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Alterations in the main steps of reverse cholesterol transport in

male patients with primary hypertriglyceridemia and low

HDL-cholesterol levels

Fernando D. Brites

_{*, Carla D. Bonavita}

_{, Catherine De Geitere}

_{, Marcelo Cloe¨s}

_,

Bernard Delfly

_{, Mario J. Yael}

_{, Jean-Charles Fruchart}

_{, Regina W. Wikinski}

_,

Graciela R. Castro

a_{Laboratory of Lipids and Lipoproteins,}_{Department of Clinical Biochemistry,}_{School of Pharmacy and Biochemistry,}

Uni6ersity of Buenos Aires,Junin 956,Capital Federal,C.P. (1113) Buenos Aires, Argentina

b_Ser₆_{ice d}_%_{Etude et de Recherche sur les Lipoprote´ines et l}_%_{Athe´roscle´rose,}_Unite´ ₃₂₅ _INSERM,_{Institut Pasteur de Lille,}_Lille, _France

Received 22 December 1998; received in revised form 5 October 1999; accepted 3 November 1999

Abstract

Hypertriglyceridemia is a complex pathological entity strongly connected to low HDL-C levels but controversially related to the risk of coronary artery disease. In this study, we evaluated the main steps of the antiatherogenic pathway called reverse cholesterol transport in a group of patients with primary hypertriglyceridemia and low HDL-C levels in comparison to normotriglyceridemic subjects with or without hypoalphalipoproteinemia. In patients with primary hypertriglyceridemia, low HDL-C levels were accompanied by decreased apo A-I and apo A-II concentrations. These reductions were manifested by a selective reduction in LpA-I:A-II particles. In addition, apo C-III Lp non B was found to be elevated and HDL lipid percentage composition showed a triglyceride enrichment and cholesterol depletion. The capacity of serum samples from hypertriglyceridemic patients to promote cellular cholesterol efflux was reduced, as evidenced by using two different cellular models, Fu5AH and J774 cells. This impaired cholesterol efflux promotion was also corroborated by incubations of isolated HDL fractions with Fu5AH cells. Lecithin:cholesterol acyltransferase (LCAT) activity, the driving force of reverse cholesterol transport, showed a tendency towards lower values in hypertriglyceridemic patients, but this difference was not statistically significant. Additionally, cholesteryl ester transfer protein (CETP) activity was increased in this group of patients. Therefore, hypertriglyceridemia was found to induce quantitative and qualitative alterations in HDL and its subclasses and, consequently, in some steps of reverse cholesterol transport. The abnormalities found in this antiatherogenic pathway and its promoters could constitute a possible connection between hypertriglyceridemia and atherosclerosis. © 2000 Elsevier Science Ireland Ltd. All rights reserved.

Keywords:Hypertriglyceridemia; High density lipoproteins (HDL); Hypoalphalipoproteinemia; Reverse cholesterol transport; Cholesterol efflux; Lecithin:cholesterol acyltransferase (LCAT); Cholesteryl ester transfer protein (CETP)

www.elsevier.com/locate/atherosclerosis

1. Introduction

Hypertriglyceridemia, defined as an increase in plasma triglyceride levels over 200 mg/dl [1], is a com-plex entity which can be of primary origin or secondary to any other factor or pathological condition capable of producing a lipid disorder.

Plasma triglyceride levels have been related to the concentration of cholesterol transported in high density lipoproteins (HDL-C) and to the risk of coronary artery disease (CAD) [2,3]. HDL-C levels and CAD risk show a strong negative association; triglyceride concentration and CAD risk exhibit a weak positive relation; and HDL-C and triglyceride levels are con-nected in a solid inverse way. Thus, while HDL-C stands as a manifest antiatherogenic factor [4], the direct intervention of triglycerides in the genesis of atherosclerosis still remains controversial. However,

re-* Corresponding author. Tel.: +54-11-4964-8297; fax: + 54-11-4508-3645.

E-mail address:[email protected] (F.D. Brites).

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sults from a meta-analysis based on 17 different studies suggest that triglycerides are a risk factor for CAD, independent of HDL-C [5]. More recently, a consensus about the treatment of hypertriglyceridemia also high-lighted the strong evidence which associates hyper-triglyceridemia and increased CAD risk [6].

In hypertriglyceridemia, diverse lipoprotein particles seem to be affected. The increase in plasma triglyceride levels reflects an accumulation of two overlapping lipo-protein families, which are comprised within chylomi-cron and VLDL flotation densities: those containing apolipoprotein (apo) B and apo C-III (LpB:C-III) and those with apo B and apo E (LpB:E). Furthermore, not only the concentration, but also the lipid and apoprotein composition of these triglyceride rich lipo-proteins were proven to be abnormal [7]. On the other hand, hypertriglyceridemia may also influence any of the two HDL subclasses: those which contain apo A-I without apo A-II (LpA-I) and those with apo A-I and apo A-II (LpA-I:A-II), two different metabolic entities [8]. HDL subclasses may also be classified according to their apo C-III or apo E content, and are identified as apo C-III Lp non B or apo E Lp non B.

