American Journal of Transplantation 2004; 4: 414–418 Blackwell Munksgaard
C Blackwell Munksgaard 2004 Copyright
doi: 10.1111/j.1600-6143.2004.00338.x
Defining Normal Plasma Citrulline in Intestinal Transplant Recipients Gabriel E. Gondolesia,∗ , Stuart S. Kaufmana , Claude Sansaricqc , Margret S. Magidb , Kimiyo Raymondc , Liesl P. Iledana , Ye Taoa , Sander S. Flormana , Neal S. LeLeikoc and Thomas M. Fishbeina a
Recanati/Miller Transplantation Institute, Department of Pathology, and c Department of Pediatrics, Mount Sinai School of Medicine, New York, NY ∗ Corresponding author: Gabriel E. Gondolesi,
[email protected] b
Biopsy is the only means to identify intestinal graft rejection. Plasma citrulline (P-Cit) has been proposed as a marker for rejection after intestinal transplant (IT), but normative data is lacking. We analyzed P-Cit in IT recipients without rejection or other histological abnormalities. In 40 patients, P-Cit was measured with a Beckman amino acid analyzer within 24 h of protocol or clinically indicated endoscopic biopsy procured > 6 and < 360 days post-IT. Measurements included for analysis corresponded to normal (or minimally abnormal) biopsies that remained so for 7 days. These criteria were met by 145 samples from 10 adults and 14 children. Overall mean P-Cit (nmol/mL) was 34.0 ± 19.9. Mean P-Cit was 22.2 ± 13.2 between 6 and 30 days post-IT, 34.9 ± 17.2 (p = 0.001) between 30 and 60 days, 43.6 ± 15.8 between 60 and 90 days (p = 0.001), then stable until the end of the first year. Plasma citrulline was lower in 13 patients with body surface area (BSA) ≤ 1 m2 vs. 11 patients with BSA ≥ 1.1 m2 (p = 0.0001). Plasma citrulline increased linearly during the first 120 days in both BSA groups (r = 0.573 and r = 0.512; p = 0.0001). Within 3 months after IT, variations in P-Cit based on body size and postop interval should be considered when evaluating the need for histological confirmation of graft dysfunction. Key words: Citrulline, dysfunction, intestine, rejection, transplant Received 17 July 2003, revised 25 September 2003 and accepted for publication 17 October 2003
Introduction Diagnosis of rejection in intestinal transplantation depends on the combination of clinical assessment and histology, 414
which requires endoscopic biopsy. There is no serum assay or other noninvasive test that indicates allograft dysfunction and the need for confirmatory endoscopy analogous to the serum creatinine in kidney transplantation or serum liver chemistries in liver transplantation. Because endoscopic biopsy is potentially hazardous, a serum marker for intestinal dysfunction and/or rejection would be useful. Recently, others and the authors have proposed that plasma concentration of citrulline, a nonprotein amino acid, may be such a marker (1,2). Although citrulline is produced by both enterocytes and hepatocytes, only the intestine contributes significantly to circulating citrulline levels. The pathway of intestinal citrulline synthesis begins with conversion of glutamate to ornithine as catalyzed by pyrroline-5-carboxylate synthetase (P5C). Ornithine is then converted to citrulline via ornithine transcarbamoylase. Following release from the enterocytes into the portal circulation, citrulline is taken up primarily by the kidneys (83%), which in turn release arginine equivalent to 75% of the citrulline taken up (3–8) (Figure 1). Because enterocytes are the primary source of circulating citrulline, plasma citrulline levels reflect functional intestinal mass (9). Patients with short gut syndrome have reduced plasma citrulline concentrations in proportion to the length of the remnant intestine. Citrulline levels vary with age, ranging from 3 to 55 nmol/mL (beginning 1 month after birth to adulthood) with a mean of 38 ± 8 nmol/mL from samples obtained in 100 healthy volunteers (10). Transplantation offers a unique model of tissue metabolism, as most energy consumption by the graft stops during organ procurement and resumes after reperfusion. It is likely that the stress of transplantation reduces graft citrulline production. To use plasma citrulline concentration as a marker of intestinal allograft dysfunction, it is first necessary to define normal post transplant levels. We determined citrulline values after transplant in intestinal graft recipients with normal histology or minor, nonspecific changes.
