Heart-type fatty acid-binding protein (H-FABP) is a low molecular weight cytoplasmic protein and present abundantly in the myocardium. When the myocardium is injured, as in the case of myocardial infarction, low molecular weight cytoplasmic proteins including H-FABP are released into the circulation and H-FABP is detectable in a blood sample. We have already developed a direct sandwich-ELISA for quantification of human H-FABP using two distinct types of monoclonal antibodies specific for human H-FABP. In this study we investigated the clinical validity of H-FABP as a biochemical diagnostic marker in the early phase of acute myocardial infarction (AMI). To evaluate the diagnostic usefulness of H-FABP in the early phase of AMI, blood samples were obtained from the following patients within 12 hours after the appearance of symptoms, and serum levels of H-FABP were compared with those of conventional diagnostic markers, such as myoglobin and creatine kinase isoenzyme MB (CK-MB). Blood samples were collected from patients with confirmed AMI (n=140), patients with chest pain who were afterwards not classified as AMI by normal CK-MB levels (non-AMI) (n=49) and normal healthy volunteers (n=75). The serum concentration of H-FABP was quantified with our direct sandwich-ELISA. The concentration of myoglobin mass was measured with a commercial RIA kit. The serum CK-MB activity was determined with an immuno-inhibition assay kit. The overall sensitivity of H-FABP, within 12 hours after the appearance of symptoms, was 92.9%, while it was 88.6% with myoglobin and 18.6% with CK-MB. The overall specificity of H-FABP was 67.3%, while it was 57.1% with myoglobin and 98.0% with CK-MB. The diagnostic efficacy rates with these markers were 86.2% (H-FABP), 80.4% (myoglobin) and 39.2% (CK-MB), respectively. The diagnostic validity of H-FABP was further assessed by receiver operating characteristic (ROC) curve analysis. The area under the curve (AUC) of H-FABP was 0.921, which was significantly greater than with myoglobin (AUC: 0.843) and CK-MB (AUC: 0.654). These parameters, such as sensitivity, specificity, diagnostic efficacy and diagnostic accuracy, obtained for patients with chest pain within 3 hours and/or 6 hours after the onset of symptoms were almost the same as those for patients within 12 hours after symptoms. H-FABP is more sensitive than both myoglobin and CK-MB, more specific than myoglobin for detecting AMI within 12 hours after the onset of symptoms, and shows the highest values for both diagnostic efficacy and ROC curve analysis. Thus, H-FABP has great potential as an excellent biochemical cardiac marker for the diagnosis of AMI in the early phase.
Geldanamycin (GM), a benzoquinone ansamycin antibiotic, is a natural product inhibitor of Hsp90 with potent and broad anti-cancer properties. Because of its adverse effects on liver, its less toxic derivatives 17-(allylamino)-17-demethoxygeldanamycin (17-AAG) and 17-(dimethylaminoethylamino)-17-demethoxygeldanamycin (17-DMAG) are currently being evaluated for the treatment of cancer. Previously, it has been demonstrated that the redox cycling of GM by NADPH-cytochrome P450 reductase leads to the formation of the GM semiquinone and superoxide radicals, the latter being identified using spin-trapping. We hypothesized that the different hepatotoxicity induced by GM, 17-AAG and 17-DMAG reflects the redox active properties of the quinone moiety and possibly the extent of superoxide formation, which may stimulate cellular oxidative injury. Our data demonstrate that superoxide can be efficiently trapped during the reduction of GM, 17-AAG and 17-DMAG by NADPH-cytochrome P450 reductase, and that superoxide formation rate followed the order 17-DMAG > 17-AAG > GM. In the absence of superoxide scavengers, the rate of NADPH oxidation followed the order 17-DMAG > GM > 17-AAG. The half-wave one-electron reduction potentials (E1/2) of GM, 17- AAG and 17-DMAG in DMSO have been determined to be −0.37, −0.13 and −0.015 V (vs. Ag/AgCl), respectively. If the same order of E1/2 follows in neutral aqueous media, thermodynamic considerations imply that 17-DMAG is more readily reduced by the P450 reductase as well as by superoxide. The order of the drug cytotoxicity toward rat primary hepatocytes, as determined by their effect on cell viability and on intracellular oxidant level, was opposite to the order of E1/2 of the respective quinone/semiquinone couples. These results suggest that hepatotoxicity exhibited by the Hsp90 inhibitors belonging to benzoquinone ansamycins could be attributed to superoxide. The apparent discrepancy between the order of toxicity and the orders of superoxide formation rate, which is correlated with E1/2, is discussed.
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