The value of combined elevation of D-dimer and neopterin as a predictive parameter for early stage acute mesenteric ischemia: An experimental study

Abstract

Background

The diagnosis of acute mesenteric ischemia is variable. Early diagnosis is important for reducing the mortality and morbidity rates.

Aim

This experimental study aims to investigate the diagnostic utility of D-dimer and neopterin as a marker for the early stage of acute mesenteric ischemia caused by occlusion of superior mesenteric artery.

Methods

The levels of D-dimer and neopterin were measured using an animal acute mesenteric ischemia model in 21 male rabbits. Superior mesenteric artery occlusion (Group 1, n = 14) and control (Group 2, n = 7) groups were identified. Blood samples at different times are collected from each rabbits. Blood samples from superior mesenteric artery occlusion group were taken 30 min after anesthesia but before laparotomy, 1, 2, and 3 h after superior mesenteric artery ligation. Blood samples from control group were taken 1 h before, 1 and 3 h after anesthesia and laparotomy. The D-dimer and neopterin levels of each blood sample were measured.

Results

The probability of acute mesenteric ischemia was found to be 36 times higher when the D-dimer level was over 0.125 ng/L, whereas the probability was 19.2 times higher when the neopterin level was over 1.25 nmol/L.

Conclusions

In this experimental study, the combined elevation of two significant markers, D-dimer and neopterin, may be helpful for the early diagnosis of acute mesenteric ischemia.

Keywords

D-dimer neopterin acute mesenteric ischemia rabbit

Introduction

Acute mesenteric vascular occlusions can cause life-threatening conditions, with high mortality rates of between 50% and 80%.^1–3 Acute bowel ischemia–related mesenteric vascular disorders represent a higher mortality rate than colon cancer, accounting for 5% of the deaths that occur in the United States on a yearly basis.^4–7 Although severe pain is an initial symptom following the occlusion of the superior mesenteric artery (SMA), the other abdominal symptoms are minimal. Therefore, at the early stage, diagnosis is difficult, even for an experienced surgeon.

Early diagnosis before the beginning of the intestinal ischemic process is crucial for prognosis in acute mesenteric ischemia (AMI). Many diagnostic tests have been employed to evaluate AMI, including elevation of lactatedehydrogenase (LDH), transamines, amylases, creatinine phosphokinase (CPK), D-dimer, D-lactate, ischemia-modified albumin, glutathione S-transferase, and intestinal fatty-acid binding protein. The levels of some markers were shown to be elevated by sixth hour of arterial occlusion, whereas the elevation of other markers takes longer. However, a few markers such as D-lactate, glutathione S-transferase, intestinal fatty acid binding protein, ischemia-modified albumin, and D-dimer were found to be effective in the early diagnosis of AMI.^8–11

The catheter angiography and biphasic mesenteric computed tomography angiography (CTA) have been defined as gold standard radiologic techniques.¹² Still both are expensive and invasive procedures, and are not readily available in every center. In recent studies, D-dimer testing has proven to be a useful diagnostic marker for AMI.^11–13 However, there is no study available demonstrating the assessment of changes occurring in the first 3 h.

Neopterin is used as a marker of cellular immune system activation and vascular endothelial damage.^14,15 Neopterin level is shown to be elevated by ischemic or septic damage of different organ systems.^16–21 Neopterin has not been studied as a marker for vascular pathologies causing AMI. Thus, we aimed to study neopterin as a marker for early AMI diagnosis.

D-dimer and neopterin as the markers related with vascular pathologies may be useful in the early diagnosis of AMI and using them together is potentially feasible. With this background, the present experimental study is organized to assess the prognostic values of the combined elevation of both markers for the early diagnosis of AMI caused by the occlusion of the SMA.

Materials and methods

Animals

In the present study, a total of 21 male rabbits (weight: 3.6–3.9 kg) were used for the experiments. Animal experiments and procedures were performed in accordance with the national guidelines for the use and care of laboratory animals, and were approved by local animal ethic committee. The rabbits were housed in standard plastic cages on sawdust bedding in an air-conditioned room at 22 ± 1℃ under lighting controls (14-h light/10-h dark cycle). Standard rabbit chow and tap water were given ad libitum.

Chemicals

Ketamine (Ketalar-500 mg-vial) was obtained from Pfizer Istanbul-Turkey. Xylazine (Rompun-50 ml-vial) was obtained from Roche Istanbul-Turkey.