Concerning the physiological function that HDL has in cholesterol transport within the organism, Glomset [9] was the first to recognize its participation in the antiatherogenic process called reverse cholesterol trans-port. This metabolic pathway is responsible for the movement of excess cholesterol from peripheral tissues to the liver for lipoprotein recycling or excretion and could be defined as a progression of closely intercon-nected events [10]. Among them, four steps are pointed out as the most relevant ones: (1) free cholesterol efflux from extrahepatic cells and its uptake by initial accep-tors [11]; (2) free cholesterol esterification by lecithin:cholesterol acyltransferase (LCAT); (3) transfer of newly synthesized cholesteryl esters from HDL to apo B-containing lipoproteins and interchange with triglycerides, carried out by the cholesteryl ester trans-fer protein (CETP); and (4) hepatic uptake of cholesteryl esters so formed [12].

In hypertriglyceridemic patients, different authors have described quantitative and qualitative variations in lipids and apolipoproteins transported in HDL and its subpopulations [13 – 15]. Moreover, in a study car-ried out in type 2 diabetic patients with moderate hypertriglyceridemia [16], we found an abnormal re-verse cholesterol transport both in fasting and post-prandial states. Nevertheless, we were not able to find out if the described disorders were due to hypertriglyce-ridemia itself or to the alterations associated with the diabetic condition. Evidence then is lacking about the different steps of reverse cholesterol transport in pri-mary hypertriglyceridemia in which no additional fac-tors can affect the lipoprotein spectrum. While it has been suggested that LCAT and CETP activities could

be determinant factors for HDL levels in hypertriglyce-ridemic patients [17], cholesterol efflux promotion has not been fully examined before. If hypertriglyceridemia demonstrably affected the whole reverse cholesterol transport, the protective role of this pathway would be deteriorated, thus contributing to the understanding of the controversial relationship between hypertriglyce-ridemia and atherogenicity.

In view of these considerations, the aim of the present study was to explore the first three steps of reverse cholesterol transport and especially the capacity to promote cholesterol efflux from two different cellular models in primary hypertriglyceridemic patients. We also characterized the lipoprotein, apolipoprotein and lipoprotein particle environment concerned in this an-tiatherogenic pathway.

2. Materials and methods 2.1. Subjects

We studied 36 male subjects aged between 21 and 65 years old. Subjects were recruited consecutively during a period of about 6 months from Hospital de Clı´nicas Jose´ de San Martı´n, University of Buenos Aires. Sub-jects were included in the present study when they satisfied the following criteria previously described [7]: (1) lack of abnormalities in carbohydrate metabolism evidenced by plasma levels of fasting glucose, HbA1c,

insulin and an oral glucose tolerance test; (2) normal thyroid function evaluated by plasma levels of T3, T4, TSH and clinical examination of the thyroid gland; (3) normal renal function evaluated by plasma levels of urea and creatinine; and (4) normal hepatic function evaluated by different biochemical hepatic parameters and absence of hepatomegalia confirmed by clinical examination. Special care was taken to avoid including subjects with additional causes of dyslipidemia such as tobacco consumption, 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 three groups: group 1, subjects with high plasma TG (]200 mg/dl) and low plasma HDL-C (535 mg/dl) levels (n=12); group 2, subjects with normal plasma TG (B200 mg/dl) and low plasma HDL-C (535 mg/dl) levels (n=12); and group 3, control subjects with normal plasma TG (B200 mg/dl) and normal plasma HDL-C (\35 mg/dl) levels (n=

12). Cut-off points for defining hypertriglyceridemia and hypoalphalipoproteinemia were chosen in accor-dance with previous reports [18,19]. Informed consent was obtained from all participants and the protocol was approved by the Ethical Committees from School of Pharmacy and Biochemistry and from Hospital de

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Clı´nicas Jose´ de San Martı´n, University of Buenos Aires.

2.2. Study protocol and samples

The day before blood extraction patients and con-trols were instructed to follow a standard diet without alcohol consumption. After a 12-h overnight fast, venous blood was drawn from the antecubital vein. Serum was separated within 30 min by centrifugation at 1500×g, for 15 min, at 4°C and immediately used for lipoprotein studies. Aliquots were stored at −20°C for apolipoprotein and lipoprotein particle determination and for the evaluation of reverse cholesterol transport. An 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 three days previous 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. HDL isolation

HDL fractions were isolated from serum by gel filtra-tion chromatography using a fast protein liquid chro-matography system (FPLC, Pharmacia, Sweden) [20]. We employed one Superose 6 and one 12 HR column placed in series (Pharmacia, Sweden). The columns were equilibrated with PBS-EDTA buffer containing 1.5×10−3 _{M NaN}

3. The absorbance of the eluate was

monitored at 280 nm. In each run, 0.2 ml of serum were injected and eluated at a constant flow rate of 12 ml/h. The cholesterol content of all the fractions was assayed and HDL fractions were pooled.

