Decreased survival in vivo of diamide-incubated dog erythrocytes. A model of oxidant-induced hemolysis.

Abstract

A B S T R A C T Erythrocytes from patients with chronic hemolytic variants of glucose-6-phosphate dehydrogenase (G-6-PD) deficiency have structural membrane protein abnormalities accompanied by decreased cell membrane deformability which we postulate represent the consequences of oxidant-induced membrane injury. To evaluate the pathophysiologic significance of oxidant-induced membrane injury, we studied the in vitro and in vivo effects of the thiol-oxidizing agent, diamide, on dog erythrocytes. In vitro incubation ofdog erythrocytes with 0.4 mM diamide in Tris-buffered saline for 90 min at 370C resulted in depletion of GSH, formation of membrane polypeptide aggregates (440,000 and >50,000,000 daltons) and decreased cell micropipette deformability, abnormalities similar to those observed in the erythrocytes of patients with chronic hemolytic variants of G-6-PD deficiency. In addition, diamide-incubated cells had increased viscosity and increased membrane specific gravity, but no change in ATP. Reinjection of 51Cr-labeled, diamideincubated cells was followed by markedly shortened in vivo survival and splenic sequestration. Further incubation of diamide-incubated cells in 4 mM dithiothreitol reversed the membrane polypeptide aggregates, normalized micropipette deformability, decreased cell viscosity, prolonged in vivo survival, and decreased splenic sequestration.These studies demonstrate that diamide induces a partially reversible erythrocyte lesion which is a useful model of oxidant-induced membrane injury. They sug-

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“…Superoxide, hydroxyl groups, and molecular hydrogen and oxygen are major products of cathodic electrochemical reactions occuring in aqueous solution. The ability of oxidant agents to promote membrane injury has been described [Johnson et al, 1980], as well as the contribution of chlorine to tissue destruction [Samuelsson and Jo Ènsson, 1980;. Superoxide radicals can also in¯uence the occurrence of cell lysis, since they are generated by cathodic reactions [Forman and Fridovich, 1972] and are ef®cient agents in destructing target cells [Saran et al, 1998;Zhou and Petty, 1993].…”

Naturally occurring anti-band-3 antibodies and complement together mediate phagocytosis of oxidatively stressed human erythrocytes.

“…This property is of particular relevance because it renders anti-band-3 antibody considerably more effective than if it induced classical pathway C3b deposition, which lacks a positive feedback. Complement requirement was either not apparent or not considered in earlier studies of phagocytosis of senescent (3-6) and oxidatively damaged red cells (15)(16)(17), though erythrocyte senescence (45) and shortened viability of stored erythrocytes (46) correlated with increased deposition of C3 fragments. The mechanism by which anti-band-3 stimulated C3 binding may involve enhanced convertase activity of C3b bound to antibody (ref.…”

“…Although this classical manipulation resulted in an IgG requirement for efficient phagocytosis, it did not allow us to identify the antigenic sites exposed in the course of storage. Therefore, erythrocyte aging was mimicked by oxidative stress, which is known to accelerate erythrocyte clearance in vivo (16)(17)(18) and to induce membrane protein oligomerization (17,(19)(20)(21)(22) and band 3 protein clustering (22). Band 3 clustering enhances binding of antibodies from autologous IgG, whether studied on skeleton-free vesicles (8) or on erythrocytes containing Heinz bodies (22,23).…”

Oxidant damage of the lipids and proteins of the erythrocyte membranes in unstable hemoglobin disease. Evidence for the role of lipid peroxidation.

“…We have shown in an in vivo model system in dogs that erythrocytes with diamide-induced membrane polypeptide aggregates and decreased deformability are preferentially removed from the circulation and sequestered in the spleen (7). Further, our patients' erythrocyte membranes had sustained damage in the form of nondissociable aggregates.…”