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Serum Citrulline Is A Simple Quantitative Marker for Small Intestinal Enterocytes Mass and Absorption Function in Short Bowel Patients
Journal of Surgical Research 127, 177–182 (2005) doi:10.1016/j.jss.2005.04.004
Serum Citrulline Is A Simple Quantitative Marker for Small Intestinal Enterocytes Mass and Absorption Function in Short Bowel Patients Gong Jianfeng, Zhu Weiming,1 Li Ning, Liu Fangnan, Tan Li, Luo Nan, and Li Jieshou Research Institute of General Surgery, Jinling Hospital, Medical School of Nanjing University, Nanjing, P.R. China Submitted for publication October 21, 2004
Background. To investigate the clinical significance of serum citrulline in evaluating the remnant small bowel enterocytes mass and absorptive function in short bowel (SB) patients. Materials and methods. Serum citrulline concentrations were determined using high-performance liquid chromatography (HPLC) in 22 SBS patients and 33 healthy controls. Five-hour urine D-xylose excretion and digestive protein absorption were measured using HPLC and micro-Kjeldahl method, respectively. Small bowel length and surface area were assessed on X-ray radiograph. Correlations between serum citrulline levels and small bowel length, small bowel surface, and nutritional substrate digestive absorption percentage were analyzed. For six patients receiving bowel rehabilitation therapy, serum citrulline, D-xylose excretion, and intestinal protein absorption were measured pre- and immediately postmanagement, and their correlations were analyzed. Results. Serum citrulline levels were significantly lower in SB patients compared with healthy controls. In SB patients, they correlated well with remnant small bowel length (r ⴝ 0.82, P < 0.001), surface area (r ⴝ 0.86, P < 0.001), 5-h urine D-xylose excretion (r ⴝ 0.56, P ⴝ 0.007), and digestive protein absorption (r ⴝ 0.48, P ⴝ 0.046). The increased percentage of serum citrulline level in six patients receiving rehabilitation therapy followed a trend of correlating with that of intestinal protein absorption (r ⴝ 0.79, P ⴝ 0.063) and urine D-xylose excretion (r ⴝ 0.81, P ⴝ 0.053). Conclusions. In patients with short bowel syndrome, serum citrulline is a simple and accurate biomarker for the severity of intestinal failure and may be a can-
1
To whom correspondence and reprint requests should be addressed at Research Institute of General Surgery, Jinling Hospital, Medical School of Nanjing University, Nanjing, P.R. China. E-mail: [email protected].
didate marker for the gut-trophic effects of bowel rehabilitation therapies. © 2005 Elsevier Inc. All rights reserved. Key Words: citrulline; short bowel syndrome; intestinal failure. INTRODUCTION
Intestinal failure, such as short bowel syndrome (SBS) and mucosal injury after small bowel transplantation, is associated with a sharp reduction of absorptive area and small intestinal function. However, unlike renal or liver failure that has functional indicators as serum creatinine or bilirubin, there lacks a reliable serum biological marker of absorptive enterocytes mass helping the diagnosis of small intestinal failure in these patients. Serum citrulline, an amino acid not incorporated into the protein, synthesized exclusively in small intestinal enterocytes from glutamine and then metabolized into arginine by the kidney, was found in recent years to be a candidate marker for small bowel enterocytes mass [1]. In this study, we determined serum citrulline levels using high performance liquid chromatography (HPLC) in 22 short bowel (SB) patients as a model of intestinal failure and evaluated the applicability of serum citrulline as a quantitative biomarker for small intestinal enterocytes mass and absorptive capacity. MATERIALS AND METHODS Patients Twenty-two patients (16 males and 6 females) with nonmalignant short bowel syndrome (defined as small bowel length less than 150 cm), followed up from July 2003 to December 2003 at our hospital, were enrolled in our study. Patients’ characteristics (mean age 37.86 ⫾ 15.07 years, ranging 13– 66) are listed in Table 1. The average length of their small intestines was 52.27 ⫾ 36.41 cm (ranging 0 to 110 cm). Subjects with one or more of the following criteria were excluded: upper gastrointestinal tract surgery (stomach, pancreas,
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TABLE 1 General and Surgical Characteristics of 22 Patients with Short Bowel Syndrome No.
Gender
Age (y)
Jejunem-ileum (cm)
Cause of resection
Colon
Survival time (m)
Route of feeding
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
M M M M M F M M F M F M M F M M M M M F M F
27 25 42 13 66 34 16 34 40 28 19 30 50 46 66 40 51 40 20 46 59 41
60 30 90 30 80 70 20 30 25 30 80 40 80 0 0 110 120 80 0 80 15 80
Small bowel volvulus Small bowel volvulus Small bowel volvulus Small bowel volvulus SMA thrombosis Small bowel volvulus Small bowel volvulus SMA thrombosis SMA thrombosis Small bowel volvulus Crohn’s disease SMA thrombosis Small bowel obstruction Small bowel volvulus SMA injury Repeated surgery Small bowel volvulus Trauma Small bowel volvulus SMA injury SMA thrombosis Small bowel obstruction
ICV(⫺) ALL ALL ACR ACR ICV(⫺) ALL ALL ICV(⫺) ICV(⫺) ICV(⫺) ALL ALL ACR ACR ALL ALL ALL ACR ACR ALL ICV(⫺)
52 12 36 40 70 113 56 40 22 68 24 24 84 24 1 2 192 24 2 0.6 9 13
EN⫹LFD EN⫹LFD EN⫹LFD EN⫹LFD EN⫹LFD EN⫹LFD EN⫹LFD EN⫹LFD EN⫹LFD EN⫹LFD EN⫹LFD EN⫹LFD EN⫹LFD TPN TPN EN⫹LFD EN⫹LFD EN⫹LFD TPN EN⫹LFD PN⫹EN EN⫹LFD
SMA: superior mesenteric artery; ICV: ileocecal valve; ACR: ascending colon resection; LFD: low fat diet.
