Lipid peroxidation and susceptibility of low-density lipoprotein to in vitro oxidation in hyperhomocysteinaemia

Abstract. The pathobiochemical mechanism of arteriosclerosis in hyperhomocysteinaemia has not yet been elucidated. In vitro studies have shown that the cytotoxic properties of 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-deficient (CSD) patients and in a control group of 10 healthy subjects of comparable age and sex. The susceptibility of low-density lipoprotein (LDL) from hyperhomo-cysteinaemic 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 of oxidation (expressed as total diene production) of LDL from CSD patients were not significantly different from those of LDL from controls. Furthermore, the time needed to reach maximal diene production, i.e. t(max), was similar for LDL from patients and controls. In addition, the vitamin E concentrations in LDL of CSD patients and controls were similar. The mean concentration (± SD) of plasma thiobarbituric acid reactive substances (TBARS), an indicator of in vivo lipid peroxidation, was 2.2 ± 0.7 μmol L^-1 in CSD patients, a lower value than that measured in the matched controls (50± 2.0 μmol L^-1). Investigation of in vivo and in vitro parameters of lipid peroxidation shows that the increased risk of arteriosclerosis in hyperhomocysteinaemia is unlikely to be due to increased lipid peroxidation.     

Vitamin E supplementation in hyperlipidaemic patients: effect of increasing doses on in vitro and in vivo low-density lipoprotein oxidation

BACKGROUND: Vitamin E supplementation is associated with a reduced risk of developing atherosclerotic events; probably because it inhibits low-density lipoprotein (LDL) oxidation, an initial step in atherosclerosis. Metal ion-dependent LDL oxidation is a commonly used method to estimate oxidizability of LDL, but the effect of antioxidant supplementation on the levels of autoantibodies to oxidised LDL (ox-LDL), an in vivo indicator of LDL oxidation, is unknown. DESIGN: This double-blind, placebo-controlled study investigated the susceptibility of LDL to copper induced oxidation and malondialdehyde (MDA) derivatized-LDL (MDA-LDL) in hyperlipidaemic patients on supplements of vitamin E. The vitamin E group (n = 20) took vitamin E 100 IU daily and the dose was doubled at six-weekly intervals to 1600 IU daily. The control group (n = 17) received placebo in the same fashion. Blood samples were obtained at baseline and each subsequent visit to measure vitamin E status and oxidation of LDL. RESULTS: A significant increase in both alpha-tocopherol levels and the lengths of lag phase was seen in the vitamin E group after first week of supplementation (100 IU day-1). This continued to rise in a dose-dependent fashion with a doubling of the lag phase on 1600 IU daily. However, the titre of antibodies to MDA-LDL was not altered. CONCLUSIONS: The results suggest that although regarded as an in vivo marker of LDL oxidation, antibodies to MDA-LDL may not be a suitable measure to evaluate the effect of short-term antioxidant supplementation. The failure of autoantibody titres to fall despite reduced oxidizability of LDL may possibly be attributable to a long half-life of the antibody or, once initiated, a continuous immunological response to ox-LDL contained in atherosclerotic lesions of the arterial wall.     

The effect of selective low-density lipoprotein apheresis on plasma lipoperoxides and antioxidant vitamins in familial hypercholesterolemic patients.

Familial hypercholesterolemia (FH) is an autosomal dominant genetic disorder characterized by a lifelong elevation in the concentration of low-density lipoprotein (LDL) bound cholesterol in blood by cholesterol deposits and by early coronary artery disease. The LDL apheresis technique has been introduced with the goal of reducing LDL cholesterol levels, thereby preventing the development of atherosclerosis. The literature on LDL apheresis reports 2 different facets, the therapeutic aspect associated with the lessening of LDL concentration and the initiation of a peroxidation process associated with the biocompatibility of the artificial membrane. Lipid and protein peroxidation gives rise to toxic and atherogenic hydroperoxide, mostly lipid hydroperoxides, and derivative compounds, which may offset the benefit of the procedure. In this paper, plasma hydroperoxide levels are determined along with the elevation of the serum and LDL antioxidant status in hypercholesterolemic patients before and following repeated LDL apheresis sessions. Hydroperoxide concentration has been expressed both in terms of plasma volume and LDL concentration. A highly significant increase in LDL lipid hydroperoxides is demonstrated when expressed in terms of LDL concentration and is associated with the LDL apheresis procedure. The usefulness of antioxidant supplementation in LDL apheresis is discussed.     

