Catherine Rice-Evans

Free radical-lipid interactions and their pathological consequences

In this review we have tried to present the current thinking on the consequences for lipids of their interactions with free radicals and the pathological implications. In particular, atherosclerosis and cancer have been addressed. In the case of the former, it is not clear whether the initial oxidative event is an enzymic or free radical-mediated process as yet. However, the importance of the antioxidants in controlling LDL oxidation, macrophage uptake of oxidatively modified LDL and progression of atheroma in animal models certainly suggests an important propagative role for free radical-mediated events. With regard to cancer, oxidative modification of cell lipids has potential consequences for tumour cell proliferation. Whilst lipid hydroperoxides can serve as an origin of prostaglandins with tumour inhibitor (or immunosuppressive) properties, they may also influence cellular growth regulatory proteins normally dependent on membrane lipid integrity. Alternatively, they may function as a source of aldehydic breakdown products capable of down-regulating cell proliferation through covalent modification of regulatory proteins. Oils rich in n-3 polyunsaturated fatty acids have toxic effects towards tumour cells. This toxicity is not mediated by prostaglandins but rather through the capacity of such agents to elevate the levels of lipid peroxides. This may be enhanced by active oxygen species released constitutively from tumour cells.

t-butyl hydroperoxide-induced perturbations of human erythrocytes as a model for oxidant stress

Erythrocytes were incubated with t-butyl hydroperoxide in the presence and absence of hemoglobin as a model system for oxidative stress and the alterations in the structure and integrity of the membranes were investigated. The results showed that in the presence of hemoglobin a significant modification in the membrane surface charge was induced but no such alteration was observed in peroxidized hemoglobin-free membranes. As increased hemoglobin oxidation occurred in the erythrocytes, membrane lipid peroxidation diminished, suggesting a protective role for methemoglobin in t-butyl hydroperoxide-induced lipid peroxidation. Electrophoresis on polyacrylamide gels showed modification of the cytoplasmic protein region but no high molecular weight aggregates formed at the concentrations of the hydroperoxide used in this work. The results suggest that the t-butyl hydroperoxide/normal erythrocyte system seems to be an instructive model for membrane perturbations characteristic of oxidative disorders.

Desferrioxamine as a lipid chain-breaking antioxidant in sickle erythrocyte membranes

The mechanism of action of desferrioxamine in the inhibition of the catalysis of iron‐induced oxidative damage has been ascribed to its ability to chelate available ferric ion (K b = 1031). However, recent work has proposed that the trihydroxamate moiety of desferrioxamine can also be involved in electron transfer reactions involving the Superoxide radical, peroxidase/hydrogen peroxide mixtures and ferryl myoglobin radicals. In this study we report evidence for the ability of desferrioxamine to inhibit peroxidative damage to pathological membranes with which non‐haem iron is associated through a mechanism of action as a lipid chain breaking antioxidant, independently of its iron chelating properties.

Studies on the altered membrane characteristics of sickle cells.

It has been shown that sickle cell anaemia causes abnormalities of the red cell membrane which are reflected in altered red cell deformability, permeability, surface area [l], elevated calcium content [2], altered protein patterns and reduced membrane sialic acid content [3 1, potassium loss and cellular dehydration [4]. The major feature of this disease is the tendency of erythrocytes to sickle when exposed to decreased oxygen tensions and to unsickle when reoxygenated. The sickle cell membrane is deformed by polymerised tibres of deoxygenated Hb-SS which adhere to the internal surface of the membrane [5]. This polymerisation is essentially reversible with oxygenation, although some of the cells do maintain their original sickle shape. These irreversibly sickled cells constitute 4-44% of cells in oxygenated capillary blood of individuals with sickle cell disease [6-81. In this communication we have investigated alterations in the fluidity of the hydrophobic lipid region and changes at the surface of the sickle cell membranes compared with those of normal erythrocytes. The results indicate that, in comparison with normal erythrocyte membranes: (i) The hydrophobic lipid region of sickle cell membrane is less fluid. (ii) This is not a consequence of alterations in the cholesterol/phospholipid ratio or fatty acid content. (iii) The negative potential at the surface of the sickle cell membrane is decreased. 2. Experimental