esophagus, etc.) other than small bowel; being unable to be fed enterally due to remnant intestinal stenosis or obstruction; dysfunction or failure of other organs such as liver, kidney, or heart; total or subtotal villous atrophy on intestinal radiograph; bowel rehabilitation therapy [2] within 1 year. Rest energy expenditure (REE) and body composition were measured in all patients on the second or third hospital day using Indirect Calorimetry and Inbody 3.0 (Biospace Co., Seoul, Korea), respectively. Routine serum biochemical indices, such as albumin, prealbumin, transferrin, fibronectin, and creatinine, were all determined automatically by a biochemical analyzer. Among the 22 patients enrolled, 18 were maintained on partial enteral nutrition (Pepti 2000 LF, Nutricia Co., Zoetermeer, Netherlands) supplemented with a low-fat diet, and the other four were maintained on either total parenteral nutrition (TPN) or parenteral/ enteral nutrition (PN/EN). For the latter four patients, the volume and composition of intravenous infusion were adjusted to personal energy expenditure requirements in accordance with 1.5⫻ REE and water-mineral losses. Six of the 22 SB patients received bowel rehabilitation therapy with a combination of recombinant human growth hormone (rhGH, Serono Inc., Geneva, Switzerland), nutrition support, glutamine, and dietary fiber as previously described [2]. On the second posttreatment day, their serum citrulline levels and 5-h urine D-xylose excretion were redetermined. Digestive protein absorption test was also reperformed from the third to fifth day after therapy.
Serum Citrulline Sampling and Determination Blood samples were collected from SB patients under stable conditions without clinical evidence of dehydration or electrolyte disturbance. Venous postabsorptive serum concentrations were measured after an overnight fast in both short bowel patients and healthy controls. Control subjects were 33 healthy volunteers (16 males and 17 females) without any known metabolic or digestive diseases, with an average age of 24.53 ⫾ 3.18 years (ranging from 19 to 54). In
patients receiving continuous EN or PN treatment, nutrition was stopped at least 8 h before sampling. Serum samples were stored at ⫺80°C until analysis. For determination of amino acids, 250 L serum was deproteinized and serum citrulline concentrations were determined using the HPLC method as previously described [3].
Remnant Small Intestinal Length and Surface Area Measurement Postduodenal remnant small bowel length was measured using radiograph films of barium meals. Briefly, a nasogastric tube was placed through the duodenojejunal flexure, and then a thin barium solution was injected followed by an opisometer. The remnant small bowel length was calculated as the length between duodenojejunal flexure and ileocecal valve (or ileum-colon junction). The mean small bowel diameter was also measured pertaining to an estimate of small bowel surface area ⫽ ⫻ length ⫻ diameter of small bowel. Radiograph barium abnormalities, i.e., modified mucosal fold, ulcerations, and nonoclusive stenosis of remnant small bowel, were also recorded.
Evaluation of D-xylose and Protein Absorption in SB Patients D-xylose absorption was used to assess the small intestinal surface area on the second hospital day. Briefly, SB patients were administrated a 5.0 g oral dose of D-xylose in 250 ml tap water after an overnight fast. A 5-h urine was collected without preservatives and the total amount was recorded. Subjects were free to drink water during the 5-h period. The concentration of D-xylose in urine samples was determined using HPLC as previously described [4]. All except four patients maintained on PN were tested for protein absorption for three continuous days. During evaluation, all subjects tested were fed with standard enteral nutrition formulas (Pepti 2000 LF) with an approximate composition of protein, fat, and carbohydrates, and the energy supply was 1.5⫻ REE. For each 24-h period, stool volume was recorded and 2 ml sample was taken. Fecal nitro-
JIANFENG ET AL.: CITRULLINE AS A MARKER FOR INTESTINAL FAILURE
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TABLE 2 Clinical and Biochemical Characteristics of Patient with Short Bowel Syndrome Subjects
Mean value
Net absorption of protein (%) a 5-h Urine D-xylose excretion (%) Body mass index (kg/m 2) Rest energy expenditure (kcal/d) Serum albumin (g/L) Serum prealbumin (mg/L) Serum transferrin (mg/L) Serum fibronectin (mg/L) Serum creatinine (mol/L)
67.37 ⫾ 16.85 (10.5–92.1) 8.41 ⫾ 6.93 (0.06–35.4) 16.88 ⫾ 2.55 (13.2–23.2) 1217.83 ⫾ 263.00 (890–1697) 40.18 ⫾ 5.18 (32.3–50.6) 203.59 ⫾ 58.15 (100–321) 2.33 ⫾ 0.41 (1.7–3.0) 162.29 ⫾ 45.58 (102–255) 85.13 ⫾ 22.92 (62–109)
Note. Results are expressed as mean ⫾ SD. a n ⫽ 18.
gen was estimated by the micro-Kjeldahl method [5]. The percentage of net digestive absorption was calculated as percentage of protein representing the proportion of oral intake not recovered in fecal output.
