Low-density-lipoprotein (LDL) oxidation may provide the crucial link between plasma LDL and atherosclerotic-lesion formation. Oxidation can be induced in vitro by incubating LDL with cells or metal ions and can be measured by continuously monitoring conjugated-diene absorbance at 234 nm. Measurement of LDL oxidizability was improved by performing the assay with 0.05 g of LDL-protein per liter of phosphate buffer containing 1 mumol of EDTA, by initiating oxidation by adding CuCl2 (5 mumol/L) at 30 degrees C, and by using a short-run ultracentrifugation method for isolating LDL, which reduced the time needed for obtaining purified LDL and thus reduced in vitro oxidation. LDL apolipoprotein analysis and oxidizability determination showed that this method is better than the longer sequential-isolation procedure. Adding butylated hydroxytoluene (BHT) to plasma as an antioxidant unpredictably increased the LDL oxidation lag time, making BHT unsuitable as an antioxidant. Adding EDTA appeared to be sufficient to prevent in vitro oxidation. Additionally, the diene production correlated highly with the concentration of thiobarbituric acid-reactive substances (r = 0.97). No relation between the vitamin E content of LDL and the oxidation lag time was found.
Abstract. The pathobiochemical mechanism of arte riosclerosis in hyperhomocysteinaemiu has not yet been elucidated. In vitro studies have shown that the cytotoxic properties o f homocysteine can be ascribed to its generation of reactive oxygen species, We studied lipid peroxidation, both in vivo and in vitro, in 10 homozygous cystathionine synthase-deficicnt (GSD) patients and in a control group of 10 healthy subjects of comparable age and sex. The susceptibility of low-density lipoprotein (LDL) from hyperhomocysteinaemic patients to oxidation was determined in vitro by continuously measuring the conjugated diene production induced by incubation with copper ions, Oxidation resistance (expressed as lag time), maximal oxidation rate, and extent o f oxidation (expressed as total diene production) of LDL from CSD patients were not significantly different from those o f LDL from controls. Furthermore, the time needed to reach maximal diene production, i.e. t(max), was similar lor LDL from patients and controls. In addition, the vitamin E concentrations in LDL o f CSD patients and controls were similar. The mean concentration (d::SD) o f plasma thiobarbituric acid reactive sub stances (TBARS), an indicator of in vivo lipid per oxidation, was 2-2 ± 0*7 //mol L 1 in CSD patients, a lower value than that measured in the matched con trols (5*0 ± 2*0 /¿mol L 1). investigation o f in vivo and in vitro parameters o f lipid peroxidation shows that the increased risk of arteriosclerosis in hyperhomo cysteinaemia is unlikely to be due to increased lipid peroxidation.Keywords. Atherosclerosis, cystathionine synthase deficiency, hyperhomocysteinaemia, lipid peroxi dation, low-density lipoprotein. Abbreviations* CSD, cystathionine /2-synthase defi ciency; LDL, low-density lipoprotein; TBARS, thio barbituric acid reactive substances.
The diet of Watanabe heritable hyperlipidemic (WHHL) rabbits was supplemented with a low dose (0.025% [wt/wt]) of the antioxidant vitamin E or probucol. The effect of 6 months of treatment on the susceptibility of low-density lipoproteins (LDLs) to oxidative modification and on established atherosclerotic lesions was studied. Vitamin E administration had no effect on plasma lipid levels; probucol supplementation decreased plasma total cholesterol. Vitamin E levels in plasma and LDL increased threefold in the course of treatment with this antioxidant. Six months of treatment with vitamin E and probucol increased the lag time of conjugateddiene formation of LDL subjected to in vitro oxidation by 54% (P<.00l) and 51% (P=.O19), respectively. In this LDL-oxidizability assay, only vitamin E reduced the maximal rate of conjugated-diene production (-24%, P<.001). Neither vita- showing that probucol attenuates the progression of atherosclerotic lesion formation in Watanabe heritable hyperlipidemic (WHHL) rabbits, an animal model for familial hypercholesterolemia, provided strong evidence that lipid peroxidation plays a prominent role in atherogenesis. On the basis of these studies and others, 35 a lipid peroxidation hypothesis has been developed to explain the process of atherogenesis in humans.6 Central in this hypothesis is the oxidation of low-density lipoproteins (LDLs), leading to characteristic physicochemical modifications. Once LDL is modified, unlimited amounts are taken up through the scavenger receptor of macrophages present in the arterial intima. The excessive accumulation of cholesterol in macrophages results in their transformation to foam cells, the hallmark of atherosclerotic lesion formation.LDL contains several natural antioxidants that provide protection against excessive oxidative modification.
Abstract. We studied the effects of the 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase inhibitors simvastatin and pravastatin on the in vitro susceptibility of low-density lipoprotein (LDL) to oxidation. Twenty-three hypercholesterolaemic patients (mean serum cholesterol 9.7 mmol 1-') were treated with increasing doses of either simvastatin or pravastatin for 18 weeks. No significant differences in effect on lipid levels between the two drugs were found. Treatment resulted in lowering of total cholesterol and LDL-cholesterol by maximally 30% and 34%, respectively. Chemical composition analysis showed that LDL particles contained relatively more protein and less free cholesterol and cholesteryl-ester after treatment. The LDL cholesterol/protein ratio decreased from 1.24 f 0.21 to 0.97 f 0.23 (n = 20). By continuous monitoring of in vitro oxidation it appeared that LDL was less susceptible to oxidation after drug treatment. Maximal rate of diene production was significantly decreased from 19.7f 3.1 to 18.5 f 3.3 nmol min-l mg-' LDL; total diene production decreased significantly from 420.3k67.6 to 380.5k49.1 nmol mg-l LDL; the lag time was unchanged throughout the study. These studies show that HMG-CoA reductase inhibitors reduce the oxidizability of LDL by altering its composition.
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