VLDL compositional changes and plasma levels of triglycerides and high density lipoprotein

Clinica Chimica Acta 269 (1998) 107–124

VLDL compositional changes and plasma levels of triglycerides and high density lipoprotein a

a

b

¨ , Mario Fernando D. Brites , Carla D. Bonavita , Marcelo Cloes a b b J. Yael , Jean-Charles Fruchart , Graciela R. Castro , Regina a, W. Wikinski * a

Laboratory of Lipids and Lipoproteins, Department of Clinical Biochemistry, School of Pharmacy and Biochemistry, University of Buenos Aires, Junin 956, C.P.(1113), Buenos Aires, Argentina b ´ ´ ´ Service d’ Etude et de Recherche sur les Lipoproteines et l’ Atherosclerose , Unite´ 325 INSERM, Institut Pasteur de Lille, Lille, France Received 17 January 1997; received in revised form 1 August 1997; accepted 3 August 1997

Abstract VLDL chemical composition is related to plasma levels of triglycerides and HDL-cholesterol. We evaluated patients with primary hypertriglyceridemia with or without hypoalphalipoproteinemia and subjects with normotriglyceridemia with hypoalphalipoproteinemia. The pattern observed in all the groups was an enrichment in the triglyceride content of VLDL and in apo B-VLDL. Compared to controls, LpC-III:B levels were higher in hypertriglyceridemic patients with low or normal HDL-cholesterol levels (7.360.6 vs. 14.961.8 and 12.362.8 mg / dl; P , 0.005 and P , 0.01, respectively) and LpE:B concentration was only increased in patients with hypertriglyceridemia and normal HDL-cholesterol levels (3.160.5 vs. 6.361.0 mg / dl; P , 0.01). The activity of the cholesteryl ester transfer protein was higher in hypertriglyceridemic patients with low HDL-cholesterol levels than in controls (380625 vs. 262614% cholesteryl esters / ml.h; P , 0.001). The most atypical VLDL particle was found in patients who combined an accumulation of VLDL particles and a reduction in HDL-cholesterol concentration. These two parameters represent both ends of the cholesteryl ester-triglyceride transfer, a crucial factor for VLDL chemical composition and HDL levels.  1998 Elsevier Science B.V. Keywords: VLDL; HDL; Hypertriglyceridemia; Hypoalphalipoproteinemia; Apolipoproteins C-III and E; CETP

*Corresponding author. 0009-8981 / 98 / $19.00  1998 Elsevier Science B.V. All rights reserved. PII S0009-8981( 97 )00193-9

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1. Introduction The relationship between hypertriglyceridemia and atherosclerosis still remains controversial. This association is only confirmed in epidemiological prospective studies on univariate analysis, but it disappears on multivariate analysis [1]. The reason for this difference is that the pathological entity called hypertriglyceridemia consists of a series of abnormalities which do not only involve triglyceride-rich lipoproteins. Several authors have reported alterations in the plasma concentration, chemical composition and metabolic functions of low density lipoproteins (LDL) [2,3] and high density lipoproteins (HDL) [4]. As regards intermediate density lipoproteins (IDL), while normolipidemic subjects exhibit low plasma levels [5], in hypertriglyceridemia IDL concentration was found to be increased [2]. Lipoprotein classes are generally classified as single homogeneous lipidprotein complexes of similar hydrated densities, but there is another criteria which takes into account the qualitatively distinct lipoprotein particles (Lp) that make up each class [6]. These particles may be classified into two families, those with apo B (Lp B) and those without apo B (Lp non-B). Within Lp B particles, there are particles which contain apo C-III (LpC-III:B) and others with apo E (LpE:B). It could be assumed that both LpC-III:B and LpE:B particles are the most abundant constituents of triglyceride rich lipoproteins, chylomicrons and VLDL. On the other hand, it has been reported that IDL are mostly formed by LpE:B particles [7]. Hypertriglyceridemia is attributable to an elevated synthesis and / or a low catabolic rate of triglyceride rich lipoproteins produced by a primary disorder or secondary to another pathology, such as diabetes, renal, thyroid or hepatic dysfunction. Recently, ‘The International Committee for the Evaluation of Hypertriglyceridemia as a Vascular Risk Factor’ and ‘The National Cholesterol Education Program’ have defined hypertriglyceridemia as an elevation of plasma triglyceride levels over 200 mg / dl [8,9]. In a study carried out in Argentina, 913 adults of both sexes were examined and the frequency of hypertriglyceridemia, defined employing this cut-off point, was about 10% [10]. Metabolism of triglyceride-rich lipoproteins is closely related to the synthesis and catabolism of HDL. By playing its main role in reverse cholesterol transport, HDL is considered to be the anti-atherogenic lipoprotein fraction [11]. The maturation of the nascent HDL particle requires the addition of surface components coming from the lipolysis of triglyceride-rich lipoproteins [12,13]. This pathway is delayed in the hypertriglyceridemic condition, thus contributing to reduced levels of mature HDL. Such abnormality, usually estimated by the plasma concentration of HDL-C, is called hypoalphalipoproteinemia [4]. This dyslipidemia can be attributed to an accelerated catabolism of apo A-I [14,15] and apo A-II [14,16], the two major HDL apolipoprotein constituents. Another

