Melatonin is a potent scavenger of reactive oxygen species or free radicals like superoxide and hydroxyl radicals. The oxidation of hemoglobin to methemoglobin (meth-Hb) by oxidizing compounds has been widely studied. The present work was designed to evaluate the ability of different concentrations of melatonin to inhibit nitrite–induced oxidation of hemoglobin. Blood samples were obtained from apparently healthy individuals from which erythrocyte hemolysate was prepared. Different concentrations of melatonin (10^-9-1.0 mg/ml) were incubated for 10 min with the hemolysate, then to the resultant mixture 1 ml of sodium nitrite (final concentration 0.6 mM) was added, and the formation of meth-Hb was measured by monitoring absorbance of light at 631 nm each min for 30 min. Control samples without melatonin were utilized for comparison. Nitrite caused rapid oxidation of hemoglobin to meth-Hb in control samples; in the presence of melatonin, the oxidation process was delayed in a dose–dependent manner. The effect of melatonin on the time course of nitrite-induced oxidation of Hb showed that melatonin has a protective effect initiated early after addition along with nitrite. Melatonin also affect the time required for the formation of meth-Hb, the   time required to convert 50% of the available Hb to meth-Hb was 4 min in the absence of melatonin, and became  17, 22, 26, 30, 114 and 383  min with increasing melatonin concentrations (10^-9, 10^-6, 0.001, 0.01, 0.1, and 1.0 mg/ml  respectively). In conclusion, melatonin in a concentration  and time dependent manner can protect Hb from oxidation by nitrite; melatonin delays the onset of autocatalytic stage and the protective effect extended over long period of time.

Key words: melatonin, erythrocytes oxidation