2.4. Analytical procedures

Fasting insulin levels were determined by a standard-ized immunoenzymatic test for determination of human insulin in plasma (Boehringer Mannheim, Germany). Within-run and between-day precision (CV) were 4.0 and 4.5%, respectively. Total cholesterol and triglyce-ride levels were quantified by standardized enzymatic methods (Boehringer Mannheim, Germany) in a Hi-tachi 717 autoanalyzer. Within-run precision (CV) was 1.1 and 1.3%, respectively. Between-day precision (CV) was 1.5 and 2.4%, respectively. HDL was isolated in the supernatant obtained following precipitation of apo B-containing lipoproteins with 20 g/l dextran sulfate (MW 50 000) and 1.0 M MgCl2 [21]. Within-run and

between-day precision (CV) were 3.2 and 3.8%, respec-tively. HDL3 was separated by precipitation of the

supernatant containing total HDL with 40 g/l dextran

sulfate (MW 50 000) and 2.0 M MgCl2[21]. Cholesterol

and triglycerides in total HDL and HDL3 fractions

were determined by standardized enzymatic methods (Boehringer Mannheim, Germany). Phospholipids in both fractions were tested following Bartlett’s method [22]. Total CV for this determination was 3.1%. HDL2

lipid components were calculated as the difference be-tween the corresponding values obtained for total HDL and HDL3. LDL-C level was determined as the

differ-ence between total cholesterol and the cholesterol con-tained in the supernatant obcon-tained after selective precipitation of LDL with 10 g/l polyvinylsulfate in polyethyleneglycol (MW 600; 2.5%; pH 6.7) [23]. Within-run and between-day precision (CV) were 4.7 and 5.0%, respectively.

VLDL-C was calculated by substracting the choles-terol concentration of the supernatant obtained after precipitation with polyvinylsulfate (VLDL+HDL) and HDL-C level. Triglycerides, total and free cholesterol, and phospholipids in HDL fractions isolated by gel filtration chromatography were measured by standard-ized enzymatic methods (Boehringer Mannheim, Ger-many). HDL cholesteryl esters were calculated as the difference between total and free cholesterol multiplied by 1.67.

2.5. Determination of apolipoprotein and lipoprotein particle plasma le6_els

Apo A-I, apo A-II, apo B100, LpA-I, total apo C-III,

total apo E, apo C-III Lp non B, and apo E Lp non B were measured by electroimmunodiffusion (Hydragel, SEBIA, France) in serum samples from patients and controls. The procedure was performed according to the manufacturer’s instructions and had previously been validated [24]. LpA-I:A-II was calculated as the difference between plasma levels of apo A-I and LpA-I. Apo C-III Lp non B and apo E Lp non B represent the apo C-III and apo E present in lipoprotein particles without apo B (HDL family), respectively. For determi-nation of apo C-III Lp non B and apo E Lp non B levels, serum samples were first treated with polyclonal anti apo B antibodies and the quantification was car-ried out in the supernatant obtained. LpB:C-III concen-tration was calculated as the difference between the levels of total apo C-III and apo C-III Lp non B. LpB:E concentration was calculated as the difference between the levels of total apo E and apo E Lp non B.

2.6. Cholesterol efflux experiments 2.6.1. Fu5AH cells

Cellular cholesterol efflux was determined using Fu5AH rat hepatoma cells following the procedure described by de la Llera Moya et al. [25]. Briefly, the cells were maintained in minimal essential medium

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con-taining 5% fetal calf serum. Approximately 25 000 Fu5AH cells/ml were plated on 24-mm multiwell plates (Inbro, Polylabo) using 2 ml/well. Then 2 days after plating, cellular cholesterol was labeled during a 72-h incubation with [3_{H]cholesterol (NEN, Dupont de}

Ne-murs) (1mCi/well). To allow equilibration of the label, the cells were washed and incubated for 24 h in mini-mal essential medium with 0.5% bovine serum albumin. Then the cells were washed with PBS and incubated at 37°C for 3 h with 2.5% diluted serum. At the end of the incubation, the medium was removed and centrifuged; the monolayer cells were washed three times with PBS and harvested with 0.5 ml of 0.1 M NaOH. Radioactiv-ity was then measured in both medium and cells, and percentage of cholesterol efflux calculated. Results were corrected by the protein content of each cellular frac-tion as determined by the method of Lowry [26]. All efflux values were averages of three determinations. Cholesterol efflux experiments were also carried out employing HDL fractions isolated by gel filtration chromatography (25 mg HDL-phospholipids/ml) as study samples and in this case the incubation at 37°C was performed during 6 h.

2.6.2. J774 cells

J774 cells were maintained in RPMI, 10% fetal calf serum. Approximately 150 000 cells/ml were plated on 24-mm multiwell plates using 2 ml/well. Then 1 day after plating, cells were washed and cellular cholesterol

was labeled during a 48-h incubation with

[3_{H]cholesterol (NEN, Dupont de Nemurs) (1}_m_Ci /well) in medium containing 2.5% bovine serum albumin (BSA). On the day of the experiment, confluent cells were washed three times with PBS and incubated for 2 h at 37°C with RPMI containing 1% BSA to allow equilibration of the [3_{H]cholesterol in the membranes.}

For determination of cholesterol efflux, the cells were washed once with PBS and incubated at 37°C for 16 h with 5% diluted serum. The rest of the procedure was the same as described for Fu5AH cells.

