Background Hemoglobin released from subarachnoid hemorrhage (SAH) is rapidly followed by the oxidation of the heme from the ferrous form (FeII) to the ferric state (FeIII). In this state of oxidation, hemoglobin can catalyze the oxidation of free or membrane-bound lipids by ferric-ferryl (FeIV) redox cycling. The reaction of oxidation is initiated by the reaction of the ferric heme with peroxides present in the milieu. Such peroxides can be hydrogen peroxide generated by activated phagocytes but can also be endogenous lipid peroxides. An accumulating body of evidence has suggested that these oxidation processes are responsible for the oxidative injuries associated with the hemolytic event following subarachnoid hemorrhage. We hypothesized that acetaminophen (ApAP) could inhibit the peroxidase activity of hemoglobin in a mechanism similar to the inhibition of prostaglandin synthases and therefore inhibit lipid peroxidation and formation of F2-isoprostanes (products of lipid peroxidation with a powerful vesoconstrictive effect). That could prevent cerebral vasoconstriction and subsequent ischemia and neuron death in SAH patients.
Method Ferryl hemoglobin was produced by oxidation of ferric hemoglobin with H2O2. Reduction of ferryl hemoglobin by ApAP was assessed spectrophotometrically by measuring the absorbance at 415 nm for ferryl hemoglobin and 406 nm for ferric hemoglobin. The ability of ApAP to inhibit hemoglobin-induced lipid peroxidation was determined by incubation of ferryl hemoprotein with [14 C] arachidonic acid. Unoxidized radiolabeled arachidonic acid and its more polar oxidized products were separated by thin layer chromatography (TLC), and counted by a radio-TLC scanner. The ratio of oxidized to residual arachidonic acid was calculated to give the percentage of the oxidized arachidonic acid.
Results ApAP inhibited the oxidation of arachidonic acid by ferryl hemoglobin with IC50 of 17 μM. Spectrophotometric analysis of hemoglobin in visible wavelengths indicated that ApAP could also reduce ferryl hemoglobin to its ferric form.
Conclusion ApAP reduces ferryl hemoglobin to ferric hemoglobin in vitro; ApAP prevents ferryl hemoglobin-induced lipid peroxidation in vitro at concentrations much lower than the normal therapeutic plasma concentration in humans; treatment with ApAP may prevent subarachnoid hemorrhage-induced vasospasm.
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