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mechanism responsible for the reduction in HDL-cholesterol (HDL-C) levels in hypertriglyceridemia could be an increased rate of cholesteryl ester transfer from HDL to triglyceride rich lipoproteins [17]. This action is carried out by the cholesteryl ester transfer protein (CETP) that interchanges cholesteryl esters and triglycerides between HDL and apo B-containing lipoproteins. On the other hand, there are some patients who show high levels of plasma triglycerides but normal levels of HDL-C and others who exhibit hypoalphalipoproteinemia with normal triglyceride concentration. Therefore, it must be noted that the relationship between triglyceride and HDL-C levels is highly heterogeneous [18]. As regards the atherogenic capacity of VLDL particles, it must be remarked that VLDL contain more total cholesterol per particle than do LDL. Therefore, the uptake of one VLDL particle by macrophages would deliver more total cholesterol than one LDL [19]. Eisenberg et al. [4,20] and Gianturco et al. [19,21] have reported that VLDL found in hypertriglyceridemic patients generally showed an increase in the content of cholesteryl esters. Taking into consideration the lipoprotein heterogeneity, these authors postulated the existence of an atypical distribution of VLDL subfractions with a preponderance of the largest fraction with the highest triglyceride content. Making a comparison with the same VLDL subfraction, obtained from normolipidemic subjects, large VLDL from hypertriglyceridemia showed one or two additional moles of apo E. Moreover, it has been proven that these subfractions can be taken up by macrophages, thus contributing to the formation of foam cells, a crucial process for the development of the atherosclerotic lesion [22]. The aim of our study was to characterize the chemical composition of VLDL, the levels of lipoprotein particles containing apo C-III and / or apo E and the CETP activity, in relation to variations in plasma triglyceride and HDL-C levels. For this purpose, we studied patients with primary hypertriglyceridemia with or without decrease in HDL-C levels, and we compared them to normotriglyceridemic subjects with or without decrease in HDL-C concentration.

2. Materials and methods

2.1. Subjects We studied 48 male subjects aged between 21 and 60 years old. Subjects were recruited consecutively during a period of about 6 months from Jose de San ´ Clinical Hospital, University of Buenos Aires and were included in the Martın present study when satisfying the following criteria: (1) lack of abnormalities in the carbohydrate metabolism evidenced by plasma levels of fasting glucose, HbA 1c , insulin and an oral glucose tolerance test; (2) normal thyroid function

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evaluated by plasma levels of T 3 , T 4 , TSH and clinical examination of the thyroid gland; (3) normal renal function evaluated by plasma levels of urea and creatinine; (4) normal hepatic function evaluated by different biochemical hepatic parameters and absence of hepatomegaly confirmed by clinical examination. Special care was taken to avoid subjects with additional causes of dyslipidemia such as excessive ethanol intake, therapy with drugs that could affect lipoprotein metabolism and familial history of diabetes mellitus. Subjects were classified according to their plasma triglyceride (TG) and HDL-C levels into four groups: group 1, subjects with hypertriglyceridemia (TG $ 200 mg / dl) and hypoalphalipoproteinemia (HDL-C # 35 mg / dl) (n 5 12); group 2, subjects with hypertriglyceridemia (TG $ 200 mg / dl) and normoalphalipoproteinemia (HDL-C . 35 mg / dl) (n 5 7); group 3, subjects with normotriglyceridemia (TG , 200 mg / dl) and hypoalphalipoproteinemia (HDL-C # 35 mg / dl) (n 5 12); and group 4, control subjects with normotriglyceridemia (TG , 200 mg / dl) and normoalphalipoproteinemia (HDL-C . 35 mg / dl) (n 5 17). Informed consent was obtained from all participants and the protocol was approved by the ethical committees from the School of Pharmacy and Biochemistry and from ´ Clinical Hospital, University of Buenos Aires. Jose de San Martın

2.2. Study protocol and samples The day before the test patients and controls were instructed to follow an isocaloric diet without alcohol intake. Body mass index and waist / hip ratio were recorded from all subjects. After a 12-h overnight fast, venous blood was drawn from the antecubital vein. Serum was separated within 30 min by centrifugation at 1500 3 g, for 15 min, at 48C and immediately used for lipoprotein studies. Aliquots were stored at 2 208C and 2 808C. The oral glucose tolerance test was performed at least 4 days after the first blood extraction. Subjects were instructed to follow a carbohydrate-rich ( . 150 g / day) diet during the 3 days prior to the test. The test was carried out after an 8-h overnight fast. Blood was drawn before and 30, 60 and 120 min after the intake of a solution containing 75 g of glucose in 375 ml of water. Samples were processed in a similar way to the basal study.

