Inhibition of Lipid Oxidation

Abstract

Oxidative modification of the low-density lipoproteins (LDL) has been shown to cause accelerated degradation of LDL via the scavenger receptor pathway. Under conditions of high serum LDL levels, LDL particles can migrate into the subendothelial space where oxidation of LDL can occur (Heinecke 1998; Jiang et al. 2011). The actual oxidation process is believed to begin with lipid peroxidation, followed by fragmentation to result in short-chain aldehydes. These aldehydes can form adducts with the lysine residues of apo B, creating a new epitope which is recognized by the scavenger receptor of macrophages.

Hans Gerhard Vogel: deceased.

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General Considerations

Oxidative modification of the low-density lipoproteins (LDL) has been shown to cause accelerated degradation of LDL via the scavenger receptor pathway. Under conditions of high serum LDL levels, LDL particles can migrate into the subendothelial space where oxidation of LDL can occur (Heinecke 1998; Jiang et al. 2011). The actual oxidation process is believed to begin with lipid peroxidation, followed by fragmentation to result in short-chain aldehydes. These aldehydes can form adducts with the lysine residues of apo B, creating a new epitope which is recognized by the scavenger receptor of macrophages.

During the same process, lecithin is converted to lysolecithin , which is a selective chemotactic agent for monocytes. The monocytes adhere to the arterial wall and penetrate through to the subendothelium. Once there, the monocyte changes to a tissue macrophage which takes up the oxidized LDL via the scavenger receptor. The uptake of oxidized LDL continues until the macrophage is engorged with cholesteryl esters ultimately forming a foam cell. Groups of these foam cells constitute a fatty streak. By inhibiting the oxidation of LDL, it is hoped that the modification of apo B and the production of chemotactic lysolecithin can be prevented.

The family of receptors for mammalian low-density proteins has been reviewed by Hussain et al. (1999).

References and Further Reading

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• Hussain MM, Strickland DK, Bakillah A (1999) The mammalian low-density lipoprotein receptor family. Annu Rev Nutr 19:141–172

• Jürgens G (1989) Modified serum lipoproteins and atherosclerosis. Ann Rep Med Chem 25:169–176

• McCarthy PA (1993) New approaches to atherosclerosis: an overview. Med Res Rev 13:139–159

• Parthasarathy S, Wieland E, Steinberg D (1989) A role for endothelial cell lipoxygenase in the oxidative modification of low density lipoprotein. Proc Natl Acad Sci U S A 86:1046–1050

• Rankin SM, Parthasarathy S, Steinberg D (1991) Evidence for a dominant role of lipoxygenase(s) in the oxidation of LDL by mouse peritoneal macrophages. J Lipid Res 32:449–456

• Steinberg D (1990) Arterial metabolism of lipoproteins in relation to atherogenesis. In: Lee KT, Onodera K, Tanaka K (eds) Atherosclerosis II: recent progress in atherosclerosis research, vol 598, Annals of the New York Academy of Sciences. pp 188–193

• Steinbrecher UP (1987) Oxidation of human low density lipoprotein results in derivatization of lysine residues of apolipoprotein B by lipid peroxide decomposition products. J Biol Chem 262:3603–3608

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Inhibition of Lipid Peroxidation of Isolated Plasma Low-Density Lipoproteins

Purpose and Rationale

Hypercholesterolemic Watanabe rabbits are considered to be a suitable model to study the effect of antioxidants as antiatherosclerotic agents (Carew et al. 1987; Kita et al. 1987; Steinberg et al. 1988; Dresel et al. 1990). Plasma of Watanabe heritable hyperlipidemic (WHHL) rabbits is used to test the inhibition of Cu²⁺-induced lipid peroxidation of isolated low-density lipoproteins (LDL).

Procedure

Animals of a modified Watanabe heritable hyperlipidemic rabbit strain (Gallagher et al. 1988) are used. The animals are fed over a period of 12 weeks with Purina rabbit chow diet with or without 1 % of test compound or standard (probucol). Plasma samples are collected in Na₂EDTA (0.1 % final concentration). LDLs are isolated from each rabbit plasma using a sequential ultracentrifugation technique at d = 1.019–1.063 g/ml (Mao et al. 1983). LDLs are then dialyzed against phosphate-buffered saline (PBS, 0.01 M sodium phosphate, 0.12 M NaCl, pH 7.4) at 4 °C for 24 h.

