Figure 1.  Comparison of troponin t levels among all groups

Group I: Control group, group II: Sham group, group III: Three hr after mesenteric ischemia, group IV: Six hr after mesenteric ischemia, group V: Three hr after peripheral ischemia and group VI: Six hr after peripheral ischemia. P

 ethics committee approved the protocol. Healthy male Wistar albino rats with an average weight of 250-300 g were procured from the Laboratory Animal Production Unit of Dicle University.

 Animal subjects

Forty-eight rats were used in the experiments. The experiments were performed in laboratories with standardized conditions at 22±2°C and 50±5% humidity. A standard diet and tap water were provided ad libitum. Twelve hours prior to the expe-rimental procedure, the rats were given only water.

 Study protocol

Rats were divided into six randomized equal groups. Ketamine 130 mg/kg (Ketalar, Pfizer) and xylasine 20 mg/kg (Rompun, Bayer) were used to induce anesthesia. Additionally, ketamine HCl               (50 mg/kg) was used during the surgeries for anesthesia maintenance. All vital signs (respiratory rate, pulse, oxygen saturation (So₂), and body temperature) were monitored.

The control group (Group I) was employed to determine the basal troponin and myoglobin levels in rats. From group I, blood samples were taken but no surgeries were performed. A sham group (Group II) was used to determine the impact of surgical incision on biochemical parameters and blood samples were taken following a simple laparotomy. Two mesenteric ischemia groups were created (group III-IV) by ligating the superior mesenteric artery (SMA) via a simple laparatomy. Intracardiac blood samples were taken 3 hr after SMA ligation in group III and 6 hr after ligation in group IV. In order to create peripheral ischemia, the left femoral artery was clamped in two groups of rats (group V-VI). Intracardiac blood samples were prepared after 3 hr of ischemia in group IV and after 6 hr in Group VI. After the experimental procedures, all rats were sacrificed.

 Laboratory analysis

Point-of-care testing (POCT) by Roche (Roche Diagnostics, Mannheim, Germany) was performed on each whole blood sample. The samples were obtained in vacuum tubes (which contained separating gel and lithium heparin) and troponin T and myoglobin levels were quantified simulta-neously using a fluorescence immunoassay.

 Troponin-T and myoglobin measurement

Myoglobin and troponin T levels were measured in whole blood by POCT using the Roche Cobas® h 232 immunoassay analyzer (Roche Diagnostics, Mannheim, Germany). The range of detection was 0.1 to 3 μg/l for troponin T and 30 to 700 μg/l for myoglobin (7, 8).

 Statistical analysis

The results were expressed as mean±standard deviation (SD). The Kolmogorov-Smirnov test was used to determine if the data were normally distributed. Analysis of variance (ANOVA) and Bonferroni post-hoc tests were used to compare the groups. All statistical procedures were performed using SPSS software version 15.0 (SPSS Inc., Chicago, IL). A P-value of ≤0.05 was considered statistically significant.

 Figure 2.  Comparison of myoglobin levels among all groups

Group I: Control group, group II: Sham group, group III: Three hr after mesenteric ischemia, group IV: Six hr after mesenteric ischemia, group V: Three hr after peripheral ischemia and group VI: six hr after peripheral ischemia. P

 Statistical analysis

The results were expressed as mean±standard deviation (SD). The Kolmogorov-Smirnov test was used to determine if the data were normally distributed. Analysis of variance (ANOVA) and Bonferroni post-hoc tests were used to compare the groups. All statistical procedures were performed using SPSS software version 15.0 (SPSS Inc., Chicago, IL). A P-value of ≤0.05 was considered statistically significant.

 Results

The mean troponin T levels in groups I, II, III, IV, V, and VI were found to be 0.18±0.026, 0.21±0.034, 1.18±0.046, 0.99±0.054, 0.42±0.037, and 0.69±0.043, respectively (Figure 1). The troponin T levels were significantly higher in the study groups compared to the control group (P<0.05). Moreover, 3 hr after SMA ligation, the mean troponin T level was higher than any other study groups (Group III). A comparison of the mean troponin T values using ANOVA and post-hoc Bonferroni tests is presented in Figure 1.

