Melatonin protects human red blood cells from oxidative hemolysis: New insights into the radical-scavenging activity

Antioxidant activity of melatonin in human erythrocytes, exposed to oxidati ve stress by cumene hydroperoxide (cumOOH), was investigated. CumOOH at 300 mu M progressively oxidized a 1% suspension of red blood cells (RBCs), lea ding to 100% hemolysis in 180 min. Malondialdehyde and protein carbonyls in the membrane showed a progressive increase, as a result of the oxidative d amage to membrane lipids and proteins, reaching peak values after 30 and 40 min, respectively. The membrane antioxidant vitamin E and the cytosolic re duced glutathione (GSH) were totally depleted in 20 min. As a consequence o f the irreversible oxidative damage to hemoglobin (Hb), hemin accumulated i nto the RBC membrane during 40 min. Sodium dodecyl sulfate (SDS) gel electr ophoresis of membrane proteins showed a progressive loss of the cytoskeleto n proteins and formation of low molecular weight bands and protein aggregat es, with an increment of the intensity of the Hb band. Melatonin at 50 mu M strongly enhanced the RBC resistance to oxidative lysis, leading to a 100% hemolysis in 330 min. Melatonin had no effect on the membrane lipid peroxi dation, nor prevented the consumption of glutathione (GSH) or vitamin E. Ho wever, it completely inhibited the formation of membrane protein carbonyls for 20 min and hemin precipitation for 10 min. The electrophoretic pattern provided further evidence that melatonin delayed modifications to the membr ane proteins and to Hb. In addition, RBCs incubated for 15 min with 300 mu M cumOOH in the presence of 50 mu M melatonin were less susceptible, when s ubmitted to osmotic lysis, than cells incubated in its absence. Extraction and high-performance liquid chromatography (HPLC) analysis showed a much mo re rapid consumption of melatonin during the first 10 min of incubation, th en melatonin slowly decreased up to 30 min and remained stable thereafter. Equilibrium partition experiments showed that 15% of the melatonin in the i ncubation mixture was recovered in the RBC cytosol, and no melatonin was ex tracted from RBC membrane. However, 35% of the added melatonin was consumed during RBC oxidation. Hydroxyl radical trapping agents, such as dimethylsu lfoxide or mannitol, added into the assay in a 1,000 times molar excess, di d not vary melatonin consumption, suggesting that hydroxyl radicals were no t involved in the indole consumption. Our results indicate that melatonin i s actively taken up into erythrocytes under oxidative stress, and is consum ed in the defence of the cell, delaying Hb denaturation and release of hemi n. RBCs are highly exposed to oxygen and can be a site for radical formatio n, under pathological conditions, which results in their destruction. A pro tective role of melatonin should be explored in hemolytic diseases.