Objective: Our aim in this study was to examine the effects of bosentan, an orally active antagonist of endothelin A and B receptors, on plasma ischemia-modified albumin (IMA) levels in mesenteric ischemia induced rats. Material and Method: In this randomized, controlled trial 36 mature female wistor rats were divided into three groups. Group 1 (n=12) was the control group which only laparotomy was performed. Group 2 (n=12) was the ischemia group and group 3 (n=12) was the bosentan pretreated (100 mg/kg-1 day, 1 gavage/day for two days before surgery) ischemia group. In the ischemia groups (GR 2 and GR 3) following laparotomy the superior mesenteric artery was clamped using a bulldog clamp during laparotomy. Blood samples were taken at 30 minutes from all groups and IMA levels were studied. Results: Plasma IMA levels in the ischemia group (GR 2) was significantly higher compared to those of the control (p<0.001) and bosentan pretreated group (GR 3) (p=0.002). Serum IMA levels were higher in GR 3 than control group but there were no statistical significance (p=0.659). Conclusion: These preliminary results suggest that bosentan might have a protective effect in mesenteric ischemic conditions

Aydın, Sinan

Gülpınar, Kamil

Korkmaz, Atilla

Özdemir, Süleyman

Öziş, Erpulat

English

Ufuk Universitesi Akademik Acikerisim Sistemi

NOBEL MEDICUS 26 | C LT: 9, SAYI: 296 THE EFFECT OF BOSENTAN ON PLASMA ISCHEMIA-MODIFIED ALBUMIN LEVELS IN ACUTE MESENTERIC ISCHEMIAKamil Gülpınar, Süleyman Özdemir, Erpulat Özi , Sinan Aydın, Atilla KorkmazUfuk University School of Medicine, General Surgery Department, AnkaraABSTRACTObjective:  Our aim in this study was to examine the effects of bosentan, an orally active antagonist of endothelin A and B receptors, on plasma ischemia-modified albumin (IMA) levels in mesenteric ischemia induced rats. Material and Method: In this randomized, controlled trial 36 mature female wistor rats were divided into three groups. Group 1 (n=12) was the control group which only laparotomy was performed. Group 2 (n=12) was the ischemia group and group 3 (n=12) was the bosentan pretreated (100 mg/kg-1 day, 1 gavage/day for two days before surgery) ischemia group. In the ischemia groups (GR 2 and GR 3) following laparotomy the superior mesenteric artery was clamped using a bulldog clamp during laparotomy. Blood samples were taken at 30 minutes from all groups and IMA levels were studied.Results: Plasma IMA levels in the ischemia group (GR 2) was significantly higher compared to those of the control (p<0.001) and bosentan pretreated group (GR 3) (p=0.002). Serum IMA levels were higher in GR 3 than control group but there were no statistical significance (p=0.659). Conclusion: These preliminary results suggest that bosentan might have a protective effect in mesenteric ischemic conditions. Key Words: Ischemia-modified albumin, bosentan, acute mesenteric ischemia  Nobel Med 2013; 9(2): 96-99AKUT MEZENTERİK İSKEMİDE, BOSENTAN’IN MODİFİYE ALBUMİN SEVİYELERİ ÜZERİNE OLAN ETKİSİÖZET Amaç: Bu deneysel hayvan çalışmasındaki amacımız; endotelin A ve B reseptör antagonisti olan Bosentan adlı preperatın, mezenterik iskemi oluşturulan rat-larda plazma iskemi modifiye albumin (İMA) üzerine olan etkilerini araştırmaktır.Materyal ve Metod: Randomize kontrollü olarak planlanan bu çalışmada 36 matür dişi wistor rat kul-lanılmış ve 3 ayrı gruba ayrılmıştır. 1. grup (n=12) sadece laparotomi uygulanan kontrol grubu, 2. grup (n=12) mezenter iskemi oluşturulan grup ve 3. grup (n=12) mezenter iskemi öncesi bosentan tedavisi (100 mg/kg-1 gün, 1 gavaj/gün, cerrahi öncesi 2 gün boyunca) alan gruptur. İskemi oluşturulan 2. ve 3. grupta laparotomi sırasında superior mezenterik arter bulldog klemp ile klemplenmiştir. Tüm gruplardan kan örnekleri iskemi sonrası 30. dakikada alınmıştır ve İMA seviyeleri çalışılmıştır.Bulgular: Plazma İMA seviyeleri 2. grupta 1. grup  ve 3. grup ile karşılaştırıldığında belirgin yüksek bulun-muştur. (sırasıyla p<0,001 ve p=0,002). Serum İMA seviyeleri 3. grup ratlarda kontrol grubundan daha yüksek bulunmasına rağmen istatistiksel olarak belir-gin fark saptanmamıştır (p=0,659).Sonuç: Yaptığımız bu ön çalışma sonuçları, bosenta-nın mezenterik iskemide koruyucu etkisinin olabile-ceğini göstermektedir.Anahtar Kelimeler: İskemi modifiye albumin, bosentan, akut  mezenterik iskemi  Nobel Med 2013; 9(2): 96-99NOBEL MEDICUS 26 | C LT: 9, SAYI: 297INTRODUCTIONMesenteric ischemia is an important clinical entity due its high mortality rate, commonly caused by occlusion, vasospasm, or hypoperfusion of