbaum J, Alam AK, Kent TH. Subclinical small-intestinal disease in East Pakistan. BMJ 1966;2:1616e9. 5. Ukabam SO, Homeida MM, Cooper BT. Small intestinal permeability in normal Sudanese subjects: evidence of tropical enteropathy. Trop Med Hyg 1986;80:204e7. 6. Iqbal TH, Lewis KO, Gearty JC, Cooper BT. Small intestinal permeability to mannitol and lactulose in the three ethnic groups resident in west Birmingham. Gut 1996;39:199e203. 7. Kelly P, Menzies I, Crane R, Zulu I, Nickols C, Feakins R, et al. Responses of small intestinal architecture and function over time to environmental factors in a tropical population. Am J Trop Med Hyg 2004;70:412e9. 8. Pappas PA, Saudubray JM, Tzakis AG, Rabier D, Carreno MY, Gomez-Marin O, et al. Serum citrulline and rejection in small bowel transplantation: a preliminary report. Transplantation 2001;72:1212e6. 9. Pappas PA, Saudubray JM, Tzakis AG, Rabier D, Carreno MY, Gomez-Marin O, et al. Serum citrulline as a marker of acute cellular rejection for intestinal transplantation. Transpl Proc 2002;34:915e7. 10. Crenn P, Vahedi K, Lavergne-Slove A, Cynober L, Matuchansky C, Messing B. Plasma citrulline: a marker of enterocyte mass in villous atrophy-associated small bowel disease. Gastroenterology 2003;124:1210e9. 11. Crenn P, de Truchis P, Neveux N, Galpérine T, Cynober L, Melchior JC. Plasma citrulline is a biomarker of enterocyte mass and an indicator of parenteral nutrition in HIV-infected patients. Am J Clin Nutr 2009;90:1e8. 12. Hozjasz KK, Szaflarska-Poplawska A, Oltarzewski M, Mazur J, Müller L, Jab3oñska E, Milanowski A. Whole blood citrulline levels in patients with coeliac disease. Pol Merkur Lekarski 2006;20:173e5. 13. Gondolesi G, Ghirardo S, Raymond K, Hoppenhauer L, Surillo D, Rumbo C, et al. The value of plasma citrulline to predict mucosal injury in intestinal allografts. Am J Transpl 2006;6:2786e90. 14. Lutgens L, Lambin P. Biomarkers for radiation induced small bowel epithelial damage: an emerging role for plasma Citrulline. World J Gastroenterol 2007;13:3033e42. 15. Papadia C, Sherwood RA, Kalantzis C, Wallis K, Volta U, Fiorini E, et al. Plasma citrulline concentration: a reliable marker of small bowel absorptive capacity independent of intestinal inflammation. Am J Gastroenterol 2007;102:1474e82. 16. Luo M, Fernandez-Estivariz C, Manatunga AK, Bazargan N, Gu LH, Jones DP, et al. Are plasma citrulline and glutamine biomarkers of intestinal absorptive function in patients with short bowel syndrome? J Parenter Enteral Nutr 2007;31:1e7. 17. Peters JH, Wierdsma NJ, Teerlink T, van Leeuwen PA, Mulder CJ, van Bodegraven AA. Poor diagnostic accuracy of a single fasting plasma citrulline concentration to assess intestinal energy absorption capacity. Am J Gastroenterol 2007;102:2814e9. 18. Miceli E, Poggi N, Missanelli A, Bianchi P, Moratti R, Corazza GR. Is serum citrulline measurement clinically useful in coeliac disease? Intern Emerg Med 2008;3:233e6.