DALI low-density lipoprotein apheresis in homozygous and heterozygous familial hypercholesterolemic patients using low-dose citrate anticoagulation.

The purpose of this study was to clarify the efficacy and safety of direct adsorption of lipoprotein low-density lipoprotein apheresis (DALI LDL apheresis) in patients with severe homozygous and heterozygous familial hypercholesterolemia who showed minor adverse effects during treatment with the usual DALI configuration (AC 1:20) through the use of a new system with low-dose citrate anticoagulation (AC 1:40) developed in order to minimize citrate-related adverse effects. Serum total cholesterol and LDL-cholesterol (LDL-C) showed a decrease of 57% to 61%, and 62% to 67%, respectively, in the 2 patients. Serum lipoprotein (a) (Lp[a]) was higher in the homozygous patient (Patient 1: MD) and within the normal range in the heterozygous patient (Patient 2: ES). In the former, Lp(a) was reduced by 52%. Serum high-density lipoprotein cholesterol (HDL-C) showed a statistically insignificant acute reduction: 15% to 19%. The observed reduction is mainly related to the well-known effect of hemodilution. The cardiovascular risk (total cholesterol/HDL-C) was reduced in both patients (46% to 54%) as expected. Serum triglycerides were reduced by 33% to 49%. The mean blood volume processed per session was 7,600 ml. Fifteen treatments for each patient have successfully been completed without the appearance of any clinically significant subjective and objective symptoms related to treatment with the new system.     

Oxidized low-density lipoprotein reduces plasma coagulation in vitro

Oxidation of low-density lipoprotein (LDL) was shown to occur in vivo and involved lipid peroxidation and apolipoprotein modification. We studied the effect of oxidized-LDL (Ox-LDL) on plasma coagulation by measuring prothrombin time (PT) and partial thromboplastin time (PTT) following the addition of Ox-LDL to normal plasma. Ox-LDL, but not native LDL, caused prolongation of PT and PTT by 30% in a dose- and time-dependent pattern. This effect was also shown to be present following lipoprotein delipidation, suggesting that it was the apolipoprotein fraction of Ox-LDL, but not its lipid fraction, that was responsible for the prolongation of PT and PTT. This was further substantiated since similar effect could be obtained by adding LDL treated with trinitrobenzenesulphonic acid to block the lysine groups, as occurs in oxidized LDL. Ox-LDL, unlike LDL, was found to reduce plasma ionized calcium by 33%. Moreover, adding calcium ions to Ox-LDL negated its effect on PT and PTT, suggesting that Ox-LDL apolipoprotein may influence coagulation by binding calcium ions.     

Oxidized low-density lipoprotein reduces plasma coagulation in vitro

Oxidation of low-density lipoprotein (LDL) was shown to occur in vivo and involved lipid peroxidation and apolipoprotein modification. We studied the effect of oxidized-LDL (Ox-LDL) on plasma coagulation by measuring prothrombin time (PT) and partial thromboplastin time (PTT) following the addition of Ox-LDL to normal plasma.

Ox-LDL, but not native LDL, caused prolongation of PT and PTT by 30% in a dose- and time-dependent pattern. This effect was also shown to be present following lipoprotein delipidation, suggesting that it was the apolipoprotein fraction of Ox-LDL, but not its lipid fraction, that was responsible for the prolongation of PT and PTT. This was further substantiated since similar effect could be obtained by adding LDL treated with trinitrobenzensulphonic acid to block the lysine groups, as occurs in oxidized LDL. Ox-LDL, unlike LDL, was found to reduce plasma ionized calcium by 33%. Moreover, adding calcium ions to Ox-LDL negated its effect on PT and PTT, suggesting that Ox-LDL apolipoprotein may influence coagulation by binding calcium ions.     