2.7. LCAT acti6_ity

LCAT activity was determined according to the ex-ogenous substrate method modified by Chen and Al-bers [27]. Briefly, an artificial proteoliposome substrate was prepared containing apo A-I, lecithin, unlabeled cholesterol, and [14_{C]cholesterol at a molar ratio of}

0.8:250:7.5:5. The LCAT activity assay was carried out by incubation of 10ml of the proteoliposome substrate with 100ml of serum from patients and controls at 37°C during 60 min. The esterification was linear during this time. The enzymatic reaction was then stopped and lipids were extracted with chloroform:methanol (1:1). Free cholesterol and cholesteryl esters were separated by thin-layer chromatography and the radioactivity of

the bands was counted. Results were expressed as per-centage of 14_{C-cholesteryl esters formed, per hour, per}

ml of plasma. Total CV for this determination was 6.5%. All the samples were tested for LCAT activity using the same proteoliposome substrate preparation.

2.8. CETP acti6_ity

Cholesteryl ester transfer activity was determined in serum samples according to the general procedure pre-viously described [28]. Briefly, the capacity of serum samples to promote the transfer of tritiated cholesteryl esters from a tracer amount of biosynthetically labeled HDL3(

H-CE-HDL3) towards serum apo B-containing

lipoproteins was evaluated. Serum samples (25ml) were incubated with 3_H-CE-HDL

3 (2.5 nmol of cholesterol)

and iodoacetate (75 nmol) in a final volume of 50 ml, during 3 h at 37°C in a shaking water bath. Since CETP activity is negligible at 0°C, each sample incu-bated at this temperature served as control. Incubations were stopped by placing the tubes on ice for about 15 min. They were then centrifuged for 5 min at low speed to remove condensed water and apo B-containing lipo-proteins were separated by ultracentrifugation. Incuba-tion mixtures (45 ml) were added to 2 ml of a KBr solution (density 1.070 g/ml) and then ultracentrifuged for 4 h at 4°C and 250 000×gin a TLA-100.4 rotor in a TL-100 ultracentrifuge. Both supernatant (containing VLDL, IDL and LDL fractions) and subnatant (con-taining HDL fraction) were recovered and radioactivity was measured in both fractions. Within-run and be-tween-day precision (CV) were 4.9 and 6.0%, respec-tively. Results were expressed as percentage of

3_{H-cholesteryl esters transferred from HDL}

3 to apo

B-containing lipoproteins, per hour, per ml of plasma.

2.9. CETP mass

The cholesteryl ester transfer protein mass was deter-mined in plasma by a sandwich-type immunoassay, as previously described by Mezdour et al. [29]. A specific monoclonal antibody that recognizes an epitope located in the C-terminal domain was used for antigen capture and an anti-CETP peptide antibody directed against the 290 – 306 residue was used for detection. Bound anti-bodies were revealed with an antibody-peroxidase con-jugate specific for IgG. Total CV for this determination was 9.2%.

2.10. Data and statistical analysis

Data are presented as the mean9S.D. When data did not follow the Gaussian distribution, the Mann – Whitney non-parametric test (U-test) was used to com-pare the different groups. Correlations between all variables were carried out by least square linear

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regres-sion. Differences were considered significant atPB0.05 in the bilateral situation.

3. Results

In this study, we evaluated a group of patients with primary hypertriglyceridemia and low HDL-C levels (group 1,n=12). Results were analyzed in comparison both to a group of subjects who exhibited normal plasma triglyceride and low HDL-C levels (group 2,

n=12) and to normotriglyceridemic subjects with nor-mal HDL-C levels (group 3, controls, n=12).

All the subjects were of similar age (47911, 40914, 41912 years, mean9S.D.; groups 1, 2 and 3, respec-tively). The body mass index was moderately higher in patients with hypertriglyceridemia and low HDL-C lev-els (group 1) than in controls (group 3) (28.093.6, 27.993.6, 24.992.6 kg/m2_{; groups 1, 2 and 3,}

respec-tively;PB0.05), whereas no statistically significant dif-ferences were detected in the waist/hip ratio (0.9690.07, 0.9190.08, 0.9290.09; groups 1, 2 and 3, respectively).

Fasting insulin levels were similar in the three groups of subjects (15.496.5, 11.594.2, 12.093.7 mIU/ml; groups 1, 2 and 3, respectively).

The lipid and lipoprotein profiles from the 36 sub-jects are described in Table 1. As expected, differences in plasma triglycerides and HDL-C levels emerged from the selection criteria and the constitution of each group. In hypertriglyceridemic patients 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. Plasma triglyceride levels from nor-motriglyceridemic subjects with low HDL-C levels (group 2) were within the reference values (B200 mg/

dl), though significantly higher than control subjects. Of 12 patients from the above mentioned group 2, nine showed plasma triglyceride levels higher than 150 mg/

dl. Total and LDL-cholesterol plasma concentrations were not significantly different in the three groups. HDL-phospholipids and the cholesterol transported in HDL subfractions, HDL2and HDL3, were significantly

reduced in both groups with low HDL-C levels (groups 1 and 2). In contrast, in the high triglyceride group (group 1), HDL-triglyceride concentration showed an elevation when compared to controls.