Statistical Analysis SPSS for Windows software (release 11.0) was used for statistical analysis. Quantitative values were expressed as mean ⫾ SD. Serum citrulline concentrations were compared between short bowel and control subgroup of patients using independent Student’s t-test. To assess a linear relationship between parameters, bivariate correlations procedure was used for calculation of Pearson’s correlation coefficient. The significance level was defined as P ⬍ 0.05.
RESULTS
The clinical and biomedical characteristics of 22 SB patients are listed in Table 2. Their mean (⫾SD) serum citrulline level was 5.94 ⫾ 2.65 mol/L, significantly lower than control subjects (16.87 ⫾ 5.97 mol/L, P ⬍ 0.001) (Fig. 1).
FIG. 1. Serum citrulline levels in short bowel patients (n ⫽ 22) and healthy control subjects (n ⫽ 33). Difference is significant between the two groups (5.94 versus 16.87 mol/L, P ⬍ 0.001).
FIG. 2. Scatter spots showing the relationships of serum citrulline with remnant small bowel length and small bowel surface area. Pearson’s correlation coefficients are r ⫽ 0.82 and r ⫽ 0.86 and the corresponding P values are ⬍0.001 and ⬍0.001, respectively. In patients without small bowel (No. 14, 15, and 19), serum citrulline concentrations were 0.92, 1.25, and 0.77 mol/L, respectively, showing organs other than small intestine only have a minor effect on serum citrulline levels.
As shown in Fig. 2, a strong correlation was observed between serum citrulline level and small bowel length (r ⫽ 0.82, P ⬍ 0.001). In addition, the association of small bowel surface area with serum citrulline levels was also significant (r ⫽ 0.86, P ⬍ 0.001). Small bowel length was correlated both with 5-h urine D-xylose excretion (r ⫽ 0.58, P ⫽ 0.005) and with net digestive protein absorption percentage (r ⫽ 0.54, P ⫽ 0.016). Serum citrulline level was correlated significantly with 5-h urine D-xylose excretion (r ⫽ 0.56, P ⫽ 0.007) and net digestive protein absorption (r ⫽ 0.48, P ⫽ 0.046) (Fig. 3), but not with nutritional parameters such as serum albumin (P ⫽ 0.62), prealbu-
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tinal protein absorption (r ⫽ 0.79, P ⫽ 0.063) and urine D-xylose excretion (r ⫽ 0.81, P ⫽ 0.053). DISCUSSION
Evaluation of the small bowel absorptive area and capacity has long been a challenge for gastroenterologists. Functional assays, such as D-xylose and 3-O-methylglucose absorption test [6, 7] and net digestive ratio of nutrients, have been developed to assess the severity of intestinal failure, but these methods are often time-consuming and cumbersome and therefore not suitable for repeated monitoring as a routine in the clinical practice. Serum citrulline is chosen as a biochemical marker for intestinal failure because of its methodological simplicity and repeatability; citrulline is an amino acid released exclusively from small bowel enterocytes [8], and its blood level is highly dependent on small bowel enterocytes mass. Recently, much work has been done on the application of this biomarker in evaluating the severity of small intestinal mucosal injury due to small bowel transplant rejection, irradiation, or high-dosage chemotherapy [9, 10, 11]. In the present study, we have also observed a significant decrease of serum citrulline level in SB patients compared with healthy controls and a strong correlation between serum citrulline levels and remnant small bowel length as well as surface area. The association between small bowel length/area and macronutrient absorption has already been shown in previous studies [12], and it was confirmed in our study. In addition, a remarkable correlation between serum citrulline level with 5-h urine D-xylose excretion and intestinal protein absorption was also found, conFIG. 3. Correlation between citrulline assay and substrate absorption test. (a) Scatter plot showing the relationship of serum citrulline concentrations and 5-h urine D-xylose excretion ratio in short bowel patients. The Pearson’s correlation coefficient is r ⫽ 0.56 and the corresponding P value is 0.007. Urine D-xylose ratios in three patients without small bowel are 0.06, 0.03, and 0.16%, respectively. (b) Correlation between serum citrulline level and net digestive protein absorption was also significant, as shown in scatter plot. The Pearson’s correlation coefficient is r ⫽ 0.48 (P ⫽ 0.046). Four patients (No. 14, 15, 19, and 21) maintained on TPN or PN/EN did not take the protein absorption test.
min (P ⫽ 0.49), fibronectin (P ⫽ 0.71), transferrin (P ⫽ 0.32), and creatinine (P ⫽ 0.33) levels or REE (P ⫽ 0.15) and body mass index (BMI) (P ⫽ 0.85). All of the six patients receiving rehabilitation therapy had increased levels of serum citrulline, digestive protein absorption, and urine D-xylose excretion, as presented in Table 3. Although not statistically significant, the percentage of increase in serum citrulline level followed a trend of correlating with that of intes-
TABLE 3 The Increased Percentage of Serum Citrulline, Intestinal Protein Absorption, and 5-h Urine D-xylose Excretion in Patients Receiving Rehabilitation Therapy Increased percentage after therapy (%) Patient No.