Experimental model

A study group and a control group were identified. The study group (group 1) included 14 rabbits, and animals in this group underwent laparotomy, SMA occlusion, and mesenteric ischemia. The control group (group 2) included 7 rabbits, and animals in this group underwent only laparotomy but no SMA occlusion. Animals in group 1 were anesthetized as mentioned below. First blood samples were collected after 30 min of anesthesia with the aim to see the effect of anesthesia on the levels of markers. There after, a median laparotomy and SMA occlusion was performed on each rabbit as described below. Second and third blood samples were collected 1, 2, and 3 h after the SMA occlusion. These blood samples from group 1 were marked as “sample 1/0,” “sample 1/1,” “sample 1/2,” and “sample 1/3,” respectively, according to their collection time.

In group 2, the first blood samples, which are the basic levels of the markers, were collected before anesthesia with the aim of comparing the post anesthesia levels of markers in study group. After that, animals were anesthetized and they underwent a median laparotomy. SMA was not occluded in control group. Second and third blood samples were collected after 1 and 3 h after the closure of laparotomy. These blood samples from group 2 were marked as “sample 2/0,” “sample 2/1,”and “sample 2/3,” respectively, according to their collection time.

Intestinal ischemia model

The rabbits were made to fast overnight, but were allowed to drink water ad libitum. They were anesthetized by the subcutaneous administration of ketamine (30 mg/kg) and xylazine (5 mg/kg) with atropine sulfate (0.15 mg/kg). The rabbits were placed on heating pads at 37℃ throughout the experiment. An intravenous (i.v.) cannula was placed in the left dorsal auricular vein and 0.9% NaCl was administered at a rate of 10 ml/kg/h until the end of the experiment to prevent dehydration. Another cannula was placed in the right dorsal auricular vein of the rabbits for blood sampling. After a median laparotomy, the animals were laid on their right side to point out the mesentery of the intestines on the level of the aorta. The SMA was separated from the aorta and ligated with a 2/0 silk suture. Next, the midline incision was sutured. To maintain an adequate anesthetic plane, ketamine was administered (5 to 10 mg/kg i.v.). The blood samples were taken from each rabbit at previously described times. After last blood collection, the animals were sacrificed. Blood samples were immediately transferred to the laboratory to facilitate the biochemical analysis.

Biochemical evaluations

Blood was collected in two different tubes (1 ml/tube). The tube containing sodium citrate was used for D-dimer testing, while a shaded tube was used for neopterin testing (light sensitivity). A Trinity Biotech Amax 190 Coagulation Analyzer (Bray, Ireland) was employed to measure the D-dimer levels. Sera obtained from the samples were stored at −80℃ until thawing at the time of assay. Neopterin (TML,TR65101) was measured from one sample with highly sensitive enzyme-linked immunoassay (ELISA) kits, according to the manufacturer’s instructions. Assays for each animal and its matched control were run in the same lot.

Statistical analysis

The SPSS for Windows computer program (version 15.0) was used for statistical analyses. All data were expressed as means, medians, and standart deviations (SD). The Mann–Whitney U test was used to compare control and intervention levels of markers between the groups. In each group, for the repeated measurements, the Friedman test and post hoc Bonferroni-adjusted Wilcoxon-signed ranks test were used. Receiver Operating Characteristic (ROC) curve analysis was used to determine the best cut-off value for the diagnosis of AMI. A p value < 0.05 was accepted as statistically significant. The power analysis with Power and Sample Size programme is done for the SMA ligation groups. The power analyses for D-dimer and neopterin were found as 0.98 and 0.93, respectively.

Results

The mean D-dimer levels in the plasma of rabbits were defined in Table 1. Mean plasma D-dimer levels in samples 1/0, 2/0, 2/1, and 2/3 were lower than those of the samples 1/1, 1/2, and 1/3. When the plasma D-dimer levels were compared statistically, the values were significantly lower in sample 1/0 than in samples 1/1 (p = 0.006), 1/2 (p = 0.009), and 1/3 (p = 0.009) (Figure 1). When we compare the control groups with intervention groups, the values for 1 h (p = 0.001) and 3 h (p = 0.000) were significant. ROC curves were calculated to determine the diagnostic value of D-dimer. For the diagnosis of acute mesenteric ischemia in its early stage, the best cutoff point was at 0.125 ng/L (area under curve = 0.976). Calculated sensitivity, specificity, positive predictive value, and negative predictive value were 85.7%, 85.7%, 94.7%, and 66.7%, respectively. The probability of acute mesenteric ischemia was found to be 36 times higher when the D-dimer level was over 0.125 ng/L.

Figure 1.

Evaluation of plasma D-dimer levels over the course of time.

Table 1.

Plasma D-dimer and neopterin levels in blood samples.

