Contribution of an in vivo oxidized LDL to LDL oxidation and its association with dense LDL subpopulations.

Oxidative modification of LDL is thought to be a radical-mediated process involving lipid peroxides. The small dense LDL subpopulations are particularly susceptible to oxidation, and individuals with high proportions of dense LDL are at a greater risk for atherosclerosis. An oxidatively modified plasma LDL, referred to as LDL-, is found largely among the dense LDL fractions. LDL- and dense LDL particles also contain much greater amounts of lipid peroxides compared with total LDL or the more buoyant LDL fractions. The content of LDL- in dense LDL particles appears to be related to copper- or heme-induced oxidative susceptibility, which may be attributable to peroxide levels. The rate of lipid peroxidation during the antioxidant-protected phase (lag period) and the length of the antioxidant-protected phase (lag time) are correlated with the LDL- content of total LDL. Once LDL oxidation enters the propagation phase, there is no relationship to the initial LDL- content or total LDL lipid peroxide or vitamin E levels. Beyond a threshold LDL- content of approximately 2%, there is a significant increase in the oxidative susceptibility of nLDL particles (ie, purified LDL that is free of LDL-), and this susceptibility becomes more pronounced as the LDL- content increases. nLDL is resistant to copper- or heme-induced oxidation. The oxidative susceptibility is not influenced by vitamin E content in LDL but is strongly inhibited by ascorbic acid in the medium. Involvement of LDL(-)-associated peroxides during the stimulated oxidation of LDL is suggested by the inhibition of nLDL oxidation when LDL- is treated with ebselen prior to its addition to nLDL. Populations of LDL enriched with LDL- appear to contain peroxides at levels approaching the threshold required for progressive radical propagation reactions. We postulate that elevated LDL- may constitute a pro-oxidant state that facilitates oxidative reactions in vascular components.

Naturally occurring modified low density lipoproteins are similar if not identical: more electronegative and desialylated lipoprotein subfractions.

Previous studies demonstrated that more electronegative low density lipoprotein (LDL) isolated from human blood by ion exchange chromatography has a chemical composition and physical properties similar to desialylated LDL obtained by lectin chromatography (Avogaro et al., 1988; Orekhov et al., 1989). In this study, sialic acid content and percentage of desialylated LDL in the electronegative LDL (LDL-) subfraction have been investigated. The sialic acid content of the LDL- was 2- to 6-fold lower than that of native LDL and close to that of desialylated LDL. In the native LDL subfraction, 83% of lipoprotein particles did not bind to the Ricinus communis agglutinin, indicating lack of terminal galactose, presumably appearing as a result of desialylation of LDL carbohydrate chains. By contrast, a major proportion of human LDL- (81%) was bound to the lectin. It was also found that the desialylated LDL subfraction consists of 88% LDL-. Native LDL did not affect the contents of free and esterified cholesterol in intimal cells cultured from grossly normal human aorta, while LDL- and desialylated LDL induced a 1.5- to 3-fold increase in the intracellular content of cholesteryl esters. Thus, LDL- is desialylated LDL which induces lipid accumulation in intimal cells. Our findings suggest that the LDL- particle is similar if not identical to the desialylated LDL particle.

Regulatory function of glucose and insulin on high-density lipoprotein cholesterol in normolipidemic subjects.

A decreased plasma concentration of high-density lipoprotein (HDL) cholesterol is associated with a higher incidence of coronary artery disease in populations. Therefore, there is intense investigation into the mechanisms responsible for the regulation of HDL cholesterol concentration in plasma. Insulin has a potent effect on HDL cholesterol, but it is unclear whether this is mediated by the primary effect insulin has on plasma triglycerides (TG). In this study, the question of the relationship between glucose, insulin, and HDL cholesterol has been addressed by investigating a cohort of nondiabetic normolipidemic men living in the Venice, Italy, area. One hundred twenty-eight men aged 30 to 69 years were initially recruited. The following parameters were measured: fasting plasma cholesterol, TG, HDL cholesterol, glucose, and insulin. One hundred seventeen of these subjects underwent an oral glucose tolerance test (OGTT), and the glucose and insulin responses were assessed. The final statistical analysis was performed on 98 nondiabetic individuals with plasma lipid levels within the 75th percentile for cholesterol and TG concentrations of the general population of the same age. The insulin response was a positive independent variable for plasma TG (P < .005) and HDL cholesterol (P < .005). On the other hand, HDL cholesterol was negatively associated with plasma TG. This relationship remained significant (P < .0001) also after controlling for age, body mass index (BMI), and glucose- and insulin-related measurements. Consistent with these results, both a stepwise variable selection analysis and a stratification analysis of the data indicated that the plasma TG concentration is the major determinant of HDL cholesterol level.(ABSTRACT TRUNCATED AT 250 WORDS)

Probucol protects low-density lipoproteins from in vitro and in vivo oxidation.