Serum citrulline
Protein absorption
5-h D-xylose excretion
5 6 11 14 16 20
13.94 8.30 10.87 8.11 28.00 20.48
19.96 6.67 4.81 13.81 23.90 18.57
16.54 7.14 8.23 7.27 22.69 32.75
Note. All six patients have increased serum citrulline levels, digestive protein absorption, and 5-h urine D-xylose excretion. The increased percentage of serum citrulline has the trend of correlating with that of intestinal protein absorption (r ⫽ 0.79, P ⫽ 0.063) and urine D-xylose excretion (r ⫽ 0.81, P ⫽ 0.053).
JIANFENG ET AL.: CITRULLINE AS A MARKER FOR INTESTINAL FAILURE
sistent with the assessment of absorption function of the small bowel with this biomarker. Being not a protein component, citrulline is incorporated neither in enteral food nor endogenous proteins, and it is not released from proteolysis as most other amino acids are [13]. Therefore, body protein composition or diet status does not have a major effect on its blood level. This point was confirmed by our observation that serum citrulline does not correlate independently with either nutritional status (BMI, serum albumin, prealbumin, transferrin, fibronectin) or energy expenditure (REE). Among the 22 SB patients, 3 patients (No. 14, 15, and 19) did not have small bowel (but the duodenum and colon remained), and their serum citrulline concentrations were 0.92, 1.25, and 0.77 mol/L, respectively (only 4.6 –7.4% of normal value), suggesting that organs or tissues other than small bowel only have a minor effect on blood level. Renal dysfunction may have an impact on serum citrulline levels, but will not be statistically significant until plasma creatinine clearance is below 50 ml/min [14], which is excluded in our patients. Bowel rehabilitation therapy, which combines administration of growth hormone, glutamine, nutritional support, and other enterotrophic factors, has been widely applied in SB patients and its gut trophic effect has been well recognized [2, 15, 16]. In this study, all six patients receiving this regimen had increased levels of serum citrulline, 5-h D-xylose excretion, and digestive protein absorption. Although not statistically significant (possibly due to limited subjects), the increased percentage of serum citrulline followed the trend of correlating with that of urine D-xylose excretion (r ⫽ 0.81, P ⫽ 0.053) and protein absorption (r ⫽ 0.79, P ⫽ 0.063), indicating that serum citrulline may also be a candidate marker for the therapeutic effect of rehabilitation therapy on patients with intestinal failure. We have chosen X-ray film and 5-h urine D-xylose excretion for assessment of small intestinal surface area in this study. However, it should be pointed out that the “gold standard” assay for small bowel mucosal area still remains. X-ray evaluation may reflect the small bowel length accurately [17], but fail to detect the height of the villous and the depth of the crypts, which may vary among individuals and are often the most responsive element to a specific therapy, while the accuracy of 5-h urine D-xylose excretion test may be compromised by intestinal microbacterial overgrowth, intestinal transit hurry, renal function, and the remnant site of the small bowel [6, 18]. The average serum citrulline level in the healthy Chinese controls is 16.87 mol/L, which is much lower than that reported by Crenn et al. in the French population (above 40 mol/L) [1, 19], possibly due to racial discrepancy or difference in long-term diet habits. In
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fact, blood citrulline may vary widely among healthy individuals. In Crenn’s study, the normal value ranges from 20 to 60 mol/L in 51 healthy controls, while in another two studies conducted by Lutgens et al. [20] and Apostol et al. [21], the levels were 25.7 and 26 mol/L, respectively. More data are needed to confirm our results in the Chinese population. In conclusion, our results demonstrate that after massive small bowel resection, the serum citrulline levels provide valuable information on the intestinal surface area available for nutrient absorption and its absorptive capacity of protein. Detection of patients’ serum citrulline level has the advantage of being simpler and less time-consuming than conventional balance study or radiological morphological evaluation and is easily repeated. Our preliminary observation of serum citrulline level changes in SB patients receiving rehabilitation therapy supports extending the application of this serum marker in monitoring the variants in intestinal absorptive area and function in SB patients receiving specific regimen. Determination of its trends over time will be easily performed by repeated measurement over a long period. To extend its diagnostic value in clinical practice, further studies are to be made on the application of this biomarker in a wider category of surgical patients, especially the critically ill such as those with severe acute pancreatitis, in whom intestinal epithelial injury is common and sometimes the major cause of mortality. REFERENCES 1.
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JOURNAL OF SURGICAL RESEARCH: VOL. 127, NO. 2, AUGUST 2005 Citrulline: a potentially simple quantitative marker of intestinal epithelial damage following myeloablative therapy. Bone Marrow Transplant. 34: 193, 2004. Lutgens, L. C., Deutz, N., Granzier-Peeters, M., et al. Plasma citrulline concentration: a surrogate end point for radiation-induced mucosal atrophy of the small bowel. A feasibility study in 23 patients. Int. J. Radiat. Oncol. Biol. Phys. 60: 275, 2004. Rodrigues, C. A., Lennard-Jones, J. E., and Thompson, D. G. Energy absorption as a measure of intestinal failure in the short bowel syndrome. Gut 30: 176, 1989. Wahren, J., Felig, P., and Hagenfeldt, L. Effect of protein ingestion on splanchnic and leg metabolism in normal man and in patients with diabetes mellitus. J. Clin. Invest. 57: 987, 1976. Ceballos, I., Chauveau, P., Guerin, V., et al. Early alterations of plasma amino acids in chronic renal failure. Clin. Chim. Acta 188: 101, 1990. Zhou, X., Li, Y. X., Li, N., et al. Glutamine enhances the guttrophic effect of growth hormone in rat after massive small bowel resection. J. Surg. Res. 99: 47, 2001 Jul.