	D-dimer (ng/L)		Neopterin (nmol/L)
Samples	Group 1	Group 2	Group 1	Group 2
1/0, 2/0	0.10 ± 0.018	0.06 ± 0.007	1.07 ± 0.097	0.91 ± 0.068
1/1, 2/1	0.16 ± 0.046	0.12 ± 0.009	1.12 ± 0.14	1.09 ± 0.019
1/2	0.20 ± 0.034		1.46 ± 0.08
1/3, 2/3	0.27 ± 0.024	0.14 ± 0.017	1.75 ± 0.25	1.14 ± 0.023

The measurements of neopterin levels in the plasma of rabbits were defined as listed in Table 1. The mean plasma neopterin levels in samples 2/0, 2/1, 2/3, and 1/0 were lower than those in samples 1/2 and 1/3. Furthermore, the plasma neopterin levels were compared statistically, and the values were statistically significantly lower in sample 1/0 than in samples 1/2 (p = 0.003) and 1/3 (p = 0.003) (Figure 2). When we compare the control groups with intervention groups, the value for 3 h (p = 0.000) were significant. But the value for 1 h (p = 0.599) was not significant. ROC curves were calculated to find the diagnostic value of neopterin. For the diagnosis of acute mesenteric ischemia in its early stage, the best cutoff point was at 1.25 nmol/L (area under curve = 0.864). And the calculated sensitivity, specificity, positive predictive value, and negative predictive value were 76.2%, 85.7%, 94.1%, and 54.5%, respectively. The probability of acute mesenteric ischemia was found to be 19.2 times higher when the neopterin level was over 1.25 nmol/L. ROC curves of D-dimer and neopterin are shown in Figure 3.

Figure 2.

Evaluation of plasma neopterin levels over the course of time.

Figure 3.

ROC curves of D-dimer and neopterin.

If both D-dimer and neopterin levels were over the cut-off point, then the sensitivity, specificity, positive predictive value, and negative predictive value were calculated as 95.2%, 85.7%, 95.2%, 85.7%, respectively. The probability of acute mesenteric ischemia was found to be 120 times higher when both D-dimer and neopterin levels were over their cut-off points.

Discussion

AMI caused by mesenteric vascular disease is more common in patients with additional cardiovascular problems.⁶ The pathophysiology of AMI depends on two major events: The first and most common one is arterial embolism (40–50%), while the second is arterial thrombosis (25–30%).⁷ Both of these events are commonly seen in conjunction with atherosclerotic and cardiac thromboembolic disease.⁶ The range of mortality and morbidity rates associated with AMI is between 30% and 90%.²² This is related to the inability to diagnose AMI in its early stages. Acosta et al. and Mamode et al. found that in only one-third of patients, AMI is correctly diagnosed prior to either a surgical procedure or death.^23,24

Early diagnosis within 6 h, before irreversible intestinal changes occur, is critical to patients’ survival; the mortality rate decreases with early diagnosis.²⁵ AMI is caused by vascular pathologies at a rate of 65%–80%. If any screening or diagnostic test can reveal this pathology, we will be able to prevent loss of organ function and even death. We studied two markers at the early stage of AMI. Both of them seem to be useful for early diagnosis of disease. However, as this is an animal experiment, it is not possible to make any direct statement for human beings. Further clinical studies may show similar results. With local intravascular activation, Schoots et al. pointed to an increase in the level of the D-dimer, thrombin, and antithrombin complex.²⁶ Altinyollar et al. compared their acute mesenteric experimental model with different times at rats with the help of D-dimer. They found a significant increase of D-dimer at 30 min and 7 h.¹¹ In our study, we aimed for one of the parameters to be pertinent to fibrin products in the vascular system. Our results were compatible with those of recent studies. One hour after the ligation of SMA, a statistically significant increase was detected for the D-dimer level, and this increase continued over time.