Twelve patients with primary hypercholesterolemia were treated for 12 weeks with probucol (500 mg b.i.d.). For each patient low density lipoproteins (LDL), isolated by ultracentrifugation were subfractionated by ion exchange high resolution chromatography in order to evaluate the content of a more electronegatively charged LDL (LDL-), a small subfraction that probably represent a circulating oxidatively modified lipoprotein. The treatment induced a 17% reduction of total LDL and 43% reduction of LDL-. By thin layer chromatography the probucol content in LDL- was a quarter of that in normally charged LDL. Under basal conditions, native LDL incubated for 24 h with 3 microM copper sulphate shows a net increase in electrophoretic mobility, an increase in relative fluorescence intensity and a reduction in vitamin E content, thus indicating peroxidative damage. After treatment with probucol, no significant changes of electrophoretic mobility, fluorescence and vitamin E content are detectable. LDL isolated from patients treated with probucol thus become resistant to oxidation by copper ions. The observed reduction of LDL- after treatment with probucol, confirms in vivo the antioxidant role of the drug and support the hypothesis that circulating LDL- may be linked to an oxidative process occurring in vivo.

Biochemical and cytotoxic characteristics of an in vivo circulating oxidized low density lipoprotein (LDL-).

Using ion exchange high pressure liquid chromatography, total plasma low density lipoprotein (LDL) from 30 hypercholesterolemic and 10 normocholesterolemic cynomolgus monkeys was subfractionated into unmodified LDL (n-LDL) and more negatively charged LDL (LDL-). In hypercholesterolemic monkeys, the absolute LDL-cholesterol level was 16.54 +/- 2.82 mg/dl (mean +/- SE) whereas in normocholesterolemic monkeys it was 2.39 +/- 0.12 mg/dl (P < 0.0001); the percentage of LDL- was 5.2 +/- 0.71% and 4.9 +/- 0.19% of the total LDL for hypercholesterolemic versus normocholesterolemic monkeys, respectively. LDL- averaged 5% and n-LDL 95% of the total plasma LDL cholesterol. To confirm and further elucidate the oxidative nature of LDL-, cholesterol and cholesterol oxide contents of LDL- and n-LDL were determined by capillary gas chromatography; 53.98 +/- 2.24% (mean +/- SE) of the LDL- cholesterol was oxidized whereas in n-LDL only 10.70 +/- 1.06% of the cholesterol was oxidized (P < 0.00001). The spectrum of oxysterols identified, which was similar for LDL- and n-LDL, suggested a free radical-mediated process for cholesterol oxidation. The principal oxysterols identified were: cholest-5-ene-3 beta, 7 alpha-diol, cholesta-3,5-diene-7-one, cholest-5-ene-3 beta, 7 beta-diol, 5,6 beta-epoxy-5 beta-cholestan-3 beta-ol, 5,6 alpha-epoxy-5 alpha-cholestan-3 beta-ol, 5 alpha-cholestan-3 beta,5,6 beta-triol, 3 beta-hydroxycholest-5-ene-7-one, and cholest-5-ene-3 beta,25-diol. To model one of the steps in the possible mechanism of atherogenesis, the cytotoxicity of LDL- was demonstrated to be greater against subconfluent than confluent aortic endothelial cells.(ABSTRACT TRUNCATED AT 250 WORDS)

Relationship of triglycerides and HDL cholesterol in hypertriglyceridemia.