mesenteric vessels that leads to reduction in intestinal blood flow. The diagnosis is challenging and acute interventions are critically important. Ischemia-modified albumin (IMA) has shown to be a sensitive marker of myocardial ischemia, skeletal muscle ischemia, pulmonary embolism and in addition it has recently been shown in animal studies that;  serum IMA levels represents a significant parameter in the early diagnosis of mesenteric ischemia. Drugs that inhibit vasoconstriction and increase blood flow are the first line medical therapy in mesenteric ischemia. Bosentan is a potent competitive antagonist of both endothelin receptors A and B and has also shown to inhibit the increase in vascular resistance in strangulated bowel. As endothelins are known to be the most potent vasoconstrictor agents, their inhibition could prevent intestinal damage and reduce morbidity and mortality rate.Our aim in this study was to examine the effects of bosentan on plasma Ischemia-modified albumin levels which is suggested to be an ischemia serum marker in the diagnosis of mesenteric ischemia. MATERIAL and METHODThis is a randomized, controlled, non-blinded interventional animal study. An approval was obtained from the animal use committee. 36 mature female wistor albino rats weighing 200 to 250 gr were used in the study. The animals were kept in cages at room temperature and were fed with standard rat cow until the start of the experiments. Rats were allocated into three experimental groups. General anesthesia was administered with 50 mg/kg ketamine and 5 mg/kg xylazine applied intramuscularly. 3 ml/kg/hour saline solution were given by femoral vein catherization during the surgeries. 4 to 5 cm midline incisions were made for laparotomy and were used for all groups. A heating pad was applied during anesthesia to maintain body temperature. Group 1 (n=12) underwent a simple laparotomy and blood samples were taken in 30 minutes thereafter. Group 2 (n=12) was the mesenteric ischemia group without any pretreatment. After the laparotomy, superior mesenteric artery (SMA) was clamped with a bulldog clamp at the aortic bifurcation. Three-milliliters of aortic blood specimens were taken from each rat after 30 min. and placed in citrated tubes and send to the laboratory for IMA levels.Group 3 (n=12) was the bosentan pretreated (100 mg/kg-1 day, 1 gavage/day for two days before surgery) mesenteric ischemia group. Bosentan was given daily by gavage at the dose of 100 mg/kg. Treatment of the animals started 2 days before the experiments. The dose was given according to recommendation in literature.1,2 After the laparotomy, superior mesenteric artery (SMA) was clamped with a bulldog clamp at the aortic bifurcation (Figure 1,2). Three-milliliters of aortic blood specimens were taken from each rat after 30 min. and placed in citrated tubes and send to the laboratory for IMA levels.IMA concentrations were analyzed by measuring the complex composed of dithiothr eitol and cobalt unbound from albumin by the colorimetric method as described by Gunduz et al.3 Specimen absorbencies were analyzed at 470 nm using a spectrophotometer and the results were given as absorbance units (ABSU).All animals were sacrificed by cervical dislocation after blood samples for IMA measurements were taken. 4 rats were died due to general anesthesia before surgery and 2 rats died during surgeries due to the blood loss. New rats were used instead to equal the number of subjects. RESULTSStatistical AnalysisData analysis was performed with SPSS for windows, version 11.5 (SPSS, Chicago, IL). Data were expressed as mean±SD. Comparison of results between experimental groups was performed one-way ANOVA followed by a Tukey`s test. A probability value of p<0.05 was considered to be statistically significant.The mean values are summarized in Table 1. Serum IMA levels in the clamped group were significantly higher compared to the levels of control group and bosentan pretreated clamped group. IMA levels in bosentan pretreated and clamped group were slightly higher than the control group but showed no statistical significance (p=0.659). IMA levels in bosentan pretreated clamped group were significantly lower than the non-treated clamped group (p=0.002). Table 2 showing the summary of comparison values between groups.DISCUSSIONMesenteric ischemia is due to a reduction in intestinal blood flow, which is most commonly caused by occlusion, vasospasm or hypoperfusion of the THE EFFECT OF BOSENTAN ON PLASMA ISCHEMIA-MODIFIED ALBUMIN LEVELS IN ACUTE MESENTERIC ISCHEMIANOBEL MEDICUS 