19. Kelly P, Davies SE, Mandanda B, Veitch A, McPhail G, Zulu I. Enteropathy in Zambians with HIV related diarrhoea: regression modelling of potential determinants of mucosal damage. Gut 1997;41:811e6. 20. Klipstein FA, Beauchamp I, Corcino JJ, Maldonado M, Tomasini JT, Maldonado N, et al. Nutritional status and intestinal function among rural populations of the West Indies. Gastroenterology 1972;63:758e67. 21. Vandgama JV, Evered DF. Characteristic of L-citrulline transport across rat small intestine in vitro. Pediatr Res 1992;32:472e8. 22. Bahri S, Curis E, Aussel C, Chaumeil J, Cynober L, Zerrouk N. Caractérisation in vitro du transport intestinal de la citrulline. Nutr Clin Metab 2006;20:S111eS112. 23. Wu G, Morris Jr SM. Arginine metabolism: nitric oxide and beyond. Biochem J 1998;336:1e17. 24. Crenn P, Coudray-Lucas C, Thuillier F, Cynober L, Messing B. Postabsorptive plasma citrulline concentrations as a marker of absorptive enterocyte mass and intestinal failure in humans. Gastroenterology 2000;119:1496e505. 25. Wakabayashi Y, Yamada E, Yoshida T, Takahashi H. Arginine becomes an essential aminoacid after massive resection of rat small intestine. J Biol Chem 1994;269:32667e71. 26. Osowska S, Moinard C, Neveux N, Loi C, Cynober L. Citrulline increases arginine pools and restores nitrogen balance after massive intestinal resection. Gut 2004;53:1781e6. 27. Miller AR, Griffin GE, Batman P, Farquar C, Forster SM, Pinching AJ, Harris JR. Jejunal mucosal architecture and fat absorption in male omosexuals infected with human immunodeficiency virus (HIV): evidence for HIV-induced enteropathy. Q J Med. 1988;69:1009e19. 28. Ullrich R, Heise W, Bergs C, L’age M, Höffken G, Riecken EO. Small intestinal structure and function in patients infected with human immunodeficiency virus (HIV) infected patients. Digestion 1989;46:302e7. 29. Ullrich R, Zeitz M, Heise W, L'age M, Ziegler K, Bergs C, Riecken EO. Mucosal atrophy is associated with loss of activated T cells in the duodenal mucosa of human immunodeficiency virus (HIV) infected patients. Digestion 1990;46:302e7. 30. Ullrich R, Heise W, Bergs C, L'age M, Riecken EO, Zeitz M. Effects of zidovudine treatment on the small intestinal mucosa in patients infected with the human immunodeficiency virus. Gastroenterology 1992;102:1483e92. 31. Sankaran S, George M, Reay E, Guadalupe M, Flamm J, Prindiville T, Dandekar S. Rapid onset of intestinal epithelial barrier dysfunction in primary human immunodeficiency virus infection is driven by an imbalance between immune response and mucosal repair and regeneration. J Virol 2008;82:538e45. 32. Epple HJ, Schneider T, Troeger H, Kunkel D, Allers K, Moos V, et al. Impairment of the intestinal barrier is evident in untreated but absent in suppressively treated HIV-infected patients. Gut 2009;58:220e7. 33. Dworkin B, Wormser GP, Rosenthal WS, Heier SK, Braunstein M, Weiss L, et al. Gastrointestinal manifestation of the acquired immunodeficiency syndrome; a review of 22 cases. Am J Gastroenterol 1985;80:774e8. 34. Sewankambo N, Mugerwa RD, Goodgame R, Carswell JW, Moody A, Lloyd G, Lucas SB. Enteropathic AIDS in Uganda. An endoscopic, histological and microbiological study. AIDS 1987;1:9e13. 35. Fleming SC, Kapembwa MS, MacDonald TT, Griffin GE, et al. Direct in vitro infection of human intestine with HIV-1. AIDS 1992;6:1099e104. 36. Batman PA, Fleming SC, Sedgwick PM, MacDonald TT, Griffin GE. HIV infection of human fetal intestinal explant cultures induces epithelial cell proliferation. AIDS 1994;8:161e7. 37. Kotler DP, Reka S, Chow K, Orenstein JM. Effect of enteric parasitoses and HIV infection upon small intestinal structure and function in patients with AIDS. J Clin Gastroenterol 1993;16:10e5. 38. Veazey RS, Marx PA, Lackner AA. The mucosal immune system: primary target for HIV infection and AIDS. Trend Immunol 2001;11:626e33. 39. Reka S, Borchich A, Cronin W, Kotler D. Diarrhoea associated with intestinal HIV infection in ARC. Clin Res 1989;37:A371. 40. Veitch AM, Higgins LM, Bajaj-Elliot M, Farthing MJ, MacDonald TT. Impaired rejection and mucosal injury of small intestinal allograft lacking the interferong receptor. Int J Exp Path 2003;84:107e13.