2.3. VLDL isolation VLDL was isolated by flotation in a Beckman XL-90 ultracentrifuge using a fixed-angle rotor type 90Ti [23]. We layered 5 ml of serum supplemented with Na 2 HEDTA (1.2 g / l) and NaN 3 (0.1 g / l), under an equal volume of a NaCl solution (0.154 M NaCl, 0.01 M Tris, 1.7 mM Na 2 HEDTA, pH 5 7.4, density 5 1.006 g / ml). Ultracentrifugation was carried out at 105 000 3 g at 158C for 18

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h. The supernatant containing the VLDL fraction was separated and its purity tested by agarose gel electrophoresis [24]. The VLDL fraction was stored at 48C.

2.4. Analytical procedures Fasting insulin levels were determined by a standardized immunoenzymatic test for determination of human insulin (Boehringer Mannheim, Germany). Within-run and between-day precision (CV) were 4.0% and 4.5%, respectively. Total serum cholesterol and triglycerides were quantified by standardized enzymatic methods (Boehringer Mannheim, Germany) in a Hitachi 717 autoanalyzer. Within-run precision (CV) was 1.1% and 1.3%, respectively. Betweenday precision (CV) was 1.5% and 2.4%, respectively. Laboratory bias was 2 1.7% and 1.1% for total cholesterol and triglycerides, respectively. HDL-C level was determined in the supernatant obtained following precipitation of apo B-containing lipoproteins with 20 g / l dextran sulfate (Mw 50 000) and 1.0 M MgCl 2 [25], by the enzymatic method previously mentioned. Within-run and between-day precision (CV) were 3.2% and 3.8%, respectively. Laboratory bias was 2 2.0%. LDL-C level was determined as the difference between total cholesterol and the cholesterol contained in the supernatant obtained after selective precipitation of LDL with 10 g / l poly(vinyl sulfate) in polyethyleneglycol (Mw 600; 2.5%; pH 5 6.7) [26]. Within-run and between-day precision (CV) were 4.7% and 5.0%, respectively. Triglycerides, total and free cholesterol in VLDL were measured by standardized enzymatic methods (Boehringer Mannheim, Germany). VLDL cholesteryl esters were calculated as the difference between total and free cholesterol multiplied by 1.67. VLDL phospholipids were determined following the Bartlett method [27]. Total CV for this determination was 3.1%. VLDL total proteins were quantified by the Lowry method [28] using a commercial standard of bovine serum albumin (Sigma). Within-run and between-day precision (CV) were 3.0% and 4.2%, respectively. In order to obtain an estimation of VLDL particle size, we calculated the ratio between the components of the core and the components of the lipoprotein surface (cholesteryl esters 1 triglycerides / free cholesterol 1 phospholipids 1 total proteins) [29]. Apo A-I, apo B, total LpC-III, LpC-III non-B, total LpE and LpE non-B were measured by electroimmunodiffusion (Hydragel, SEBIA, France). Within-run precision (CV) for apo A-I and apo B was 2.5% and 2.6%, respectively and between-day precision (CV) was 3.7% and 4.0%, respectively. For determination of LpC-III non-B and LpE non-B levels, serum samples were first treated with polyclonal anti-apo B antibodies. Within-run precision (CV) for total LpC-III, LpC-III non-B, total LpE and LpE non-B was 5.0%, 5.4%, 5.1% and 5.5%, respectively and between-day precision (CV) was 10.1%, 10.5%, 10.0% and 9.8%, respectively. LpC-III:B and LpE:B concentrations were calculated as

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the difference between the corresponding levels of total Lp and Lp non-B particles.