26 | C LT: 9, SAYI: 298mesenteric vessels. The superior mesenteric artery (SMA) is most commonly affected.4The underlying causes vary as follows; most commonly occlusive arterial obstruction by emboli or thrombosis of mesenteric arteries, occlusive venous obstruction by segmental strangulation or thrombosis, non-occlusive mesenteric ischemia due severe systemic illness with reduced cardiac output or intestinal vasospasm by cocaine, ergot poisoning, digoxin use. In addition, hypotension from cardiac failure, myocardial infarction, sepsis, severe liver or renal disease might predispose to mesenteric ischemia.5-8The morbidity and mortality rates are very high unless diagnosis and treatment is done rapidly.9 The overall prevalence of acute mesenteric ischemia is 0.1% of all hospital administrations but the mortality rate might be as high as 90% if the diagnosis and treatment is missed until infarction.10 Additionally, even with appropriate treatment up to 50-80% of the patients die.10,11 As early diagnosis of SMA occlusion before the inception of irreversible intestinal ischemia is extremely important, a specific serum marker is still sought.12  An increase white blood cell count or serum lactate concentration as well as the high C-reactive protein levels are insensitive and non-specific in the early stages of disease.13In recent years many studies of IMA levels in acute ischemic conditions has been reported. IMA in serum has been shown to be a rapid and low-cost technique in various ischemic conditions such as cerebral ischemia, myocardial ischemia, mesenteric ischemia, and pulmonary ischemia.3,14-16 Human serum albumin consists of 585 amino acids. The first 3 amino acids in the N-terminus, Asp-Ala-His, constitute a specific binding site for transition metals such as cobalt, copper and nickel. Rat serum albumin resembles human serum albumin in its amino acid sequence therefore it has been used in studies based on the decrease in the cobalt binding capacity of the N-terminus of human serum albumin during ischemia to rat serum albumin.17,18 During acute ischemic conditions, albumin`s metal binding capacity for transition metals are reduced thus giving rise to a metabolic variant of the protein known as IMA.16 Recently, studies focusing in ischemia modified albumin (IMA) have been published.3,18 In these experimental animal studies, IMA levels were shown to represent a significant parameter in the diagnosis of acute mesenteric ischemia. In our study we used IMA as a marker of mesenteric ischemia and its levels to compare bosentan drug effect in mesenteric ischemia.It has been shown that, structural changes of mucosal cells of intestine occur only after 10 minutes of the onset of mesenteric ischemia caused by superior mesenteric occlusion.19 Later on the mucosal and serosae layers are effected and the risk of perforation and stricture takes place.20 So as soon as the diagnosis is made, preventive cautions should be commenced. In our study, we first sought bosentan drug effect after 30 minutes of mesenteric ischemia which is enough for acute necrosis and our following studies would be made on time dependent experiments for chronic ischemia results.In mesenteric ischemia, inhibition of vasoconstriction and increase blood flow may contribute to gain time for surgery and prevent infarction. Many vasodilatotors and anti-inflammatory drugs were studied for this effect. In our study we used bosentan which is a competitive antagonist of both endothelin receptors ETa and ETb. We considered bosentan might be a suitable drug for these conditions as it inhibits the increase in vascular resistance in strangulated small bowel.21 In addition Johnson RJ et al. have demonstrated that mesenteric vessels have endothelin a and b receptors and it is highly possible that their inhibition by bosentan could prevent intestinal damage and reduce morbidity and mortality rate.22 Results of our study supported the idea, as we have shown low IMA levels in bosentan pretreated group. These preliminary results suggest that bosentan has  F1 F2Figure 1: Laparotomy was done and superior mesenteric artery was foundFigure 2. Superior mesenteric artery was clamped with a bulldog clamp at the aortic bifurcation and mesenteric ischemia was generated.Table 1: The mean values of IMA in groupsMean (absorbance units) Std Dev SEMGroup 1 0.509 0.0804 0.0195Group 2 0.726 0.146 0.0355Group 3 0.551 0.182 0.0442SEM: Standard error of the mean, IMA: Ischemia modified albuminTable 2: All pairwise multiple comparison proceduresComparison Diff of Means p p<0.050Group 2 vs Group 1 0.217 <0.001 yesGroup 2 vs Group 3 0.174 0.002 yesGroup 3 vs Group 1 0.0428 0.659 noNOBEL MEDICUS 26 | C LT: 9, SAYI: 299THE EFFECT OF BOSENTAN ON PLASMA ISCHEMIA-MODIFIED ALBUMIN LEVELS IN ACUTE MESENTERIC ISCHEMIAa protective effect of mesenteric ischemia by endothelin receptor blockage that results in low IMA levels which suggests less tissue damage in many ischemic conditions. The underlying mechanism in how the blockage of endothelin receptors might prevent the albumin N-terminus reduction of transition metal binding needs extreme molecular studies.  