Contents lists available at ScienceDirect
Clinical Nutrition journal homepage: http://www.elsevier.com/locate/clnu
Original Article
Plasma citrulline as a quantitative biomarker of HIV-associated villous atrophy in a tropical enteropathy population Cinzia Papadia a, b, Paul Kelly c, d, Saverio Caini e, Gino Roberto Corazza f, Tamara Shawa c, d, Angelo Franzè b, Alastair Forbes a, *, Antonio Di Sabatino f a
Department of Gastroenterology, University College Hospital, London, United Kingdom Department of Gastroenterology, University of Parma, Parma, Italy Institute of Cell and Molecular Science, Barts & The London School of Medicine, Queen Mary University of London, United Kingdom d Tropical Gastroenterology and Nutrition Group, University of Zambia School of Medicine, Lusaka, Zambia e Molecular and Nutritional Epidemiology Unit, Istituto per lo Studio e la Prevenzione Oncologica, Florence, Italy f First Department of Medicine, S. Matteo Hospital, University of Pavia, Pavia, Italy b c
a r t i c l e i n f o
s u m m a r y
Article history: Received 8 December 2009 Accepted 13 April 2010
Background & aims: Studies have shown that the circulating citrulline concentration is decreased in patients with proximal small bowel villous atrophy from coeliac disease and more so in patients with extensive damage to the intestinal mucosa, but there have been few data on HIV enteritis and tropical enteropathy (TE). Our primary aim was to correlate serum citrulline with the degree of reduction of the enterocyte mass in HIV-infected patients with TE. Methods: Postabsorptive fasting serum citrulline was measured in 150 TE pts, 44 of whom had HIV infection, using reverse phase, high performance liquid chromatography. Absorptive capacity and permeability were measured after intrajejunal instillation of 4 sugars (5 g lactulose, 1 g L-rhamnose, 0.5 g D-xylose, 0.2 g 3-O methyl Dglucose) with assay by thin-layer chromatography. Morphometric analysis was carried out on jejunal biopsies. Results: In HIV positive patients, the median serum citrulline was significantly lower (median 19, interquartile range (IQR) 17e24 mmol/L) than in HIV negative patients (median 27, IQR 23e33 mmol/L; p < 0.001). There were statistically significant correlations (p < 0.005) between citrulline and: crypt depth; villous height/crypt depth ratio; Shenk-Klipstein score; and xylose absoption, only in the HIV positive. Conclusions: Serum citrulline concentration appears to be a quantitative biomarker of small bowel mass integrity in HIV positive enteropathy and desrves assessment as a surrogate for monitoring anti-retroviral therapy. Ó 2010 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved.
Keywords: Citrulline HIV Tropical enteropathy Sprue Celiac Biomarker
1. Introduction Tropical enteropathy (TE) is a population shift in intestinal mucosal morphology and function which resembles an asymptomatic villous atrophy of the small bowel. It is characterised by subclinical malabsorption and increased permeability, but its clinical significance is unknown. It is not even clear whether it is truly an abnormality or an adaptive response to environmental factors. It is most prevalent in the developing world within 30 of either side * Corresponding author at: Department of Gastroenterology and Clinical Nutrition, Maple House, University College Hospital, 235 Euston Road, London, NW1 2BU, United Kingdom. Tel.: þ44 (0) 845 155 5000x9011; fax: þ44 (0) 20 7380 9699. E-mail address: [email protected] (A. Forbes).
of the equator, but it is not limited to indigenous populations as it also affects European and American expatriates living in tropical regions. Mild reduction in villous height, broadening of the villi and increased crypt depth are the most common histological features described in TE.1 An increase in inflammatory cell infiltrate may occur, as well as mucosal T cell activation and crypt hyperplasia.2 Functional changes have been described, including subclinical malabsorption of carbohydrates,3 fat and vitamin B12,4 and increased mucosal permeability.5,6 TE is observed in asymptomatic individuals, which distinguishes it from tropical sprue, a severe but now rare clinical illness. The histopathological changes of TE resemble those occurring in the early stages of coeliac disease and in other immune mediated enteropathies. It is likely that mucosal immune activation, perhaps
0261-5614/$ e see front matter Ó 2010 Elsevier Ltd and European Society for Clinical Nutrition and Metabolism. All rights reserved. doi:10.1016/j.clnu.2010.04.008
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in response to repeated intestinal infections, results in the observed microscopic changes described in TE. We have previously observed that Citrobacter rodentium may play such a role.7 In many tropical countries, the high prevalence of HIV leads to HIV enteropathy being superimposed on TE. On the whole the morphological and functional changes become significant in advanced AIDS.7 There are surprisingly few data on the responsiveness of the enteropathy to treatment of intestinal infection and to treatment with anti-retroviral drugs, and a non-invasive marker of intestinal mucosal health would be very useful in analysing the response of the mucosa to treatment. A relationship between plasma citrulline and intestinal function has been proposed from studies examining rejection of small bowel allografts, in which plasma citrulline concentrations were found to be reduced in cases of mucosal damage, and with an inverse correlation to severity on biopsy.8,9 Lower plasma citrulline concentrations have also been recorded in patients with villous atrophy than in healthy subjects or patients with anorexia nervosa. A range of thresholds was suggested: <10 mmol/L representing diffuse total villous atrophy; through 10e30 mmol/L for nonextensive or partial villous atrophy; to normal subjects who have levels >30 mmol/L10 One of the most exciting potential applications is the use of the circulating citrulline concentration as an objective tool for determining the need for and route of nutritional support.10e12 Evidence from human and animal