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Serum Intestinal Fatty Acid Binding Protein and Phosphate Levels in the Diagnosis of Acute Intestinal Ischemia: An Experimental Study in Rabbits
The Journal of Emergency Medicine, Vol. 42, No. 6, pp. 741–747, 2012 Copyright Ó 2012 Elsevier Inc. Printed in the USA. All rights reserved 0736-4679/$ - see front matter
doi:10.1016/j.jemermed.2011.05.051
Brief Reports SERUM INTESTINAL FATTY ACID BINDING PROTEIN AND PHOSPHATE LEVELS IN THE DIAGNOSIS OF ACUTE INTESTINAL ISCHEMIA: AN EXPERIMENTAL STUDY IN RABBITS Zerrin Defne Dundar, MD,* Basar Cander,† Mehmet Gul,† Keziban Ucar Karabulut, MD,† Sedat Kocak,† Sadik Girisgin,† _Idris Mehmetoglu,‡ and Hatice Toy§ *Department of Emergency Medicine, Konya Training and Research Hospital, Konya, Turkey, †Department of Emergency Medicine, ‡Department of Biochemistry, and §Department of Pathology, Selcuk University Meram Faculty of Medicine, Konya, Turkey Reprint Address: Zerrin Defne Dundar, MD, Department of Emergency Medicine, Konya Training and Research Hospital, Konya, Turkey
, Keywords—IFABP; intestinal ischemia; phosphate; rabbit; abdominal pain
, Abstract—Background: Acute intestinal ischemia is a serious clinical disorder with mesenteric infarction, which has high mortality. It is important to establish a biochemical marker for the early diagnosis of acute intestinal ischemia. Objectives: The aim of this experimental study was to assess the changes in the serum levels of intestinal fatty acid binding protein (IFABP) and phosphate by time using the acute intestinal ischemia model in rabbits. Methods: In this study, 21 New Zealand rabbits were randomly divided into three groups. Blood samples were obtained at 0, 1, 3, and 6 h in the control group. Blood samples were obtained at 0, 1, 3, and 6 h in the sham group after simple laparotomy. Blood samples were obtained at the same hours in the ischemia group after simple laparotomy and ligation of the superior mesenteric artery. Results: There was no significant difference between the control, the sham, and the ischemia groups in terms of serum IFABP levels at any time (p > 0.05). Serum phosphate levels significantly increased in the ischemia group (p < 0.001). Studies on IFABP have begun emerging in the literature, and there is no standard approach for the technique to measure the IFABP level. No studies on IFABP were found in the literature on rabbits. Conclusion: Based on our results, the role that IFABP levels play in the diagnosis of acute intestinal ischemia is unclear at this time. Serum phosphate levels continued to rise as the duration of ischemia was prolonged. These findings support the suggestion that serum phosphate levels are valuable for the diagnosis of acute intestinal ischemia. Ó 2012 Elsevier Inc.
INTRODUCTION Acute intestinal ischemia is a potentially fatal vascular emergency with overall mortality of 60–80% (1). Although acute intestinal ischemia constitutes 1–2% of all gastrointestinal diseases, the incidence has recently increased with the increasing age of the population (2). Intestinal ischemia is associated with high morbidity and mortality because the diagnosis is typically difficult and delayed due to non-specific results of biological and radiological tests (1,3,4). In a study performed by Luther et al., only in 26 patients (41%) was a preoperative diagnostic work-up, including angiography (n = 12) and computed tomography (n = 14) performed, whereas in 42 cases the intestinal ischemia was diagnosed during surgical exploration (5). In conclusion, delays in the diagnosis and operation have led to an in-hospital mortality rate as high as 67% (5). Early diagnosis before the development of intestinal infarct and peritonitis is essential in patient survival (1,6). However, there is no specific diagnostic tool yet for early diagnosis (7). A biochemical marker is highly desirable for the early diagnosis of acute intestinal
RECEIVED: 6 October 2010; FINAL SUBMISSION RECEIVED: 27 November 2010; ACCEPTED: 25 May 2011 741
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ischemia. It is also important that it can be measured in an easily obtained sample such as venous blood. Fatty acid binding proteins (FABPs) display a high affinity for long-chain fatty acids and seem to function in metabolism and intracellular transport of lipids. Two distinct FABPs are expressed in the small intestine: the liver fatty acid binding protein encoded by the FABP1 gene and the intestinal fatty acid binding protein (IFABP) encoded by the FABP2 gene. The function of these proteins is still under investigation. The presence of two structurally distinct, independently regulated FABPs in the intestine has led to the speculation that these proteins assume unique roles in intestinal fatty acid metabolism (8,9). IFABP is an intracellular epithelial protein in the stomach and the small and large intestinal mucosa. IFABP appears in the circulation after epithelial damage. There are findings suggesting that plasma IFABP levels are useful markers for the early diagnosis of acute intestinal ischemia (10,11). Serum phosphate levels begin to increase just after mesenteric artery occlusion due to release of intracellular phosphate into the circulation after cellular damage in acute intestinal ischemia (12). Although this has been known for a long time, it has had no common practical use. The aim of this experimental study was to assess the changes in the serum levels of IFABP and phosphate by time using the acute intestinal ischemia model developing after superior mesenteric artery occlusion in rabbits.
Vacutainer (Becton, Dickinson and Company, Sparks, MD) tube for biochemical tests. After the blood sampling, the abdominal region was shaved and cleaned using 10% Povidine iodine. Laparotomy was performed through a midline incision. After passing the peritoneum, the abdominal wall and the peritoneum were sutured using 2/0 silk. After the operation, 5-mL blood samples were obtained at 1, 3, and 6 h for biochemical tests and 5 mL 0.9% saline were administered through the same vessel after each blood sampling. No tissue sample was obtained in this group.
MATERIAL AND METHODS
Each 5-mL blood sample obtained in a gelose Vacutainer tube was kept for 30 min for clotting, and then centrifuged at 3000 rpm for 10 min. The obtained serum samples were stored in Eppendorf tubes (Eppendorf, Hamburg, Germany) by pipetting. The samples were kept at 20 C for biochemical tests. Ten-centimeter specimens of the distal ileum were washed using saline and fixed in 10% formaldehyde solution for histopathological examination and were embedded in paraffin blocks after the routine xylolalcohol series.