Hypoalphalipoproteinemia (plasma HDL-cholesterol concentration at or below 35 mg/dl as reported in the National Cholesterol Education Program Guidelines) is a well known risk factor for premature coronary artery disease (CAD). In hypertriglyceridemic patients, hypoalphalipoproteinemia is commonly believed to be linked to the derangement of triglyceride metabolism. In this study the occurrence of primary hypoalphalipoproteinemia has been investigated in a cohort of hypertriglyceridemic patients whose plasma triglyceride concentration had been normalized either through diet or diet plus drug treatment. Following the initial visit, 115 hypertriglyceridemic patients received dietary advice and returned for the second visit four months later. Diet reduced plasma triglycerides in all the patients. HDL-cholesterol increased in 76 patients whereas in the others, it remained unchanged or even decreased. Plasma triglyceride concentration was normalized (less than 200 mg/dl) in 54 patients by diet alone, but among these 11 remained hypoalphalipoproteinemics. Patients in whom, despite dietary restrictions, triglycerides exceeded 200 mg/dl, were considered for pharmacological treatment with Bezafibrate (300 mg t.i.d.) for 4 months. Thirty-nine concluded the study. Treatment significantly decreased plasma triglyceride concentration in all the subjects. Normalization was achieved in 32 patients. Four of them, however, remained hypoalphalipoproteinemic. These results indicate that a subgroup of hypertriglyceridemic patients remained hypoalphalipoproteinemic even after normalization of triglyceride levels. In these patients hypertriglyceridemia and hypoalphalipoproteinemia may occur as expression of two distinct primary metabolic defects.

Some questions concerning a small, more electronegative LDL circulating in human plasma.

Atherosclerosis and its complications are prevalent worldwide with a high prevalence in western societies. The disease may sometimes be explained by a defect of low density lipoprotein (LDL) specific receptors. However, the prevalence of receptor defect is rather rare and a large body of evidence supports the possibility that an alternative pathway, the so-called "scavenger pathway", constitutes the gate through which cholesterol enters into the parietal wall and gives origin to the "foam cell". Experimental work has clearly demonstrated that LDL may be modified under the action of chemical and biological offenders, all of which make the LDL an "alien". Some papers suggest that the modifications of LDL may occur also "in vivo" in the microenvironment of the vascular vall. In 1988 we were able to record two LDL subfractions in human plasma; the more electronegative minor subfraction shares many of the peculiar traits of LDLs modified "in vitro". The present article stresses all the points which support the hypothesis that the small more electronegative LDL circulating modified LDL, may represent a certain amount of possibly oxidative in origin.

Characterization of a more electronegatively charged LDL subfraction by ion exchange HPLC.

Low density lipoproteins (LDL), collected from 32 normal male subjects (aged 30-60), were subfractionated by high resolution ion exchange chromatography (IE-HPLC). By this procedure two LDL subfractions were eluted. The first corresponds to normal LDL (nLDL); while the second one corresponds to a more electronegative subfraction, called LDL-. The mean percentage contribution of LDL- to native plasma LDL was of 3.9% (range 0.5-9.8%). The percentage concentration of LDL- in total native LDL did not correlate with plasma total cholesterol, triglycerides, and LDL cholesterol, whereas a significant negative correlation with high density lipoprotein cholesterol was found (r = -.38; p less than .05). LDL- was negatively correlated with LDL phospholipids (r = -.59; p less than .001), and with the LDL vitamin E content (r = -.63; p less than .001), and positively correlated with LDL proteins (r = -.35; p less than .05) and the content of thiobarbituric acid reactive substances (TBARS) in total LDL (r = .43; p less than .05). The TBARS molar content of LDL- was three times higher than in nLDL, with a mean concentration in LDL- of 7.3 mol/mol lipoprotein. By preparative IE-HPLC significant differences of the LDL- chemical composition were observed. The percentage content of cholesterol esters and of phospholipids was decreased, whereas proteins and free cholesterol were increased. Analysis by sodium dodecyl sulphate polyacrylamide gel electrophoresis revealed that besides apolipoprotein B-100 there was evidence of peptides with a higher molecular weight in LDL-.(ABSTRACT TRUNCATED AT 250 WORDS)

Pravastatin vs gemfibrozil in the treatment of primary hypercholesterolemia. The Italian Multicenter Pravastatin Study I.

