Electron paramagnetic resonance study of lipid and protein membrane components of erythrocytes oxidized with hydrogen peroxide

Abstract

Electron paramagnetic resonance (EPR) spectroscopy of spin labels was used to monitor membrane dynamic changes in erythrocytes subjected to oxidative stress with hydrogen peroxide (H 2 O 2 ). The lipid spin label, 5-doxyl stearic acid, responded to dramatic reductions in membrane fluidity, which was correlated with increases in the protein content of the membrane. Membrane rigidity, associated with the binding of hemoglobin (Hb) to the erythrocyte membrane, was also indicated by a spin-labeled maleimide, 5-MSL, covalently bound to the sulfhydryl groups of membrane proteins. At 2% hematocrit, these alterations in membrane occurred at very low concentrations of H 2 O 2 (50 µM) after only 5 min of incubation at 37°C in azide phosphate buffer, pH 7.4. Lipid peroxidation, suggested by oxidative hemolysis and malondialdehyde formation, started at 300 µM H 2 O 2 (for incubation of 3 h), which is a concentration about six times higher than those detected with the probes. Ascorbic acid and α-tocopherol protected the membrane against lipoperoxidation, but did not prevent the binding of proteins to the erythrocyte membrane. Moreover, the antioxidant (+)-catechin, which also failed to prevent the cross-linking of cytoskeletal proteins with Hb, was very effective in protecting erythrocyte ghosts from lipid peroxidation induced by the Fenton reaction. This study also showed that EPR spectroscopy can be useful to assess the molecular dynamics of red blood cell membranes in both the lipid and protein domains and examine oxidation processes in a system that is so vulnerable to oxidation.

d‐α‐tocopheryl polyethylene glycol 1000 succinate‐containing vehicles provide no detectable chemoprotection from oxidative damage

“…Whole blood treated with H 2 O 2 to produce oxidative stress was largely resistant to lipid peroxidation producing only minimal increases in MDA (product of lipid peroxidation) adduct formation. Similar to previous reports (Mendanha et al, 2012), when treated with NaAz to block catalase activity, MDA adduct formation increased dramatically, confirming a loss of this endogenous protective effect against H 2 O 2 -mediated oxidative damage. It is likely this was largely due to endogenous catalase activity, which is important in the protection against H 2 O 2 -mediated damage at a cellular level (Gaetani et al, 1989;Scott et al, 1991).…”

Effect of Nitric Oxide Metabolites on the Formation of Membranebound Hemoglobin Under Carbonyl Stress

“…Этот факт указывает на то, что снижение количества восстановлен-ных тиолов может быть связано с уменьшением их доступности в результате экранирования мо-лекулами hb примембранного слоя. в ряде ра-бот было показано, что при низких концентра-циях h 2 O 2 количество доступных тиоловых групп коррелирует с содержанием белка в мембра-не эритроцитов [Sharma, Premachandra, 1991;Mendanha et al, 2012]. нельзя исключить и возможность образования кова-лентных аддуктов мембранных сульфгидрилов с метилглиоксалем .…”

“…с одной стороны, NO 2 − в реакции с oxyhb приводит к образованию метаболитов с высо-кой окислительной способностью (NO 2 ▪ и h 2 O 2 ), стимулирующих Пол (перекисное окисление липидов) и окисление гемоглобина до hb- [Fe IV =о] [Zavodnik et al, 1999;Gow et al, 1999;. Это способствует встра-иванию гемоглобина, а также продуктов его окисления и деструкции (железа, гема и дена-турированного hb) в эритроцитарную мембра-ну [Sharma, Premachandra, 1991;Mendanha et al, 2012]. с другой стороны, нитритные ионы восстанавливаются интерме-диатами реакции Майяра до NO, который в ре-акции с основаниями Шиффа приводит к обра-зованию менее реакционноспособных нитро-зопроизводных, купируя дальнейшее участие оснований Шиффа в свободнорадикальных превращениях с участием MG и O 2 .…”

Effects of Vitamin E and Vitamin C on Hydrogen Peroxide-Induced Hemolysis in Moroccan Dromedary Camels (Camelus Dromedarius)

“…Further, H 2 O 2 is largely known by its toxicity due to its ready conversion to the reactive hydroxyl radical (OH -), either by exposure to ultraviolet light or by interaction with a range of transition metal ions, of which the most important in vivo is probably iron (Djordjevic, 2004). In addition, oxidized hemoglobin may bind to the RBC membrane and increase its rigidity (Mendanha et al, 2012). On the other hand, a significant increase of LP, TBARS production and hemolysis was recently observed in camel with OS inducted by Sarcoptic mange (Saleh et al, 2011) or liver cystic echinococcosis (Heidarpour et al, 2012).…”

“…The benefits of this vitamin have been reported in human RBC when challenged with phenylhydrazine hydrochloride (Claro et al, 2006). However, according to Mendanha et al, (2012), AA and α-tocopherol are able to prevent oxidative hemolysis and TBARS production, but can not decrease the membrane rigidity when RBCs are exposed to H 2 O 2 .…”