The Ethics Committee for Experimental Animal Studies of Selcuk University approved this experimental study. Study Protocol In this study, a total of 21 adult female New Zealand rabbits with body weights ranging between 2500 and 3000 g were used. All rabbits were kept under the same environment and feeding conditions. The rabbits were not fed for 12 h before the experiment and only allowed to drink water. They were randomly divided into three groups with seven in each. Control group (Group I). After undergoing anesthesia with 50 mg/kg ketamine and 15 mg/kg xylasine, vascular access was established through dorsal auricular veins. Blood samples of 5 mL were obtained at 0, 1, 3, and 6 h for biochemical tests and 5 mL 0.9% saline was administered through the same vessel after each blood sampling. No tissue sample was obtained in this group. Sham group (Group II).After being anesthetized, vascular access was established through the dorsal auricular veins. Blood sample of 5 mL was obtained at 0 h in a gelose
Ischemia group (Group III). After similar preparations in the sham group, laparotomy was performed through a midline incision. The superior mesenteric artery was found and ligated using 0 silk. The abdominal wall and the peritoneum were sutured using 2/0 silk. After the operation, 5-mL blood samples were obtained at 1, 3, and 6 h for biochemical tests and 5 mL 0.9% saline was administered through the same vessel after each blood sampling. After 6 h of ischemia, the rabbits were sacrificed. Ten-centimeter specimens of the distal ileum were washed using saline and stored in 10% formaldehyde solution for histopathological examination. Storing the Specimens
Assessing the Samples Biochemical assessment. The appropriate enzyme-linked immunosorbent assay (ELISA) kit was used to determine the serum IFABP levels (Rabbit IFABP ELISA kit, CSB-E06911RB, Cusabio Biotech Co. Ltd., Newark, DE). Serum phosphate levels were measured using the routine biochemical kits. Histopathological assessment. Sections of 5-micron thickness were prepared from the tissue samples embedded in paraffin blocks using the microtome. The specimens were stained by hematoxylin-eosin and examined under the light microscope at 100 magnification.
Serum IFABP and Phosphate in Acute Intestinal Ischemia
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Table 1. The Mean Serum IFABP Levels (ng/mL)
Control group Sham group Ischemia group
0h
1h
3h
6h
Mean 6 SD
Mean 6 SD
Mean 6 SD
Mean 6 SD
2.37 6 0.70 2.53 6 1.04 2.44 6 0.84
2.40 6 0.68 2.58 6 1.09 2.46 6 0.89
2.39 6 0.67 2.48 6 0.96 2.35 6 1.03
2.42 6 0.66 2.56 6 0.86 2.50 6 0.88
IFABP = intestinal fatty acid binding protein.
Mucosal injury was classified using the scoring system by Chiu et al. (13): Grade 0: Normal villus Grade 1: Subepithelial widening, capillary congestion in the villus apex Grade 2: Subepithelial congestion extending to the villus base Grade 3: Ulceration on top of few villi, diffuse subepithelial congestion Grade 4: Ulceration in the villus, dilatated capillaries in the lamina propria Grade 5: Irregularity, hemorrhage, and ulceration in the lamina propria Statistics. The data were installed and analyzed using SPSS 16.0 software (SPSS Inc., Chicago, IL). The Kruskal-Wallis variance analysis and Mann-Whitney U test (with Bonferroni correction) were used for the comparison of the groups. The Friedman and Wilcoxon tests (with Bonferroni correction) were used for the assessment of changes in markers by time. RESULTS Biochemical Markers Serum IFABP and phosphate levels were measured in all (n = 21) rabbits in the blood samples obtained at 0, 1, 3, and 6 h. The mean serum IFABP levels of the groups are presented in Table 1 and the change in serum IFABP levels by time is presented in Figure 1. There was no significant difference between the control, sham, and ischemia groups for serum IFABP levels at 0, 1, 3, and 6 h (p > 0.05 for all hours). There was no significant difference between the control, sham, and ischemia groups for changes in serum IFABP levels by time (p > 0.05 for all groups). The mean serum phosphate levels for the control, sham, and ischemia groups are presented in Table 2. The change in serum phosphate levels by time is presented in Figure 2. There was no significant difference between the control, sham, and ischemia groups in serum phosphate levels
at 0 h (p > 0.05). Serum phosphate levels at 1 h in the sham and ischemia groups were significantly higher than that of the control group (p = 0.002 for the sham group; p = 0.003 for the ischemia group). No significant difference between the sham and ischemia groups was found in serum phosphate levels at 1 h (p > 0.05). The serum phosphate levels at 3 h were significantly higher in the sham group than the control group (p = 0.002). Similarly, serum phosphate levels at 3 h were significantly higher in the ischemia group compared to the sham and the control groups (p = 0.003). Serum phosphate levels showed the tendency to significantly increase continuously in the ischemia group at 0, 1, 3, and 6 h (p < 0.001). Serum phosphate levels in the sham group were significantly higher at 1 and 3 h (p = 0.018 for 0–1 h and 0–3 h). They regressed to basal levels at 6 h (p > 0.05 for 0–6 h). Histopathological Results Intestinal tissue specimens of all rabbits were concordant with ischemia macroscopically after 6 h survival in the ischemia group (Figure 3). Areas in the intestinal tissue specimens, which were concordant with grade 4 and 5 ischemia, were observed under the light microscope with 100 magnification (Figure 4). DISCUSSION The most important step in the management of acute intestinal ischemia is no doubt an early diagnosis before the
Figure 1. Time-dependent changes of serum intestinal fatty acid binding protein (IFABP) levels.
