VANESSA OLEARY, ANNE'ITE GRAHAM, VICTOR. DARLEY-USMAR and DAVID STONE. 04-. 8. ;. 2. 9. 0 2-. 0 0-p. Biochemical Sciences, Wellcome Research ...
Biochemical Society Transactions ( 1 993) 21
The effect of lipid hydroperoxides on the copper dependent oxidation of low density lipoprotein. VANESSA OLEARY, ANNE'ITE GRAHAM, VICTOR DARLEY-USMAR and DAVID STONE Biochemical Sciences, Wellcome Research Laboratories, Langley Court, Beckenham, Kent, BR3 3BS, UK. There is growing evidence that the oxidation of low density lipoprotein (LDL) in the artery wall is of primary importance in the pathogenesis of atherosclerosis. While the mechanism of LDL oxidation in vivo is not yet proven, it has been suggested that lipoxygenases, specifically 15-lipoxygenase, may play a role and a number of mechanisms for the pro-oxidant action of this enzyme have been proposed. The enzyme could act directly to oxygenate the fatty acid side chains of the particle, or oxidants generated during the catalytic cycle of the enzyme could be released and initiate peroxidation. Alternatively, the enzyme could generate lipid-derived peroxides that partition into the LDL particle, where in the presence of transition metals they promote lipid peroxidation. Other investigators have shown that treatment of LDL with soybean lipoxygenase leads to formation of an oxidised and cytotoxic complex, this process requiring H202 (1) Another group has shown the treatment gave little or no oxidation of fatty acids unless phospholipase A2 was present to produce free fatty acids as substrate (2). Having previously shown that exogenously added 15-lipoxygenase derived fatty acid peroxides enhance the rate of copper dependent oxidation of LDL (3), in this study we looked at the effect of treatment with soybean lipoxygenase on this process.
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Figure 1: Samples of native LDL (lmg/ml) were incubated at 37°C in the presence of 40pg/ml soybean lipoxygenase. A. The samples were extracted and saponified, then the total fatty acid oxygenation products identified using a Merck Lichrocart column (25Ommx4mm) with hexane/propan-2-ol/acetic acid (98.7/1.2/0.1, v/v/v) as the mobile phase, flow rate lmvmin and monitoring the eluate at 235nm. B. The oxygenated products formed in the cholesteryl ester fraction of the LDL were identified by reverse phase chromatography on a Beckman Ultrasphere C18 column (25Ommx4.6mm) using a linear gradient of water (3%-0%) in acetonitrile/tetrahydrofuran (65/35, v/v) as eluting solvent, flow rate 2mVmin. Separation of cholesteryl esters was achieved and the peaks monitored at 213nm (not shown). Oxidation products were identified by monitoring at 234nm. Soybean 15-lipoxygenase (4Opg/ml) was added to solutions of LDL and incubated at 37°C for 90 minutes. The effect of this treatment on the oxidisability of the LDL was assessed by measurement of the lag phase for conjugated diene formation after the addition of copper. The antioxidant status of the LDL was determined by HPLC measurement of the vitamin E content. The peroxide products formed were also analysed by HPLC (Fig. 1). Treatment of LDL with .:rirL,ean lipoxygenase resulted in a small increase in conjugate< ~ X which, S in the absence of copper, was complete and s!&k after 90 minutes at 37°C. Subsequent addition of copper resulted in a rapid increase in conjugated diene formation. A reduction in lag phase from 53.5 minutes in untreated LDL to 14.5 minutes in lipoxygenase treated LDL was observed, whereas vitamin E levels decreased by just 30%, from 19.2 to 13.5nmol/mg. Both the total fatty acid pool and the cholesteryl ester fraction of the lipoprotein were analysed for oxidation products after 15-lipoxygenase treatment. In the total fatty acid pool, the major products were identified as 13-HODE and 13-HPODE (Fig.lA). The cholesteryl esters in native LDL were separated and identified by monitoring the absorbance of the eluate at 2 13nm, while the 15-lipoxygenase oxygenated cholesterol ester products were identified by monitoring at 234nm (Fig.1B). This fraction was collected, saponified and the fatty acid products identified as 13/9-HPODE and 13/9-HODE. The generation of oxidation products in the cholesteryl ester fraction of LDL was dependent on the concentration of lipoxygenase added to the LDL, and the increase in these products was again associated with a decrease in the lag phase for oxidation on addition of copper. These results demonstrate that LDL enriched with 15lipoxygenase derived peroxides shows an increased susceptibility to oxidation on the addition of copper. The formation of oxidation products in both the total fatty acid pool and, more interestingly in the cholesteryl ester fraction of the molecule was established. In contrast to previous results showing a requirement for phospholipase activity to release free fatty acids as substrates (2), these results show that the action of lipoxygenase alone generates low levels of peroxides and that the cholesteryl ester core of the lipoprotein can act as a substrate.
References 1. Cathcart,M.K., McNally,A.K. and Chiso1m.G.M. (1991) J.Lipid Res. 32,63-70. 2. Sparrow,C.P., Parthasarathy,S. and Steinberg,D. (1988) J.Lipid Res. 29,145-752. 3. OLeary,V.J., Darley-Usmar,V.M., Russel1,L.J. and St0ne.D. (1992) Bi0chem.J. 282,631-634.
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