Materials and Methods Between 10/1/2001 and 8/30/2002, we prospectively measured plasma citrulline concentration (P-Cit) in 18 patients before the transplant and in 40 intestinal transplant recipients within 24 h of endoscopic biopsy, which was
Plasma Citrulline in Small Bowel Recipients Muscle
Enterocyte Alanine Proline
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Ornithine
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Arginine
was significantly lower than in the six patients with BSA > 1 m2 (22.5 ± 3.9 nmol/mL; p = 0.05). Patients evaluated for short gut syndrome (SGS) had a P-Cit of 18.5 ± 12.1 nmol/mL while those with no-short gut (NSGS) had a PCit of 16.6 ± 8.3 nmol/mL (vs. 18.5 ± 12.1 nmol/mL, p > 0.05). Correction of P-Cit for BSA did not affect these results.
P5CS* CP
NH3 HCO3
OTC
Citrulline
Kidney
Figure 1: Citrulline metabolism. P5CS, Pyrroline-5-carboxylate synthetase; CP, carbamyl phosphate; OTC, ornithine transcarbamoylase.
performed using a GIF XP-160 (5.9 mm) or GIF 160 (8.5 mm) endoscope (Olympus America Inc, Melville, NY). Plasma citrulline was measured using a Beckman Model 6300 Amino Acid Analyzer (Palo Alto, CA) (ion-exchange chromatography), as previously described (2,11). Biopsies were blindly analyzed for acute cellular rejection, viral enteritis, or other pathology, based on established criteria (12). Plasma citrulline levels measured at the time of biopsy were included in this study only if three conditions were met. First, biopsy specimens were either normal or demonstrated only minor abnormalities, including minimal nonspecific increases in chronic inflammatory cells and apoptosis insufficient to qualify as graft rejection. Second, biopsies obtained within 7 days appeared similar to the index specimen. Third, a minimum of two P-Cit/biopsy pairs was available from each patient. All patients received a quadruple drug regimen consisting of tacrolimus, steroids, basiliximab, and sirolimus, as previously described (13). All patients received similar antibiotic prophylaxis (Unasyn or Zosyn) post transplant. By protocol, enteral feeds were started between days 5 and 7 post intestinal transplant in all patients. In the early post transplant period, pediatric patients received Vivonex RTF (5 g/dL of free amino acid) and the adults, Peptinex (5 g/dL of whey hydrolysate). Neither formula contains glutamine, and no patient received glutamine supplementation. Creatinine as well as tacrolimus levels were correlated with the corresponding P-Cit value.
Of the 40 transplant recipients, 24 (14 children and 10 adults) met criteria for inclusion in this study. Ages of pediatric donors were not significantly lower than recipients (0.98 ± 1.5 years vs. 2.19 ± 2.52 years; 95%CI = 0–2.8, p = 0.13). Mean ages of donors for adult recipients were significantly lower than recipients (16.1 ± 6.1 years vs. 36.5 ± 9.76 years; 95% CI = 12.7–28, p = 0.0001). However, donor body surface area (BSA) to recipient BSA ratio in the pediatric (0.83 ± 0.15) and adult patients (0.81 ± 0.15) was the same. A total of 145 P-Cit determinations were obtained from the 24 patients (6.0 ± 4.1 P-Cit determinations per patient). Mean P-Cit was 34.0 ± 19.9 nmol/mL during the study. Mean and intraindividual P-Cit is shown in Figure 2. Between postoperative days 6 and 30, P-Cit was 22.2 ± 13.2 nmol/mL (n = 55), increasing to 34 ± 17.2 (n = 38) between days 30 and 60 (p = 0.0001), and to 43.6 ± 15.8 nmol/mL (n = 26) between days 60 and 90 (p = 0.0001). Plasma citrulline then leveled off between 90 and 180 days (54.3 ± 22.4 nmol/mL, p = 0.08; n = 15) and remained stable for the remainder of the first year (38.2 ± 26 nmol/mL; n = 11, days 180–360; p = NS). As shown in Figures 2, 3 and 4, two cohorts contributed to the overall trend in P-Cit, based on body size at the time
90 80 70
Donor and recipient body surface area (BSA) was estimated using Mosteller’s formula (14):
60
BSA(m ) = sqrt [body weight (kg) × height (cm)/3600]
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Values were expressed as mean ± SD. Student’s t-test for paired differences and Pearson’s and Spearman’s rho correlation analyses were performed as indicated. Statistical analysis was performed with Software Package for Social Sciences (SPSS) for Windows (Release 10.0, Chicago). This study was reviewed and approved by the IRB of the Mount Sinai School of Medicine.