This study now needs to be supported by further experimental studies for confirmation and also a time dependent variations with reperfusion studies should be sought.REFERENCES1. Ostrowski RP, Januszewski S, Kowalska Z, et al. Effect of endothelin receptor antagonist bosentan on plasma leptin concentration in acute myocardial infarction in rats. Pathophysiology 2003; 9: 249-256.2. Mulder P, Richard V, Derumeaux G, et al. Role of endogenous endothelin in chronic heart failure: effect of long-term treatment with an endothelin antagonist on survival, hemodynamics, and cardiac remodeling. Circulation 1997; 96: 1976-1982.3. Gunduz A, Turedi S, Mentese A, et al. Ischemia-modified albumin in the diagnosis of acute mesenteric ischemia: a preliminary study. Am J Emerg Med 2008; 26: 202-205.4. Bingol H, Zeybek N, Cingoz F, et al. Surgical therapy for acute superior mesenteric artery embolism. Am J Surg 2004; 188: 68-70.5. Endress C, Gray DG, Wollschlaeger G. Bowel ischemia and perforation after cocaine use. AJR Am J Roentgenol 1992; 159: 73-75.6. Bassiouny HS. Nonocclusive mesenteric ischemia. Surg Clin North Am 1997; 77: 319-326.7. Cappell MS. Intestinal (mesenteric) vasculopathy. I. Acute superior mesenteric arteriopathy and venopathy. Gastroenterol Clin North Am 1998; 27: 783-825.8. Liu JJ, Ardolf JC. Sumatriptan-associated mesenteric ischemia. Ann Intern Med 2000; 132: 597.9. Boley SJ, Brandt LJ, Sammartano RJ. History of mesenteric ischemia. The evolution of a diagnosis and management. Surg Clin North Am 1997; 77: 275-288.10. Stamatakos M, Stefanaki C, Mastrokalos D, et al. Mesenteric ischemia: still a deadly puzzle for the medical community. Tohoku J Exp Med 2008; 216: 197-204.11. Acosta S, Nilsson TK, Bjorck M. D-dimer testing in patients with suspected acute thromboembolic occlusion of the superior mesenteric artery. Br J Surg 2004; 91: 991-994.12. Sreenarasimhaiah J. Diagnosis and management of intestinal ischaemic disorders. BMJ 2003; 326: 1372-1376.13. Troxler M, Thompson D, Homer-Vanniasinkam S. Ischaemic skeletal muscle increases serum ischaemia modified albumin. Eur J Vasc Endovasc Surg 2006; 31: 164-169.14. Bar-Or D, Lau E, Winkler JV. A novel assay for cobalt-albumin binding and its potential as a marker for myocardial ischemia-a preliminary report. J Emerg Med 2000; 19: 311-315.15. Mitsudo S, Brandt LJ. Pathology of intestinal ischemia. Surg Clin North Am 1992; 72: 43-63.16. Turedi S, Patan T, Gunduz A, et al. Ischemia-modified albumin in the diagnosis of pulmonary embolism: an experimental study. Am J Emerg Med 2009; 27: 635-640.17. Livaoglu M, Kerimoglu S, Karahan SC, et al. Ischemia-modified albumin and flap viability. Eur Surg Res 2009; 42: 87-90.18. Gunduz A, Turkmen S, Turedi S, et al. Time-dependent variations in ischemia-modified albumin levels in mesenteric ischemia. Acad Emerg Med 2009; 16: 539-543.19. Altinyollar H, Boyabatli M, Berberoglu U. D-dimer as a marker for early diagnosis of acute mesenteric ischemia. Thromb Res 2006; 117: 463-467.20. Patel A, Kaleya RN, Sammartano RJ. Pathophysiology of mesenteric ischemia. Surg Clin North Am 1992; 72: 31-41.21. Fevang J, Ovrebo K, Myking O, et al. Role of endothelin in the circulatory changes associated with small bowel strangulation obstruction in pigs: effects of the endothelin receptor antagonist bosentan. J Surg Res 2001; 96: 224-232.22. Johnson RJ, Fink GD, Galligan JJ. Mechanisms of endothelin-induced venoconstriction in isolated guinea pig mesentery. J Pharmacol Exp Ther 1999; 289: 762-767.CORRESPONDING AUTHOR:  Kamil Gülpınar  Mevlana Bulvarı No:86-88 06520 Balgat Ankara  kamilgulpinar@gmail.comDELIVERING DATE: 09 / 04 / 2012 • ACCEPTED DATE:  27 / 11 / 2012

The effect of bosentan on plasma ischemia-modified albumin levels in acute mesenteric ischemia

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The effect of bosentan on plasma ischemia-modified albumin levels in acute mesenteric ischemia

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