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Table 2. The Mean Serum Phosphate Levels (mg/dL)
Control group Sham group Ischemia group
0h
1h
3h
6h
Mean 6 SD
Mean 6 SD
Mean 6 SD
Mean 6 SD
4.17 6 0.37 4.63 6 0.45 4.01 6 0.82
4.33 6 0.39 5.94 6 0.52 6.26 6 1.67
4.46 6 0.61 6.26 6 0.77 9.00 6 1.83
4.56 6 0.57 4.83 6 0.54 14.34 6 1.76
development of intestinal infarct. The survival rate of patients diagnosed within 24 h is 60%, whereas the survival rate of patients diagnosed after 24 h is 30% (1,6). Therefore, studies on diagnostic tools for acute intestinal ischemia have recently gained speed. The common outcome of these studies is lack of a marker with high sensitivity and specificity in acute intestinal ischemia enabling early diagnosis that would increase the survival rate (4,14,15). There are several reasons for the lack of a useful biochemical marker: the first problem is related to anatomical structure of the intestine; mucosa, submucosa, and smooth muscle layers. An ideal marker should be able to reflect this complicated structure. The marker should diagnose the damage limited to the mucosa before converting to full layer infarct. The second problem is related to the vascular pathway providing blood flow to the intestine passing through the liver through the portal vein. The venous blood coming from the intestines could enter the systemic circulation after exposure to the first pass effect in the liver. The third problem is that the overlapping protein expression of the liver and intestine complicates the determination of an organspecific marker (14,15). FABPs are small intracellular proteins. Most tissues express more than one type of FABP. IFABP is found only in the epithelial cells of the stomach and in the mucosa of the small and large bowel. IFABP can be detected in plasma and urine after damage of cells (11). IFABP is a promising serum marker for intestinal ischemic injury (16).
Figure 2. Time-dependent changes of serum phosphate levels.
Studies on IFABP began to emerge in the literature in the 1990s. In a retrospective study including 61 patients with acute abdominal pain, five cases were diagnosed as intestinal ischemia and eight as strangulated intestinal obstruction. Serum IFABP levels were significantly higher in patients with ischemic intestinal disease than in patients without ischemic disease. The cutoff point was 100 ng/mL. Seven patients had true-positive elevation and 6 had false-negative results. The sensitivity of IFABP levels was 54% and the specificity was 88%, and the IFABP levels were measured by enzyme immunoassay (16). In another study aimed at developing an assay for IFABP, sera from 7 patients with the suspected diagnosis of intestinal ischemia and who later, upon surgery, proved to have intestinal ischemia, showed true-positive IFABP increase, whereas 10 cases had false-positive results. Serum IFABP levels were measured by radioimmunoassay and the threshold of detection was 1.87 ng/mL (17). Another study included a cohort of 21 patients admitted with a clinical diagnosis of mechanical small bowel obstruction. Of these patients, only 3 had intestinal necrosis. Plasma IFABP levels were positive in 3 of 3 patients with necrosis, whereas the levels were elevated in only 3 of 18 patients without necrosis. IFABP levels were measured using the human ELISA kit, and the cutoff was accepted as 100 pg/mL (18).
Figure 3. Intestinal tissue specimens of all rabbits were concordant with ischemia macroscopically after 6 h survival in the ischemia group.
Serum IFABP and Phosphate in Acute Intestinal Ischemia
Figure 4. Areas in the intestinal tissue specimens that were concordant with grade 4 and 5 ischemia were observed under the light microscope with 100 magnification.
In a recent study on diagnostic markers for acute intestinal ischemia, 10 patients with the diagnosis of acute intestinal ischemia out of 71 patients were included. No significant difference was found between the groups with and without intestinal ischemia for serum IFABP levels, and the serum IFABP levels were measured by the human ELISA kit (15). In a meta-analysis of 20 studies on 18 diagnostic biochemical markers for the diagnosis of acute intestinal ischemia, the three studies on IFABP mentioned above were included. The sensitivity and specificity of serum IFABP levels in the diagnosis of intestinal ischemia were reported as 72% (95% confidence intervals [CI] 51–88%) and 73% (95% CI 62–83%), respectively (14). The clinical IFABP studies in the literature usually have a small number of patients. The biochemical measurement techniques, units, and cutoff points show variations. Further and larger studies with more standardized methods are required. There is a small number of experimental studies in the literature for IFABP levels. In an experimental study with pigs, it was reported that IFABP levels showed an increase after clamping of the intestinal artery; however, the serum IFABP levels were measured by human IFABP ELISA kit in pg/mL (11). There has been no study on IFABP levels in rabbits. We could not find a significant difference between the control, sham, and ischemia groups for serum IFABP levels in our experimental study. There was no significant difference between 0, 1, 3, and 6 h in the ischemia group. Human IFABP kits do not demonstrate a reaction with rabbit serum as they are prepared using rabbit antibodies. Therefore, the rabbit IFABP ELISA kit was used to measure the serum IFABP levels in our experimental study, and the range of the kit was 0.3–1000 ng/mL. This should be tested with further studies to find whether or not the
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lack of a significant difference is due to the range of the kit or any difference in the levels or metabolism of IFABP in rabbits. Serum phosphate levels begin to increase just after mesenteric artery occlusion due to release of intracellular phosphate into the circulation after cellular damage in acute intestinal ischemia (12). There have been two studies on serum phosphate in a meta-analysis for diagnostic markers in acute intestinal ischemia (14). In the first one, which included 42 patients, the sensitivity and specificity of serum phosphate for acute intestinal ischemia were reported as 94% and 100%, respectively (19). In the second study including 50 patients, the sensitivity of serum phosphate was 26% and the specificity was 82% (20). In some studies, it was reported that the serum phosphate level increases within the first hour of intestinal ischemia, whereas some others stated that it increases after 3–4 h of ischemia (21,22). Despite the studies for years, the utilization of the serum phosphate level in the early diagnosis of acute intestinal ischemia is still controversial and is not a routine diagnostic test (14). In our experimental study, the serum phosphate levels began to increase in the blood at the first hour of ischemia and this increase continued for the 6 h of ischemia. However, the serum phosphate