A simple chemiluminescence assay for detecting oxidative stress in ischemic limb injury

This study was designed to evaluate a simple, sensitive, nonstimulated chemiluminescence assay to measure the oxidative stress production in the whole blood of rabbits after an ischemic insult. By using an ultra-sensitive chemiluminescence (CL) analyzer and lucigenin amplification, the assay system can be performed without leukocyte isolation and stimulant administration. The blood CL levels of healthy rabbits were 122 +/- 18 counts/10 s. After 8 h of ischemia, the CL levels of whole blood immediately after reperfusion, 1 h, 2 h, 24 h, 48 h, 7 days, and 14 days of reperfusion were 409 +/- 78 counts/10 s, 283 +/- 55 counts/10 s, 256 +/- 43 counts/10 s, 228 +/- 33 counts/10 s, 185 +/- 32 counts/10 s, 160 +/- 16 counts/10 s, and 119 +/- 15 counts/10 s. The differences were statistically significant between the control and samples obtained up to 24 h after reperfusion. The corresponding creatine phosphate kinase (CPK) levels of the rabbits in this study were 938 +/- 43 U/l for the control blood sample and 5921 +/- 498 U/l, 6948 +/- 427 U/l, 6860 +/- 1115 U/l, 5763 +/- 516 U/l, 1545 +/- 291 U/l, 478 +/- 60 U/l, and 458 +/- 48 U/l for the experimental blood samples. The CPK levels of the blood samples before 24 h and after 7 days of reperfusion were significantly different to that of the control blood sample. The changes in the CL and CPK levels were quite similar before 48 h reperfusion. This assay has proved to be valuable in the quantitative measurement of ischemic insults of skeletal muscles.