The results from the apo and lipoprotein particle plasma levels are presented in Table 2. HDL major apolipoproteins, apo A-I and apo A-II, exhibited sig-nificantly lower levels in both hypoalphalipoproteine-mic groups (groups 1 and 2) than in controls. These reductions were due to a selective decrease in LpA-I:A-II and not in LpA-I particle concentrations. Apo B100

was not different in the three groups. Total apo C-III was only increased in patients with high triglyceride levels (group 1) in comparison to both normotriglyceri-demic groups. This elevation reflects higher levels of both fractions, apo C-III Lp non B and LpB:C-III in the hypertriglyceridemic group of patients as compared to normotriglyceridemic subjects. Concerning apo E-containing lipoproteins, total apo E plasma concentra-tion was higher in the group with hypertriglyceridemia than in controls, but in this case, the increment was only attributable to the LpB:E fraction.

We also calculated the molar ratio HDL-C/apo A-I+apo A-II, which was defined by Brinton et al. [30] as

Table 1

Lipid and lipoprotein profile of patients and control subjects (mg/dl; mean9S.D)a

High TG, low HDL-C Normal TG, low HDL-C Normal TG, normal HDL-C (group 2,n=12) (group 3,n=12)

(group 1,n=12)

3419110b,c

TG 167922c ₁₀₂₉₃₈

227932

TC 224940 227934

66933b,c

VLDL-C 3397e ₂₅₉₁₁

150930

LDL-C 134939 162939

2796c

HDL-C 2994c ₅₃₉₈

HDL2-C 392d 292c 994

2496c

HDL3-C 2794c 4496

8.892.2c

TC/HDL-C 7.891.6c _4.3₉_0.4

HDL-PL 40922c ₅₀₉₁₄d ₇₅₉₁₈

1796e ₁₃₉₃

HDL-TG 1295

a_{HDL, high density lipoprotein; LDL, low density lipoprotein; PL, phospholipids; TC, total cholesterol; TG, triglycerides; VLDL, very low}

density lipoprotein.

b_P_B_{0.001 versus group 2, by Mann–Whitney}_U-test. c_P_B_{0.001 versus group 3, by Mann–Whitney}_U-test. d_P_B_{0.005 versus group 3, by Mann–Whitney}_U-test. e_P_B_{0.05 versus group 3, by Mann–Whitney}_U-test.

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Table 2

Apolipoprotein and lipoprotein particle levels of patients and control subjects (mg/dl; mean9S.D.)a

Normal TG, low HDL-C

High TG, low HDL-C Normal TG, normal HDL-C

(group 1,n=12) (group 2,n=12) (group 3,n=12)

apo A-I 117917b ₁₀₉₉₁₄h ₁₄₁₉₂₂

1993c

2094c ₂₉₉₃

apo A-II

86915

apo B100 93911 90913

4297

5099d ₄₇₉₁₀

Lp A-I

67913c

Lp A-I:A-II 67910c ₉₄₉₁₉

2.290.3

3.891.1c,e _2.2₉_0.5

Total apo C-III

1.590.3

apo C-III Lp non B 2.290.6b,g 1.690.4

0.790.2

1.690.7c,e _0.6₉_0.2

LpB:C-III

8.092.0 7.992.0

Total apo E 10.994.0d

4.291.8

4.792.0 4.991.5

apo E Lp non B

4.092.7 3.292.0

LpB:E 6.694.0f

a_{apo, apolipoprotein; HDL-C, high density lipoprotein cholesterol; Lp, lipoprotein particle; TG, triglycerides.} b_P_B_{0.01 versus group 3, by Mann–Whitney}_U-test.

c_P_B_{0.001 versus group 3, by Mann–Whitney}_U-test. d_P_B_{0.05 versus group 2, by Mann–Whitney}_U-test. e_P_B_{0.001 versus group 2, by Mann–Whitney}_U-test. f_P_B_{0.05 versus group 3, by Mann–Whitney}_U-test. g_P_B_{0.005 versus group 2, by Mann–Whitney}_U-test. h_P_B_{0.005 versus group 3, by Mann–Whitney}_U-test.

