Gemma Gianfranceschi

Triglycerides Are Major Determinants of Cholesterol Esterification/Transfer and HDL Remodeling in Human Plasma

Lecithin:cholesterol acyltransferase (LCAT) and cholesteryl ester transfer protein (CETP) are responsible for the esterification of cell-derived cholesterol and for the transfer of newly synthesized cholesteryl esters (CE) from HDL to apoB-containing lipoproteins in human plasma. LCAT and CETP are also crucial factors in HDL remodeling, a process by which HDL particles with a high capacity for cell cholesterol uptake are generated in plasma. In the present study, cholesterol esterification and transfer were evaluated in 60 patients with isolated hypercholesterolemia (HC, n = 20) and isolated (HTG, n = 20) or mixed hypertriglyceridemia (MHTG, n = 20) and in 20 normolipidemic healthy individuals (NL). Cholesterol esterification rate (CER) and net CE transfer rate (CETR) were measured in whole plasma. LCAT and CETP concentrations were determined by specific immunoassays. HDL remodeling was analyzed by monitoring changes in HDL particle size distribution during incubation of whole plasma at 37 degrees C. Mean CER and CETR were 48% and 73% higher, respectively, in hypertriglyceridemic (HTG + MHTG) versus normotriglyceridemic individuals. HDL remodeling was also significantly accelerated in plasma from hypertriglyceridemic patients. Strong positive correlations were found in the total sample between plasma and VLDL triglyceride levels and CER (r = .722 and r = .642, respectively), CETR (r = .510 and r = .491, respectively), and HDL remodeling (r = .625 and r = .620, respectively). No differences in plasma LCAT and CETP concentrations were found among the various groups except for a tendency toward higher CETP levels in hypercholesterolemic patients (+51% in MHTG and +20% in HC) versus control subjects (NL). By stepwise regression analysis, VLDL triglyceride level was the sole significant predictor of CER and CETR and contributed significantly together with baseline HDL particle distribution to HDL remodeling. These results indicate that plasma triglyceride level is a major factor in the regulation of cholesterol esterification/transfer and HDL remodeling in human plasma, whereas LCAT/CETP concentrations play a minor role in the modulation of reverse cholesterol transport.

Mechanisms of HDL reduction after probucol. Changes in HDL subfractions and increased reverse cholesteryl ester transfer.

Treatment with probucol, a widely used lipid-lowering agent, is associated with a significant reduction of high density lipoprotein (HDL) cholesterol levels, but with an apparently improved removal of cholesteryl esters from tissues (e.g., from tendon xanthomas). The effects of probucol (500 mg twice daily) on HDL subfraction distribution and cholesteryl ester transfer activity were tested in 12 patients with stable type II hyperlipidemia [low density lipoprotein (LDL) cholesterol greater than 180 mg/dl] after a placebo-controlled cross-over trial. Probucol significantly lowered total cholesterol (-13.8%), LDL cholesterol (-9.1%), and HDL cholesterol (-30%). By rate zonal ultracentrifugation, a marked reduction of HDL2 cholesterol (-68%) was shown, whereas changes in HDL3 were less significant (-21%). These findings were confirmed by polyacrylamide gradient gel electrophoresis, typically showing a reduction or disappearance of HDL2b particles and the prevalence of particles in the HDL3a range. Cholesteryl ester transfer from HDL to lower density lipoproteins was significantly increased (30%) in all patients. These findings suggest that, in addition to the well-documented in vitro changes (prevention of LDL peroxidation and macrophage uptake), probucol characteristically modifies HDL particle distribution in vivo, and is associated with a significant increase of cholesteryl ester transfer activity.

Pravastatin effectively lowers LDL cholesterol in familial combined hyperlipidemia without changing LDL subclass pattern.

Familial combined hyperlipidemia (FCHL) is the most common genetic lipid disorder among young survivors of myocardial infarction. Elevations of plasma total and low-density lipoprotein (LDL) cholesterol and the prevalence of small, dense LDL particles are both involved in the high coronary risk of FCHL patients. We investigated the ability of pravastatin to favorably correct plasma lipid and lipoprotein levels and LDL structure in FCHL patients. Twelve patients with FCHL, documented by studies of first-degree relatives, received pravastatin (40 mg/d) for 12 weeks. Pravastatin significantly lowered plasma total and LDL cholesterol levels by 21% and 32%, respectively. Triglyceride levels did not change, and apolipoprotein B (apoB) concentrations decreased by 9% (P = NS). High-density lipoprotein (HDL) cholesterol increased by 6% because of a significant 73% rise of HDL2 cholesterol. LDL were smaller (diameter, 24.5 +/- 0.5 nm), less buoyant, and apoB-rich (cholesteryl ester-apoB ratio, 1.64 +/- 0.46) in the selected patients compared with patients with familial hypercholesterolemia or healthy control subjects. LDL became even smaller (23.8 +/- 0.6 nm) and richer in apoB (cholesteryl ester-apoB ratio, 1.27 +/- 0.52) after pravastatin treatment. Although pravastatin favorably altered plasma lipid and lipoprotein levels in FCHL patients, the abnormal LDL particle distribution and composition were not affected. Because of the apparent resistance of the small, dense LDL to drug-induced modifications, a maximal lipid-lowering effect is needed to reduce coronary risk in FCHL patients.

