INFLUENCE OF FREE RADICAL -MEDIATED MEMBRANE DAMAGE ON ERYTHROCYTE Ca²⁺-HOMEOSTASIS IN CERTAIN PATHOLOGICAL STATES

Abstract

The possible nature of toxic oxygen from radical species generated in vivo under pathological conditions as well as the relationship between oxygen free radicals and/or lipid peroxidation and the abnormal regulation of intrecellular free calcium in erythrocytes have been investigated in sickle cell anaemia, essential hypertension and kwashiokor disease. The release of iron, from haemoglobin and myoglobin following incubation with varying concentrations of H₂0₂, was suppressed in the presence of membranes which underwent lipid peroxidation. Moreover, formation of ferrrylhaemoprotein species was suppressed by electron donating trihydroxamate moiety of desferrioxamine. In contrast, ascorbate, a physiological antioxidant suppressed the formation of the ferryl species and reduced the iron in the haemoprotein to the Iron (II) state. Estimation of the amount of membrane-bound, non-haem iron and the extent of lipid peroxidation in the ghost membranes indicate that the values obtained for majority of sickle cell membranes were comparatively higher than in normal membranes. The levels of membrane-bound non haem iron and the extent of membrane lipid peroxidation were significantly higher in kwashiorkor disease when compared to erthrocytes from healthy children the same age bracket. In contrast, the erythrocytes obtained from patients having essential hypertension (HTN) neither possess any appreciable amount of membrane-bound non-haem iron nor endogenous products of lipid peroxidation when compared with the values obtained for membranes prepared from erythrocytes of normotensive (NTN) controls. Results of the assay of the basal activity of the Ca²⁺-pump in these membranes show that Ca²⁺-pump activity was reduced by about 50% in ghost membranes of SCA patients when compared to normal membranes. The basal activity of the pump was almost negligible in irreversibly sickled cells (ISC) membranes while the activity in reversibly sickled cells (RSC) was comparable to that in normal membranes. Calmodulin stimulated the Ca²⁺-pump by at least four- fold in normal cell membranes and by about 2-3-fold in unseparated sickle cell membranes. The ISC ensyme was not sensitive at all to Calmodulin while the RSC enzyme was still stimulable by at least 3-fold by calmodulin. The pump is highly susceptible to oxygen free radicals in all cases and was inhibited by more than 60% while the enzyme activity in normal membranes was inhibited by almost 40%. Furthermore, exposure of RSC and ISC to oxygen free radicals resulted in a near total loss and a complete loss of activity, respectively. The basal Ca²⁺-ATPase activity in the erythrocycte membranes of both HTN and NTN individual are comparable. In contrast the calmodulin-responsiveness of the enzyme in the erythrocyte membranes of HTN individuals was only about 60% that of normal membranes. The activity of the membranes Ca²⁺-pump in HTN and NTN was inhibited by oxygen free radicals to a comparable extent. In the absence of calmodulin, the specific activity of Ca²⁺ +Mg-ATPase of erythrocyte membrane from kwashiorkor erythrocytes was more than 40% lower than the specific activity of the enzyme in normal individuals, the specific activity of which was increased by at least four-fold by calmodulin. A determination of the effect of oxygen free radicals on the Ca²⁺-pump enzyme in the intact erythrocytes and erythrocyte ghost membranes of KWA subjects, reveal that the Ca²⁺-pump enzyme was highly susceptible to inhibition by oxygen free radicals. Whereas oxidatively damaged red cell membrane protein components are reported to be more susceptible to proteolysis, electophoretic separation of erthrocyte membrane proteins and of purified Ca²⁺-pumping ATPase of erthrocytes in the three pathological states did not indicate the presence of any fragment. The findings reported in this thesis suggest that although the production of toxic free radicals may be responsible for the defective erthrocyte calcium-pumping activity in SCA and in KWA, the absence of end products of lipid peroxidation and negligible amounts of membrane-bound non-haem iron, in the membranes of patients having essential hypertension subject that the mechanism of decreased Ca²⁺-pumping in erythrocytes of HTN probably differ from that in KWA and SCA.