A. van Tol

Postprandial lipoprotein metabolism in normolipidemic men with and without coronary artery disease.

A delayed clearance of postprandial lipoproteins from the plasma may play a role in the etiology of premature coronary atherosclerosis. To address this hypothesis, we studied chylomicron (remnant) metabolism in two groups of 20 selected normolipidemic men aged 35-65 years, a group of coronary artery disease (CAD) patients, and a matched control group with documented minimal coronary atherosclerosis. Subjects received an oral fat load supplemented with cholesterol and retinyl palmitate. Plasma samples obtained during the next 24-hour period were analyzed for total as well as d less than 1.019 g/ml and d greater than 1.019 g/ml triacylglycerol, cholesterol, and retinyl ester concentrations. Although both groups of patients responded identically in terms of the appearance of gut-derived lipids in the plasma, CAD patients showed a marked delay in the clearance of retinyl esters as well as in the normalization of plasma triacylglycerol concentrations. Postheparin plasma hepatic lipase activity was significantly lower in the CAD group. Apolipoprotein E phenotype measurements did not reveal marked differences in frequency between both groups. The frequency distribution was not unusual in comparison with the normal Dutch population. The magnitude of the postprandial responses of triacylglycerol and retinyl esters was correlated positively with the fasting levels of plasma triacylglycerol and negatively with high density lipoprotein subfraction 2 cholesterol concentrations. These data indicate that the clearance of postprandial lipoproteins in normolipidemic CAD patients as selected in the present study is delayed as compared with that of controls without coronary atherosclerosis and suggest that postprandial lipoproteins may play a role in the etiology of their disease.

On the metabolic function of heparin-releasable liver lipase

Abstract Intravenous administration of specific antibody against heparin-releasable liver lipase (liver lipase) induced a 75% inhibition of the enzyme activity in situ . Administration of the antibody resulted in an increase of high density lipoprotein (density range 1.050–1.13 g/ml; HDL2) phospholipid levels (20% after 1 h; 54% after 4 h). Short-term (1 h) treatment with antibody had no significant effect on any of the other lipoprotein components. After long-term (4 h) treatment the free cholesterol level of HDL2 and all components in the very low density lipoprotein (VLDL) + intermediate density lipoprotein (IDL) fraction were elevated (1.5–2.0 fold). In the low density lipoprotein (LDL) fraction only the phospholipid level was affected (increased by 72%). All lipid components in the HDL3 fraction were decreased by the antibody treatment, but this decrease was only statistically significant for the cholesterolesters. The rate of removal of iodine-labeled high density lipoprotein (HDL) and LDL from serum was not affected by the antibody treatment. These results suggest that liver lipase may promote phospholipid removal in vivo and show that a lowering of liver lipase in situ has profound consequences for serum lipoprotein metabolism.

Daily moderate alcohol consumption increases serum paraoxonase activity : a diet-controlled, randomised intervention study in middle-aged men

Moderate alcohol consumption is associated with a reduced risk of coronary heart disease. Part of this inverse association may be explained by its effects on HDL. Paraoxonase, an HDL-associated enzyme, has been suggested to protect against LDL oxidation. We examined the effects of moderate consumption of red wine, beer and spirits in comparison with mineral water on paraoxonase activity in serum. In this diet-controlled, randomised, cross-over study 11 healthy middle-aged men consumed each of the beverages with evening dinner for 3 weeks. At the end of each 3 week period, blood samples were collected pre- and postprandially and after an overnight fast. Fasting paraoxonase activity was higher after intake of wine (P<0. 001), beer (P<0.001), and spirits (P<0.001) than after water consumption (149.4+/-111.1, 152.6+/-113.1, 152.8+/-116.5 and 143. 1+/-107.9 U/l serum), but did not differ significantly between the 3 alcoholic beverages. Similar effects were observed pre- and postprandially. The increases in paraoxonase activity were strongly correlated with coincident increases in concentrations of HDL-C and apo A-I (r=0.60, P<0.05 and r=0.70, P<0.05). These data suggest that increased serum paraoxonase may be one of the biological mechanisms underlying the reduced coronary heart disease risk in moderate alcohol consumers

Fibrates influence the expression of genes involved in lipoprotein metabolism in a tissue-selective manner in the rat.