Table 3

Percentual lipid composition of HDL isolated by gel filtration chromatography from patients and control subjects (mean9S.D.)a

Normal TG, low HDL-C

High TG, low HDL-C Normal TG, normal HDL-C

(group 1,n=12) (group 2,n=12) (group 3,n=12)

2.891.1b

FC 3.291.2c _4.2₉_0.5

34.695.7d,e

CE 40.894.6 41.295.0

32.2910.3

37.2915.2 37.094.6

23.9911.0 17.596.8

TG 25.4910.4c

a_{CE, cholesteryl esters; FC, free cholesterol; HDL-C, high density lipoprotein cholesterol; PL, phospholipids; TG, triglycerides. Results are}

expressed as percentages of the lipid mass.

b_P_B_{0.001 versus group 3, by Mann–Whitney}_U-test. c_P_B_{0.05 versus group 3, by Mann–Whitney}_U-test. d_P_B_{0.01 versus group 2, by Mann–Whitney}_U-test. e_P_B_{0.01 versus group 3, by Mann–Whitney}_U-test.

a predictor of apo A-I fractional catabolic rate (13.79

2.8, 15.591.8, 21.092.8; groups 1, 2 and 3, respec-tively). This ratio was significantly lower in both groups with hypoalphalipoproteinemia than in control subjects (groups 1 and 2 vs. group 3; PB0.001), thus suggesting an increased catabolism of apo A-I in pa-tients from groups 1 and 2. In addition, plasma triglyc-eride levels showed a negative correlation with the ratio HDL-C/apo A-I+apo A-II (r= −0.69, PB0.001,

n=36).

The alteration spectrum was not only revealed in HDL apolipoproteins and HDL subclasses, but also in HDL lipid composition (Table 3). Free cholesterol and cholesteryl ester percentages were reduced in HDL particles, whereas the triglyceride content was signifi-cantly increased in the hypertriglyceridemic group (group 1) in comparison to controls.

Cholesterol efflux experiments were carried out using two different cellular models (Fu5AH rat hepatoma and J774 macrophage cells) and two different kind of samples (whole serum and HDL fractions) (Fig. 1). As shown in Fig. 1A and B, serum samples from both hypoalphalipoproteinemic groups (groups 1 and 2) were less efficient in promoting cellular cholesterol efflux than samples from control subjects in both cell types. Results obtained employing these two cellular systems incubated with whole serum exhibited a strong direct correlation (r=0.54,PB0.001,n=36). In order to get further information about factors influencing the cholesterol efflux process, we analyzed the correlations of the capacity to induce cholesterol efflux from Fu5AH and J774 cells with different parameters. Re-sults are shown in Table 4. As exhibited in Fig. 1C, only HDL fraction from hypertriglyceridemic patients

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Fig. 1. Cholesterol efflux from monolayer cultured cells (Fu5AH rat hepatoma cells or J774 mouse macrophage cells) induced by samples (whole serum or HDL fractions) from hypertriglyceridemic patients in comparison to normotriglyceridemic subjects with or without low HDL-C levels. HDL-C, high density lipoprotein-cholesterol; TG, triglycerides.a_P_B_0.001,b_P_B_0.05, c_P_B_{0.01 versus normal TG, normal HDL-C group.}

(group 1) induced cholesterol efflux from Fu5AH cells to a lesser extent than HDL fractions from subjects with normal triglyceride levels.

LCAT activity was lower in both groups with hy-poalphalipoproteinemia (groups 1 and 2) than in con-trols, but this difference was only statistically significant for the group of subjects with normal triglyceride and low HDL-C levels (Table 5).

CETP was evaluated by measuring its activity and its mass. As represented in Table 5, CETP activity was increased in hypertriglyceridemic patients (group 1) in comparison to both groups with normal triglyceride levels, while CETP mass (Table 5) did not show any statistical difference between groups.

We also evaluated the correlations between plasma triglyceride levels and HDL lipids, apolipoproteins, lipoprotein particles, cholesterol efflux promotion, LCAT activity, and CETP mass and activity (Table 6).

4. Discussion

In this study, we evaluated diverse parameters in-volved in reverse cholesterol transport and the first

three steps that make up this antiatherogenic pathway in a group of patients with primary hypertriglyce-ridemia and low HDL-C levels (group 1), in compari-son to normotriglyceridemic subjects with or without

Table 4

Correlation coefficients of HDL related lipids, apolipoproteins and lipoprotein particles with cholesterol efflux promotion from Fu5AH rat hepatoma cells and J774 mouse macrophage cells (n=36)a

Fu5AH cells J774 cells

r PB r PB

0.001 0.62

0.001

HDL-C 0.77

HDL2-C 0.59 0.001 0.26

0.76 0.001

HDL3-C 0.66 0.001

0.001 0.52

0.001

HDL-PL 0.68

−0.14 NS

HDL-TG −0.10 NS

0.001 0.49 0.01

apo A-I 0.68

0.77 0.001

apo A-II 0.48 0.01

NS 0.27 0.01

0.42 LpA-I

0.61 0.001

LpA-I:A-II 0.45 0.01

HDL-C/A-I+A-II 0.58 0.001 0.51 0.001

a_{apo, apolipoprotein; C, cholesterol; HDL, high density}

lipo-protein; Lp, lipoprotein particle; NS, not significant (P\0.05); PL, phospholipids; TG, triglycerides.