levels of the sham group increased as much as the increase in the ischemia group during the first hour of ischemia. During the later hours of ischemia, serum phosphate levels in the sham group returned to normal levels; however, the increase in the ischemia group continued. These results support the hypothesis that serum phosphate level, which can be rapidly measured in many laboratories, can be used in the diagnosis of acute intestinal ischemia. According to our results, in clinical practice it seems that serum phosphate levels are useful only if elevated in intestinal ischemia diagnosis and could be repeated in patients with normal levels. On the other hand, the minimal elevations may also be doubtful because we found that the serum phosphate levels of the sham group increased initially and returned to normal levels during the later hours of ischemia. The available literature on this issue is inadequate and further studies should be performed. Limitations This study was designed to investigate the changes in serum biomarker levels by time in cases of acute mesenteric ischemia due to superior mesenteric artery occlusion, but different causes of mesenteric ischemia may exhibit different marker responses. Our findings are inadequate for stating that ‘‘IFABP levels have no role in early diagnosis of acute intestinal
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ischemia’’ and further studies are required. But, we believe that IFABP is not useful in a rabbit model of intestinal ischemia with the available tools. Further animal and human studies are required to find the changes in the blood levels of phosphate in other causes of abdominal pain and to decide the adequacy of specificity. CONCLUSIONS This experimental study is the first measuring the serum IFABP levels in the rabbit model. No significant difference was found in serum IFABP levels in the control, sham, and ischemia groups. Studies on IFABP have been gradually increasing in number in recent years and there is no standard approach in the measurement techniques for the levels in these studies. Serum phosphate levels have continued to increase in the blood in our study as the duration of ischemia increased in acute intestinal ischemia. These findings are valuable in supporting the hypothesis that acute intestinal ischemia is a systemic event from the onset, and serum phosphate levels can be used as a diagnostic tool in acute intestinal ischemia. REFERENCES 1. Oldenburg WA, Lau LL, Rodenberg TJ, Edmonds HJ, Burger CD. Acute mesenteric ischemia: a clinical review. Arch Intern Med 2004;164:1054–62. 2. Yasuhara H. Acute mesenteric ischemia: the challenge of gastroenterology. Surg Today 2005;35:185–95. 3. Chang JB, Stein TA. Mesenteric ischemia: acute and chronic. Ann Vasc Surg 2003;17:323–8. 4. Abboud B, Daher R, Boujaoude J. Acute mesenteric ischemia after cardiopulmonary bypass surgery. World Gastroenterol 2008;14: 5361–70. 5. Luther B, Moussazadeh K, Mu¨ler BT, et al. The acute mesenteric ischemia—not understood or incurable [German]. Zentralbl Chir Du¨sseldorf 2002;127:674–84.
6. American Gastroenterology Association. Technical review on intestinal ischemia. Gastroenterology 2000;118:954–68. 7. Ujiki M, Kibbe MR. Mesenteric ischemia. Perspect Vasc Surg Endovasc Ther 2005;17:309–18. 8. Montoudis A, Seidman E, Boudreau F, et al. Intestinal fatty acid binding protein regulates mitochondrion betaoxidation and cholesterol uptake. J Lipid Res 2008;49:961–72. 9. Agellon LB, Toth MJ, Thomson AB. Intracellular lipid binding proteins of the small intestine. Mol Cell Biochem 2002;239:79–82. 10. Ono T. Studies of the FABP family: a retrospective. Mol Cell Biochem 2005;277:1–6. 11. Niewold TA, Meinen M, van der Meulen J. Plasma intestinal fatty acid binding protein (I-FABP) concentrations increase following intestinal ischemia in pigs. Res Vet Sci 2004;77:89–91. 12. Lores ME, Canizares O, Rosselo PJ. The significance of elevation of serum phosphate levels in experimental intestinal ischemia. Surg Gynecol Obstet 1981;152:593–6. 13. Chiu CJ, McArdle AH, Brown R, Scott HJ, Gurd FN. Intestinal mucosal lesion in low-flow states. I. A morphological, hemodynamic, and metabolic reappraisal. Arch Surg 1970;101:478–83. 14. Evennett NJ, Petrov MS, Mittal A, Windsor JA. Systematic review and pooled estimates for diagnostic accuracy of serological markers for intestinal ischemia. World J Surg 2009;33:1374–83. 15. Block T, Nilsson TK, Bjo¨rck M, Acosta S. Diagnostic accuracy of plasma biomarkers for intestinal ischaemia. Scand J Clin Lab Invest 2008;68:242–8. 16. Kanda T, Fujii H, Tani T, et al. Intestinal fatty acid-binding protein is a useful diagnostic marker for mesenteric infarction in humans. Gastroenterology 1996;110:339–43. 17. Lieberman JM, Sacchettini J, Marks C, Marks WH. Human intestinal fatty acid binding protein: report of an assay with studies in normal volunteers and intestinal ischemia. Surgery 1997;121:335–42. 18. Cronk DR, Houseworth TP, Cuadrado DG, Herbert GS, McNutt PM, Azarow KS. Intestinal fatty acid binding protein (I-FABP) for the detection of strangulated mechanical small bowel obstruction. Curr Surg 2006;63:322–5. 19. Feretis CB, Koborozos BA, Vyssoulis GP, Manouras AJ, Apostolidis NS, Golematis BC. Serum phosphate levels in acute bowel ischemia. An aid to early diagnosis. Am Surg 1985;51: 242–4. 20. Leo PJ, Simonian HG. The role of serum phosphate level and acute ischemic bowel disease. Am J Emerg Med 1996;14:377–9. 21. Sodhi D, Arora N. Serum inorganic phosphate levels in experimental intestinal ischaemia. Indian J Med Sci 1993;47:259–63. _ ¸ o¨l Y, Aker Y. Diagnosis of intestinal ischemia 22. Uncu H, Uncu G, Ilc by measurement of serum phosphate and enzyme changes and the effectiveness of vitamin E treatment. Turk J Gastroenterol 1999; 10:272–5.
Serum IFABP and Phosphate in Acute Intestinal Ischemia
ARTICLE SUMMARY 1. Why is this topic important? Intestinal ischemia is a fatal abdominal emergency with 70–80% mortality. It is important to establish a biochemical marker for early diagnosis of acute intestinal ischemia. 2. What does this study attempt to show? In this study, we aimed to assess the changes in the serum level of intestinal fatty acid binding protein (IFABP) and phosphate by time using the acute intestinal ischemia model in rabbits. Our study is the first in which the serum IFABP levels were measured in a rabbit model. 3. What are the key findings? As a result, there was no significant difference among the three groups in terms of serum IFABP levels. Serum phosphate levels significantly increased in the ischemia group by time. 4. How is patient care impacted? Based on our results, the role that IFABP levels play in the diagnosis of acute intestinal ischemia is unclear at this time and elevated serum phosphate levels are valuable in supporting the hypothesis that acute intestinal ischemia is a systemic event from the onset and serum phosphate levels can be used as a diagnostic tool in acute intestinal ischemia.