Metformin Improves Peripheral Vascular Flow in Nonhyperlipidemic Patients with Arterial Disease

The clinical activity of metformin (N,N-dimethyl biguanide), a widely used antidiabetic agent, on arterial blood flow was evaluated in 15 patients with peripheral atherosclerosis. Flow was determined by quantitative strain-gauge plethysmography; plasma lipid, lipoprotein, and apoprotein levels were repeatedly tested during the cross-over trial, comparing 6 months of drug and placebo administration. Metformin (850 mg tid) significantly increased arterial flow after a standardized ischemia (+ 17.3% after 3 months and + 40.0% after 6 months). The increase in arterial flow was reversible after the switch to placebo was made. The drug was similarly effective, although to a lesser extent (+ 18.6% after 6 months), when given after the placebo. A highly significant effect of drug treatment, as well as of the sequence of administration, could be established by analysis of variance. In spite of the minimal changes of plasma lipid levels during metformin, a highly significant increase of high density lipoprotein cholesterol (+ 8.3% during the whole treatment) was demonstrated; plasma levels of isoprotein AI-1 were also raised during the metformin period. This controlled experiment confirms data from previous open studies, as well as from a longstanding clinical experience. Although the mechanism of the metformin effect cannot, at present, be defined, the reported results indicate that treatments not markedly affecting plasma lipid-lipoprotein levels may improve vascular function in selected arterial districts.

Plasma triglycerides and lipoprotein(a): inverse relationship in a hyperlipidemic Italian population.

The relationship between plasma lipoprotein(a) (Lp(a)) levels and other clinical/biochemical variables was investigated in 1200 consecutive hyperlipidemic patients. Plasma Lp(a) concentrations were measured by a sandwich-ELISA method, while the patients were either on diet or diet plus lipid-lowering drugs; 38% of them had a plasma Lp(a) level > 30 mg/dl. The median plasma Lp(a) concentration and the frequency of Lp(a) > 30 mg/dl were significantly lower in individuals with severe hypertriglyceridemia vs. hypercholesterolemics (HC) or mixed hyperlipidemics (M-HLP), but similar to normolipidemic healthy controls. Patients with isolated moderate hypertriglyceridemia had Lp(a) levels intermediate between HC and M-HLP subjects. The in vitro addition of triglyceride-rich lipoproteins to normotriglyceridemic plasma did not affect the Lp(a) measurement. Plasma Lp(a) concentrations in the whole hyperlipidemic population correlated negatively with triglycerides and positively with total cholesterol, HDL-cholesterol and age, being unrelated to either body mass index or lipid-lowering treatment. In HC patients, the presence of tendon xanthomas was associated with twofold higher Lp(a) levels. These findings argue for a regulatory role of triglycerides on plasma Lp(a) levels in hyperlipidemic patients.

Bezafibrate lowers plasma lipids, fibrinogen and platelet aggregability in hypertriglyceridaemia

SummaryThe effects of bezafibrate 400 mg/day (slow release formulation) on plasma lipids/lipoproteins and on selected haemostatic parameters were evaluated in a double blind cross-over study in patients with Type IIb and IV hyperlipoproteinaemia.Placebo treatment did not influence any of those parameters, but the drug significantly reduced plasma triglycerides (−45%) and VLDL cholesterol, as well as causing a 12 % fall in total cholesterol and a 20 % increase in HDL cholesterol. Apo AI levels were significantly increased following bezafibrate and Apo B was reduced by about 20 %. In addition to changes in the plasma lipid profile, bezafibrate reduced the sensitivity of platelets to the aggregatory effect of collagen, with no effect on TXB2 production. Fibrinogen levels after bezafibrate treatment were significantly lowered, the effect being more marked in patients with hyperfibrinogenaemia. Bezafibrate did not influence the plasma fibrinolytic profile.It is concluded that bezafibrate, besides its beneficial effects on the plasma lipoprotein profile, can exert beneficial changes on specific haemostatic parameters.

Mechanisms of high-density lipoprotein reduction after probucol treatment: changes in plasma cholesterol esterification/transfer and lipase activities.