studies suggests that citrulline may prove useful as a more general marker of small bowel epithelial damage regardless of cause.13,14 There are few data on citrulline levels in humans. In our previous work we reported a quadratic (non-linear) correlation between fasting citrulline concentrations and remnant small bowel length, indicating that decreased citrulline concentrations predominantly occur when intestinal disease (or loss) is severe.15 This observation supported the earlier work from Paris and elsewhere.10,13,14. However, Luo et al.16 reported no relation between fasting plasma citrulline concentrations and intestinal absorptive capacity in short bowel syndrome, Peters et al.17 described poor diagnostic value of citrulline concentrations in intestinal energy absorption in enterocyte damage, and Miceli et al.18 were unable to confirm the value of plasma citrulline as a sensitive marker of intestinal atrophy. The position of citrulline as a marker of intestinal function cannot therefore be considered fully established. In the current study we hypothesize that serum citrulline concentration might be a reliable marker of early morphological and functional changes in HIV enteropathy. 2. Methods 2.1. Patients Endoscopic jejunal biopsy specimens and peripheral blood samples were collected from 150 adult patients with TE to investigate whether serum citrulline concentrations mirrored the degree of mucosal injury. TE was defined histologically from the presence of reduction in villous height, broadening of the villi and increased crypt depth in the absence of serological evidence for coeliac disease or documentable gastrointestinal infection TE.1 We included both HIV seropositive and HIV seronegative TE patients. A cohort study had already been set up in a poor sector of Misisi township in Lusaka, the capital of Zambia, and we have previously reported the morphometric and functional data to which reference is made here.7,19 The study was reviewed and approved by the Research Ethics Committees of both the University of Zambia and the London School of Hygiene and Tropical Medicine.7 Patient characteristics at entry to the study are listed in Table 1. Participants
were enrolled in a three year program of investigation and treatment of each episode of ill-health. They were then asked to attend once a year for investigation, blood tests, enteroscopy with jejunal biopsy, and sugar absorption/permeability tests, all performed in the course of a single day in the endoscopy unit. Patients also underwent monthly stool sample examination to document parasitic and bacterial infections. Venous blood sampling after an overnight fast was carried out. Standard serum biochemical analyses (liver function tests albumin, urea, creatinine, electrolytes, C-reactive protein), and a full blood count were performed. Serum was saved for citrulline assay (see below). Testing for HIV was conducted and CD4 counts were determined using a FACSCount analyzer (Becton Dickinson, Franklin Lakes, NJ) according to the manufacturer’s instructions. Pregnant or lactating patients were temporarily excluded from the study. Endoscopy was also deferred in participants who had experienced diarrhoea and had taken antibiotics or non-steroidal anti-inflammatory drugs until one month had elapsed. The incidence of diarrhoea was assessed by recall using a simple questionnaire administered every two weeks, and by active case-finding while offering investigation and treatment of each episode. Participants then submitted a stool sample that was analyzed for the presence of protozoa, bacteria, and helminths. At the time of recruitment highly active anti-retroviral therapy was not available in Zambia. 2.2. Reverse phase liquid chromatography with UV detection for serum citrulline The blood samples were centrifuged (2500 g, 10 min) at room temperature. Aliquots of serum were then stored at 80 C until batched derivatization and reverse-phase HPLC. A 1 mmol/L stock solution of citrulline (Sigma) was diluted to 1, 5, 10, 50, and 100 mmol/L concentrations, and used as standards for assay calibration. Phenyl-isothiocyanate was selected as the derivatization reagent as it combines high sensitivity with ready detection by its ultraviolet (UV) absorption at 254 nm. The Thermo Analytical P4000 quaternary pump, AS3000 autosampler, 5 mm guard column 5 mm i.d., C18 (ODS) analytical column 25 mm long 5 mm i.d., the Uv 2000 UV detector, and the CHROMQUEST computing integrator (Thermo, St. Albans, UK) were employed. The blood samples were deproteinized with 10% sulfosalicylic acid, and 750 mmol/L norleucine was used as an internal standard. Supernatants were diluted with acetonitrile and phenyl-isothiocyanate coupling reagent for 20 min at room temperature, evaporated to dryness on a rotary evaporator (Genevac EZ2, Dublin, Ireland), and then resuspended in the mobile phase. Gradient elution was achieved with sodium acetate/acetonitrile. The gradient profile was modified to enable adequate separation of citrulline from taurine. At baseline, there was 90% buffer 1: sodium acetate 0.01 M, and 10% buffer 2: acetonitrile/sodium acetate (60% v/v) 0.01 M. The injection volume was 180 mL and the column flow rate was 1.0 mL/min. At 40 min, i.e. after the citrulline peak and the internal standard had eluted from the column (at approximately 12.3 min and at approximately 39 min, respectively) the percentage of acetonitrile was rapidly increased to 100% with the expectation of eluting all remaining amino acids within 5 min. The complete run time was therefore 45 min. The solvent gradient was returned to its starting composition over the following 5 min, and allowed to re-equilibrate, thus permitting introduction of the next sample 25 min later. 2.3. Sugar testing of absorption and permeability The four sugar test solution instilled into the small intestine contained 0.5 g of xylose, 1 g of rhamnose, 5 g of lactulose, and 0.2 g of 3-O-methyl Dglucose (Sigma Chemicals, Poole, UK) in 100 mL of
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797
Table 1 Median values (IQR) of age, anthropometric measures and malabsorption indices.