747
doi:10.1016/j.jemermed.2011.05.051
Brief Reports SERUM INTESTINAL FATTY ACID BINDING PROTEIN AND PHOSPHATE LEVELS IN THE DIAGNOSIS OF ACUTE INTESTINAL ISCHEMIA: AN EXPERIMENTAL STUDY IN RABBITS Zerrin Defne Dundar, MD,* Basar Cander,† Mehmet Gul,† Keziban Ucar Karabulut, MD,† Sedat Kocak,† Sadik Girisgin,† _Idris Mehmetoglu,‡ and Hatice Toy§ *Department of Emergency Medicine, Konya Training and Research Hospital, Konya, Turkey, †Department of Emergency Medicine, ‡Department of Biochemistry, and §Department of Pathology, Selcuk University Meram Faculty of Medicine, Konya, Turkey Reprint Address: Zerrin Defne Dundar, MD, Department of Emergency Medicine, Konya Training and Research Hospital, Konya, Turkey
, Keywords—IFABP; intestinal ischemia; phosphate; rabbit; abdominal pain
, Abstract—Background: Acute intestinal ischemia is a serious clinical disorder with mesenteric infarction, which has high mortality. It is important to establish a biochemical marker for the early diagnosis of acute intestinal ischemia. Objectives: The aim of this experimental study was to assess the changes in the serum levels of intestinal fatty acid binding protein (IFABP) and phosphate by time using the acute intestinal ischemia model in rabbits. Methods: In this study, 21 New Zealand rabbits were randomly divided into three groups. Blood samples were obtained at 0, 1, 3, and 6 h in the control group. Blood samples were obtained at 0, 1, 3, and 6 h in the sham group after simple laparotomy. Blood samples were obtained at the same hours in the ischemia group after simple laparotomy and ligation of the superior mesenteric artery. Results: There was no significant difference between the control, the sham, and the ischemia groups in terms of serum IFABP levels at any time (p > 0.05). Serum phosphate levels significantly increased in the ischemia group (p < 0.001). Studies on IFABP have begun emerging in the literature, and there is no standard approach for the technique to measure the IFABP level. No studies on IFABP were found in the literature on rabbits. Conclusion: Based on our results, the role that IFABP levels play in the diagnosis of acute intestinal ischemia is unclear at this time. Serum phosphate levels continued to rise as the duration of ischemia was prolonged. These findings support the suggestion that serum phosphate levels are valuable for the diagnosis of acute intestinal ischemia. Ó 2012 Elsevier Inc.
INTRODUCTION Acute intestinal ischemia is a potentially fatal vascular emergency with overall mortality of 60–80% (1). Although acute intestinal ischemia constitutes 1–2% of all gastrointestinal diseases, the incidence has recently increased with the increasing age of the population (2). Intestinal ischemia is associated with high morbidity and mortality because the diagnosis is typically difficult and delayed due to non-specific results of biological and radiological tests (1,3,4). In a study performed by Luther et al., only in 26 patients (41%) was a preoperative diagnostic work-up, including angiography (n = 12) and computed tomography (n = 14) performed, whereas in 42 cases the intestinal ischemia was diagnosed during surgical exploration (5). In conclusion, delays in the diagnosis and operation have led to an in-hospital mortality rate as high as 67% (5). Early diagnosis before the development of intestinal infarct and peritonitis is essential in patient survival (1,6). However, there is no specific diagnostic tool yet for early diagnosis (7). A biochemical marker is highly desirable for the early diagnosis of acute intestinal
RECEIVED: 6 October 2010; FINAL SUBMISSION RECEIVED: 27 November 2010; ACCEPTED: 25 May 2011 741
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Z. D. Dundar et al.
ischemia. It is also important that it can be measured in an easily obtained sample such as venous blood. Fatty acid binding proteins (FABPs) display a high affinity for long-chain fatty acids and seem to function in metabolism and intracellular transport of lipids. Two distinct FABPs are expressed in the small intestine: the liver fatty acid binding protein encoded by the FABP1 gene and the intestinal fatty acid binding protein (IFABP) encoded by the FABP2 gene. The function of these proteins is still under investigation. The presence of two structurally distinct, independently regulated FABPs in the intestine has led to the speculation that these proteins assume unique roles in intestinal fatty acid metabolism (8,9). IFABP is an intracellular epithelial protein in the stomach and the small and large intestinal mucosa. IFABP appears in the circulation after epithelial damage. There are findings suggesting that plasma IFABP levels are useful markers for the early diagnosis of acute intestinal ischemia (10,11). Serum phosphate levels begin to increase just after mesenteric artery occlusion due to release of intracellular phosphate into the circulation after cellular damage in acute intestinal ischemia (12). Although this has been known for a long time, it has had no common practical use. The aim of this experimental study was to assess the changes in the serum levels of IFABP and phosphate by time using the acute intestinal ischemia model developing after superior mesenteric artery occlusion in rabbits.
Vacutainer (Becton, Dickinson and Company, Sparks, MD) tube for biochemical tests. After the blood sampling, the abdominal region was shaved and cleaned using 10% Povidine iodine. Laparotomy was performed through a midline incision. After passing the peritoneum, the abdominal wall and the peritoneum were sutured using 2/0 silk. After the operation, 5-mL blood samples were obtained at 1, 3, and 6 h for biochemical tests and 5 mL 0.9% saline were administered through the same vessel after each blood sampling. No tissue sample was obtained in this group.
MATERIAL AND METHODS
Each 5-mL blood sample obtained in a gelose Vacutainer tube was kept for 30 min for clotting, and then centrifuged at 3000 rpm for 10 min. The obtained serum samples were stored in Eppendorf tubes (Eppendorf, Hamburg, Germany) by pipetting. The samples were kept at 20 C for biochemical tests. Ten-centimeter specimens of the distal ileum were washed using saline and fixed in 10% formaldehyde solution for histopathological examination and were embedded in paraffin blocks after the routine xylolalcohol series.