Neopterin (D-erythro-neopterin) was selected, because it is an inflammatory marker related to vascular endothelial damage.¹⁵ Recently, neopterin was found to be an important marker of atherosclerotic plaque instability in both coronary and carotid atherosclerotic lesions, acting as a pro-oxidant and promoting cell death and atherosclerotic plaque.^20,27,28 On average, two-thirds of AMIs are caused by vascular disorders.⁷ Oxidant formations and cell death were seen in AMI over time, giving rise to ischemic conditions in the mesenteric structures. From this perspective, we have studied neopterin in the diagnosis of early AMI. In our study, neopterin levels became statistically significant 2 h after the ligation of SMA. This situation continued when the increasing levels of neopterin were evaluated 3 h after the ligation of SMA. The evaluation methods used for the diagnosis of AMI include both serological markers and radiological techniques. LDH, transamines, amylases, CPK, D-dimer, D-lactate, glutathione S-transferase, intestinal fatty-acid binding protein, CPK, amylase, alanine aminotransferase, aspartate aminotransferase, and leukocytes have all been used. Increases in the levels of these markers have been detected in relation with changes in the intestinal wall. In some of these, such as CPK, LDH, amylase, AST, ALT, and leukocytes, the increase was significant at 6–12 h after the occlusion of SMA.²⁹ This can be related to irreversible changes in the intestinal wall. Another parameter is that some of these markers can be affected by hepatic clearance before interfering with the systemic circulation. Because of this, some of them cannot reach a measurable level. Another point has to do with the affected layer of the intestine. The effect of an infarction of the intestinal wall can be different from damage occurring at the mucosa layer of the intestine. Furthermore, the increase in the levels of serological markers can be different in relation to the state of the ischemia. Among all of these negative states, a few serological markers have been detected for effective diagnosis, specifically D-lactate, glutathione-S transferase, intestinal fatty-acid binding protein, and D-dimer.^8–11

In terms of radiological techniques, Doppler ultrasonography, selective mesenteric angiography, and mesenteric CTA have been used effectively. Among these, selective mesenteric angiography and mesenteric CTA are defined as the gold standard methods for the diagnosis of acute arterial occlusion.^5,25,30 The sensitivity of angiography was found to be 87% in a study conducted by Klein et al. in 1995.³¹ The sensitivity and specificity of multidetector-row CT were found to be 96% and 94%, respectively.³² However, there are some limitations to these techniques. The invasive approach has the potential to cause organ insufficiencies, allergic reaction, respiratory disorders, congestive heart failure, or even an anaphylactic reaction due to usage of radiopaque fluid. These are expensive tests when it comes to the management of the apparatus. Experienced staff members are needed to carry out the procedure reliably. Finally, the necessary equipment cannot be found in every center.^7,8 The aim of this animal study was to investigate the serum level changes of D-dimer and neopterin over time periods in early stage of AMI, following occlusion of SMA. But we have some limitations for this study. As this is the first trial of neopterin for this condition, the biomarker levels can show different attitudes in mesenteric ischemia for animals and human beings. Moreover, we have used the routine mesenteric ischemia model by ligation. The levels of biomarkers may react differently according to the model. Therefore, clinical trials with larger groups are needed to evaluate their impression. Meanwhile, the effect of oxidative stress, other systemic vascular occlusive disorders, and inflammatory or chronic diseases on serum levels should be kept in mind for evaluation of biomarkers’ level changes in the course of time.

Conclusion

This experimental study provides valuable information related to the clinical assessment of AMI. Using the combined elevation of D-dimer and neopterin may be helpful for the early diagnosis of AMI. However, the diagnostic value of these markers should be analyzed with larger study groups for a definitive decision in clinical studies.

Footnotes

Authors’ contributions

Conception and design: AKC, ZH, AO; Acquisition of data: AKC, ZH, YB, FD, TC, OM; Analysis and interpretation of data: AKC, ZH, AH, TY; Drafting the article: AKC, AH, TY; Revising it critically for important intellectual content: ZH, AO, BSO, OK; Final approval of the version: AKC, ZH, YP.

Acknowledgments

The authors greatly appreciate the contributions of Dr. Turker for the statistical analysis. The Animal Ethical Approval was given by Gulhane local animal ethic committee.

Declaration of conflicting interests

The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Funding

The author(s) received no financial support for the research, authorship, and/or publication of this article.

References

Jrvinen

Laurikka

Salenius

. Acute intestinal ischaemia. A review of 214 cases. Ann Chir Gynaecol 1994; 83: 22–25.

Bradbury

Brittenden

McBride

. Mesenteric ischaemia: a multidisciplinary approach. Br J Surg 1995; 82: 1446–1459.

Martin

. Prevention of gastrointestinal complications in the critically ill patient. AACN Adv Crit Care 2007; 18: 158–166.

Levine

Jacobson

. Intestinal ischemic disorders. Dig Dis 1995; 13: 3–24.

Wiesner

Hauser

Steinbrich

. Accuracy of multidetector row computed tomography for the diagnosis of acute bowel ischemia in a non-selected study population. Eur Radiol 2004; 14: 2347–2356.

Wyers

. Acute mesenteric ischemia: diagnostic approach and surgical treatment. Semin Vasc Surg 2010; 23: 9–20.

Lock

. Acute intestinal ischaemia. Best Pract Res Clin Gastroenterol 2001; 15: 83–98.