Number of Patients Age (yrs) BMI (kg/m2) MUAC (cm) Citrulline (mmol/L) D-Xylose (%) L/R ratio Villous height (mm) Crypt depth (mm) VH/CD ratio
pa
All
HIV þ
HIV
145 35 (27e44) 20.5 (18.7e23.5) 26.5 (25.5e29) 25 (19e31) 19.7 (15e24) 0.07 (0.04e0.10) 258 (229e296) 153 (137e175) 1.71 (1.47e1.95)
44 28 (24e34) 20.8 (19.5e22.1) 26 (25.5e28.5) 19 (17e24) 18.3 (13.2e21.2) 0.07 (0.05e0.10) 238.5 (224e279) 162 (146.5e193) 1.53 (1.26e1.72)
101 39 (29e48) 20.2 (18.7e24.6) 27 (25.5e29) 27 (23e33) 20.3 (15.3e24.6) 0.06 (0.04e0.10) 264 (234e306) 146 (132e170) 1.81 (1.61e2.01)
<0.001 0.698 0.675 <0.001 0.177 0.434 0.018 0.001 <0.001
Men
Women
62 37 (28e49) 19.7 (18.0e20.9) 26 (25e28) 28 (22e34) 20.8 (15.3e25) 0.06 (0.04e0.10) 253.5 (228e286) 147.5 (131e175) 1.71 (1.50e1.95)
83 32 (26e41) 21.6 (19.9e25.2) 28 (26e29) 24 (18e29) 18.5 (13.9e23.6) 0.07 (0.05e0.10) 261 (231e315) 157 (138e175) 1.71 (1.44e1.95)
pa 0.025 <0.001 0.004 0.001 0.209 0.299 0.103 0.212 0.903
BMI, body mass index; MUAC, mid-upper arm circumference; L/R, lactulose-to-rhamnose ratio; VH/CD, villous height-to-crypt depth ratio. a p-values are for Wilcoxon rank-sum test comparing medians.
distilled water. The same solution was given orally in those not undergoing endoscopy. Urine samples were collected prior to endoscopy and for exactly 5 h afterwards and preserved with merthiolate (final concentration of approximately 0.04%). Urine samples were frozen at 80 C for transport and analyzed by thinlayer chromatography.
villi, and grade 4 is flat. For cellular infiltration: grade 0 is normal, grade 1 shows a mild increase, grade 2 a moderate increase, and grade 3 a marked increase. To ensure that the morphometric technique was reproducible, measurements were repeated in triplicate from the same areas of 8 jejunal biopsies. The inter-assay variation was 13% indicating that variation seen between individuals is unlikely to be explained by effects of sampling.
