Comparison of human red cell lysis by hypochlorous and hypobromous acids: insights into the mechanism of lysis

Abstract

Human red blood cells are lysed by the neutrophil-derived oxidant hypochlorous acid (HOCl), although the mechanism of lysis is unknown. Hypobromous acid (HOBr), a similarly reactive oxidant, lysed red cells approx. 10-fold faster than HOCl. Therefore we compared the effects of these oxidants on thiols, membrane lipids and proteins to determine which reactions are associated with lysis. There was no difference in the loss of reduced glutathione or membrane thiols with either oxidant, but HOBr reacted more readily with membrane lipids and proteins. Bromohydrin derivatives of phospholipids and cholesterol were seen at approx. one-tenth the level of oxidant than chlorohydrins were. However, these products were detected only with high concentrations of HOCl or HOBr, which caused instant haemolysis. Membrane protein modification occurred at much lower doses of oxidant and was more closely correlated with lysis. SDS/PAGE analysis showed that band 3, the anion transport protein, was lost at the lowest dose of HOBr and at the higher concentrations of HOCl. Labelling the red cells with eosin 5-maleimide, a fluorescent label for band 3, suggested possible clustering of this protein in oxidant-exposed cells. There was also irreversible cross-linking of all the major membrane proteins; this reaction occurred more readily with HOBr. The results indicate that membrane protein modification is the reaction responsible for HOCl-mediated lysis. These effects, and particularly cross-link formation, might result in clustering of band 3 and other membrane and cytoskeletal proteins to form haemolytic pores.

Myeloperoxidase-derived Hypochlorous Acid Antagonizes the Oxidative Stress-mediated Activation of Iron Regulatory Protein 1

“…HOCl is cell permeable and reacts readily with thiols, thioethers and amino groups (11,12). Exposure of red cells or endothelial cells to HOCl results in an early decrease of the glutathione pool (13,14). This suggests that HOCl could modulate cell processes in a manner similar to that seen with H 2 O 2 or peroxynitrite.…”

Ozone treatment of ballast water on the oil tanker S/T Tonsina: chemistry, biology and toxicity

“…In other studies (data not shown), we observed a decrease in total numbers of microorganisms in seawater samples exposed to ozone that were stained with a nucleic acid stain and examined by epifluorescence microscopy or flow cytometry. Vissers et al (1998) explained the mechanism of human red cell lysis by hypobromous acid by stating that it reacts with membrane lipids and proteins. Ozone has been used for many years to maintain water quality in seawater aquaculture settings.…”

Protective Effect of Philesia Magellanica (Coicopihue) From Chilean Patagonia Against Oxidative Damage

“…HClO treatment of erythrocyte membrane results in changes of membrane fluidity, surface area, and morphological transformations, events that precede cell lysis. 21,31,37 Our SEM observations showed that 50 µM HClO induced morphological alterations to the red cells, from a discoid to a spherocytic form. According to the bilayer couple hypothesis shape changes induced in erythrocytes by foreign molecules are due to differential expansion of the two monolayers of the red cell membrane.…”

“…21 However, because it readily reacts with a range of biological targets it has been difficult to identify which reactions are critical for its cytotoxic effects. 37 Human erythrocytes are a reliable and easily obtainable mechanism to detect oxidative stress. 11 Although the exact mechanism is not clear the cell membrane is considered the primary site for reaction.…”