The influence of different fibrates on apolipoprotein metabolism was investigated. Administration of fenofibrate provoked a dose-dependent decrease in plasma cholesterol concentration that was already evident after 1 day. Intestinal apolipoprotein (apo) A-I and apo A-IV mRNA levels remained fairly constant. In contrast, liver apo A-I, apo A-II, and apo A-IV mRNA levels decreased in a dose-dependent fashion, which was associated with a lower transcription rate of the apo A-I but not the apo A-II gene. The decline in hepatic apo A-I, apo A-II, and apo A-IV mRNA had already started after 1 day and was associated with a drop in plasma apo A-I and apo A-IV concentrations. Plasma apo E had already decreased after 1 day of fenofibrate, whereas apo B initially remained constant and increased only after 14 days of fenofibrate at the highest dose. Hepatic and intestinal apo B mRNA contents and liver, heart, kidney, and testis apo E mRNA contents were only marginally affected after treatment with fenofibrate. Liver low density lipoprotein receptor mRNA levels rose slightly after a 3-day administration of the highest dose of fenofibrate. Both clofibrate and gemfibrozil had effects comparable to those of fenofibrate on liver and intestinal apolipoprotein mRNA levels except for liver apo A-II mRNA, which decreased only marginally. Compared with fenofibrate, clofibrate caused similar changes in plasma cholesterol, apo A-I, apo A-IV, and apo E concentrations, whereas gemfibrozil increased plasma cholesterol and apo E without changing apo A-I and apo A-IV concentrations.(ABSTRACT TRUNCATED AT 250 WORDS)

Different locations of cholesteryl ester transfer protein and phospholipid transfer protein activities in plasma

Activities of cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP) were measured in plasma of four vertebrate species: man, rabbit, pig, and rat. The activities were measured in the absence and presence of antibodies raised against purified human CETP. PLTP activities were present in all four species with highest values in pig (11.7 +/- 1.2 U/ml) and human plasma (9.2 +/- 1.6 U/ml). Considerable lower activities were found in rabbit (3.5 +/- 0.6 U/ml) and rat plasma (1.6 +/- 0.7 U/ml). These activities were not affected significantly by antibody against human CETP. CETP activities could be measured in human (0.23 +/- 0.05 U/ml) and in rabbit plasma (0.19 +/- 0.03 U/ml). CETP activity in human plasma was inhibited over 97% by antibody against human CETP. Plasma was chromatographed on a Superose 6 gel filtration column. Average HDL particle sizes in the four species differed notably and decreased in the order: rat HDL greater than rabbit HDL greater than human HDL greater than pig HDL. A separation of the two lipid transfer activities was evident after gel filtration chromatography. The peak of the PLTP activity coeluted with a fraction of HDL particles with the size of human HDL2 (particle weights 300-375 kDa). CETP activity in human and rabbit plasma coeluted largely with relatively small HDL particles (particle weights 140-180 kDa). These results show that CETP and PLTP activities are located in different macromolecular complexes.

Postprandial triglyceride response in young adult men and familial risk for coronary atherosclerosis.