Pistachio intake increases high density lipoprotein levels and inhibits low-density lipoprotein oxidation in rats

There is increasing evidence that nuts have protective effects against coronary artery disease by improving lipid profile and inhibiting lipid oxidation. However, data about pistachio nuts are limited, and to our knowledge, there is no study investigating the effects of pistachio intake on lipid oxidation and serum antioxidant levels. This study, therefore, sought to determine the effects of pistachio intake on serum lipids and determine whether consumption of pistachio would alter serum antioxidant levels. Rats were randomly divided into three groups (n=12 for each): control group fed basic diet for 10 weeks and treated groups fed basic diet plus pistachio which constituted 20% and 40% of daily caloric intake, respectively. Consumption of pistachio as 20% of daily caloric intake increased high-density lipoprotein (HDL) levels and decreased total cholesterol (TC)/HDL ratio, compared with those not taking pistachio. However, TC, low-density lipoprotein (LDL) cholesterol and triglyceride levels were unaffected by pistachio consumption. Consumption of pistachio as 20% of daily caloric intake increased serum paraoxonase activity by 35% and arylesterase activity by 60%, which are known to inhibit LDL cholesterol oxidation, compared with the control group. However, increased antioxidant activity was blunted when pistachio intake was increased to 40% of daily caloric intake. In conclusion, the present results show that consumption of pistachio as 20% of daily caloric intake leads to significant improvement in HDL and TC/HDL ratio and inhibits LDL cholesterol oxidation. These results suggest that pistachio may be beneficial for both prevention and treatment of coronary artery disease.     

Effect of ethyl esterification of phenolic acids on low-density lipoprotein oxidation.

Inhibition of copper-induced low-density lipoprotein (LDL) oxidation by phenolic acids and their ethyl esters was investigated. LDL oxidation was evaluated by the hydroperoxide concentration and the chromatographic pattern of apoprotein fractions after fast protein liquid chromatography (FPLC). Antiradical properties against 1,1-diphenyl-2-picryl hydrazyl (DPPH) radical and 2,2'-azobis(2-amidinopropane)dihydrochloride (AAPH) were also investigated, and lipophilicity determined by thin-layer chromatography. Caffeic acid at 5 microM and sinapic acid at 10 microM protected LDL against oxidation, inhibiting both hydroperoxide formation and the increase of apoprotein negative charge. Ferulic, gallic and p-hydroxy cinnamic acids were ineffective. Ethyl esterification increased the lipophilicity of the five acids, and enhanced the antioxidant properties of caffeic, sinapic and ferulic acids. Ethyl caffeate was protective at 1 microM. In contrast, gallic and p-hydroxy cinnamic ethyl esters were ineffective. Our results indicate that ethyl esterification of phenolic acids increases lipophilicity of their ethyl esters and may enable a better incorporation into the lipid layer of the LDL particle and the exertion of their antioxidant effect in the true site of lipoperoxidation. However, increasing lipophilicity is not the only mechanism able to potentiate preexisting antioxidant properties of molecules, and probably other mechanisms are implicated.     

Increased low-density lipoprotein oxidation,but not total plasma protein oxidation,in Alzheimer's disease

ObjectivesThe two most common forms of dementia are Alzheimer's disease (AD), and vascular dementia (VaD). In the overlap of biochemical processes which have been identified in AD and VaD, oxidative stress is believed to contribute to the numerous pathologies of both dementias.Design and methodsThis study assessed oxidative damage in total plasma proteins, and isolated LDL in AD patients and age matched controls, in addition total antioxidant capacity (TAC) was measured.ResultsSignificantly higher LDL protein carbonylation was observed in AD compared to age-matched controls (AD: 4.17 ± 0.73 vs. control: 3.85 ± 0.86 nmol/mg LDL; p = 0.05, 2-tailed Mann–Whitney), in addition to reduced TAC (AD: 924.708 ± 174.429 vs. control: 1078.536 ± 252.633 μM; p = 0.001, 2-tailed Mann–Whitney). No differences were seen in total plasma protein carbonyl content (AD: 3.88 ± 0.31 vs. control: 3.98 ± 0.48 nmol/mg protein).ConclusionThe results further support the view that oxidation events in AD may be specific in nature, and represent functional changes to proteins, rather than random global events.     

