IT is thought that the deleterious properties of thalassaemia (thal) and glucose-6-phosphate dehydrogenase deficiency (G6PD−) are balanced in the population by a conferred protection against malaria, and that some characteristic of the variant red cell acts directly to inhibit the invasion or intracellular development of the malaria parasite. Indeed, such is the case for sickle cell disorders1,2. Recently, Pasvol et al.3, having shown that fetal haemoglobin (HbF) (in both fetal and adult cells) inhibits malaria parasite development, have suggested that the slight persistance of HbF in children with thal trait is responsible for the selective advantage of the thal gene. They have reported no difference in parasite development in the red cells of adult carriers of thal. Evidence is presented here that both α- and β-thal trait red cells from adults are refractory to parasite development because of oxidant sensitivity, and, furthermore, that fetal red cells and G6PD− cells are refractory for the same reason.

Oxidant damage mediates variant red cell resistance to malaria

CHAPTER 5 – Peroxidative Reactions in Red Cell Biology

Exposure of target cells to a bolus of H2O2 induced cell lysis after a latent period of several hours, which was prevented only when the H2O2 was removed within the first 30 min of injury by addition of catalase. This indicated that early metabolic events take place that are important in the fate of the cell exposed to oxidants. In this study, we described two early and independent events of H2O2-induced injury in P388D1 macrophagelike tumor cells: activation of the glutathione cycle and depletion of cellular NAD. Glutathione cycle and hexose monophosphate shunt (HMPS) were activated within seconds after the addition of H2O2. High HMPS activity maintained glutathione that was largely reduced. However, when HMPS activity was inhibited--by glucose depletion or by incubation at 4 degrees C--glutathione remained in the oxidized state. Total pyridine nucleotide levels were diminished when cells were exposed to H2O2, and the breakdown product, nicotinamide, was recovered in the extracellular medium. Intracellular NAD levels fell by 80% within 20 min of exposure of cells to H2O2. The loss of NADP(H) and stimulation of the HMPS could be prevented when the glutathione cycle was inhibited by either blocking glutathione synthesis with buthionine sulfoximine (BSO) or by inhibiting glutathione reductase with (1,3-bis) 2 chlorethyl-1-nitrosourea. The loss of NAD developed independently of glutathione cycle and HMPS activity, as it also occurred in BSO-treated cells.

https://dm5migu4zj3pb.cloudfront.net/manuscripts/112000/112068/JCI85112068.pdf

Glutathione cycle activity and pyridine nucleotide levels in oxidant-induced injury of cells.

Since unstable hemoglobins have been considered a source of reactive oxygen radicals, and oxidative membrane damage a prehemolytic event, we examined the erythrocyte membranes of six patients (three splenectomized) with hemoglobin Koln disease. In the hydrogen peroxide stress test, the patients' erythrocytes generated more than twice the malonyldialdehyde (a lipid peroxidative product) than control erythrocytes. Fluorescence spectra of lipid extracts of the patients' erythrocytes showed an excitation maximum at 400 nm and an emission maximum of 460 nm, characteristic of malonyldialdehyde lipid adducts. Two types of membrane polypeptide aggregates were found in the erythrocytes of the splenectomized patients. The first, which were dissociable by treatment with mercaptoethanol, contained disulfide-linked spectrin, band 3 and globin. The second, not dissociable by mercaptoethanol, had an amino acid composition similar to that of erythrocyte membranes and spectrin (unlike globin) and like that of aggregates produced by the action of malonyldialdehyde on normal erythrocyte membranes. Atomic absorption spectroscopy of hemoglobin Koln erythrocytes showed no increase in calcium content implying that these cross-links were not due to calcium-stimulated transglutaminase. Using a micropipette technique, we demonstrated that erythrocytes containing membrane aggregates from splenectomized patients were less deformable while aggregate-free erythrocytes from non-splenectomized patients had normal deformability. We conclude that the erythrocyte membranes in hemoglobin Koln disease show evidence of lipid peroxidation with production of malonyldialdehyde, and that the nondissociable membrane aggregates formed in this disease are likely cross-linked by malonyldialdehyde. Because the erythrocytes containing membrane aggregates from splenectomized patients with unstable hemoglobin disease show decreased membrane deformability, we hypothesize that this abnormality results in premature erythrocyte destruction in vivo.

/pdf/oxidant-damage-of-the-lipids-and-proteins-of-the-erythrocyte-rmm3rp5w7l.pdf

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

Red-cell membranes from patients with glucose-6-phosphate dehydrogenase deficiency were studied with polyacrylamide gel electrophoresis and gel filtration chromatography in sodium dodecyl sulfate. Membranes from each of five such patients who also had chronic hemolytic disease contained polypeptide aggregates within two molecular-weight ranges (4.4 X 105 and >50 X 106 daltons). The 4.4 X 105 dalton aggregates were not detectable in red-cell membranes of patients with the enzyme deficiency without chronic hemolysis or in membranes from normal subjects, and the >50 X 106 dalton aggregates were not found in appreciable amounts in these cells. The aggregates were dissociated by mercaptoethanol or dithiothreitol — indicating that they were formed by intermolecular disulfide bonds. The polypeptide aggregates contained spectrin but not globin. Red-cell deformability was decreased in aggregate-containing cells. We postulate that the polypeptide aggregates are indicators of oxidant damage to the red-cell ...

Red-cell-membrane polypeptide aggregates in glucose-6-phosphate dehydrogenase mutants with chronic hemolytic disease. A clue to the mechanism of hemolysis.

Kaplan Me

Papers

Membrane polypeptide aggregates in glucose 6-phosphate dehydrogenase-deficient and in vitro aged red blood cells.