2.5. CETP activity 2.5.1. HDL3 isolation and radiolabel As fully described by Lagrost and Barter [30] radiolabeled HDL 3 were prepared by a method derived from that of Tollefson and Albers [31]. A volume of 20 ml of normal human plasma was adjusted to density 1.130 g / ml with solid KBr, aliquoted in tubes containing 2 ml final volume and ultracentrifuged for 7 h at 48C and 250 000 3 g in a TLA.4 rotor by using a TL-100 ultracentrifuge (Beckman, USA). The plasma fraction of density . 1.130 g / ml was recovered and dialyzed overnight at 48C against a TBS buffer containing 10 mmol / l Tris, 150 mmol / l NaCl, 5 mmol / l Na 2 HEDTA and 3 mmol / l NaN 3 (pH 5 7.4). A quantity of 10 nmol of [1a,2a(n)- 3 H]cholesterol (specific activity, 46.3 Ci / mmol) was evaporated to dryness under a stream of nitrogen gas and then redissolved in 50 m l of ethanol. The plasma fraction of density . 1.130 g / ml was then added to the radiolabeled cholesterol solution under gentle stirring. The mixture was then incubated for 24 h at 378C in a shaking water bath to allow cholesterol esterification by the lecithin:cholesterol acyltransferase. After incubation, the solution was adjusted to density 1.130 g / ml with solid KBr and subjected to ultracentrifugation as described above. The aim of this last ultracentrifugation was to remove any radiolabeled lipoproteins of density , 1.130 g / ml, possibly generated during the incubation. The resulting infranatant was adjusted to density 1.210 g / ml; the fraction of density , 1.210 g / ml, containing HDL 3 , was recovered after two sequential 7-h spins at 48C and 250 000 3 g in a TLA-100.4 rotor. The radiolabeled HDL 3 were finally dialyzed against TBS. The purity of the HDL fraction was tested by agarose gel electrophoresis [24]. As judged by thin-layer chromatography, about 98% of total radioactivity of HDL 3 resided in the cholesteryl ester moiety. All the samples were tested for CETP activity using the same radiolabeled HDL 3 preparation. 2.5.2. Cholesteryl ester transfer assay CETP activity [32] was evaluated as the capacity of serum samples to promote the transfer of tritiated cholesteryl esters from a tracer amount of biosynthetically labeled HDL 3 ( 3 H-CE-HDL 3 ) towards serum apo B-containing lipoproteins. Serum samples (25 m l) were incubated with 3 H-CE-HDL 3 (2.5 nmol of cholesterol) and iodoacetate (75 nmol) in a final volume of 50 m l, during 3 h at 378C in a shaking water bath. A blank of each sample was carried out by incubating the mixtures at 08C. Incubations were stopped placing the tubes on ice for about 15 min, they were centrifuged for 5 min at low speed to

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remove condensed water and apo B-containing lipoproteins were separated by using ultracentrifugation. Incubation mixtures (45 m l) were added to 2 ml of a KBr solution (density 1.070 g / ml) and then ultracentrifuged for 4 h at 48C and 100 000 rev. / min in a TLA-100.4 rotor in a TL-100 ultracentrifuge. Both supernatant (containing VLDL, IDL and LDL fractions) and subnatant (containing HDL fraction) were recovered and radioactivity was measured in both fractions. Within-run and between-day precision (CV) were 4.9% and 6.0%, respectively. Results were expressed as mmol of 3 H-cholesteryl esters transferred from HDL 3 to apo B-containing lipoproteins, per ml, per h.

2.6. Data and statistical analysis Data are presented as the mean6standard error. When data did not follow the Gaussian distribution, the Mann-Whitney non-parametric test (U test) was used to compare the different groups. Correlations between all variables were carried out by least square linear regression. Differences were considered significant at P , 0.05 in the bilateral situation.

3. Results We studied four groups of subjects; two of them presented primary hypertriglyceridemia associated to low HDL-C levels (group 1) or to normal HDL-C levels (group 2); and the other two groups exhibited normotriglyceridemia also associated to low HDL-C levels (group 3) or to normal HDL-C levels (group 4, controls). Subjects from the four groups were of similar age and also showed similar waist / hip ratio (Table 1). The body mass index was moderately higher in patients with hypertriglyceridemia and hypoalphalipoproteinemia (group 1) in comparison to controls (group 4) (Table 1). Fasting insulin levels were not significantly different in the four groups of Table 1 Anthropometric characteristics from patients and control subjects (media6S.E.M.) Age (years) HTG Hypoalpha (n 5 12) HTG Normoalpha (n 5 7) NTG Hypoalpha (n 5 12) NTG Normoalpha (n 5 17)

4763 4864 4064 4363

B.M.I. (kg / m 2 ) 29.561.6 27.261.5 28.561.1 25.960.7

a

Waist / hip 0.9660.01 0.9660.01 0.9160.01 0.9360.02

HTG, hypertriglyceridemia; NTG, normotriglyceridemia; Hypoalpha, hypoalphalipoproteinemia; Normoalpha, normoalphalipoproteinemia; B.M.I., body mass index. a P , 0.05 vs. NTG Normoalpha by Mann-Whitney U-test.