This study compared the efficacy and safety of pravastatin and gemfibrozil in the treatment of primary hypercholesterolemia. Three hundred eighty-five outpatients from 13 lipid clinics in Italy participated in this randomized double-blind study. Patients were assigned to receive either 40 mg once daily of pravastatin or 600 mg of gemfibrozil twice daily after an initial diet lead-in period. After 24 weeks, mean reductions from baseline values of plasma total and low-density lipoprotein cholesterol were, respectively, 23% and 30% with pravastatin and 14% and 17% with gemfibrozil. Significant lipid-lowering effects were noted within 4 weeks. Apolipoprotein B decrease was 21% with pravastatin and 13% with gemfibrozil. A statistically significant increase of high-density lipoprotein cholesterol of 5% was achieved with pravastatin compared with a 13% increase for gemfibrozil. Serum triglyceride values decreased 5% with pravastatin and 37% with gemfibrozil. Familial and polygenic hypercholesterolemic patients were also examined separately. Pravastatin effectiveness in reducing low-density lipoprotein cholesterol was greater by 6% in polygenic than in familial hypercholesterolemic patients. Treatment for 25 patients (eight treated with pravastatin and 17 treated with gemfibrozil) was discontinued during the study. The incidence of clinical symptoms and laboratory alterations was low for both treatment groups. Pravastatin and gemfibrozil were well tolerated, but pravastatin was significantly more effective in reducing total and low-density lipoprotein cholesterol levels in primary (either familial or polygenic) hypercholesterolemias than gemfibrozil.

A cross-over controlled study on beclobrate versus bezafibrate in the treatment of type IIb hyperlipoproteinaemia.

The effects of the new fibric acid derivative beclobrate (100 mg) on some plasma lipidic and apoproteic parameters in 20 patients with type IIb hyperlipoproteinaemia were matched in a randomized cross-over study to a sustained release formulation of bezafibrate (400 mg). Inclusion criteria were: total cholesterol (TC) plasma levels greater than 260 mg/dl and triglyceride (TG) levels greater than 200 mg/dl, after a 2-month period of isocaloric diet. The drugs were administered once a day for 8 weeks, and then crossed-over after 8 weeks of wash-out for another 8 week period of treatment. Both drugs were significantly effective in reducing TC and TG, and in increasing HDL-C plasma levels, but only beclobrate significantly decreased Apo B and LDL-C whereas bezafibrate seemed to be more active in increasing HDL-C levels. The electrophoretic pattern of LP in patients treated with beclobrate revealed a decrease of VLDL, a normalization of the LDL fraction and an increase of HDL. The tolerability was generally good.

Plasma lipid lowering activity of acipimox in patients with type II and type IV hyperlipoproteinemia. Results of a multicenter trial.

A multicenter study was carried out in 130 out-patients to assess the plasma lipid lowering activity of acipimox in type IIa, IIb and IV hyperlipoproteinemia. The study consisted of two periods, an 8-week randomized, double-blind comparison of active drug versus placebo and a 16-week open follow-up with acipimox (400 mg and 250 mg t.i.d., respectively, in type II and IV patients). During the double-blind phase acipimox, compared to placebo, showed a highly significant triglyceride lowering effect in type IV patients (-43% vs. +4%, P less than 0.01), while reducing plasma cholesterol significantly in type II patients (-7% vs. -3%, P less than 0.05). Further reductions in plasma lipids were obtained in both types of hyperlipoproteinemia after the 16-week follow-up. In type II patients, total cholesterol fell by 9% in the former acipimox group and 17% in the former placebo group, whereas a 34% reduction in triglycerides was found in type IV patients previously treated with placebo. Treatment had to be discontinued in 4 patients during the double-blind phase and in 5 patients during follow-up, because of adverse events such as skin reactions and gastric disturbances. Statistical analysis of hematological and biochemical variables expressing safety did not show any significant change during treatment.

Presence of a modified low density lipoprotein in humans.