Atherosclerosis starts early in life [1, 2]. Postprandial lipoprotein metabolism is proposed to be involved in this process [3]. Cholesteryl ester-rich remnants of triglyceride-rich lipoproteins may directly promote accumulation of cholesteryl esters in the arterial wall [4-6]. Further, it has been reported [7, 8] that the protective effect of increased levels of high-density lipoprotein (HDL) cholesterol on the risk for coronary artery disease may not only be explained by their role in reverse cholesterol transport but also by the relation between HDL and triglyceride metabolism [9]. Plasma triglycerides have an effect on HDL composition and HDL cholesterol levels [10]; an inverse relation between HDL2 levels and postprandial triglyceride levels has been shown [11]. Levels and composition of HDL could thus be a reflection of the effectiveness of triglyceride-rich lipoprotein catabolism. A deranged metabolism of triglyceride-rich lipoproteins in plasma has been reported in familial dysbetalipoproteinemia [12, 13], a disease associated with premature coronary atherosclerosis. Studies comparing patients with coronary artery disease and persons without the disease have shown that differences with respect to postprandial hypertriglyceridemia [14-16] and postprandial retinyl palmitate concentrations (a marker of chylomicrons and their remnants) are detectable after an oral lipid load [14, 15]. These results suggest a delayed clearance of these lipoproteins in patients with coronary artery disease. Thus, accumulating evidence indicates that postprandial lipemia plays an important role in causing coronary artery disease, and the implications with respect to treatment and primary prevention are increasingly being recognized [17]. Postprandial lipoprotein metabolism has not yet been studied in children and young adults with an increased risk for coronary atherosclerosis. In our study, male offspring of men with clinical manifestations of and angiographically proven coronary atherosclerosis were compared with male offspring of men who did not have coronary atherosclerosis (negative result after angiography). This approach enabled us to study a group of healthy young adults at high familial risk for developing clinical manifestations of coronary artery disease later in life. We assessed whether changes in the triglyceride response to a standardized oral lipid load, as reported in patients with coronary artery disease, can also be measured in healthy young male offspring of such patients. Methods Participants Men with very severe coronary artery disease (patients), defined as more than 70% occlusion in at least three major coronary vessels, were selected from coronary angiography databases of cardiology departments of the Zuiderziekenhuis Rotterdam (1988 to 1991), the University Hospital Rotterdam (1988 to 1989), the Refaja Ziekenhuis in Dordrecht (1990 to 1991), and the Antonius Ziekenhuis in Nieuwegein (1992), all hospitals situated in the Netherlands. Simultaneously, a reference group of men [controls] was selected who at coronary angiography had no or, at most, only minor lesions, defined as 20% stenosis or less in all coronary vessels. Further, participants were selected according to the following additional criteria: 1) age between 45 and 65 years; 2) blood pressure not exceeding 160/100 mm Hg; 3) absence of liver disease, diabetes mellitus, thyroid disease, and renal disease; 4) first coronary angiography within 2 years before examination for our study; and 5) first consultation of a physician for cardiac symptoms within 5 years before the examination for our study. Eligible participants were sent a letter asking whether they had a son 15 to 30 years of age and, if so, whether the son was willing to participate in the study. These sons (identified by their fathers) received a separate letter inviting them to have an oral lipid loading test. Participants also received a short questionnaire about smoking habits, alcohol intake, physical activity, and fat intake. We screened medical files of 629 patients whose coronary angiographic data met the criteria. Of these patients, 19 had died, 46 had diabetes mellitus, 6 had renal disease, 2 had thyroid disease, 183 had either no son or sons outside the required age range, 58 could not be contacted, 17 had no contact with their children, 78 had a cardiac history exceeding 5 years, and 63 could not be invited for other reasons (hospitalization, other serious diseases). Of 157 families (fathers and sons) who met all the criteria, 55 (either father or son) refused to participate in the study (response, 65%), leaving 102 fathers and 139 sons. The latter had oral lipid loading tests. The study protocol was approved by the medical ethics committees of the Zuiderziekenhuis Rotterdam and the University Hospital Rotterdam. Informed consent forms were obtained from all participants in the study. Baseline Measurements Fathers were asked to visit the hospital at 9:00 a.m. after fasting for at least 12 hours. Fathers responded to a questionnaire about the number of first-degree relatives who had had myocardial infarctions and about medication use at the time of the examination (medication was taken to the hospital where the examination took place). Systolic blood pressure and diastolic blood pressure were measured using a random zero sphygmomanometer (Hawksley, Lancing, United Kingdom). Fasting serum blood samples were drawn by antecubital venipuncture for measurement of levels of triglycerides, total cholesterol, low-density lipoprotein (LDL) cholesterol, and HDL cholesterol (and its subfractions HDL2 and HDL3). Height and weight were measured without shoes and without heavy clothing. The sons were invited to come to the hospital, after the same period of fasting, on a separate day to have an oral lipid loading test. For sons, questionnaires were used to obtain data about use of medication, fat intake, alcohol intake, and smoking habits, referring to a 1-month period before the examination for this study. Daily total fat intake was calculated from an 81-item semiquantitative food frequency questionnaire by using a computerized food-composition table [18]. In the sons, blood samples were taken by antecubital venipuncture for measurement of baseline levels of serum triglycerides, total cholesterol, LDL cholesterol, HDL cholesterol (and its subfractions HDL2 and HDL3), apoprotein A-1, apoprotein A-2, apoprotein B, and retinyl palmitate concentrations. This baseline measurement of lipid levels was taken as the starting point (t0) for the oral lipid loading test. In all of the sons, apolipoprotein E was phenotyped. Oral Lipid Loading Test Sons of patients and sons of controls came to the hospital at 7:45 a.m. after an overnight fasting of 12 hours. Height and weight were measured first to calculate body surface area. Five minutes after the venipuncture for obtaining baseline lipid levels (at t0), all participants received a liquid lipid load, which consisted of a mixture of dairy cream (40% fat), egg yolk, milk powder, and retinyl palmitate (in aqueous solution) [15]. Participants received the lipid load in a dose based on their individual body surface area (77.5 g fat, 0.5 g cholesterol, and 27 000 IU of retinyl palmitate per square meter of body surface area). The mixture was consumed within 15 minutes. The participants received an antecubital venous catheter (Venflon Viggo AB, Helsingborg, Sweden), which was kept open during the test period by means of disposable obturators (Venflon). Through this catheter, blood samples were drawn at 2 (t2), 4 (t4), 5 (t5), 6 (t6), 7 (t7), 8 (t8), 10 (t10), and 12 (t12) hours after starting consumption of the oral lipid load. Total cholesterol, triglyceride, and retinyl palmitate concentrations were determined in serum isolated from these samples. During the 12-hour period, other sources of calories were withheld from participants. Because postprandial exercise has been reported to decrease postprandial lipemia [19], participants stayed in the hospital and were asked to refrain from heavy physical activity during the test period. Laboratory Analyses Serum total cholesterol levels were measured using an automated enzymatic method (Boehringer Mannheim, Mannheim, Germany) CHOD-PAP reagent kit [20]. Levels of HDL cholesterol and LDL cholesterol were measured by the same method after precipitation. For HDL cholesterol, the phosphotungstate method according to Burstein [21], with a minor modification as described by Grove [22], was used. For LDL cholesterol, precipitation was carried out with polyvinylsulfate (Boehringer Mannheim). Throughout the entire study period, results of total cholesterol and HDL cholesterol determinations were within limits of the quality control program of the World Health Organization Regional Lipid Reference Centre (Prague, Czechoslovakia). Levels of apoprotein A-1 and B were assayed using an automated immunoturbidimetric method (Kone Diagnostics, Espoo, Finland). Levels of apoprotein A-2 were determined by radial immunodiffusion against specific antiserum (Boehringer Mannheim, Germany) according to Cheung and Albers [23], with slight modifications. All automated analyses were carried out on the Kone Specific Analyzer (Kone Instruments) using frozen ( 20C) serum samples. High-density lipoprotein2 and HDL3 in serum were assayed as described by Gidez and colleagues [24] with slight modifications. High-density lipoprotein2 and HDL3 were separated using stepwise precipitation of apoprotein B containing lipoproteins with heparin/Mn2+ and HDL2 with dextran sulfate. Apolipoprotein E phenotyping was done by isoelectric focusing of delipidated serum followed by immunoblotting, using apolipoprotein E antiserum as first antibodies [25]. Retinyl palmitate analyses were done as described previously by Groot and colleagues [15]. Statistical Analysis Means SDs were calculated for baseline characteristics of all family members. The total cholestero