Beneficial effect of aggressive low-density lipoprotein apheresis in a familial hypercholesterolemic patient with severe diabetic scleredema.

We present a 59-year-old woman with severe diabetic scleredema (DS) associated with heterozygous familial hypercholesterolemia (FH). She had been treated with drugs to lower blood glucose, with insulin for diabetes mellitus (DM), and with low-density lipoprotein (LDL) apheresis therapy monthly or every 2 weeks in addition to drugs to lower serum lipids for FH. However, her scleredema had not improved. After we had tried weekly LDL apheresis therapy for a period of 3 years to treat her hyperlipidemia, the levels of her serum lipids were reduced to normal ranges, and scleredema in her nape improved. We also demonstrated the histopathological improvement in dermis of her cervical skin. We conclude that weekly LDL apheresis therapy is effective for diabetic scleredema that is resistant to conventional treatments.     

Effects of direct adsorption of lipoproteins apheresis on lipoproteins, low-density lipoprotein subtypes, and hemorheology in hypercholesterolemic patients with coronary artery disease.

Direct adsorption of lipoproteins (DALI) apheresis has been shown to reduce effectively low-density lipoprotein (LDL) cholesterol and lipoprotein (a) concentrations. However, the effects on nontraditional risk indicators such as hemorheology and LDL subtypes have not been investigated so far. Five patients (2 women, 3 men, age 53 +/- 8 years) with coronary artery disease and severe LDL hypercholesterolemia regularly treated with other LDL apheresis devices entered the study and were then treated with DALI for the first time. Hemorheological and lipoprotein parameters were measured before and immediately after the initial DALI apheresis as well as before the fourth DALI apheresis. Compared to baseline (before the first DALI apheresis), the following parameters were significantly improved (p < 0.05) after the first DALI apheresis: LDL cholesterol (69 +/- 28 versus 208 +/- 82 mg/dl) and cholesterol in each LDL subfraction as well as plasma viscosity (1.23 +/- 0.04 versus 1.37 +/- 0.06 mPa), C-reactive protein, native blood viscosity, red cell aggregation, and red cell deformability. When parameters before the fourth DALI apheresis were compared to baseline, LDL cholesterol was still lower, and red cell deformability was still improved while cholesterol in each subfraction showed a statistical trend to lower concentrations (0.08 < p < 0.14). In conclusion, DALI apheresis not only reduces LDL cholesterol but also induced a significant reduction of cholesterol in all LDL subfractions and improved various hemorheological parameters.     

Proprotein convertase substilisin/kexin type 9 antagonism reduces low-density lipoprotein cholesterol in statin-treated hypercholesterolemic nonhuman primates

Proprotein convertase substilisin/kexin type 9 (PCSK9) promotes the degradation of low-density lipoprotein (LDL) receptor (LDLR) and thereby increases serum LDL-cholesterol (LDL-C). We have developed a humanized monoclonal antibody that recognizes the LDLR binding domain of PCSK9. This antibody, J16, and its precursor mouse antibody, J10, potently inhibit PCSK9 binding to the LDLR extracellular domain and PCSK9-mediated down-regulation of LDLR in vitro. In vivo, J10 effectively reduces serum cholesterol in C57BL/6 mice fed normal chow. J16 reduces LDL-C in healthy and diet-induced hypercholesterolemic cynomologous monkeys, but does not significantly affect high-density lipoprotein-cholesterol. Furthermore, J16 greatly lowered LDL-C in hypercholesterolemic monkeys treated with the HMG-CoA reductase inhibitor simvastatin. Our data demonstrate that anti-PCSK9 antibody is a promising LDL-C-lowering agent that is both efficacious and potentially additive to current therapies.     