2.4. Morphometric analysis 2.5. Statistical analysis Morphometric analysis was carried out as previously described.7,19 Slides were evaluated by a senior pathologist before morphometry was performed by a single observer. The following variables were then measured: 1) cross-sectional area of the villous compartment, which is representative of villous compartment volume (VCV); 2) total length of epithelium, which is used as a measure of epithelial surface area (ESA); 3) villous height (VH), which is measured from villus tip to crypt-villus junction as judged by eye (care was taken to include every recognizable villous unit of well orientated mucosa); 4) crypt depth (CD), which was measured in the same way, one for one with each villus; and 5) villous width (VW), which was measured automatically as the maximum width of the villus along a line perpendicular to VH in each unit of the villous compartment. The criteria suggested by Schenk and Klipstein20 were used to grade histological sections for villous abnormalities and cellular infiltrations. For villous appearance: grade 0 is normal, grades 1e3 show progressive broadening and flattening of
Patients were studied according to the following criteria: gender, HIV status, degree of small bowel mucosa atrophy. To compare medians and interquartile ranges (IQR) of continuous variables, the Wilcoxon rank-sum test and KruskaleWallis test were used, with a p-value less than 0.05 considered to confer statistical significance. Permeability was expressed as lactuloseto-L-rhamnose ratio (where normal subjects have a ratio of <0.06%) and absorption determined by percentage urinary retrieval of D-xylose at 5 h (which is conventionally considered to be normal if greater than 20%). Correlations between citrulline and the other measured variables including permeability, absorption, villus height-to-crypt depth ratio, and CD4 counts, were also studied. A non-parametric correlation method (Spearman’s rank test) was applied to analyze these relationships. A Bonferroni correction test for multiple comparisons was then carried out. Sensitivity and specificity were calculated using
Table 2 Median values of citrulline according to Klipstein index and HIV status.
HIV negative (N ¼ 65) HIV positive (N ¼ 32) ALL (N ¼ 97)
a
N median citrulline (mmol/l) N median citrulline (mmol/l) N median citrulline (mmol/l)
Normal mucosa
Shortened villi
Partial atrophy
Subtotal atrophy
Flat mucosa
pa
0-1-2
3e4
pa
0
1
2
3
4
e e
6 25.0
50 29.0
9 31.0
e e
0.316
56 28.0
9 31.0
0.954
e e
1 25.0
16 21.0
14 18.0
1 18.0
0.784
17 22.0
15 18.0
0.496
e e
7 25.0
66 27.0
23 21.0
1 18.0
0.110
73 26.0
24 21.0
0.027
p-values are for KruskaleWallis test and Wilcoxon rank-sum test comparing medians.
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permeability (p ¼ 0.01) in both HIV positive and HIV negative patients, however this significance was not confirmed after Bonferroni’s correction test for multiple correlations (Table 3). A significant correlation between citrulline concentrations and the xylose absorption test was however confirmed with the Bonferroni method (Table 3). Although the specificity was 69%, the sensitivity and predictive values were disappointingly low (<50%) 4. Discussion
Fig. 1. Correlation between D-xylose urinary test and serum citrulline concentration in 101 HIV negative and 44 HIV-positive patients with tropical enteropathy.
a citrulline threshold of 20 mmol/l below which intestinal insufficiency was to be diagnosed. All analyses were performed using Stata 9.0 SE. The clinical study was registered as a trial with clinicaltrials.gov/ ct2/show/results/NCT00816842.
3. Results Our data set included 150 subjects (64 men and 86 women) affected by TE. HIV status was available for 145 subjects and only these individuals are considered further. Some 30.3% of the total sample were HIV seropositive (44 of 145; 21% in men:13 of 62; 37.3% in women: 31 of 83). One quarter (n ¼ 11) of the HIV seropositive patients had a CD4 count below 200. Citrulline concentrations were very similar in all HIV seronegative subjects (Table 1). The median villus-height-to-crypt depth ratio was significantly lower in HIV positive (1.53 mm IQR 1.26e1.72 mm) than in HIV negative patients (1.81 mm; IQR 1.61e2.01 mm). Citrulline concentrations were significantly lower (p < 0.001) in HIV positive patients (median 19 mmol/L; IQR 17e2 mmol/L) than in HIV negative patients (median 27 mm; IQR 23e33 mm). The percentage of patients with citrulline concentrations lower than 20 mmol/L was 14.9% and 52.3% respectively in HIV negative and HIV positive patients. Serum citrulline concentration showed a statistically significant negative correlation with the Schenk and Klipstein score in HIV positive patients (p < 0.004). Moreover the percentage of patients with a citrulline concentration below 20 mmol/L was inversely correlated with the Schenk and Klipstein score (Table 2). There was a sensitivity of 60%, a specificity of 59%, and positive and negative predictive values of 56% and 63% respectively in the HIV positive. Serum citrulline concentration also showed a statistically significant (p < 0.001) positive correlation with xylose absorption (Fig. 1) in the HIV positive patients, but the sensitivity, specificity and predictive values were too low to be of clinical value. Citrulline levels correlated significantly with mid-upper arm circumference (p ¼ 0.04) in the whole group, and were significantly reduced in all those with diarrhoea (p ¼ 0.003). A statistically significant positive correlation was found between citrulline concentrations and