Higher plasma lipid transfer protein activities and unfavorable lipoprotein changes in cigarette-smoking men.

The mechanism responsible for the atherogenic lipoprotein changes associated with cigarette smoking are largely unknown. Lecithin: cholesterol acyltransferase (LCAT) and cholesteryl ester transfer protein (CETP) are key factors in the esterification of plasma cholesterol and the transfer of cholesteryl ester from high-density lipoproteins (HDLs) toward very-low- and low-density lipoproteins (VLDLs+LDLs). Another transfer factor, phospholipid transfer protein (PLTP), recently has been shown to be involved in the interconversion of HDL particles in vitro, but its physiological function is not yet clear. We measured the activities of LCAT, CETP (as cholesteryl ester exchange activity), and PLTP using exogenous substrate assays as well as lipoprotein profiles in the plasma of 21 normolipidemic cigarette-smoking men (total plasma cholesterol below 6.5 mmol/L and triglyceride below 2.5 mmol/L) and 21 individually matched nonsmoking control subjects. HDL cholesterol, HDL cholesteryl ester, and plasma apolipoprotein A-I levels were lower in the smokers than in the control subjects (P < or = .05 for all parameters). Median plasma CETP activity was 18% higher (P < .02) and median plasma PLTP activity was 8% higher (P < .05) in the smokers compared with the nonsmokers. LCAT activity was not different between the groups. HDL cholesteryl ester concentration was positively related to LCAT activity in control subjects but not in smokers. By contrast, there was an inverse relation of CETP activity with HDL cholesteryl ester in smokers but not in nonsmokers. Multiple regression analysis demonstrated that the lowering effect of smoking on HDL cholesteryl ester could be explained by its influence on CETP activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Plasma phospholipid transfer protein activity is related to insulin resistance : impaired acute lowering by insulin in obese Type II diabetic patients