Endothelial dysfunction, oxidation of low-density lipoprotein, and cardiovascular disease.

The oxidative modification of low-density lipoprotein (LDL) may be dependent or independent of lipid peroxidation. This peroxidation may be initiated by metal ions, possibly in association with phospholipase activity or catalyzed by myeloperoxidase independent of metal ions. It results in the generation of aldehydes, which substitute lysine residues in the apolipoprotein B-100 moiety and thus in the generation of oxidized LDL. Endothelial injury, associated with increased production of free radicals during oxidative stress, is associated with increased prostaglandin synthesis and platelet adhesion/activation. These processes are associated with the release of aldehydes, which induce the oxidative modification of LDL in the absence of lipid peroxidation and thus in the generation of malondialdehyde (MDA)-modified LDL. We have demonstrated an association between coronary artery disease (CAD) and increased plasma levels of oxidized LDL. The increase of circulating oxidized LDL is most probably independent of plaque instability. Indeed, plasma levels of oxidized LDL were very similar for patients with stable CAD and for patients with acute coronary syndromes. Acute coronary syndromes, however, were associated with increased release of MDA-modified LDL that was independent of the necrosis of myocardial cells. These data suggest that oxidized LDL is a marker of coronary atherosclerosis whereas MDA-modified LDL is a marker of plaque instability. Recently, a prospective study in cardiac transplant patients suggested an active role of oxidized LDL in the development of CAD. Oxidized LDL may contribute to the progression of atherosclerosis by enhancing endothelial injury by inducing foam cell generation and smooth muscle proliferation.     

Sialic acid content of low-density lipoprotein in women with coronary artery disease

A low sialic acid content in low-density lipoprotein (LDL) has been associated with coronary artery disease (CAD) in studies that have included mostly male subjects. We compared the sialic acid-to-apolipoprotein B ratios of total LDL and its subfractions in middle-aged women with CAD (CAD+, n = 22) with those ratios in healthy female control subjects (CAD-, n = 11). CAD+ subjects had a lower sialic acid ratio in total LDL and in its subfractions as compared with results in CAD- subjects. In total, light, and dense LDL, the sialic acid ratio was negatively correlated with the respective cholesterol and phospholipid concentrations, and in very dense LDL, it was negatively correlated with triglyceride concentration. In multivariate analysis, CAD and LDL cholesterol contributed to the explanation of the variability of LDL sialic acid ratios. In summary, a low sialic acid-to-apolipoprotein B ratio in LDL was associated with the presence of CAD in middle-aged women with mild to moderate hypercholesterolemia.     

Decreased in vitro oxidizability of low-density lipoprotein in hypercholesterolaemic patients treated with 3-hydroxy-3-methyIgIutaryl-CoA reductase inhibitors

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^-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 ± 0.21 to 0.97 ± 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 19.7 ± 3.1 to 18.5 ± 3.3 nmol min^-1 mg^-1 LDL; total diene production decreased significantly from 420.3 ± 67.6 to 380.5 ± 49.1 nmol mg-¹ 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.     

Abnormal low-density lipoprotein subfraction profile in patients with untreated hypertension