Citrulline contained in food is absorbed by the intestine. Its presence in the lumen, leads to citrulline accumulation in enterocytes, especially in the middle and lower ileum.21 A broad set of transporters seems to be able to transport citrulline from the lumen to the cell.22 Most of the circulating citrulline however comes from glutamine conversion in enterocytes.23 Citrulline is excreted by the kidneys after conversion to arginine. In severe villous atrophy or in massive intestinal resection the main site of net citrulline production is greatly reduced. Probably as a consequence of the diminished circulating citrulline, decreased arginine is also observed,24,25 and it is suggested that arginine becomes an essential amino acid after massive intestinal resection.25 In this metabolic context the citrulline pathway might be thought to be a more important alternative route for arginine metabolism. Citrulline might therefore better express small bowel enterocyte functioning mass in the presence of poor nitrogen balance, since in this condition it becomes the most important amino acid substrate for protein synthesis in enterocytes. Osowska et al. tested this possibility in animals with strongly supportive results.26 However, while these concepts are becoming well established in intestinal failure and short bowel syndrome, their application to functional intestinal failure (ie intestinal failure in the absence of massive resection) has been more controversial and was one of the drivers for the present study. The pioneering study by Crenn et al.10 showed that in chronic villous atrophy (e.g. coeliac disease, tropical enteropathy and various infectious enteritides) citrulline is decreased (<20 mmol/L) in patients with destructive lesions of the proximal small intestinal villous architecture. It is more severely decreased in patients with extensive (proximal and distal) impairment of intestinal mucosa, and in these patients, a citrulline value below 10 mmol/L was highly predictive of the need for parenteral nutrition. Similar results were recently reported in patients with HIV enteropathy or severe intestinal infectious disease.11 In our study serum citrulline concentrations showed a significant inverse correlation with the Schenk and Klipstein score only in HIV positive patients. Our data are in agreement with previous studies10,11 where citrulline levels correlated with severity and extent of villous atrophy according to the modified Marsh classification in coeliac disease patients and to the degree of villous enteropathy in HIV positive patients. However our data show an important distinction between the behaviour of those with and without HIV infection, with a definite reduction of serum citrulline in those with infection compared to those without. This corresponded to worse evaluations on Schenk and Klipstein scoring and villus height-to-crypt depth ratio. Citrulline concentrations significantly correlated with xylose absorption test results but at a level too low for clinical application.
Table 3 Spearman’s ranks correlation coefficients (p-value) for citrulline versus xylose test, lactulose-to-rhamnose ratio, and villous height-to-crypt depth ratio according to Bonferroni’s method in the group of HIV positive patients.
Citrulline Xylose test Lactulose-to-rhamnose ratio Villous height-to-crypt depth ratio
Citrulline
Xylose test
Lactulose-to-rhamnose ratio
Villous height-to-crypt depth ratio
1000 0.431 (0.05) 0.076 (1.00) 0.038 (1.00)
1000 0.073 (1.00) 0.064 (1.00)
1000 0.192 (1.00)
1000
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In patients with short bowel syndrome, there is a positive relationship between citrulline and absorptive enterocyte mass, but our previous data indicated that this relationship was less pronounced at higher citrulline values and in patients with borderline status.14 This might help to explain why citrulline concentrations correlate relatively weakly with xylose absorption, but the notoriously poor reproducibility of the xylose test may also be partly to blame. This concept might be extended to the potential use of serum or plasma citrulline concentration as an objective tool for the indication for and route of nutritional support as has been previously suggested in HIV-associated intestinal disease.11 The eight of Crenn’s patients who needed parenteral nutrition included all six patients with citrulline levels below 10 mmol/L, and citrulline normalized with recovery from enteropathy through specific treatment.11 However, another study17 using energy absorptiometry to assess the severity of enterocyte damage reported contradictory results in showing that plasma citrulline concentrations yielded poor diagnostic accuracy for the detection of decreased intestinal absorption capacity. In interpreting and integrating these results it should be taken into account that citrulline concentration cannot alone reflect overall digestive function. Citrulline appears however to be a reliable marker of enterocyte absorptive mass, especially in severe malabsorption, while small bowel energy absorption incorporates (inter alia) other mucosal functions, bilio-pancreatic secretions, and digestive motility. The gold-standard in this context might be considered to come from balance studies, but these are time consuming and highly dependent on unusual levels of patient compliance (especially in an outpatient setting). Against this, citrulline compares well, with the important reservation that it is more reliable for severe malabsorption than for milder disease.15 