Evennett

Petrov

Mittal

. Systematic review and pooled estimates for the diagnostic accuracy of serological markers for intestinal ischemia. World J Surg 2009; 33: 1374–1383.

Gunduz

Turedi

Mentese

. Ischemia-modified albumin in the diagnosis of acute mesenteric ischemia: a preliminary study. Am J Emerg Med 2008; 26: 202–205.

10.

Jager

Bollinger

Valli

. Measurement of mesenteric blood flow by duplex scanning. J Vasc Surg 1986; 3: 462–469.

11.

Altinyollar

Boyabatli

Berberoğlu

. D-dimer as a marker for early diagnosis of acute mesenteric ischemia. Thromb Res 2006; 117: 463–467.

12.

Akyildiz

Akcan

Ozturk

. The correlation of the D-dimer test and biphasic computed tomography with mesenteric computed tomography angiography in the diagnosis of acute mesenteric ischemia. Am J Surg 2009; 197: 429–433.

13.

Ver Elst

Jochmans

De Pauw

. Plasma D-dimer concentrations in different clinical conditions. Acta Clin Belg 2002; 57: 325–330.

14.

Oettl

Reibnegger

. Pteridines as inhibitors of xanthine oxidase: structural requirements. Biochim Biophys Acta 1999; 1430: 387–395.

15.

Pierrakos

Vincent

. Sepsis biomarkers: a review. Crit Care 2010; 14: R15–R15.

16.

Hamerlinck

. Neopterin: a review. Exp Dermatol 1999; 8: 167–176.

17.

Bertz

Barani

Gottsater

. Are there differences of inflammatory bio-markers between diabetic and non-diabetic patients with critical limb ischemia? Int Angiol 2006; 25: 370–377.

18.

Ekman

Gottsater

Lindblad

. Plasma concentrations of Gas6 and soluble Axl correlate with disease and predict mortality in patients with critical limb ischemia. Clin Biochem 2010; 43: 873–876.

19.

Melichar

Gregor

Solichova

. Increased urinary neopterin in acute myocardial infarction. Clin Chem 1994; 40: 338–339.

20.

Sugioka

Naruko

Matsumura

. Neopterin and atherosclerotic plaque instability in coronary and carotid arteries. J Atheroscler Thromb 2010; 17: 1115–1121.

21.

Weiss

Willeit

Kiechl

. Increased concentrations of neopterin in carotid atherosclerosis. Atherosclerosis 1994; 106: 263–271.

22.

Falkensammer

Oldenburg

. Surgical and medical management of mesenteric ischemia. Curr Treat Options Cardiovasc Med 2006; 8: 137–143.

23.

Acosta

Ogren

Sternby

. Incidence of acute thrombo-embolic occlusion of the superior mesenteric artery–a population-based study. Eur J Vasc Endovasc Surg 2004; 27: 145–150.

24.

Mamode

Pickford

Leiberman

. Failure to improve outcome in acute mesenteric ischaemia: seven-year review. Eur J Surg 1999; 165: 203–208.

25.

Brandt

Boley

. AGA technical review on intestinal ischemia. American Gastrointestinal Association. Gastroenterology 2000; 118: 954–968.

26.

Schoots

Levi

Roossink

. Local intravascular coagulation and fibrin deposition on intestinal ischemia-reperfusion in rats. Surgery 2003; 133: 411–419.

27.

Andreesen

Scheibenbogen

Brugger

. Adoptive transfer of tumor cytotoxic macrophages generated in vitro from circulating blood monocytes: a new approach to cancer immunotherapy. Cancer Res 1990; 50: 7450–7456.

28.

Gieseg

Crone

Flavall

. Potential to inhibit growth of atherosclerotic plaque development through modulation of macrophage neopterin/7,8-dihydroneopterin synthesis. Br J Pharmacol 2008; 153: 627–635.

29.

Kurt

Akin

Demirbas

. D-dimer in the early diagnosis of acute mesenteric ischemia secondary to arterial occlusion in rats. Eur Surg Res 2005; 37: 216–219.

30.

Wiesner

. [Is multidetector computerized tomography currently the primary diagnostic method of choice in diagnostic imaging of acute intestinal ischemia?]. Praxis 2003; 92: 1315–1317.

31.

Klein

Lensing

Klosterhalfen

. Diagnostic imaging of mesenteric infarction. Radiology 1995; 197: 79–82.

32.

Kirkpatrick

Kroeker

Greenberg

. Biphasic CT with mesenteric CT angiography in the evaluation of acute mesenteric ischemia: initial experience. Radiology 2003; 229: 91–98.