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Table 2 Lipid, lipoprotein and apolipoprotein concentrations in patients and control subjects (mg / dl; media6S.E.M.) TG HTG Hypoalpha (n 5 12) HTG Normoalpha (n 5 7) NTG Hypoalpha (n 5 12) NTG Normoalpha (n 5 17)

TC a,b

299627 277627 a,b 16268 e 116610

22467 260624 219613 23468

HDL-C b,c

2961 4161 a,f 3061 b 5162

LDL-C 14269 173623 158613 15867

apo A-I d,e

11665 13364 g 11064 b 13965

apo B 9163 9967 8665 9363

HTG, hypertriglyceridemia; NTG, normotriglyceridemia; Hypoalpha, hypoalphalipoproteinemia; Normoalpha, normoalphalipoproteinemia; TG, triglycerides; TC, total cholesterol; HDL-C, high density lipoprotein-cholesterol; LDL-C, low density lipoprotein-cholesterol; apo, apolipoprotein. a P , 0.001 vs. NTG Hypoalpha. b P , 0.001 vs. NTG Normoalpha. c P , 0.001 vs. HTG Normoalpha. d P , 0.01 vs. HTG Normoalpha. e P , 0.01 vs. NTG Normoalpha. f P , 0.05 vs. NTG Normoalpha. g P , 0.005 vs. NTG Hypoalpha by Mann-Whitney U-test.

subjects (16.262.1, 10.661.3, 11.961.1 and 11.661.0 m UI / ml, in groups 1, 2, 3 and 4, respectively). Plasma triglyceride and HDL-C levels are shown in Table 2. As expected, both hypertriglyceridemic groups (groups 1 and 2) presented significantly higher plasma triglyceride levels than the other two groups (groups 3 and 4). In addition, subjects from group 3 had triglyceride levels within the reference values ( , 200 mg / dl), but they were still significantly higher than in control subjects (P , 0.01). In a similar way, both hypoalphalipoproteinemic groups (groups 1 and 3) showed significantly lower HDL-C concentration than the two groups with normoalphalipoproteinemia (groups 2 and 4). In this case, in subjects with hypertriglyceridemia and normoalphalipoproteinemia (group 2) HDL-C levels were normal but significantly lower in comparison to controls (group 4) (P , 0.05). As shown in Table 2, there were no significant differences in plasma levels of total cholesterol, LDL-cholesterol and apo B among the four groups studied. On the other hand, apo A-I concentration was significantly reduced in the groups with hypoalphalipoproteinemia (groups 1 and 3) compared to subjects with normal HDL-C (groups 2 and 4). In hypertriglyceridemic patients (groups 1 and 2), the elevation of plasma triglyceride levels was only due to an increased VLDL concentration and not to the presence of chylomicrons. This was evidenced by an increased band in the preb position (VLDL) and absence of band in the origin (chylomicron) of an electrophoretic run in agarose gel for lipoproteins.

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Table 3 Characterization of VLDL from patients and control subjects (media6S.E.M.) Mass % FC

CE

5.960.7

a,b

HTG Normoalpha (n 5 7)

5.460.2

b

NTG Hypoalpha (n 5 12)

4.560.3

NTG Normoalpha (n 5 17)

4.460.3

12.060.4

HTG Hypoalpha (n 5 12)

TG

PL

61.161.1

c,d,e

11.661.0

56.461.8

i

10.361.1

11.360.8

CE 1 TG ]]] FC 1 PL 1 TP

Apo B (mg/dl)

2.760.2 c,d,e

10.961.3 d,h

TP

10.860.9

f,a,e

14.861.1

b

10.560.8 g,e 11.860.6

55.861.5 h

14.061.1 h

15.660.7

2.060.1 i

5.560.7 b

48.261.3

19.361.0

16.261.0

1.660.1

4.060.5

d,h

2.260.2

h

11.761.3 d,e

HTG, hypertriglyceridemia; NTG, normotriglyceridemia; Hypoalpha, hypoalphalipoproteinemia; Normoalpha, normoalphalipoproteinemia; FC, free cholesterol; CE, cholesteryl esters; TG, triglycerides; PL, phospholipids; TP, total proteins; apo, apolipoprotein. a P , 0.05 vs. NTG Hypoalpha. b P , 0.05 vs. NTG Normoalpha. c P , 0.05 vs. HTG Normoalpha. d P , 0.005 vs. NTG Hypoalpha. e P , 0.001 vs. NTG Normoalpha. f P , 0.01 vs. HTG Normoalpha. g P , 0.001 vs. NTG Hypoalpha. h P , 0.005 vs. NTG Normoalpha. i P , 0.01 vs. NTG Normoalpha by Mann-Whitney U-test.