BACKGROUND: Low-density lipoprotein (LDL) consists of a heterogeneous group of particles of varying size and electrophoretic mobility. A predominance of small, more mobile particles is a risk factor for cardiovascular disease. AIM: To investigate the hypothesis that untreated patients with essential hypertension in the absence of vascular disease may exhibit abnormalities of LDL subfractions. SETTING: Specialist hypertension clinic. DESIGN: Cross-sectional study. METHODS: Following disc polyacrylamide gel electrophoresis, the mean (LDL locus) and heterogeneity (LDL spread) of mobility was recorded in 41 patients (mean age 52.6 years, 24 men) presenting with untreated essential hypertension (in the absence of vascular disease or diabetes mellitus) and in 45 healthy controls (age 56.9 years, 22 men) recruited from primary-care lists. RESULTS: Although there were no significant differences in total, low- or high-density lipoprotein cholesterol concentrations, LDL locus was significantly greater in the hypertensive group: mean (95%CI) 36.7 (35.7-37.6) vs. 34.8 (34.1-35.5), p=0.002. LDL locus was significantly elevated even in hypertensives with triglyceride concentrations below the median (<1.25 mmol/l). LDL spread was also greater in the hypertensive group, but not significantly: 5.6 (5.2-6.0) vs. 5.5 (5.3-5.8), p=0.10. DISCUSSION: Hypertensive patients have a preponderance of smaller LDL subfractions. This dyslipidaemia is not readily detected by conventional lipid assays.     

Atheroprotective effect of exogenous heparin-derivative treatment on the aortic disturbances and lipoprotein oxidation in hypercholesterolemic diet fed rats

BACKGROUND: The present work explores the myriad of biochemical and cellular changes that are featured in the early stages of atherosclerosis; if unchecked these changes lead to the complicated atherosclerotic plaque formation. The influence of a low-molecular-weight heparin derivative on the aortic aberrations and lipoprotein oxidation has been assessed in an experimental model of hypercholesterolemic atherogenesis. METHODS: Two groups of male Wistar rats (140+/-10 g) were fed a hypercholesterolemic atherogenic diet (rat chow supplemented with 4% cholesterol, 1% cholic acid and 0.5% thiouracil; CCT diet) for 2 weeks; one of these groups received LMWH (Certoparin) treatment of 300 microg/day/rat, s.c. for 7 days. An untreated control and a LMWH drug control group were also included. RESULTS: Abnormal increase in the aortic lipids -glycosaminoglycans levels (p<0.001) in CCT-diet fed group was circumvented by the exogenous glycosaminoglycan (LMWH) treatment (p<0.001). The escalation of oxidative stress (markers-lipid peroxidation and thiol levels, superoxide dismutase and catalase activities) in the atherogenic aorta was minimised by LMWH treatment. Further, an increased susceptibility of the apo B-containing lipoproteins (LDL+VLDL) to oxidation in vitro, induced by copper ions and Fenton's reagent, was observed in the untreated CCT diet fed group. This paper reports the favorable modulation of these oxidative changes by LMWH administration. Vascular protection by LMWH is further substantiated by the normal aortic histologic findings as against the appearance of foam cells in the untreated atherogenic group. CONCLUSION: The exogenous heparin-derivative (LMWH) treatment attempted in this experimental model of hypercholesterolemic atherogenesis affords substantial protection against abnormal levels of aortic lipids and glycosaminoglycans, aortic oxidative stress and also stunts the lipoprotein peroxidative process, thereby proving its multi-faceted anti-atherogenic effects.     

Soybean protein diet increases low density lipoprotein receptor activity in mononuclear cells from hypercholesterolemic patients.

The effect of two diets containing different protein sources (animal vs. soybean) on the low density lipoprotein (LDL) receptor activity was tested in freshly isolated mononuclear cells from 12 individuals with severe type II hyperlipoproteinemia. The two diets, both taken for 4 wk in a crossover design were of otherwise identical composition. During the soybean protein diet period, total cholesterol was reduced by 15.9% and LDL-cholesterol by 16.4%. The diet containing animal proteins exerted no significant change in plasma lipid levels vs. the baseline findings. The soybean diet regimen dramatically affected the degradation of LDL by mononuclear cells. Degradation was increased 16-fold vs. the basal activity and 8-fold compared with the standard low lipid diet with animal proteins. There was, however, no clear relationship between the reduction of total and LDL-cholesterolemia and the increased LDL degradation. These findings confirm similar data previously obtained in cholesterol-fed rats and suggest that some factor/s, most likely of a protein nature, may regulate the expression of lipoprotein receptors in peripheral cells, particularly when receptor activity is suppressed by experimental diets and/or spontaneous hypercholesterolemia.