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Table 5

LCAT activity, CETP activity and mass in serum samples from patients and control subjects (mean9S.D.)a

Normal TG, low HDL-C

High TG, low HDL-C Normal TG, normal HDL-C

(group 1,n=12) (group 2,n=12) (group 3,n=12)

8.695.3 5.793.1b _11.6₉_5.8

LCAT activity (%/h per ml)

369989c,d ₂₅₆₉₈₁

CETP activity 254959

(%/h per ml)

9679308 9149225

CETP mass (ng/ml) 9899245

a_{C, cholesterol; CETP, cholesteryl ester transfer protein; HDL, high density lipoprotein; LCAT, lecithin:cholesterol acyltransferase; TG,}

triglycerides.

b_P_B_{0.05 versus group 3, by Mann–Whitney}_U-test. c_P_B_{0.005 versus group 3, by Mann–Whitney}_U-test. d_P_B_{0.01 versus group 2, by Mann–Whitney}_U-test.

hypoalphalipoproteinemia (groups 2 and 3, respec-tively). Hypertriglyceridemia was found to induce quantitative and qualitative alterations in HDL and its subclasses and, consequently, in all the steps of reverse cholesterol transport that we were able to evaluate.

All the patients selected for this study were primary hypertriglyceridemic subjects, and even if obese patients were not included in this study, the group of subjects with hypertriglyceridemia and low HDL-C levels was the only one which presented a moderately higher body mass index in comparison to controls. Nevertheless, the waist/hip ratio, a better indicator of abdominal fat [31], and fasting insulin levels, an estimate of insulin resis-tance, were not different in the three groups.

In both groups with low HDL-C levels, a diminution in apo A-I and apo A-II plasma levels was observed. The reduction in HDL-C was due to a decrease in the concentration of HDL2-C and in HDL3-C. In addition,

HDL particles from group 1 were depleted in free and esterified cholesterol, and significantly enriched in triglycerides in comparison to controls. But in patients from group 2, HDL lipid composition was not signifi-cantly different from controls. This difference between both hypoalphalipoproteinemic groups reflects a possi-ble influence of plasma triglyceride concentration in the regulation of HDL lipid composition.

A specific evaluation of HDL subclasses with respect to the apolipoprotein content revealed that hyper-triglyceridemic patients showed a selective diminution in LpA-I:A-II and not in LpA-I particle levels. As we found similar results in group 2, this pattern seems to be related more to the low HDL-C syndrome than to hypertriglyceridemia itself. Nevertheless, this is a some-what controversial topic which does not seem to result from the methodology employed to measure LpA-I and LpA-I:A-II levels. Among the few techniques available for carrying out this determination, electroimmunodif-fusion was selected because it was the only one exact and precise enough as to clinically quantify plasma concentrations.

As regards data previously reported, Genest et al. [32] studied a group of patients with hypertriglyce-ridemia and CAD who presented low levels of both LpA-I and LpA-I:A-II particles, determined by elec-troimmunodiffusion and ELISA, respectively. On the other hand, Montali et al. [33] described a more signifi-cant reduction in LpA-I than in LpA-I:A-II when they studied patients with hypoalphalipoproteinemia associ-ated or not with hypertriglyceridemia. Similarly to our study, these authors employed electroimmunodiffusion for both measurements. However, the studies from Genest [32] and Montali [33] included smokers and

Table 6

Correlation coefficients of HDL related lipids, apolipoproteins, lipo-protein particles and different steps of reverse cholesterol transport with plasma triglyceride levels (n=36)a

r PB

HDL-C −0.56 0.001

−0.25 HDL2-C

HDL3-C −0.63 0.001

HDL-PL −0.50 0.001

−0.56 0.001

HDL-TG

apo A-I −0.07 NS

−0.32

apo A-II 0.05

apo B100 0.15 NS

NS 0.23

LpA-I

−0.20

LpA-I:A-II NS

Total apo C-III 0.89 0.001

0.001 0.79

apo C-III Lp non B

0.84

LpB:C-III 0.001

Total apo E 0.60 0.001

apo E Lp non B 0.08 NS

0.53

LpB:E 0.001

−0.32 0.05

Efflux Fu5AH

0.01

−0.43 Efflux J774

−0.10

LCAT activity NS

0.55

CETP activity 0.001

CETP mass 0.12 NS

a_{apo, apolipoprotein; C, cholesterol; CETP, cholesteryl ester}

trans-fer protein; HDL, high density lipoprotein; LCAT, lecithin:cholesterol acyltransferase; Lp, lipoprotein particle; NS, not significant (P\0.05); PL, phospholipids; TG, triglycerides.

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patients treated with b-blockers, two factors known to reduce HDL levels and its subpopulations beyond hy-pertriglyceridemia [34], unlike our study which exclu-sively selected primary hypertriglyceridemic male subjects. Furthermore, in a previous study [35], we evaluated HDL subfractions in a group of type II diabetic patients with moderate hypertriglyceridemia. Employing the same electroimmunoduffusion method-ology and following exactly the same conditions, in that research we were able to point out that in the diabetic patients the decrease in HDL levels was due to LpA-I subfraction and not to LpA-I:A-II particles, a com-pletely different pattern to the one observed in patients with primary hypertriglyceridemia. These data clearly show that results obtained do not depend on the analyt-ical procedure employed, and other factors could be more relevant.