VLDL chemical composition is shown in Table 3. VLDL from patients with hypertriglyceridemia and hypoalphalipoproteinemia (group 1) was triglycerideenriched and phospholipid-depleted in comparison to VLDL from the other groups. Moreover, while free cholesterol content was higher, total proteins were lower in VLDL from group 1 than in subjects with normal plasma triglyceride levels (groups 3 and 4). VLDL from subjects with hypertriglyceridemia and normal HDL-C levels (group 2) showed the same alterations found in VLDL from patients with hypertriglyceridemia and low levels of HDL-C, in comparison to controls. Nevertheless, these abnormalities were less significant. When making a comparison between subjects with hypertriglyceridemia and normal HDL-C (group 2) and patients with normotriglyceridemia and low HDL-C (group 3), the only difference was a lower protein content in VLDL from group 2 (P , 0.005). Finally, in subjects with normotriglyceridemia and low HDL-C levels (group 3), VLDL was triglyceride-enriched and phospholipid-depleted in comparison to control subjects. In Table 3, we show the ratio between the components of the core and the surface of VLDL particles, which estimates the particle size. The highest ratio was obtained for patients with hypertriglyceridemia and hypoalphalipoproteinemia, while VLDL from control subjects showed the lowest ratio. VLDL apo B was significantly higher in the three groups of patients (groups

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1, 2 and 3) in comparison to controls (group 4) and it also turned out to be elevated in patients with hypertriglyceridemia when compared to normotriglyceridemic-hypoalphalipoproteinemic subjects (Table 3). Fig. 1 and Fig. 2 exhibit the results obtained from the determination of plasma levels of total LpC-III, LpC-III:B, total LpE, and LpE:B. All patients with hypertriglyceridemia (groups 1 and 2) showed a significant increase in total LpC-III levels. This elevation could be partially attributed to the increase observed in LpC-III:B concentration. As regards LpE particles, both hypertriglyceridemic groups (groups 1 and 2) had higher total LpE levels, though this difference was only statistically significant for patients with hypertriglyceridemia and normal HDL-C levels (group 2) in comparison to control subjects

Fig. 1. Total LpC-III and LpC-III:B levels in patients and control subjects determined by electroimmunodiffusion. HTG, hypertriglyceridemia; NTG, normotriglyceridemia; Hypoalpha, hypoalphalipoproteinemia; Normoalpha, normoalphalipoproteinemia. a P , 0.001 vs. NTG Hypoalpha; b P , 0.005 vs. NTG Normoalpha; c P , 0.001 vs. NTG Normoalpha; d P , 0.005 vs. NTG Hypoalpha; e P , 0.01 vs. NTG Normoalpha; f P , 0.05 vs. NTG Hypoalpha; g P , 0.05 vs. NTG Normoalpha.

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Fig. 2. Total LpE and LpE:B levels in patients and control subjects determined by electroimmunodiffusion. HTG, hypertriglyceridemia; NTG, normotriglyceridemia; Hypoalpha, hypoalphalipoproteinemia; Normoalpha, normoalphalipoproteinemia. a P , 0.005 vs. NTG Normoalpha; b P , 0.01 vs. NTG Normoalpha.

(P , 0.005). In this case, the elevation in total LpE could be also attributed to an increase in LpE:B levels (P , 0.01). LpC-III:B showed a positive correlation with VLDL free cholesterol (r 5 0.51; P , 0.001; n 5 48) and triglyceride contents (r 5 0.42; P , 0.005; n 5 48) and a negative correlation with phospholipid (r 5 2 0.36; P , 0.05; n 5 48) and protein contents (r 5 2 0.55; P , 0.001; n 5 48). A positive correlation was also observed between LpC-III:B and the ratio described above as an estimator of VLDL particle size (r 5 0.49; P , 0.001; n 5 48). On the other hand, LpE:B correlated positively with VLDL triglyceride content (r 5 0.031; P , 0.05; n 5 48) and negatively with VLDL phospholipid (r 5 2 0.32; P , 0.05; n 5 48) and protein contents (r 5 2 0.31; P , 0.05; n 5 48). CETP activity (Fig. 3) was significantly higher in patients with hypertriglyceridemia and hypoalphalipoproteinemia (group 1) in comparison to normot-

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Fig. 3. CETP activity evaluated by the capacity of serum samples to promote the transfer of tritiated cholesteryl esters (CE) from a tracer amount of biosynthetically labeled HDL 3 towards serum apo B-containing lipoproteins. HTG, hypertriglyceridemia; NTG, normotriglyceridemia; Hypoalpha, hypoalphalipoproteinemia; Normoalpha, normoalphalipoproteinemia. a P , 0.005 vs. NTG Hypoalpha; b P , 0.001 vs. NTG Normoalpha.

riglyceridemic subjects with low (P , 0.05) or normal (P , 0.001) HDL-C levels.