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Gu, Y., Wu, Z. H., Xie, J. X., et al. Effects of growth hormone (rhGH) and glutamine supplemented parenteral nutrition on intestinal adaptation in short bowel rats. Clin. Nutr. 20: 159, 2001 Apr.
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Apostol, A. T., and Tayek, J. A. A decrease in glucose production is associated with an increase in plasma citrulline response to oral arginine in normal volunteers. Metabolism 52: 1512, 2003.
Serum Citrulline Is A Simple Quantitative Marker for Small Intestinal Enterocytes Mass and Absorption Function in Short Bowel Patients Gong Jianfeng, Zhu Weiming,1 Li Ning, Liu Fangnan, Tan Li, Luo Nan, and Li Jieshou Research Institute of General Surgery, Jinling Hospital, Medical School of Nanjing University, Nanjing, P.R. China Submitted for publication October 21, 2004
Background. To investigate the clinical significance of serum citrulline in evaluating the remnant small bowel enterocytes mass and absorptive function in short bowel (SB) patients. Materials and methods. Serum citrulline concentrations were determined using high-performance liquid chromatography (HPLC) in 22 SBS patients and 33 healthy controls. Five-hour urine D-xylose excretion and digestive protein absorption were measured using HPLC and micro-Kjeldahl method, respectively. Small bowel length and surface area were assessed on X-ray radiograph. Correlations between serum citrulline levels and small bowel length, small bowel surface, and nutritional substrate digestive absorption percentage were analyzed. For six patients receiving bowel rehabilitation therapy, serum citrulline, D-xylose excretion, and intestinal protein absorption were measured pre- and immediately postmanagement, and their correlations were analyzed. Results. Serum citrulline levels were significantly lower in SB patients compared with healthy controls. In SB patients, they correlated well with remnant small bowel length (r ⴝ 0.82, P < 0.001), surface area (r ⴝ 0.86, P < 0.001), 5-h urine D-xylose excretion (r ⴝ 0.56, P ⴝ 0.007), and digestive protein absorption (r ⴝ 0.48, P ⴝ 0.046). The increased percentage of serum citrulline level in six patients receiving rehabilitation therapy followed a trend of correlating with that of intestinal protein absorption (r ⴝ 0.79, P ⴝ 0.063) and urine D-xylose excretion (r ⴝ 0.81, P ⴝ 0.053). Conclusions. In patients with short bowel syndrome, serum citrulline is a simple and accurate biomarker for the severity of intestinal failure and may be a can-
1
To whom correspondence and reprint requests should be addressed at Research Institute of General Surgery, Jinling Hospital, Medical School of Nanjing University, Nanjing, P.R. China. E-mail: [email protected].
didate marker for the gut-trophic effects of bowel rehabilitation therapies. © 2005 Elsevier Inc. All rights reserved. Key Words: citrulline; short bowel syndrome; intestinal failure. INTRODUCTION
Intestinal failure, such as short bowel syndrome (SBS) and mucosal injury after small bowel transplantation, is associated with a sharp reduction of absorptive area and small intestinal function. However, unlike renal or liver failure that has functional indicators as serum creatinine or bilirubin, there lacks a reliable serum biological marker of absorptive enterocytes mass helping the diagnosis of small intestinal failure in these patients. Serum citrulline, an amino acid not incorporated into the protein, synthesized exclusively in small intestinal enterocytes from glutamine and then metabolized into arginine by the kidney, was found in recent years to be a candidate marker for small bowel enterocytes mass [1]. In this study, we determined serum citrulline levels using high performance liquid chromatography (HPLC) in 22 short bowel (SB) patients as a model of intestinal failure and evaluated the applicability of serum citrulline as a quantitative biomarker for small intestinal enterocytes mass and absorptive capacity. MATERIALS AND METHODS Patients Twenty-two patients (16 males and 6 females) with nonmalignant short bowel syndrome (defined as small bowel length less than 150 cm), followed up from July 2003 to December 2003 at our hospital, were enrolled in our study. Patients’ characteristics (mean age 37.86 ⫾ 15.07 years, ranging 13– 66) are listed in Table 1. The average length of their small intestines was 52.27 ⫾ 36.41 cm (ranging 0 to 110 cm). Subjects with one or more of the following criteria were excluded: upper gastrointestinal tract surgery (stomach, pancreas,
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TABLE 1 General and Surgical Characteristics of 22 Patients with Short Bowel Syndrome No.