Probucol treatment results in a significant reduction of plasma high-density lipoprotein (HDL) levels. Since the remodeling of HDL within the plasma compartment is a crucial determinant of HDL levels, the activities of several factors participating in the process, ie, lecithin:cholesterol acyltransferase (LCAT), cholesteryl ester transfer protein (CETP), and lipoprotein and hepatic lipases (LPL, HL), were evaluated in 15 hypercholesterolemic patients treated with probucol (1 g/d) for 8 weeks. Drug treatment was associated with significant reductions of HDL cholesterol ([HDL-C] -32%), HDL2-C (-65%), HDL3-C (-22%), apolipoprotein (apo)A-I (-27%), and apo A-II (-11%) levels and with the accumulation of small HDL in plasma. CETP activity increased by 48%, with minor changes in LCAT (-7%), LPL (+4%), and HL (-7%) activities. By linear regression analysis, CETP activity correlated inversely with HDL-C, HDL2-C, and apo A-I levels (r = -.63, -.52, and -.73, respectively) and with HDL particle size. In multivariate analysis, CETP activity was the strongest predictor of HDL-C levels, apo A-I levels, and HDL particle size. The hypothetical mechanism of probucol is a stimulation of CETP activity, resulting in the formation of triglyceride (TG)-enriched HDL. These are acted on by HL, leading to the accumulation of small HDL in plasma.

Decreased high density lipoprotein-cholesterol levels in male patients with transient ischemic attacks

Plasma lipid and lipoproteins levels were determined in a continuous series of 50 patients (36 males and 14 females), mean age around 50 years, with a clinical diagnosis of transient ischemic attacks (TIAs). TIA was defined as a sudden episode of focal cerebrovascular insufficiency, with complete resolution of the symptoms within 24 h. TIAs are considered an important prognostic symptom for ischemic cerebrovascular diseases, being manifest in approximately 45% of the patients later undergoing a complete stroke. Plasma total cholesterol levels did not differ in these patients, when compared with a similar series of patients of the same age and sex, free of cerebrovascular lesions. A slight elevation of mean triglyceride levels was detected in the patients of both sexes, as well as higher incidence of type IV hyperlipoproteinemia. The most significant finding, however, observed only in male TIA patients, was that of significantly reduced high density lipoprotein (HDL)-cholesterol levels. This reduction (-19.7% compared to the control group) is similar to that recently reported for patients with clear-cut ischemic cerebrovascular disease. The detection of decreased HDL-cholesterol levels in male TIA patients may be of considerable significance for a prognostic evaluation of this biochemical parameter.

Prolonged retention of doubly labeled phosphatidylcholine in human plasma and erythrocytes after oral administration

The plasma kinetics of a preparation of dilinoleoyl phosphatidylcholine (DLPC) specifically labeled with3H in the choline moiety and with14C in the 2-fatty acid (FA) were evaluated in six healthy volunteers after oral administration. Retention of both isotopes in plasma exceeded expectations, with a half-life in the elimination phase of 172.2 h for3H and 69.7 h for14C. Up to 60 d after administration, there were still significant levels of radioactivity present in plasma. The relative stability of the [14C]FA label was demonstrated by the retention for more than 12 h of an isotope ratio close to that of the compound administered. The14C label of DLPC remained in position-2, as assessed by cleavage of plasma phospholipids with phospholipase A2. The [3H]choline label showed an early incorporation into high density lipoproteins and subsequently into low density lipoproteins (LDL); conversely, the14C radioactivity was rapidly incorporated into triacylglycerols that were mainly associated with very low density lipoproteins. Radioactivity measurements revealed that both isotopes remained the longest time in LDL. In red blood cell (RBC) lipids, [3H]choline radioactivity accumulated over time, with a plateau after 48 h, whereas FA radioactivity accumulated more rapidly and was followed by a progressive decay. Analysis of the isotope ratio in these cells suggested an early incorporation of lyso products followed by rapid transfer of FA from plasma. The RBC maintained considerable radioactivity for a prolonged time, thus acting as a possible reservoir for the DLPC administered. Our study showed that dilinoleoyl PC remained in plasma longer than predicted based on earlier studies, and that after absorption the FA label was found in position 2.

Prostacyclin-lipoprotein interactions: studies on human platelet aggregation and adenylate cyclase

The in vitro effects of different lipoprotein fractions (VLDL, LDL and HDL) on human washed platelet aggregation, induced by collagen and thrombin, were evaluated in the presence and absence of PGI2. Although VLDL and LDL increased the platelet aggregation while HDL showed an opposite effect, none of the tested lipoprotein fractions affected the potency of PGI2 as inhibitor of platelet aggregation (IC50). In addition, studies were performed to evaluate the effects of lipoproteins on adenylate cyclase activity in human platelet membranes. The three lipoprotein classes inhibited both basal and PGI2-stimulated adenylate cyclase without affecting the EC50 for PGI2. This inhibitory activity was not specifically elicited by any protein or lipid since neither bovine serum albumin nor a lipid emulsion (Intralipid) displayed any inhibition. The effect on adenylate cyclase elicited by VLDL, LDL and HDL does not seem to be correlated with the activity on platelet aggregation. It is concluded that mediators other than cAMP might be involved in the control of platelet function by lipoproteins.