A spectrofluorimetric study of the interaction of glycerol mono-oleate with human erythrocyte ghosts

It is now well established by the work of Lucy et al., that various unsaturated fatty acids and other chemical reagents (e.g. certain esters, polyols, dimethyl sulphoxide) will induce cell fusion for different types of cell under appropriate conditions [l-5] . Cell fusion may also be induced by other means including heat [2] and high pH with a high concentration of calcium ions [6]. It has been suggested that it may be necessary, in addition to other requirements, for lipids in natural membranes to be in a fluid condition for fusion to occur [4,5]. Kosower et al., have suggested that some increase in local fluidity favours cell fusion [7] from work involving membrane mobility agents which promote motion through cell membranes [8] by inducing local disorder and which actively promote the fusion of hen erythrocytes under similar conditions to those used by Lucy et al. In the present communication evidence will be presented which indicates that treatment of human erythrocyte ghost membranes with the fusogenic lipid glycerol mono-oleate leads to an increase in the fluidity of the membrane lipids, whereas the chemically related nonfusogenic lipid glycerol monostearate has no effect.

The Effects of Iron-Mediated Oxidative Stress in Isolated Renal Cortical Brush Border Membrane Vesicles at Normothermic and Hypothermic Temperatures

Experiments on renal cortical brush border membrane vesicles have been undertaken in order to assess the involvement of iron in oxidative stress at physiological temperatures and under conditions of hypothermia. A decrease in temperature stimulated iron-induced lipid peroxidation. The results are discussed in relation to the role of the oxidation state of the iron and iron(II)/iron(III) ratios in the initiation of peroxidative events.

Erythrocyte membrane abnormalities in glucose-6-phosphate dehydrogenase deficiency of the mediterranean and A-types

The red cells of patients with inherited deficiencies of glucose-6-phosphate dehydrogenase (G-6-PD) are sensitive to the haemolytic effects of a wide variety of drugs such as phenylhydrazine, primaquine, acetylsalicylic acid and favabean [I]. The oxidative damage induced by such haemolytic agents may be caused by the generation of hydrogen peroxide, superoxide and hydroxyl radicals from the autoxidation of the active drug or its metabolites, or via a linked oxidation between oxyhaemoglobin and the drug or its metabelites [2-4]. These reactive species have the capacity to initiate the peroxidation of unsaturated fatty acids in the erythrocyte membrane phospholipids [5,6]. Oxidation of reduced glutathione is the major pathway of hydrogen peroxide metabolism in intact erythrocytes [7]. Continuous reduction of the oxidised glutathione is dependent on glutathione reductase, which itself is (NADPH + H)-dependent. The reduced form of this coenzyme is in short supply in individuals deficient in glucose-6-phosphate dehydrogenase and the resulting failure to maintain normal concentrations of reduced glutathione and the accompanying intracellular accumulation of hydrogen peroxide and oxidising radicals leads to the oxidation of free sulphydryl groups. The red cell membranes from G-6-PD-deficient individuals with accompanying chronic haemolytic disease may contain polypeptide aggregates involving spectrin, dissociable by disulphide reducing agents [8,9]. These aggregates are considered to be indicators of oxidant damage to the red cell membrane leading to decreased deformability and haemolysis. This work involves the erythrocytes from patients with the less severe and more common G-6-PD mutant of the Aand Mediterranean-types which are characterised clinically by intermittent haemolysis associated with drug treatment or infection; their catalytic activity is ~20-40% of the average activity of normal fresh human red cells. The results indicate the following membrane changes in such erythrocytes: (i) The bulk lipid fluidity is increased; (ii) The cholesterol:phospholipid ratio is decreased; (lii) The lipid peroxidation level is elevated.

The effects of hydrazine on sickle cells

Abstract The response of the red cells from patients with sickle cell disease to hydrazine treatment in vitro is to inhibit the sickled morphology, while the metabolic characteristics and the osmotic fragility of the cells remain unaltered. However, the oxygen affinity of the sickle cell haemoglobin is decreased.