4. Discussion This study is focused on the frequent association between hypertriglyceridemia and hypoalphalipoproteinemia, as well as the less usual combinations hypertriglyceridemia-normoalphalipoproteinemia and normotriglyceridemiahypoalphalipoproteinemia in comparison to control subjects. The pattern observed in the three groups of patients was an enrichment in VLDL triglycerides

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which could be linked to the apo C-III content. Moreover, hypertriglyceridemia was in fact related to LpC-III:B and LpE:B levels. Hypertriglyceridemia and hypoalphalipoproteinemia have been defined as syndromes closely related to an increase in abdominal fat and to an insulin resistant state [18]. The group of patients with hypertriglyceridemia and hypoalphalipoproteinemia was the only one which exhibited a moderate increase in the body mass index only when comparing them to controls. Nevertheless, this group of patients showed a waist / hip ratio that was not different from the other subjects. It is noteworthy that unlike B.M.I, waist / hip ratio is a good indicator of abdominal obesity, which is related to alterations in lipoprotein metabolism [33]. As regards fasting insulin levels, there were no significant differences among the four groups. On the one hand, these data confirm that all the patients fulfilled the inclusion criteria which pointed to the selection of primary dyslipidemia. On the other hand, these patients presented a profile where hypoalphalipoproteinemia and / or hypertriglyceridemia were not associated to hyperinsulinemia, a pattern already described in a model of mice transgenic for the human apo C-III which exhibited hypertriglyceridemia without hyperinsulinemia or insulin resistance [34]. Regarding the lipid and lipoprotein profile, subjects from the four groups presented similar plasma levels of total cholesterol, LDL-C and apo B. In agreement with the study protocol, differences were found in plasma triglyceride and HDL-C levels. Even though these differences were raised from the inclusion criteria, we also found that subjects with normotriglyceridemia and hypoalphalipoproteinemia showed triglyceride levels within the reference values ( , 200 mg / dl), but significantly higher than control subjects. In addition, eight out of 12 patients from the above mentioned group showed plasma triglyceride levels higher than 150 mg / dl, the upper limit proposed by Austin et al. [35] for the outcome of the association between increased levels of VLDL and small and dense LDL. Therefore, our two groups with hypoalphalipoproteinemia should not be different in their LDL characteristics. It is noteworthy that in the PROCAM Study [36], men with plasma triglyceride levels between 150 and 199 mg / dl and HDL-C levels lower than 35 mg / dl presented similar incidence of coronary heart disease over a 6-year period (112 per 1000 subjects) than men with plasma triglyceride levels higher than 200 mg / dl and HDL-C levels lower than 35 mg / dl, (128 per 1000 subjects). In addition to the significant reduction in HDL-C levels observed in the groups with hypoalphalipoproteinemia, our patients with hypertriglyceridemia and normoalphalipoproteinemia also presented lower HDL-C concentration compared to control subjects. This decrease was not associated to low plasma apo A-I concentration, which was only evidenced in hypoalphalipoproteinemic subjects. This reduction in HDL-C levels, but not in its main apolipoprotein, apo A-I, could indicate an alteration in the role that these lipoproteins play as acceptors of free cholesterol flowing from

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extrahepatic tissues. However, this hypothesis could only be confirmed by a systematic study of cholesterol efflux promotion and of the reverse cholesterol transport pathway in this group of patients. The electrophoretic analysis of triglyceride rich lipoproteins isolated by ultracentrifugation demonstrated that they exclusively consisted of VLDL. In patients with hypertriglyceridemia and hypoalphalipoproteinemia, the increase in plasma triglyceride levels was due to a higher number of VLDL particles as indicated by apo B levels in the VLDL fraction. Such VLDL particles were, in turn, triglyceride-enriched and significantly larger than VLDL from the other groups. These particles also showed an elevation in the content of free cholesterol and a proportional reduction in phospholipids and total proteins. As regards the levels of lipoprotein particles, LpC-III:B concentration was significantly higher in this group of patients as compared to the other groups. This increase could be attributed either to a larger number of apo C-III molecules per VLDL particle or merely to the presence of a higher number of VLDL particles. LpE:B levels were also increased in these patients but this difference was not statistically significant. VLDL particles from patients with high triglyceride and normal HDL-C levels were also increased in number, richer in triglycerides and larger than VLDL particles from controls, with both LpC-III:B and LpE:B levels significantly increased. In patients with normotriglyceridemia and hypoalphalipoproteinemia, the higher plasma triglyceride level in comparison to control subjects was due to a higher number of triglyceride-enriched VLDL particles. This increment was less important than in hypertriglyceridemic patients. In addition, the triglyceride content and VLDL particle size were similar to hypertriglyceridemic patients with normal HDL-C levels. In this group of subjects, LpC-III:B and LpE:B were not different from controls. The enrichment in the triglyceride content and the larger size of VLDL particles were the most remarkable abnormalities found in the three groups of patients in comparison to the control group. It must be noted that the alterations previously described affected VLDL particles from hypertriglyceridemic patients with low HDL-C levels to a larger extent than VLDL from subjects with hypertriglyceridemia and normal HDL-C levels. Additionally, modifications in VLDL particles from the latter group were in turn of higher degree than in normotriglyceridemic subjects with low HDL-C levels. Manzato et al. [37] isolated and characterized VLDL subclasses from patients with moderate hypertriglyceridemia and control subjects. From their results, we recalculated the ratio between the components of the core and the components of the surface of each VLDL subfraction and we compared them with our own results. Both control groups showed similar ratios, while the value obtained for VLDL from our hypertriglyceridemic and hypoalphalipoproteinemic patients