The Ethics Committee for Experimental Animal Studies of Selcuk University approved this experimental study. Study Protocol In this study, a total of 21 adult female New Zealand rabbits with body weights ranging between 2500 and 3000 g were used. All rabbits were kept under the same environment and feeding conditions. The rabbits were not fed for 12 h before the experiment and only allowed to drink water. They were randomly divided into three groups with seven in each. Control group (Group I). After undergoing anesthesia with 50 mg/kg ketamine and 15 mg/kg xylasine, vascular access was established through dorsal auricular veins. Blood samples of 5 mL were obtained at 0, 1, 3, and 6 h for biochemical tests and 5 mL 0.9% saline was administered through the same vessel after each blood sampling. No tissue sample was obtained in this group. Sham group (Group II).After being anesthetized, vascular access was established through the dorsal auricular veins. Blood sample of 5 mL was obtained at 0 h in a gelose
Ischemia group (Group III). After similar preparations in the sham group, laparotomy was performed through a midline incision. The superior mesenteric artery was found and ligated using 0 silk. The abdominal wall and the peritoneum were sutured using 2/0 silk. After the operation, 5-mL blood samples were obtained at 1, 3, and 6 h for biochemical tests and 5 mL 0.9% saline was administered through the same vessel after each blood sampling. After 6 h of ischemia, the rabbits were sacrificed. Ten-centimeter specimens of the distal ileum were washed using saline and stored in 10% formaldehyde solution for histopathological examination. Storing the Specimens
Assessing the Samples Biochemical assessment. The appropriate enzyme-linked immunosorbent assay (ELISA) kit was used to determine the serum IFABP levels (Rabbit IFABP ELISA kit, CSB-E06911RB, Cusabio Biotech Co. Ltd., Newark, DE). Serum phosphate levels were measured using the routine biochemical kits. Histopathological assessment. Sections of 5-micron thickness were prepared from the tissue samples embedded in paraffin blocks using the microtome. The specimens were stained by hematoxylin-eosin and examined under the light microscope at 100 magnification.
Serum IFABP and Phosphate in Acute Intestinal Ischemia
743
Table 1. The Mean Serum IFABP Levels (ng/mL)
Control group Sham group Ischemia group
0h
1h
3h
6h
Mean 6 SD
Mean 6 SD
Mean 6 SD
Mean 6 SD
2.37 6 0.70 2.53 6 1.04 2.44 6 0.84
2.40 6 0.68 2.58 6 1.09 2.46 6 0.89
2.39 6 0.67 2.48 6 0.96 2.35 6 1.03
2.42 6 0.66 2.56 6 0.86 2.50 6 0.88
IFABP = intestinal fatty acid binding protein.
Mucosal injury was classified using the scoring system by Chiu et al. (13): Grade 0: Normal villus Grade 1: Subepithelial widening, capillary congestion in the villus apex Grade 2: Subepithelial congestion extending to the villus base Grade 3: Ulceration on top of few villi, diffuse subepithelial congestion Grade 4: Ulceration in the villus, dilatated capillaries in the lamina propria Grade 5: Irregularity, hemorrhage, and ulceration in the lamina propria Statistics. The data were installed and analyzed using SPSS 16.0 software (SPSS Inc., Chicago, IL). The Kruskal-Wallis variance analysis and Mann-Whitney U test (with Bonferroni correction) were used for the comparison of the groups. The Friedman and Wilcoxon tests (with Bonferroni correction) were used for the assessment of changes in markers by time. RESULTS Biochemical Markers Serum IFABP and phosphate levels were measured in all (n = 21) rabbits in the blood samples obtained at 0, 1, 3, and 6 h. The mean serum IFABP levels of the groups are presented in Table 1 and the change in serum IFABP levels by time is presented in Figure 1. There was no significant difference between the control, sham, and ischemia groups for serum IFABP levels at 0, 1, 3, and 6 h (p > 0.05 for all hours). There was no significant difference between the control, sham, and ischemia groups for changes in serum IFABP levels by time (p > 0.05 for all groups). The mean serum phosphate levels for the control, sham, and ischemia groups are presented in Table 2. The change in serum phosphate levels by time is presented in Figure 2. There was no significant difference between the control, sham, and ischemia groups in serum phosphate levels
at 0 h (p > 0.05). Serum phosphate levels at 1 h in the sham and ischemia groups were significantly higher than that of the control group (p = 0.002 for the sham group; p = 0.003 for the ischemia group). No significant difference between the sham and ischemia groups was found in serum phosphate levels at 1 h (p > 0.05). The serum phosphate levels at 3 h were significantly higher in the sham group than the control group (p = 0.002). Similarly, serum phosphate levels at 3 h were significantly higher in the ischemia group compared to the sham and the control groups (p = 0.003). Serum phosphate levels showed the tendency to significantly increase continuously in the ischemia group at 0, 1, 3, and 6 h (p < 0.001). Serum phosphate levels in the sham group were significantly higher at 1 and 3 h (p = 0.018 for 0–1 h and 0–3 h). They regressed to basal levels at 6 h (p > 0.05 for 0–6 h). Histopathological Results Intestinal tissue specimens of all rabbits were concordant with ischemia macroscopically after 6 h survival in the ischemia group (Figure 3). Areas in the intestinal tissue specimens, which were concordant with grade 4 and 5 ischemia, were observed under the light microscope with 100 magnification (Figure 4). DISCUSSION The most important step in the management of acute intestinal ischemia is no doubt an early diagnosis before the
Figure 1. Time-dependent changes of serum intestinal fatty acid binding protein (IFABP) levels.