P-Cit
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Results Plasma citrulline was obtained before intestinal transplant in 18 patients. The mean value for the entire group of 18 was 16.4 ± 9.6 nmol/mL. Twelve patients with a BSA < 1 m2 had a P-Cit equal to 13.3 ± 10.3 nmol/mL; this value American Journal of Transplantation 2004; 4: 414–418
10 3
4
13 15 7 5 4 8
7 9 2
3
3 3 2
8
9 10 8 3
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Figure 2: Intra-individual variability of plasma citrulline (P-Cit) in the 24 patients. The numbers below the x-axis represent the number of samples. Lines represent the mean value for each BSA group.
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16.6 nmol/mL by 60–90 days after transplant (p = 0.0001; n = 8) and remained stable for the remainder of the first year (Figure 3).
80
P-Cit (nm/mL)
60
In patients with a BSA ≥ 1.1 m2 , P-Cit increased from 27.3 ± 13.9 nmol/mL during days 6–30 (n = 32) to 44.0 ± 13.4 during days 30–60 (n = 26; p = 0.0001). Plasma citrulline did not significantly increase between days 60–90 (47.3 ± 14.6; n = 18). In this cohort, the later increase in P-Cit to 61.4 ± 18.9 nmol/mL did not achieve significance n = 12; days 90–180, p = NS).
40
20
BSA > 1.1 < 1.0
0 0
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40
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Figure 3: Plasma citrulline (P-Cit) during the first 120 days after intestinal transplant (IT) in patients with normal biopsies and according to body surface area (BSA). Lines represent the correlation coefficient. 120
Figure 3 demonstrates the linear regression between PCit and time after transplant during the first 120 days in recipients with BSA ≤ 1.0 m2 (P-Cit = 0.57 × number of days post-Tx + 9.3, p = 0.0001) or BSA ≥ 1.1 m2 (P-Cit = 0.51 × number of days post-Tx + 25, p = 0.0001). Thirteen patients received an isolated intestinal transplant. In those patients P-Cit was higher (37.4 ± 20.7 nmol/mL) than in the 11 patients receiving a combined liver-intestine transplant (26.16 ± 20.7 nmol/mL; p = 0.001); however, 10 recipients of an isolated graft had a BSA ≥ 1.1 m2 , while only one recipient of a combined graft had a BSA ≥ 1.1 m2 . We observed no significant difference in P-Cit levels between patients who required interruption of enteral feeding owing to re-operation or other complications and those who did not. Mean time for TPN discontinuation in pediatric patients was 28.0 ± 22.6 days, which was not significantly different from the mean time to TPN discontinuation in adults (15.3 ± 27.6 days, p = 0.1).
100
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40
BSA
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< 1.0 0
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Figure 4: Plasma citrulline (P-Cit) during the first 360 days after intestinal transplant (IT) in patients with normal biopsies and according to body surface area (BSA). Lines represent the mean (95% confidence interval) for each BSA group.
of transplant. Thirteen pediatric patients (93%) had a BSA ≤ 1.0 m2 . In this group with 57 P-Cit samples, mean P-Cit was 22.4 ± 14.7 nmol/mL (95% CI: 19.4–25.37 nmol/mL). The remaining child and all 10 adults each had a BSA ≥ 1.1 m2 . In this group, mean P-Cit was 41.6 ± 19.2 nmol/mL (n = 88, 95% CI: 38.8–44.5 nmol/mL; p = 0.0001). In patients with a BSA ≤ 1.0 m2 , P-Cit did not significantly vary during the first 60 postoperative days, equaling 15.0 ± 8.0 nmol/mL (days 6–30, n = 22) and 16.9 ± 6.0 (days 30–60, n = 13). Plasma citrulline increased to 35.0 ± 416
When we analyzed all the P-cit and the corresponding creatinine values, we found a positive correlation between creatinine levels and P-Cit (Spearman’s rho = 0.49; Rsp = 0.11; p = 0.01). However this correlation was not constant within the individual cases. There was no correlation between tacrolimus level and P-Cit (Spearman’s rho = – 0.6; Rsp = 0.0001; p = NS).