Low density lipoproteins (LDL) collected from 18 fasting humans were subjected to ion exchange chromatography on DEAE Sepharose. By this procedure, a LDL subfraction was isolated with an electric charge more negative than the LDL bulk. This LDL appeared to be mainly characterized by low phospholipid content, high free cholesterol and protein content, low esterified/free cholesterol ratio, and a high content of conjugated dienes, particularly of cholesterol esters. This subfraction, in an amount ranging from 5% to 20% of total LDL, was characterized by the presence of apo B-100 and protein aggregates that were reactive to anti-apo B monoclonal antibodies. Electron microscopy showed the more electronegative LDL to be heterogeneous in size with a tendency to aggregate. This LDL had low binding capacity with high affinity receptors of fibroblasts and low immunoreactivity with the monoclonal antibodies that recognize the receptor binding domain of apo B. Finally, the incubation of this LDL subfraction with cultured macrophages led to a higher increase in cellular cholesterol in spite of a lower rate of uptake as compared to the LDL bulk and to acetyl-LDL. The more electronegative LDL subfraction that we isolated for chemico-physical behavior and conjugated diene content may represent the peroxidized aliquot of human LDL.

Plasma malondialdehyde in coronary patients and in "normal" people at rest and after exercise.

The behavior of plasma lipid peroxides, expressed as plasma malondialdehyde, was studied in 27 patients with documented coronary artery disease and 17 volunteers without coronary artery disease (henceforth designated "normal"), after a standardized exercise on a bicycle ergometer. In the control group, the basal values of malondialdehyde were significantly lower than in coronary patients. Persons in the control group did not show any significant variation of malondialdehyde after exercise and recovery, whereas patients with coronary artery disease showed malondialdehyde levels significantly higher than the baseline, both after exercise and after recovery. In the control group, a significant inverse correlation between the malondialdehyde variations during the exercise and the total work produced was ten times lower than in coronary patients. It seems probable that the higher levels of lipid peroxides in these patients may leave some long-term unwanted effects. Furthermore, the increased values of lipid peroxides after exercise may be regarded as a possible trigger of fatal myocardial malfunction occurring during physical activity.

Effects of pantethine on in-vitro peroxidation of low density lipoproteins.

The effects of pantethine on LDL peroxidation in vitro are reported. LDL isolation by density gradient ultracentrifugation from 12 normal subjects were dialyzed 48 h under conditions allowing oxidation. The LDL peroxides were assayed for the presence of malondialdehyde (MDA) on the lipoprotein. The effect of peroxidation on the LDL protein moiety (apo B) was studied by SDS-gel electrophoresis. The presence in the dialysis buffer of 1 mM reduced glutathione or of an equimolar concentration of pantethine markedly inhibited the MDA formation in LDL. Less effective were 0.5 and 2 mM pantethine, while 10 mM pantethine did not prevent the LDL peroxidation. Both glutathione and pantethine (1 or 2 mM) preserved the original LDL electrophoretic mobility. The electronegative charge of LDL was correlated to the MDA production during the dialysis procedures. Freshly prepared LDL showed a single apo B band by SDS-gel electrophoresis (apo B-100). Following peroxidation 2 or 3 bands with higher molecular weight appeared. Both glutathione and pantethine (1 or 2 mM) strongly inhibited the appearance of higher molecular weight peptides. In appropriate concentrations therefore pantethine inhibits the LDL peroxidation in vitro, thus preserving the molecular integrity of apo B.

Concentration, composition and apolipoprotein B species of very low density lipoprotein subfractions from normolipidemic and hypertriglyceridemic humans.

Lipoproteins in the d less than 1.006 g/ml density range obtained form 13 healthy normolipidemic subjects and from 15 patients affected by primary endogenous hypertriglyceridemia after 14-h fasting were subfractionated by filtration in Biogel A-15 M columns. The mass values and chemical composition of very low density lipoprotein (VLDL) subfractions 1 and 2 thus obtained were studied. In each subfraction the behavior of apolipoprotein B (Apo B) was tested by sodium dodecyl-sulfate polyacrylamide gel electrophoresis. VLDL2 was higher and richer in cholesterol and proteins than VLDL1, while the percentage content of triglycerides was lower. In hypertriglyceridemic patients both VLDL1 and VLDL2 were higher than in normolipidemic subjects, the difference being particularly evident for VLDL1. In both VLDL1 and VLDL2 of nearly all the subjects studied the presence in electrophoretic gels of a large Apo B-100 band and of a minor Apo B-48 band with the appropriate mobility of lymph chylomicrons was detected. The Apo B-100/Apo B-48 ratio was about 6 in VLDL1 and 24 in VLDL2. A trend of a reduced Apo B-100/Apo B-48 ratio was observed in VLDL1 of hypertriglyceridemic patients.