Gender
Age (y)
Jejunem-ileum (cm)
Cause of resection
Colon
Survival time (m)
Route of feeding
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
M M M M M F M M F M F M M F M M M M M F M F
27 25 42 13 66 34 16 34 40 28 19 30 50 46 66 40 51 40 20 46 59 41
60 30 90 30 80 70 20 30 25 30 80 40 80 0 0 110 120 80 0 80 15 80
Small bowel volvulus Small bowel volvulus Small bowel volvulus Small bowel volvulus SMA thrombosis Small bowel volvulus Small bowel volvulus SMA thrombosis SMA thrombosis Small bowel volvulus Crohn’s disease SMA thrombosis Small bowel obstruction Small bowel volvulus SMA injury Repeated surgery Small bowel volvulus Trauma Small bowel volvulus SMA injury SMA thrombosis Small bowel obstruction
ICV(⫺) ALL ALL ACR ACR ICV(⫺) ALL ALL ICV(⫺) ICV(⫺) ICV(⫺) ALL ALL ACR ACR ALL ALL ALL ACR ACR ALL ICV(⫺)
52 12 36 40 70 113 56 40 22 68 24 24 84 24 1 2 192 24 2 0.6 9 13
EN⫹LFD EN⫹LFD EN⫹LFD EN⫹LFD EN⫹LFD EN⫹LFD EN⫹LFD EN⫹LFD EN⫹LFD EN⫹LFD EN⫹LFD EN⫹LFD EN⫹LFD TPN TPN EN⫹LFD EN⫹LFD EN⫹LFD TPN EN⫹LFD PN⫹EN EN⫹LFD
SMA: superior mesenteric artery; ICV: ileocecal valve; ACR: ascending colon resection; LFD: low fat diet.
esophagus, etc.) other than small bowel; being unable to be fed enterally due to remnant intestinal stenosis or obstruction; dysfunction or failure of other organs such as liver, kidney, or heart; total or subtotal villous atrophy on intestinal radiograph; bowel rehabilitation therapy [2] within 1 year. Rest energy expenditure (REE) and body composition were measured in all patients on the second or third hospital day using Indirect Calorimetry and Inbody 3.0 (Biospace Co., Seoul, Korea), respectively. Routine serum biochemical indices, such as albumin, prealbumin, transferrin, fibronectin, and creatinine, were all determined automatically by a biochemical analyzer. Among the 22 patients enrolled, 18 were maintained on partial enteral nutrition (Pepti 2000 LF, Nutricia Co., Zoetermeer, Netherlands) supplemented with a low-fat diet, and the other four were maintained on either total parenteral nutrition (TPN) or parenteral/ enteral nutrition (PN/EN). For the latter four patients, the volume and composition of intravenous infusion were adjusted to personal energy expenditure requirements in accordance with 1.5⫻ REE and water-mineral losses. Six of the 22 SB patients received bowel rehabilitation therapy with a combination of recombinant human growth hormone (rhGH, Serono Inc., Geneva, Switzerland), nutrition support, glutamine, and dietary fiber as previously described [2]. On the second posttreatment day, their serum citrulline levels and 5-h urine D-xylose excretion were redetermined. Digestive protein absorption test was also reperformed from the third to fifth day after therapy.
Serum Citrulline Sampling and Determination Blood samples were collected from SB patients under stable conditions without clinical evidence of dehydration or electrolyte disturbance. Venous postabsorptive serum concentrations were measured after an overnight fast in both short bowel patients and healthy controls. Control subjects were 33 healthy volunteers (16 males and 17 females) without any known metabolic or digestive diseases, with an average age of 24.53 ⫾ 3.18 years (ranging from 19 to 54). In
patients receiving continuous EN or PN treatment, nutrition was stopped at least 8 h before sampling. Serum samples were stored at ⫺80°C until analysis. For determination of amino acids, 250 L serum was deproteinized and serum citrulline concentrations were determined using the HPLC method as previously described [3].
Remnant Small Intestinal Length and Surface Area Measurement Postduodenal remnant small bowel length was measured using radiograph films of barium meals. Briefly, a nasogastric tube was placed through the duodenojejunal flexure, and then a thin barium solution was injected followed by an opisometer. The remnant small bowel length was calculated as the length between duodenojejunal flexure and ileocecal valve (or ileum-colon junction). The mean small bowel diameter was also measured pertaining to an estimate of small bowel surface area ⫽ ⫻ length ⫻ diameter of small bowel. Radiograph barium abnormalities, i.e., modified mucosal fold, ulcerations, and nonoclusive stenosis of remnant small bowel, were also recorded.
Evaluation of D-xylose and Protein Absorption in SB Patients D-xylose absorption was used to assess the small intestinal surface area on the second hospital day. Briefly, SB patients were administrated a 5.0 g oral dose of D-xylose in 250 ml tap water after an overnight fast. A 5-h urine was collected without preservatives and the total amount was recorded. Subjects were free to drink water during the 5-h period. The concentration of D-xylose in urine samples was determined using HPLC as previously described [4]. All except four patients maintained on PN were tested for protein absorption for three continuous days. During evaluation, all subjects tested were fed with standard enteral nutrition formulas (Pepti 2000 LF) with an approximate composition of protein, fat, and carbohydrates, and the energy supply was 1.5⫻ REE. For each 24-h period, stool volume was recorded and 2 ml sample was taken. Fecal nitro-
JIANFENG ET AL.: CITRULLINE AS A MARKER FOR INTESTINAL FAILURE
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TABLE 2 Clinical and Biochemical Characteristics of Patient with Short Bowel Syndrome Subjects
Mean value
Net absorption of protein (%) a 5-h Urine D-xylose excretion (%) Body mass index (kg/m 2) Rest energy expenditure (kcal/d) Serum albumin (g/L) Serum prealbumin (mg/L) Serum transferrin (mg/L) Serum fibronectin (mg/L) Serum creatinine (mol/L)
67.37 ⫾ 16.85 (10.5–92.1) 8.41 ⫾ 6.93 (0.06–35.4) 16.88 ⫾ 2.55 (13.2–23.2) 1217.83 ⫾ 263.00 (890–1697) 40.18 ⫾ 5.18 (32.3–50.6) 203.59 ⫾ 58.15 (100–321) 2.33 ⫾ 0.41 (1.7–3.0) 162.29 ⫾ 45.58 (102–255) 85.13 ⫾ 22.92 (62–109)
Note. Results are expressed as mean ⫾ SD. a n ⫽ 18.