Discussion Deriving meaningful conclusions about amino acid metabolism based on plasma concentrations in various conditions is difficult, because the plasma pool of amino acids is minute in comparison with the free intracellular and protein-bound pools (5). Until the 1980s, it was assumed that amino acids absorbed by the intestinal mucosa entered the portal circulation intact (3,4). It is now recognized that the intestinal mucosa extensively metabolizes nonessential amino acids utilizing enzymes such as P5C. In contrast with the liver and kidney, the urea cycle is incomplete in the intestinal mucosa; most citrulline produced by the bowel is released into the circulation. Unlike enterocytes, colonic epithelial cells do not appear to be a significant source of P-Cit (7). Renal clearance of enterocyte American Journal of Transplantation 2004; 4: 414–418
Plasma Citrulline in Small Bowel Recipients
citrulline completes the urea cycle, with the normal circulating half-life of P-Cit being 3–4 h. Reduced kidney function is also associated with increased P-Cit, presumably by reduced clearance (15,16); our results are consistent with this impression. Plasma citrulline normally rises with age, implying that intestinal secretion increases more than renal clearance over time. Enterocyte synthesis of citrulline is highly energy-demanding (Km = 1.93). Conditions or disorders associated with ATP depletion and lactic acidosis, such as sepsis and mucosal ischemia, reduce enterocyte citrulline synthesis and secretion (15). We have shown that P-Cit is at its nadir after the first week after intestinal transplantation and progressively rises thereafter, regardless of body size and, by implication, age of the patient. Early ischemia/reperfusion injury or sepsis may explain initial low P-Cit, which is consistently found in the absence of histological abnormality. Local accumulation of lactate may contribute to low P-Cit by inhibiting P5C (15). The rise in P-Cit commencing in the second postoperative week likely reflects recovery of intracellular function after ischemia and reperfusion. Induction of P5C by pharmacological doses of glucocorticosteroids early after transplant as well as the increasing intake of dietary nitrogen may accentuate this phenomenon (5). The plateau in P-Cit after 90 days may result from usual reduction in glucocorticosteroid therapy in the setting of a regenerated mucosa. Once steady-state P-Cit was achieved 3 months post transplantation, levels in our smaller and larger patients were similar to levels in age-matched, healthy counterparts, as previously reported (3). We used BSA rather than length or chronological age for comparisons based on body size for two reasons. First, pediatric patients with intestinal failure are often smaller than children of comparable chronological age (17). Second, the source of P-Cit is the allograft, which is size-matched rather than age-matched to the recipient. The difference in time to reach the plateau may be related to the difference in graft size between those recipients with BSA ≥ 1.1 m2 vs. those with BSA ≤ 1 m2 . Previous publications suggest that P-Cit level is a marker of functional intestinal mass, being lower in patients with short gut syndrome in proportion to the length of remnant gut (9). Furthermore, evidence suggests that in persons with normal gut length, P-Cit reflects quality of mucosal function (9). Plasma citrulline also correlates well with dietary protein and fat absorption. There are no data that demonstrate a reduced P-Cit in diffuse mucosal inflammatory disorders such as untreated celiac disease or extensive inflammatory bowel disease. However, our preliminary data (2) suggest that levels of P-Cit may be lower than expected with moderate or severe rejection and posttransplant viral enteropathy (9–18). The real value of routine P-Cit determination after intestinal transplantation will lie in its ability to identify patients in American Journal of Transplantation 2004; 4: 414–418
whom endoscopy with biopsy is indicated. This approach will potentially reduce the number of unnecessary invasive procedures. Clarification of normal P-Cit values represents the first step in this process. The next step will be to determine the extent to which P-Cit falls as a result of graft injury rejection or other inflammatory processes. Then, the true, lower limit of normal P-Cit can be defined at various times after transplant. The present study emphasizes that during the first 3 months post IT, P-Cit is lower than expected for nontransplanted individuals and varies based on BSA. Recognition of typically reduced P-Cit within the first months after IT is particularly important, as low levels might be erroneously attributed to graft rejection, which is common during this time. Following the transient post IT depression of P-Cit, levels are similar to those in healthy individuals, emphasizing that the histologically normal intestinal graft functions like the native bowel (19).
Acknowledgments The authors wish to acknowledge Mr Aguila Alejandro for his expertise in processing the citrulline. We also thank our intestinal transplant nurse coordinators Rachel Spencer and Jill Robinson for their assistance with blood and tissue sample collection, Dr Raffaella Morotti for her assistance with interpretation of biopsy specimens, and Nancy Ehrlich Lapid for editorial assistance.
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American Journal of Transplantation 2004; 4: 414–418