The mean levels of myoglobin in groups I, II, III, IV, V, and VI were found to be 92±6.35, 104±4.34, 184±6.22, 211±8.57, 139±6.72 and 171±7.61, res-pectively (Figure 2). The myoglobin levels were significantly higher in study groups compared to the control group (P<0.05). Furthermore, higher levels were observed in the mesenteric ischemia groups at the 3^rd and 6^th hours (Group III-IV). Figure 2 shows the myoglobin values for groups following the statistical analysis using ANOVA and post-hoc Bonferroni tests.

As shown in Figures 1 and 2, troponin T and myoglobin have similar levels in both peripheral and mesenteric ischemia in the first six hours after the onset of ischemia. Nevertheless, the highest mean troponin T level was observed 3 hours after the onset of mesenteric ischemia. Six hours after the onset of mesenteric ischemia, the mean troponin T level (Group IV) was lower than its level at 3 hours (Group III).

 Discussion

Myoglobin is an essential striated muscle O₂ storage protein that was demonstrated to be highly reactive with peroxidate in the presence of H₂O₂ mediated oxidation (9). It is responsible for the release of O₂ during periods of hypoxia and anoxia. Therefore, myoglobin was investigated as a marker of ischemic muscle (9, 10). A study by van Weel et al suggested that myoglobin may lead to improved vascularization in patients with arterial obstructive disease who were treated with therapies designed to increase angiogenesis (5). These results suggest that myoglobin is an important protein in the setting of muscle tissue hypoxia and reorganization. Troponin is a contractile regulatory muscle protein complex that binds to calcium in the actin-myosin bundles within muscle fibers. There are three types of troponin, classified as troponin C, T, and I. The C type is associated with striated muscles. However, troponin I and T are closely associated with cardiac muscles (11). Although troponin I and T are specific for cardiac muscle, significant changes in both of these troponin subtypes have been reported during ischemia of other organs (12). Our findings support the idea that myoglobin and troponin T levels are increased in both mesenteric and peripheral ischemia.

Troponin T levels were investigated in patients undergoing surgery for an abdominal aortic aneurysm or lower extremity arterial obstruction (13). The authors suggested that elevated troponin T levels may predict late mortality. In another study, it was found that troponin T was increased in patients with acute limb ischemia. Additionally, in that study it was suggested that troponin T levels may be an early marker of overall disease severity and a predictor of outcome (14). Also, it was reported that elevated troponin I is a common finding in patients with acute mesenteric ischemia (3).  Myoglobin has also been investigated as a biomarker in ischemic tissues, especially in ischemic muscles (9, 10). Although the specific role of myoglobin in hypoxia is unclear, previous reports have suggested that hypoxia regulates myoglobin expression and myoglobin prodution is an adaptive response to hypoxic conditions (15, 16). Yang et al studied myoglobin expression in an experimental hindlimb ischemia model in mice. They concluded that elevated myoglobin expression in ischemic skeletal muscle reduces the endogenous perfusion recovery (16). The role of vascular myoglobin in hypoxic conditions was studied and it was suggested that myoglobin contributes to nitrite-induced vasodi-lation in these conditions, significantly (17). In this study, we created experimental acute mesenteric and peripheral ischemia by clamping sustaining arteries and then drew blood samples three and six hr after the ischemic event. Troponin T and myoglobin values were measured in these blood samples. We found that these two markers were significantly elevated (P<0.05) in both peripheral and mesenteric ischemia. The highest troponin T levels were observed in the samples taken three hr after the onset of mesenteric ischemia and the highest myoglobin levels were seen in samples taken six hr after mesenteric ischemia. Other findings were similar in both peripheral and mesenteric ischemia experimental groups.

 Conclusion

We could mention that troponin T and myoglobin are not specific markers for mesenteric or peripheral ischemia. Therefore, there is no diagnostic value when they are in normal ranges. However, in patients with positive troponin or myoglobin without an underlying cardiac cause, it is likely that ischemia is present in another tissue.

Acknowledgment

The authors received no financial support for the research and/or authorship of this article.

 Declaration of interest

The authors declare no conflicts of interest with respect to the authorship and/or publication of this article.