gen was estimated by the micro-Kjeldahl method [5]. The percentage of net digestive absorption was calculated as percentage of protein representing the proportion of oral intake not recovered in fecal output.
Statistical Analysis SPSS for Windows software (release 11.0) was used for statistical analysis. Quantitative values were expressed as mean ⫾ SD. Serum citrulline concentrations were compared between short bowel and control subgroup of patients using independent Student’s t-test. To assess a linear relationship between parameters, bivariate correlations procedure was used for calculation of Pearson’s correlation coefficient. The significance level was defined as P ⬍ 0.05.
RESULTS
The clinical and biomedical characteristics of 22 SB patients are listed in Table 2. Their mean (⫾SD) serum citrulline level was 5.94 ⫾ 2.65 mol/L, significantly lower than control subjects (16.87 ⫾ 5.97 mol/L, P ⬍ 0.001) (Fig. 1).
FIG. 1. Serum citrulline levels in short bowel patients (n ⫽ 22) and healthy control subjects (n ⫽ 33). Difference is significant between the two groups (5.94 versus 16.87 mol/L, P ⬍ 0.001).
FIG. 2. Scatter spots showing the relationships of serum citrulline with remnant small bowel length and small bowel surface area. Pearson’s correlation coefficients are r ⫽ 0.82 and r ⫽ 0.86 and the corresponding P values are ⬍0.001 and ⬍0.001, respectively. In patients without small bowel (No. 14, 15, and 19), serum citrulline concentrations were 0.92, 1.25, and 0.77 mol/L, respectively, showing organs other than small intestine only have a minor effect on serum citrulline levels.
As shown in Fig. 2, a strong correlation was observed between serum citrulline level and small bowel length (r ⫽ 0.82, P ⬍ 0.001). In addition, the association of small bowel surface area with serum citrulline levels was also significant (r ⫽ 0.86, P ⬍ 0.001). Small bowel length was correlated both with 5-h urine D-xylose excretion (r ⫽ 0.58, P ⫽ 0.005) and with net digestive protein absorption percentage (r ⫽ 0.54, P ⫽ 0.016). Serum citrulline level was correlated significantly with 5-h urine D-xylose excretion (r ⫽ 0.56, P ⫽ 0.007) and net digestive protein absorption (r ⫽ 0.48, P ⫽ 0.046) (Fig. 3), but not with nutritional parameters such as serum albumin (P ⫽ 0.62), prealbu-
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tinal protein absorption (r ⫽ 0.79, P ⫽ 0.063) and urine D-xylose excretion (r ⫽ 0.81, P ⫽ 0.053). DISCUSSION
Evaluation of the small bowel absorptive area and capacity has long been a challenge for gastroenterologists. Functional assays, such as D-xylose and 3-O-methylglucose absorption test [6, 7] and net digestive ratio of nutrients, have been developed to assess the severity of intestinal failure, but these methods are often time-consuming and cumbersome and therefore not suitable for repeated monitoring as a routine in the clinical practice. Serum citrulline is chosen as a biochemical marker for intestinal failure because of its methodological simplicity and repeatability; citrulline is an amino acid released exclusively from small bowel enterocytes [8], and its blood level is highly dependent on small bowel enterocytes mass. Recently, much work has been done on the application of this biomarker in evaluating the severity of small intestinal mucosal injury due to small bowel transplant rejection, irradiation, or high-dosage chemotherapy [9, 10, 11]. In the present study, we have also observed a significant decrease of serum citrulline level in SB patients compared with healthy controls and a strong correlation between serum citrulline levels and remnant small bowel length as well as surface area. The association between small bowel length/area and macronutrient absorption has already been shown in previous studies [12], and it was confirmed in our study. In addition, a remarkable correlation between serum citrulline level with 5-h urine D-xylose excretion and intestinal protein absorption was also found, conFIG. 3. Correlation between citrulline assay and substrate absorption test. (a) Scatter plot showing the relationship of serum citrulline concentrations and 5-h urine D-xylose excretion ratio in short bowel patients. The Pearson’s correlation coefficient is r ⫽ 0.56 and the corresponding P value is 0.007. Urine D-xylose ratios in three patients without small bowel are 0.06, 0.03, and 0.16%, respectively. (b) Correlation between serum citrulline level and net digestive protein absorption was also significant, as shown in scatter plot. The Pearson’s correlation coefficient is r ⫽ 0.48 (P ⫽ 0.046). Four patients (No. 14, 15, 19, and 21) maintained on TPN or PN/EN did not take the protein absorption test.