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Table 2. The Mean Serum Phosphate Levels (mg/dL)
Control group Sham group Ischemia group
0h
1h
3h
6h
Mean 6 SD
Mean 6 SD
Mean 6 SD
Mean 6 SD
4.17 6 0.37 4.63 6 0.45 4.01 6 0.82
4.33 6 0.39 5.94 6 0.52 6.26 6 1.67
4.46 6 0.61 6.26 6 0.77 9.00 6 1.83
4.56 6 0.57 4.83 6 0.54 14.34 6 1.76
development of intestinal infarct. The survival rate of patients diagnosed within 24 h is 60%, whereas the survival rate of patients diagnosed after 24 h is 30% (1,6). Therefore, studies on diagnostic tools for acute intestinal ischemia have recently gained speed. The common outcome of these studies is lack of a marker with high sensitivity and specificity in acute intestinal ischemia enabling early diagnosis that would increase the survival rate (4,14,15). There are several reasons for the lack of a useful biochemical marker: the first problem is related to anatomical structure of the intestine; mucosa, submucosa, and smooth muscle layers. An ideal marker should be able to reflect this complicated structure. The marker should diagnose the damage limited to the mucosa before converting to full layer infarct. The second problem is related to the vascular pathway providing blood flow to the intestine passing through the liver through the portal vein. The venous blood coming from the intestines could enter the systemic circulation after exposure to the first pass effect in the liver. The third problem is that the overlapping protein expression of the liver and intestine complicates the determination of an organspecific marker (14,15). FABPs are small intracellular proteins. Most tissues express more than one type of FABP. IFABP is found only in the epithelial cells of the stomach and in the mucosa of the small and large bowel. IFABP can be detected in plasma and urine after damage of cells (11). IFABP is a promising serum marker for intestinal ischemic injury (16).
Figure 2. Time-dependent changes of serum phosphate levels.
Studies on IFABP began to emerge in the literature in the 1990s. In a retrospective study including 61 patients with acute abdominal pain, five cases were diagnosed as intestinal ischemia and eight as strangulated intestinal obstruction. Serum IFABP levels were significantly higher in patients with ischemic intestinal disease than in patients without ischemic disease. The cutoff point was 100 ng/mL. Seven patients had true-positive elevation and 6 had false-negative results. The sensitivity of IFABP levels was 54% and the specificity was 88%, and the IFABP levels were measured by enzyme immunoassay (16). In another study aimed at developing an assay for IFABP, sera from 7 patients with the suspected diagnosis of intestinal ischemia and who later, upon surgery, proved to have intestinal ischemia, showed true-positive IFABP increase, whereas 10 cases had false-positive results. Serum IFABP levels were measured by radioimmunoassay and the threshold of detection was 1.87 ng/mL (17). Another study included a cohort of 21 patients admitted with a clinical diagnosis of mechanical small bowel obstruction. Of these patients, only 3 had intestinal necrosis. Plasma IFABP levels were positive in 3 of 3 patients with necrosis, whereas the levels were elevated in only 3 of 18 patients without necrosis. IFABP levels were measured using the human ELISA kit, and the cutoff was accepted as 100 pg/mL (18).
Figure 3. Intestinal tissue specimens of all rabbits were concordant with ischemia macroscopically after 6 h survival in the ischemia group.
Serum IFABP and Phosphate in Acute Intestinal Ischemia
Figure 4. Areas in the intestinal tissue specimens that were concordant with grade 4 and 5 ischemia were observed under the light microscope with 100 magnification.
In a recent study on diagnostic markers for acute intestinal ischemia, 10 patients with the diagnosis of acute intestinal ischemia out of 71 patients were included. No significant difference was found between the groups with and without intestinal ischemia for serum IFABP levels, and the serum IFABP levels were measured by the human ELISA kit (15). In a meta-analysis of 20 studies on 18 diagnostic biochemical markers for the diagnosis of acute intestinal ischemia, the three studies on IFABP mentioned above were included. The sensitivity and specificity of serum IFABP levels in the diagnosis of intestinal ischemia were reported as 72% (95% confidence intervals [CI] 51–88%) and 73% (95% CI 62–83%), respectively (14). The clinical IFABP studies in the literature usually have a small number of patients. The biochemical measurement techniques, units, and cutoff points show variations. Further and larger studies with more standardized methods are required. There is a small number of experimental studies in the literature for IFABP levels. In an experimental study with pigs, it was reported that IFABP levels showed an increase after clamping of the intestinal artery; however, the serum IFABP levels were measured by human IFABP ELISA kit in pg/mL (11). There has been no study on IFABP levels in rabbits. We could not find a significant difference between the control, sham, and ischemia groups for serum IFABP levels in our experimental study. There was no significant difference between 0, 1, 3, and 6 h in the ischemia group. Human IFABP kits do not demonstrate a reaction with rabbit serum as they are prepared using rabbit antibodies. Therefore, the rabbit IFABP ELISA kit was used to measure the serum IFABP levels in our experimental study, and the range of the kit was 0.3–1000 ng/mL. This should be tested with further studies to find whether or not the
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lack of a significant difference is due to the range of the kit or any difference in the levels or metabolism of IFABP in rabbits. Serum phosphate levels begin to increase just after mesenteric artery occlusion due to release of intracellular phosphate into the circulation after cellular damage in acute intestinal ischemia (12). There have been two studies on serum phosphate in a meta-analysis for diagnostic markers in acute intestinal ischemia (14). In the first one, which included 42 patients, the sensitivity and specificity of serum phosphate for acute intestinal ischemia were reported as 94% and 100%, respectively (19). In the second study including 50 patients, the sensitivity of serum phosphate was 26% and the specificity was 82% (20). In some studies, it was reported that the serum phosphate level increases within the first hour of intestinal ischemia, whereas some others stated that it increases after 3–4 h of ischemia (21,22). Despite the studies for years, the utilization of the serum phosphate level in the early diagnosis of acute intestinal ischemia is still controversial and is not a routine diagnostic test (14). In our experimental study, the serum phosphate levels began to increase in the blood at the first hour of ischemia and this increase continued for the 6 h of ischemia. However, the serum phosphate levels of the sham