Summary Cholesteryl ester transfer protein (CETP) and phospholipid transfer protein (PLTP) have important functions in high density lipoprotein (HDL) metabolism. We determined the association of plasma CETP and PLTP activities (measured with exogenous substrate assays) with insulin resistance, plasma triglycerides (TG) and non-esterified fatty acids (NEFA), and assessed the lipid transfer protein response to insulin during a 6–7 h hyperinsulinaemic euglycaemic clamp in non-obese and obese healthy subjects and patients with Type II (non-insulin-dependent) diabetes mellitus (n = 8 per group). Plasma PLTP activity was higher in obese healthy subjects and obese Type II diabetic patients compared with non-obese healthy subjects (p < 0.05 to 0.01) and was correlated with insulin resistance, plasma TG and NEFA (p = 0.02 to < 0.01). In non-obese healthy subjects, insulin decreased plasma TG and increased the HDL cholesteryl ester (CE)/TG ratio (p < 0.01 compared with saline infusion). Plasma PLTP activity fell by 14 % at the end of the clamp (p < 0.01 compared with saline) but CETP activity did not change. The decreases in plasma NEFA, TG and PLTP activity and the rise in HDL CE/TG were smaller in obese Type II diabetic patients than in non-obese healthy subjects (p < 0.01 for all). Baseline HDL CE/TG was negatively correlated with plasma TG (p < 0.001, n = 32) and PLTP activity (p < 0.01) but not with CETP activity. Likewise, the rise in HDL CE/TG during the clamp was related to the fall in plasma TG (p < 0.001) and in PLTP activity (p < 0.02). It is concluded that plasma PLTP, but not CETP, is regulated by insulin in an acute setting. High plasma PLTP activity is associated with insulin resistance in conjunction with altered NEFA and triglyceride metabolism. High plasma TG and PLTP activity have coordinate effects on HDL metabolism. [Diabetologia (1998) 41: 929–934]

Plasma apolipoprotein A5 and triglycerides in type 2 diabetes

Aims/hypothesisVariation in the human apolipoprotein (APO) A5 gene (APOA5) is associated with elevated plasma triglycerides. However, data on the exact role of plasma concentrations of APOA5 in human triglyceride homeostasis are lacking. In the present study, we estimated plasma APOA5 levels in patients with type 2 diabetes at baseline and during atorvastatin treatment, a lipid-lowering treatment that results in a reduction in plasma triglycerides and APOC3.Subjects, materials and methodsPlasma APOA5 concentration was measured by ELISA in 215 subjects with type 2 diabetes, who were taken from the Diabetes Atorvastatin Lipid-lowering Intervention (DALI) study, a 30-week randomised, double-blind, placebo-controlled study, and given atorvastatin 10 mg or 80 mg daily.ResultsAt baseline, average plasma APOA5 concentration was 25.7±15.6 μg/100 ml. Plasma APOA5 (Rs=0.40), APOC3 (Rs=0.72) and APOE (Rs=0.45) were positively correlated with plasma triglyceride levels (all p<0.001). In multiple linear regression analysis, adjusted for age and sex, the variation in plasma triglycerides was explained mostly by APOC3 (52%) and only to a small extent by APOA5 (6%) and APOE (1%). Atorvastatin treatment decreased plasma triglycerides, APOA5, APOC3 and APOE (all p<0.0001). After treatment, APOC3 remained the major determinant of plasma triglyceride levels (59%), while the contributions of APOA5 and APOE were insignificant (2 and 3%).Conclusions/interpretationOur findings reveal a positive association between plasma APOA5 and triglycerides in patients with type 2 diabetes. Treatment with atorvastatin decreased plasma APOA5, APOC3, APOE and triglycerides. In contrast to APOC3, APOA5 is not a major determinant of triglyceride metabolism in these patients.

Degradation of high density lipoprotein by heparin-releasable liver lipase

Summary In vivo inhibition of heparin-releasable liver lipase (liver lipase) induces a change in the chemical composition of a subfraction of high density lipoprotein (HDL 2 ; density range 1.05–1.13 g/ml). HDL 2 becomes rich in phospholipids and relatively poor in protein and cholesterolesters. Incubation of this phospholipid-rich HDL 2 or control HDL 2 with purified liver lipase at pH 7.4 gives a marked hydrolysis of phosphatidylcholine and phosphatidylethanolamine, but not of sphingomyelin and cholesterolesters. The bulk of lysophosphatidylcholine formed during incubation is recovered in the fraction of density > 1.21 g/ml. Phospholipid-rich HDL 2 is not converted to HDL 3 (density range 1.13–1.21 g/ml), but to a cholesterol(ester)—rich HDL 2 . This is probably due to the absence of an acceptor for cholesterol(esters) in this in vitro system. In vivo, however, liver lipase could function in the conversion of HDL 2 into HDL 3 , because the cholesterol(esters) can be transferred to other lipoproteins and/or tissues.