Heart-Type Fatty Acid Binding Protein As A Marker For Postmortem Diagnosis Of Myocardial Infarction

Objective: Heart-type fatty acid binding protein (H-FABP) is a low molecular weight protein (14-15kDa) with special tissue distribution. As tnyocardial cell membrane damaged by ischemia, H-FABP leaks to the extracellular space and enters the blood circulation very easily and quickly due to its small size and water soluble. So it is proposed as an early indicator of AMI. Our experiment is mainly about the influence of autolysis on the concentration of H-FABP in the urine as well as the Immunohistochemical staining on the myocardial cell.Method: Animal model of AMI was produced by ligating the left anterior descending coronary artery (LAD) of the rats. Briefly, 66 Wistar rats weighting 250-350g of either sex, were randomly distributed into 5 study groups, 5 control groups and 1 sham-operated group. They were anesthetized with 10% chloral hydrate and artificially ventilated with animal ventilator. After opening the skin and the left 4th intercostal space, LAD was ligated at its proximal. Successful ligation was confirmed by visual inspection for pallor of the involved myocardium and ST segment elevation≥0.1mv on electrocardiogram (ECG). Sham-operated control group rats underwent the same surgical protocol as the study group except LAD ligation. Four hours after the study group rats were ligated, they were killed.The urine sample were collected and their hearts were dissected 0h, 6h, 12h, 24h and 48h separately after their death. The urine sample were tested with the rat ELISA kit and the detection limit of the assay was 0.1μl/L, and the inter- and intra-assay analytical imprecision were below 10%. The hearts were fixed in 10% formalin in PBS over 24h. The tissue blocks were taken transversely through the center of the infarcted area and embedded in paraffin according to routine histological procedures. We used the streptavidin -peroxidase conjugated method.Results:1. The urine concentration of H-FABP in normal group was 83.93±50.33pμg/L. Six hours after death, the urine concentration of H-FABP in normal group was 282.71±164.86ug/L, which was the first peak value. Twelve hours after death, the urine concentration of H-FABP in normal group was down to 68.68±59.81ug/L. Twenty-four hours after death, the urine concentration of H-FABP in normal group was up to 327.96±300.87ug/L. Forty-eight hours after death, the urine concentration of H-FABP in normal group was up to the second peak value, 339.29±144.33|ig/. The urine concentration of H-FABP in experiment group of Oh was 263.44±67.95jxg/L. With the postmortem time prolong, the concentration was soaring wavily. Six hours after death, it came up to the first peak value, 1098.77±630.43ng/L. Twelve hours after death, it came down to 857.83±401.81 p.g/L. Twenty-four hours after death, it came down to 71O.18±228.28fig/L. Forty-eight hours after death, the urine concentration of H-FABP was 1166.06±334.47 jig/L.2. In normal control group of Oh, cytoplasm of all cardiac cells were stained homogeneously brown. After counterstaining with hematoxylin, the nuclei were stained light blue. No positive expression of H-FABP was found in interstitial tissue or other cell component. Four hours after ligation, completely depletion involving almost all myocardium which was distinct from normal cell started and with the prolongation of the postmortem time, cardiac cells autolyzed from partly to completly. After 48 hours, cytoplasm of all cardiac cells were stained asymmetrically brown.Conclusion:1. When the rats die, their blood do not circulate. If there is no H-FABP in the bladder cells releasing due to autolysis, the curve we get shall be a moving-down one. Because H-FABP in the urine is continuously decomposed with the postmortem time, the urine concentration of H-FABP we test shall be declining . On the other hand, the volume of urine in the bladder is different, and to meet the need of experiment, we dilute the sample differently, and that may influence the result. Therefore, we can draw a conclusion when the urine is drawn after death for some time, the concentration of H-FABP in urine can not be used to deduce the myocardial ischemia to be the cause of death due to autolysis.2. H-FABP leaks to the extracellular space quickly when ischemia happens. S-P staining shows that the different level of depletion of H-FABP can be observed in theischemic or infarcted myocardial cells of animal. With the prolongation of the postmortem time, the cardiomyocytes autoVyze. H-FABP is stained asymmetrically brown inside and outside the membrane with the streptavidin-peroxidase conjugated method.