group increased as much as the increase in the ischemia group during the first hour of ischemia. During the later hours of ischemia, serum phosphate levels in the sham group returned to normal levels; however, the increase in the ischemia group continued. These results support the hypothesis that serum phosphate level, which can be rapidly measured in many laboratories, can be used in the diagnosis of acute intestinal ischemia. According to our results, in clinical practice it seems that serum phosphate levels are useful only if elevated in intestinal ischemia diagnosis and could be repeated in patients with normal levels. On the other hand, the minimal elevations may also be doubtful because we found that the serum phosphate levels of the sham group increased initially and returned to normal levels during the later hours of ischemia. The available literature on this issue is inadequate and further studies should be performed. Limitations This study was designed to investigate the changes in serum biomarker levels by time in cases of acute mesenteric ischemia due to superior mesenteric artery occlusion, but different causes of mesenteric ischemia may exhibit different marker responses. Our findings are inadequate for stating that ‘‘IFABP levels have no role in early diagnosis of acute intestinal
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ischemia’’ and further studies are required. But, we believe that IFABP is not useful in a rabbit model of intestinal ischemia with the available tools. Further animal and human studies are required to find the changes in the blood levels of phosphate in other causes of abdominal pain and to decide the adequacy of specificity. CONCLUSIONS This experimental study is the first measuring the serum IFABP levels in the rabbit model. No significant difference was found in serum IFABP levels in the control, sham, and ischemia groups. Studies on IFABP have been gradually increasing in number in recent years and there is no standard approach in the measurement techniques for the levels in these studies. Serum phosphate levels have continued to increase in the blood in our study as the duration of ischemia increased in acute intestinal ischemia. These findings are valuable in supporting the hypothesis that acute intestinal ischemia is a systemic event from the onset, and serum phosphate levels can be used as a diagnostic tool in acute intestinal ischemia. REFERENCES 1. Oldenburg WA, Lau LL, Rodenberg TJ, Edmonds HJ, Burger CD. Acute mesenteric ischemia: a clinical review. Arch Intern Med 2004;164:1054–62. 2. Yasuhara H. Acute mesenteric ischemia: the challenge of gastroenterology. Surg Today 2005;35:185–95. 3. Chang JB, Stein TA. Mesenteric ischemia: acute and chronic. Ann Vasc Surg 2003;17:323–8. 4. Abboud B, Daher R, Boujaoude J. Acute mesenteric ischemia after cardiopulmonary bypass surgery. World Gastroenterol 2008;14: 5361–70. 5. Luther B, Moussazadeh K, Mu¨ler BT, et al. The acute mesenteric ischemia—not understood or incurable [German]. Zentralbl Chir Du¨sseldorf 2002;127:674–84.
6. American Gastroenterology Association. Technical review on intestinal ischemia. Gastroenterology 2000;118:954–68. 7. Ujiki M, Kibbe MR. Mesenteric ischemia. Perspect Vasc Surg Endovasc Ther 2005;17:309–18. 8. Montoudis A, Seidman E, Boudreau F, et al. Intestinal fatty acid binding protein regulates mitochondrion betaoxidation and cholesterol uptake. J Lipid Res 2008;49:961–72. 9. Agellon LB, Toth MJ, Thomson AB. Intracellular lipid binding proteins of the small intestine. Mol Cell Biochem 2002;239:79–82. 10. Ono T. Studies of the FABP family: a retrospective. Mol Cell Biochem 2005;277:1–6. 11. Niewold TA, Meinen M, van der Meulen J. Plasma intestinal fatty acid binding protein (I-FABP) concentrations increase following intestinal ischemia in pigs. Res Vet Sci 2004;77:89–91. 12. Lores ME, Canizares O, Rosselo PJ. The significance of elevation of serum phosphate levels in experimental intestinal ischemia. Surg Gynecol Obstet 1981;152:593–6. 13. Chiu CJ, McArdle AH, Brown R, Scott HJ, Gurd FN. Intestinal mucosal lesion in low-flow states. I. A morphological, hemodynamic, and metabolic reappraisal. Arch Surg 1970;101:478–83. 14. Evennett NJ, Petrov MS, Mittal A, Windsor JA. Systematic review and pooled estimates for diagnostic accuracy of serological markers for intestinal ischemia. World J Surg 2009;33:1374–83. 15. Block T, Nilsson TK, Bjo¨rck M, Acosta S. Diagnostic accuracy of plasma biomarkers for intestinal ischaemia. Scand J Clin Lab Invest 2008;68:242–8. 16. Kanda T, Fujii H, Tani T, et al. Intestinal fatty acid-binding protein is a useful diagnostic marker for mesenteric infarction in humans. Gastroenterology 1996;110:339–43. 17. Lieberman JM, Sacchettini J, Marks C, Marks WH. Human intestinal fatty acid binding protein: report of an assay with studies in normal volunteers and intestinal ischemia. Surgery 1997;121:335–42. 18. Cronk DR, Houseworth TP, Cuadrado DG, Herbert GS, McNutt PM, Azarow KS. Intestinal fatty acid binding protein (I-FABP) for the detection of strangulated mechanical small bowel obstruction. Curr Surg 2006;63:322–5. 19. Feretis CB, Koborozos BA, Vyssoulis GP, Manouras AJ, Apostolidis NS, Golematis BC. Serum phosphate levels in acute bowel ischemia. An aid to early diagnosis. Am Surg 1985;51: 242–4. 20. Leo PJ, Simonian HG. The role of serum phosphate level and acute ischemic bowel disease. Am J Emerg Med 1996;14:377–9. 21. Sodhi D, Arora N. Serum inorganic phosphate levels in experimental intestinal ischaemia. Indian J Med Sci 1993;47:259–63. _ ¸ o¨l Y, Aker Y. Diagnosis of intestinal ischemia 22. Uncu H, Uncu G, Ilc by measurement of serum phosphate and enzyme changes and the effectiveness of vitamin E treatment. Turk J Gastroenterol 1999; 10:272–5.
Serum IFABP and Phosphate in Acute Intestinal Ischemia
ARTICLE SUMMARY 1. Why is this topic important? Intestinal ischemia is a fatal abdominal emergency with 70–80% mortality. It is important to establish a biochemical marker for early diagnosis of acute intestinal ischemia. 2. What does this study attempt to show? In this study, we aimed to assess the changes in the serum level of intestinal fatty acid binding protein (IFABP) and phosphate by time using the acute intestinal ischemia model in rabbits. Our study is the first in which the serum IFABP levels were measured in a rabbit model. 3. What are the key findings? As a result, there was no significant difference among the three groups in terms of serum IFABP levels. Serum phosphate levels significantly increased in the ischemia group by time. 4. How is patient care impacted? Based on our results, the role that IFABP levels play in the diagnosis of acute intestinal ischemia is unclear at this time and elevated serum phosphate levels are valuable in supporting the hypothesis that acute intestinal ischemia is a systemic event from the onset and serum phosphate levels can be used as a diagnostic tool in acute intestinal ischemia.
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