min (P ⫽ 0.49), fibronectin (P ⫽ 0.71), transferrin (P ⫽ 0.32), and creatinine (P ⫽ 0.33) levels or REE (P ⫽ 0.15) and body mass index (BMI) (P ⫽ 0.85). All of the six patients receiving rehabilitation therapy had increased levels of serum citrulline, digestive protein absorption, and urine D-xylose excretion, as presented in Table 3. Although not statistically significant, the percentage of increase in serum citrulline level followed a trend of correlating with that of intes-
TABLE 3 The Increased Percentage of Serum Citrulline, Intestinal Protein Absorption, and 5-h Urine D-xylose Excretion in Patients Receiving Rehabilitation Therapy Increased percentage after therapy (%) Patient No.
Serum citrulline
Protein absorption
5-h D-xylose excretion
5 6 11 14 16 20
13.94 8.30 10.87 8.11 28.00 20.48
19.96 6.67 4.81 13.81 23.90 18.57
16.54 7.14 8.23 7.27 22.69 32.75
Note. All six patients have increased serum citrulline levels, digestive protein absorption, and 5-h urine D-xylose excretion. The increased percentage of serum citrulline has the trend of correlating with that of intestinal protein absorption (r ⫽ 0.79, P ⫽ 0.063) and urine D-xylose excretion (r ⫽ 0.81, P ⫽ 0.053).
JIANFENG ET AL.: CITRULLINE AS A MARKER FOR INTESTINAL FAILURE
sistent with the assessment of absorption function of the small bowel with this biomarker. Being not a protein component, citrulline is incorporated neither in enteral food nor endogenous proteins, and it is not released from proteolysis as most other amino acids are [13]. Therefore, body protein composition or diet status does not have a major effect on its blood level. This point was confirmed by our observation that serum citrulline does not correlate independently with either nutritional status (BMI, serum albumin, prealbumin, transferrin, fibronectin) or energy expenditure (REE). Among the 22 SB patients, 3 patients (No. 14, 15, and 19) did not have small bowel (but the duodenum and colon remained), and their serum citrulline concentrations were 0.92, 1.25, and 0.77 mol/L, respectively (only 4.6 –7.4% of normal value), suggesting that organs or tissues other than small bowel only have a minor effect on blood level. Renal dysfunction may have an impact on serum citrulline levels, but will not be statistically significant until plasma creatinine clearance is below 50 ml/min [14], which is excluded in our patients. Bowel rehabilitation therapy, which combines administration of growth hormone, glutamine, nutritional support, and other enterotrophic factors, has been widely applied in SB patients and its gut trophic effect has been well recognized [2, 15, 16]. In this study, all six patients receiving this regimen had increased levels of serum citrulline, 5-h D-xylose excretion, and digestive protein absorption. Although not statistically significant (possibly due to limited subjects), the increased percentage of serum citrulline followed the trend of correlating with that of urine D-xylose excretion (r ⫽ 0.81, P ⫽ 0.053) and protein absorption (r ⫽ 0.79, P ⫽ 0.063), indicating that serum citrulline may also be a candidate marker for the therapeutic effect of rehabilitation therapy on patients with intestinal failure. We have chosen X-ray film and 5-h urine D-xylose excretion for assessment of small intestinal surface area in this study. However, it should be pointed out that the “gold standard” assay for small bowel mucosal area still remains. X-ray evaluation may reflect the small bowel length accurately [17], but fail to detect the height of the villous and the depth of the crypts, which may vary among individuals and are often the most responsive element to a specific therapy, while the accuracy of 5-h urine D-xylose excretion test may be compromised by intestinal microbacterial overgrowth, intestinal transit hurry, renal function, and the remnant site of the small bowel [6, 18]. The average serum citrulline level in the healthy Chinese controls is 16.87 mol/L, which is much lower than that reported by Crenn et al. in the French population (above 40 mol/L) [1, 19], possibly due to racial discrepancy or difference in long-term diet habits. In
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fact, blood citrulline may vary widely among healthy individuals. In Crenn’s study, the normal value ranges from 20 to 60 mol/L in 51 healthy controls, while in another two studies conducted by Lutgens et al. [20] and Apostol et al. [21], the levels were 25.7 and 26 mol/L, respectively. More data are needed to confirm our results in the Chinese population. In conclusion, our results demonstrate that after massive small bowel resection, the serum citrulline levels provide valuable information on the intestinal surface area available for nutrient absorption and its absorptive capacity of protein. Detection of patients’ serum citrulline level has the advantage of being simpler and less time-consuming than conventional balance study or radiological morphological evaluation and is easily repeated. Our preliminary observation of serum citrulline level changes in SB patients receiving rehabilitation therapy supports extending the application of this serum marker in monitoring the variants in intestinal absorptive area and function in SB patients receiving specific regimen. Determination of its trends over time will be easily performed by repeated measurement over a long period. To extend its diagnostic value in clinical practice, further studies are to be made on the application of this biomarker in a wider category of surgical patients, especially the critically ill such as those with severe acute pancreatitis, in whom intestinal epithelial injury is common and sometimes the major cause of mortality. REFERENCES 1.
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