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(2.760.2) was similar to the corresponding ratios obtained for larger VLDL subspecies from hypertriglyceridemic patients from the study carried out by Manzato et al. (VLDL Sf , 200 5 3.0; VLDL Sf 100–200 5 2.4). Van Barlingen et al. [38] studied patients with familial hypertriglyceridemia and found that VLDL had higher content of triglycerides, cholesterol and phospholipids than controls. On the other hand, when they studied VLDL from patients with familial combined hyperlipidemia, they only reported an increase in the cholesterol and phospholipid content. Triglyceride enrichment of VLDL from our patients with primary dyslipoproteinemia makes them similar to VLDL from the subjects with familial hypertriglyceridemia reported by van Barlingen et al. [38]. In both groups with hypertriglyceridemia, we found higher levels of LpCIII:B. In fact, the role of apo C-III as an inhibitor of lipoprotein lipase could be partially responsible for the increase in the number of triglyceride-enriched VLDL particles. The hypertriglyceridemic effect of apo C-III has been clearly demonstrated by creating transgenic mice for human apo C-III which develop severe hypertriglyceridemia [39], and on the other hand, by generating mice lacking apo C-III which are hypotriglyceridemic [40]. The correlations between LpC-III:B and different VLDL components suggest that apo C-III could also condition the enrichment in triglycerides and free cholesterol and the depletion in phospholipids and total proteins. In addition, the elevation in LpE:B levels in hypertriglyceridemia could be determinant for the metabolism of these VLDL particles via specific receptors [41]. These findings could be of particular interest taking into consideration the increasing evidence which connects LpC-III:B and LpE:B particles with atherosclerosis [42,43]. In a study carried out in male patients, 24 h before coronary bypass surgery, LpC-III:B was a more powerful discriminant, in a stepwise analysis, than triglycerides and apo B [42]. Therefore, LpC-III:B was suggested to be a specific indicator of the dyslipoproteinemia observed in the coronary bypass population. More recently, in the ECTIM Study, LpC-III:B and LpE:B levels were also evaluated in two different populations from Northern Ireland and France [43]. The higher levels of LpC-III:B in survivors of myocardial infarction, as compared to control subjects, was one of the differences found in both countries. LpE:B levels were only increased in Irish survivors of myocardial infarction. It is known that the Irish population is characterized by a threefold increase in the mortality and in the incidence of coronary heart disease in comparison to the French population. As a result, control subjects from both countries were compared and LpC-III:B and LpE:B proved to be higher in Irish people, though these parameters lost significance in multivariate analysis when triglycerides were taken into account. This large multicentric study suggests that the distribution of apo C-III among lipoproteins could play a role in the different susceptibility of the two populations to the

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atherogenic process. All this evidence contributes to the idea of a relationship among high LpC-III:B levels, hypertriglyceridemia and atherosclerosis. In this study, we evaluated CETP activity by incubating serum samples with HDL 3 containing 3 H-cholesteryl esters as a tracer. Patients with hypertriglyceridemia and hypoalphalipoproteinemia showed a significant increase in CETP activity, while in hypertriglyceridemic patients with normal HDL-C levels, CETP activity was not different from normotriglyceridemic subjects. This increment in CETP activity would cause a reduction in HDL cholesteryl esters which are being interchanged by triglycerides coming from VLDL. Such interchange would not produce a significant enrichment in cholesteryl esters in VLDL due to the existence of a large number of VLDL particles, acceptors of the cholesteryl esters. On the other hand, triglycerides from VLDL would be incorporated into a low number of HDL particles, thus promoting dissociation of free apo A-I, which can be easily catabolized. In hypertriglyceridemic patients with normal HDL-C levels, the fact that CETP activity was not increased, could be responsible for the triglyceride enrichment of VLDL and the normal levels of HDL-C and apo A-I in these patients. We describe three groups of patients who presented several abnormalities in VLDL chemical composition, in comparison to control subjects. The pattern observed in all the patients was an enrichment in VLDL triglycerides which could be linked to the apo C-III content. In addition, hypertriglyceridemia was related to LpC-III:B and LpE:B levels, potential markers of risk of atherosclerosis. It is interesting to note that the most atypical VLDL particle was found in patients who combined an accumulation of VLDL particles and a reduction in HDL-C concentration. These two parameters represent both ends of the cholesteryl ester-triglyceride transfer carried out by CETP, which acts as a crucial factor for VLDL chemical composition and HDL levels.

Acknowledgements Fernando D. Brites is a Research Fellow from the University of Buenos Aires. This work was supported by a grant from the same University (FA 035) and it was part of the INSERM-CONICET International Cooperation Program.

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