Erythrocyte-induced oxidation of low density lipoproteins: control by antioxidant nutrients.

Abstract

Several reports have observed that when haemoglobin is released via micro bleeding processes it becomes toxic[l]. Microbleeding has been observed in the eye causing retinal damage [2], in the brain [3] and at sites of inflammation [4]. We are interested in haemorrhaging within atherosclerotic lesions and in the interactions of haem proteins [5] with low density lipoproteins (LDL). Our previous studies [6] have investigated the effects of haemoglobin released from ruptured erythrocytes on LDL. The results revealed that the onset of oxidative modification of LDL is related to the time during which the oxyhaemoglobin [HX-Fe ... O, ] becomes activated to the ferry1 state (HX-FelV=O] via the deoxy form, as well as to the antioxidant status of the LDL. Currently we are comparing mechanisms of protection of LDL from oxidative modification induced by ruptured erythrocyte lysate, by enhancing the antioxidant status of the lysate, on the one hand, and of the LDL, on the other hand. Fresh human blood was obtained from volunteers (with informed consent). The plasma was used to isolate the LDL [7] and the erythrocytes were lysed and the membranes discarded. The response of the haemolysate to interaction with LDL was monitored spectroscopically and the level of oxyhaemoglobin determined from the extinction coefficient. Oxidative modification of LDL was assessed by measuring the altered surface charge on agarose gels using the Beckman paragon lip0 electrophoresis system and by applying the thiobarbituric acid acid assay with appropriate controls. The results show that the lysate soluble antioxidant, ascorbate (50 uM), is ineffective in protecting the LDL (0.5 mglml LDL protein) from oxidative modification by erythrocyte lysate (10 uM), whereas enhancing the antioxidant level within the LDL itself with atocopherol is protective [Figs la,lb] (even down to applying 20 uM data not shown). The a-tocopherol delays the rapid oxidative conversion of haemoglobin [Fig l c ] and inhibits LDL oxidation. The combined actions of ascorbate and atocopherol behave synergistically to completely suppress the oxidation of LDL for up to 22 hours. This study shows that the antioxidant level in the aqueous phase of the erythrocyte lysate is less effective than that of the nonpolar environment within the LDL particle, in protecting the latter from oxidation by erythrocyte lysate. This confirms that the interaction between preformed lipid hydroperoxides in LDL and the oxyhaemoglobin is the mechanistic driving force for the haemoglobin-LDL reaction. We acknowlege financial support from St Thomas' Special trustees and the British Heart Foundation.