Pietro Avogaro

Lipoprotein Lp(a) and the risk for myocardial infarction

Abstract The serum lipoprotein Lp(a) concentration was measured in 76 male post-myocardial infarction (MI) patients aged between 40 and 60 years, and in 107 control subjects of the same age and sex. Quantitation was performed by the Laurell technique. It was sensitive in the range 1–60 mg/dl with a day to day C. V. of less than 4%. A considerable variation of Lp(a) concentration was noticed in the whole population with a frequency distribution of higher order. Conventional statistical methods could therefore not be applied to evaluate a possible MI risk of Lp(a). In addition to Lp(a), several other risk and anti-risk factors for atherosclerosis were assayed. The whole population was divided into normolipemics (NL) and Type IIa, IIb and IV phenotypes. In addition, the subjects were grouped into two or three Lp(a)-types, selecting several different cut-off points for Lp(a) concentrations. The results can be summarized as follows: (1) NL-controls had significantly lower total cholesterol and low density lipoprotein (LDL)-cholesterol but higher high density lipoprotein (HDL)-cholesterol values compared to the MI-patients. The HDL-cholesterol concentration was also significantly different between Type IIa controls and MI-patients. (2) Eleven per cent of the NL-controls but 25% of the NL-MI-patients exhibited Lp(a) values exceeding 50 mg/dl. Thus Lp(a) concentration above this value represents a 2.3-fold relative risk for MI. Similar findings were obtained in the Type IIa and IIb populations but not in Type IV hyperlipemics. (3) Taking 30 mg/dl as the cut-off point, Lp(a) represents a relative risk of 1.75 for MI in the NL population. (4) Hyperlipemics in general (controls + MI) exhibited higher Lp(a) values compared to NL. (5) Statistical evaluation of all the data failed to reveal any correlation between Lp(a) levels and other risk or anti-risk factors for atherosclerosis. (6) It is concluded that Lp(a) represents an independent addi tional risk factor for MI with a possible threshold value of approx. 30 mg/dl in NL.

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.

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)

Familial hyper-HDL-(α)-cholesterolemia

In one family the mother, two children and a maternal aunt showed a hypercholesterolemic state characterized by a unusually high percentage of cholesterol in ultracentrifugally-recovered HDL-(a)-LP. An intensely stained a-band was present in all these subjects both in plasma and in ultracentrifugal fraction, with d greater than 1063. Symptoms or signs due to the hypercholesterolemic state were not present. The father was affected by Type IIA hypercholesterolemia. This lipid defect was also present in one of the two children.

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.

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.

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.

Distribution and concentration of apolipoprotein E in high and low density lipoproteins.

SummaryAn electroimmunoassay of apolipoprotein E (Apo E) in total human plasma and in the supernatant following precipitation with sodium phosphotungstate in the presence of MgCl2 of the apolipoprotein B (Apo B)-containing lipoproteins is described. The assay is specific and sensitive enough to record also low levels of Apo E found in isolated lipoprotein fractions. In 30 normal male subjects, the method gave a total plasma Apo E concentration of 6.67±1.92 mg/dl and a HDL Apo E concentration of 2.58±0.91 mg/dl. Total Apo E is positively correlated with total plasma triglycerides. The values of Apo E in the lipoproteins obtained by ultracentrifugation were lower than the corresponding values obtained by a precipitation procedure, with a mean Apo E loss of 12.1% in the low density lipoproteins (VLDL+LDL) and of 34.3% in HDL. After ultracentrifugation, a substantial amount of Apo E was detected in the d>1.210 g/ml fraction. The proposed method allows to study the Apo E concentration and distribution between low and high density lipoproteins in large samples, avoiding the problems connected with high salt concentrations and centrifugal forces.

Isolation and Partial Characterization of an Oxidized LDL in Humans

Foam cells that accumulate in the earliest of atherosclerotic lesions, the so-called fatty streak, arise from two cellular sources: the arterial smooth muscle cells (SMC) and the monocyte-derived macrophage (MM).2 The latter cell type in culture takes up only a small amount of native low density lipoproteins (LDL) by receptor-mediated endocytosis, but has a distinct receptor system that binds and degrades the more negatively charged LDL.2,3 Incubation of cultured MM with LDL modified by acetylation (acyl-LDL) results in an accumulation of cholesteryl esters (CE) within the cells thus forming foam cells.2 However such chemical modification in vivo seems unlikely. Recently, two possible mechanisms by which the more negatively charged LDL can be produced in vivo have been reported. The interaction of LDL with malondialdehyde (MDA) released by aggregating platelets or produced by peroxidation of fatty acids can lead to the formation of MDA-LDL that increase the CE deposition in cultured MM.4 Moreover, the interaction of LDL with endothelial cells also alters LDL (EC-LDL), thus allowing their uptake by the MM.5

Chemical composition of ultracentrifugal fractions in different patterns of human atherosclerosis

The chemical composition of ultracentrifugal fractions of VLDL (d less than 1006), LDL (d 1006-1063) and HDL (d less than 1063) has been studied in males affected by atherosclerosis of different vascular beds. Thirty-seven subjects affected by post-infarction cardiopathy (M.I.) showed significantly higher values of total-C, VLDL-C and LDL-C when compared to 52 controls. Twenty-three patients affected by non-occlusive ischaemic heart disease (I.H.D.) showed higher values than controls of total-C, VLCL-C, LDL-C, total TG, VLDL-TG, and GDL-TG. Twenty-three patients with atherosclerosis of the inferior limbs (P.A.) were characterized by increased levels of total-TG, VLDL-TG, VLDL-C, HDL-C. A group of patients who had suffered a stroke from cerebro-vascular disease (C.V.D.) did not show any significant difference from controls. In the M.I. group, 56% of the patients had a high level of C-VLDL. Patients with I.H.D. were characterized mostly by an increase in C-LDL, Patients with P.A. showed the highest values of total -TG, VLDL-TG and LDL-TG. Some of the observed differences are probably due to different metabolic backgrounds. Some other differences may be due to variations in dietary habits after heart infarction. Patients with levels of plasma cholesterol and triglyceride beyond the 90th percentile of the normal group showed many abnormalities in the chemical composition of their lipoproteins. It is noteworthy that increased amounts of cholesterol may collect in lipoprotein classes different from LDL while increased amounts of triglyceride may collect in classes different from VLDL.