For determination of LDL lipid peroxidation induced by Cu²⁺, 100 μg of each LDL sample is adjusted to a volume of 1.5 ml with distilled water. Lipid peroxidation is initiated by addition of CuSO₄ to a final concentration of 5 μM followed by an incubation at 37 °C for 3 h. The reaction is stopped by adding 100 μl of 50 mM Na₂EDTA. Fifty micrograms of LDL from the reaction mixture are added to 1.5 ml of 20 % trichloroacetic acid and vortexed. Finally, 1.5 ml of 0.67 % thiobarbituric acid (TBA) in 0.05 N NaOH is added, and the mixture is incubated at 90 °C for 30 min. Samples are centrifuged at 1,500 rpm for 10 min. The absorbance of the supernatant fractions is determined at 532 nm to estimate the content of lipid peroxides (thiobarbituric acid-reactive substances). A standard curve (0–5 nmol) of malondialdehyde is generated using malondialdehyde bis(dimethyl acetal) as reference to determine the lipid peroxidation content in Cu²⁺-treated LDL.

Evaluation

The content of lipid peroxide in LDL is plotted against the drug concentration in LDL fractions. The extent of Cu²⁺-induced peroxidation decreases with increasing drug concentrations. The effects of test compounds are compared to the standard.

Modifications of the Method

Inhibition of iron-dependent lipid peroxidation by test compounds was measured by Braughler et al. (1987) and Yoshioka et al. 1989).

Yamamoto et al. (1986) studied the effects of probucol on lipid storage in macrophages in vitro in the presence of acetylated low-density lipoprotein using macrophage-like cells (UE-12) established from a human histiocytic lymphoma cell line.

Barnhart et al. (1989) used LDL from human plasma to study the concentration-dependent antioxidant activity of probucol.

Parthasarathy et al. (1986) incubated LDL from human plasma samples with rabbit aortic endothelial cells and measured the increase in electrophoretic mobility, the increase in peroxides, and the increase in subsequent susceptibility to macrophage degradation.

Mansuy et al. (1986) studied the inhibition of lipid peroxidation induced in liver microsomes either chemically by FeSO₄ and reducing agents (cysteine or ascorbate) or enzymatically by NADPH and CCl₄.

References and Further Reading

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• Braughler JM, Pregenzer JF, Chase RL, Duncan LA, Jacobsen EJ, McCall JM (1987) Novel 21-amino steroids as potent inhibitors of iron-dependent lipid peroxidation. J Biol Chem 262:10438–10440

• Carew TE, Schwenke DC, Steinberg D (1987) Antiatherogenic effect of probucol unrelated to its hypocholesterolemic effect: evidence that antioxidants in vivo can selectively inhibit low density lipoprotein degradation in macrophage-rich fatty streaks and slow the progression of atherosclerosis in the Watanabe heritable hyperlipidemic rabbit. Proc Natl Acad Sci U S A 84:7725–7729

• Dresel HA, Deigner HP, Frübis J, Strein K, Schettler G (1990) LDL-metabolism of the arterial wall – new implications for atherogenesis. Z Kardiol 79(Suppl 3):9–16

• Gallagher PJ, Nanjee MN, Richards T, Roche WR, Miller NE (1988) Biochemical and pathological features of a modified strain of Watanabe heritable hyperlipidemic rabbits. Atherosclerosis 71:173–183

• Heinecke JW (1998) Oxidants and antioxidants in the pathogenesis of atherosclerosis: implications for the oxidized low density lipoprotein hypothesis. Atherosclerosis 141:1–15

• Jiang X, Yang Z, Chandrakala AN, Pressley D, Parthasarathy S (2011) Oxidized low density lipoproteins–do we know enough about them? Cardiovasc Drugs Ther 25:367–377

• Kita T (1991) Oxidized lipoproteins and probucol. Curr Opin Lipidol 2:35–38

• Kita T, Nagano Y, Yokode M, Ishii K, Kume N, Ooshima A, Yoshida H, Kawai C (1987) Probucol prevents the progression of atherosclerosis in Watanabe heritable hyperlipidemic rabbit, an animal model for familial hypercholesterolemia. Proc Natl Acad Sci U S A 84:5928–5931

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• Parthasarathy S, Young SG, Witztum JL, Pittman RC, Steinberg D (1986) Probucol inhibits oxidative modification of low density lipoprotein. J Clin Invest 77:641–644

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• Yoshioka T, Fujita T, Kanai T, Aizawa Y, Kurumada T, Hasegawa K, Horikoshi H (1989) Studies with hindered phenols and analogues. 1. Hypolipidemic and hypoglycemic agents with ability to inhibit lipid peroxidation. J Med Chem 32:421–428

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