Citrulline was significantly reduced in our adults who had recently experienced diarrhoea. However, diarrhoea is partially independent of intestinal function and small bowel mucosal architectural changes in HIV related malabsorption.27e30 Ullrich et al.28 have also demonstrated a reduction in villus length and surface area in HIV positive patients compared to unmatched, presumably HIV negative controls, but with no relationship to intestinal infection or CD4 count. The crypt length was increased only in patients with intestinal infection However, enteropathy is largely a feature of late stage disease, and the characteristics of early pathological events, and their timing during HIV infection, remain largely unknown.31e33 In our study the citrulline concentration correlated significantly with crypt depth in all subjects affected by tropical enteropathy. However there was a statistically significant correlation between citrulline and crypt depth, villous height/crypt depth ratio and Klipstein score only in the HIV positive. We have previously concluded that increased crypt depth is the earliest significant change in the small bowel mucosa in HIV-infected adults,7 and can now - in the light of the present data - suggest that circulating citrulline is a reliable marker of early morphological and functional changes in HIV-infected small bowel mucosa. In our study HIV-infected patients without known gastrointestinal pathogens had shortened villi compared to age- and sexmatched controls, as well as having abnormal D-xylose absorption tests. These phenomena were accounted for mainly by those with low CD counts. Pathogen-negative HIV positive enteropathy thus reflects malabsorption resulting from small bowel architectural and functional abnormalities, but extrapolation to a causal link with the HIV itself, in keeping with the concept of “AIDS enteropathy” of chronic pathogen-negative diarrhoea and associated histological and functional changes would be inappropriate.34,35 Explants of small intestine from aborted 14- to 21- week human fetuses survive in culture for up to 14 days and can be used to study
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the gut mucosa and the effects of various stimuli on small bowel cell turnover and differentiation.36 Exposure of explants to HIV results in infection of lamina propria lymphocytes and induction of epithelial crypt cell proliferation,37 but the enterocytes themselves are not infected. Any enteropathic effect of HIV itself is therefore likely to be mediated by cytokine production from infected cells in the gut associated lymphoid tissue.32,37e39 This would explain the lack of correlation of severity of intestinal injury with the intensity of mucosal HIV infection. As crypt cell hyperplasia and villous atrophy are predominantly T cell dependent,40 it is unsurprising that these are affected in HIV infection of the gut mucosal T cells. It is possible that estimation of the circulating citrulline is acting as a close surrogate for the measurement of these parameters and the consequent functional changes in HIV positive patients. In conclusion serum citrulline concentration appears to be a reliable marker of the structural and functional integrity of the small intestine in those with HIV infection. As measuring citrulline is relatively easy, inexpensive and repeatable, it deserves attention as a potentially valuable tool in monitoring the effects of antiretroviral therapy on the gut. Its putative value is may indeed be increased by the frequent discordance of the presence of diarrhoea and features of malnutrition with the severity of HIV-related small bowel enteropathy. 5. What is current knowledge Circulating citrulline concentration correlates with reduced enterocyte mass independently of nutritional and inflammatory status. Citrulline concentration does not seem to be a sufficiently sensitive marker for rejection or viral enteritis as its values decline only in more severe intestinal mucosal damage. The time course of blood citrulline following radiation is in agreement with well known radiation effects on intestinal mucosa and clinical observations of acute intestinal injury.
6. What is new here The citrulline concentration appears to be a reliable marker of small bowel structural and functional integrity. It warrants attention as a possible surrogate for evaluation of HIV related enteropathy. Citrulline concentrations show a significant inverse correlation with the Schenk and Klipstein score in HIV positive patients Serum citrulline measurement is feasible and repeatable. It has the potential to be used as a marker of response to therapy in patients with HIV infection.
Statement of authorship All authors made a substantial contribution to the conception and design of the study, and to the interpretation of the results. All were involved in the drafting and/or revision of the manuscript, and all gave their approval for the submitted manuscript. Conflict of interest None of the authors has any conflict of interest to declare. Acknowledgements We gratefully acknowledge Roy Sherwood from King’s College Hospital who devised the citrulline assay method and carried out all the measurements. Many thanks to Simone Bartolini and Winnie
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Dhaliwal for their kind help. This study was supported in part by the Italian Government Research Fund. The support of the UK NIHR via the Comprehensive Biomedical Research Centre at UCL/UCLH is also gratefully acknowledged.
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