In summary, analysis of HDL subfractions in hyper-triglyceridemic patients, revealed a significant decrease in HDL2-C, HDL3-C and in LpA-I:A-II particle levels.

Moreover, if the contribution of HDL2 subpopulation

to the whole HDL fraction was calculated as a percent-age instead of showing the results in concentration units, only a slight reduction could be observed in hypertriglyceridemic patients in comparison to control subjects, and this difference was not statistically signifi-cant (HDL2-C=11.1911.2% vs. 16.997.2%,

respec-tively; P\0.05). The same analysis carried out with HDL3 subfraction also revealed no statistically

signifi-cant difference between patients and controls (HDL3

-C=88.9911.2% vs. 84.296.8%, respectively;

P\0.05). Even if different authors described a positive correlation between HDL2 and LpA-I levels, and

be-tween HDL3 and LpA-I:A-II levels, it is interesting to

note that these data came up from determinations in normolipemic samples. Thus, in dyslipemic individuals, as those analyzed in our study, the distribution of the different HDL subspecies could be modified. Therefore, a decrease in HDL2subfraction may not always imply

a reduction of the same magnitude in LpA-I subfrac-tion. Another fact which renders the comprehension of HDL heterogeneity more difficult is that both HDL3

and LpA-I subpopulations have been proposed to be the best cholesterol efflux promoters [36,37] and not the pair HDL2-LpA-I or HDL3-LpA-I:A-II, as would have

been expected.

Alterations in the composition of the HDL fraction were also evidenced in its minor apolipoprotein con-stituents. The concentration of apo C-III Lp non B was significantly increased in hypertriglyceridemic patients compared to normotriglyceridemic subjects. This eleva-tion is still highlighted by the significant reduceleva-tion in HDL levels. As was expected in hypertriglyceridemia, the rise in apo C-III Lp non B concentration was parallel to an increment in LpB:C-III. These results are supported by the direct correlations found between

plasma triglyceride levels and total apo C-III, apo C-III Lp non B and LpB:C-III. The concentration of apo E Lp non B was not different in the three groups, even though LpB:E levels were higher in hypertriglyceri-demic patients and were positively associated with plasma triglyceride levels.

HDL and its subclasses have been proposed to play a role in each step of reverse cholesterol transport. We evaluated the first step of this metabolic pathway, cholesterol efflux from peripheral cells, using two differ-ent cell models, Fu5AH rat hepatoma cells and J774 mouse macrophages. The purpose of carrying out the experiments in more than one cell type was to avoid limiting our conclusions to the specific behavior of a determined cellular class. Rothblat et al. reported that free cholesterol is rapidly removed from Fu5AH rat hepatoma cells and with intermediate efficacy from J774 mouse macrophage cells [38]. In our experiments, there was a strong positive correlation between the results obtained by incubating serum samples with the hepatoma cells for 3 h and with the macrophages for 16 h.

Serum samples from both groups with low HDL-C levels exhibited a significantly lower capacity to pro-mote cellular cholesterol efflux than samples obtained from controls. Our results from cholesterol efflux pro-motion employing both cellular models correlated nega-tively with plasma triglyceride levels and posinega-tively with HDL-C, HDL3-C, HDL-phospholipids, apo A-I, apo

A-II and LpA-I:A-II plasma levels. Similarly to de la Llera Moya et al. [25], one of the highest correlations to cholesterol efflux was obtained with HDL-C plasma concentration. In addition, the correlation coefficient with efflux results was much larger for LpA-I:A-II than for LpA-I. Even if there is no doubt that LpA-I is a better promoter of cholesterol than LpA-I:A-II efflux [8], in samples from our hypoalphalipoproteinemic pa-tients, LpA-I:A-II, which was actually in low concen-tration, appeared to be playing an active role. This is consistent with the results reported by Syva¨nne et al. [39] who evaluated cholesterol efflux determinants in four groups of men with or without type 2 diabetes and CAD. In this work, the authors directly postulated that the expected positive association between efflux poten-tial and LpA-I concentration was absent, while phos-pholipid transfer protein activity and LpA-I:A-II levels appeared to be the main determinants of efflux poten-tial from Fu5AH cells.

Cholesterol efflux results obtained using whole di-luted serum reflect the conjunction of many factors involved in reverse cholesterol transport, such as plasma levels and composition of cellular cholesterol acceptors (including preb-HDL particles), LCAT activ-ity, CETP activity and the concentration of apo B-con-taining lipoproteins. However, they provide no specific information about the role played by the different

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lipoprotein acceptors of free cholesterol effluxed from cells. For that reason, we isolated HDL fractions from patients and controls and we incubated the cells with equal quantities of HDL, measured by their phospho-lipid content. Experiments employing Fu5AH cells evi-denced that only HDL from patients with high triglyceride and low HDL-C levels were less effective in promoting cholesterol efflux than samples from control subjects.

Among the different methodologies available for lipoprotein isolation, gel filtration chromatography was chosen taking into account that this technique scarcely alters the delicate structure of lipoprotein particles, due to the short time employed and because it does not require the use of aggressive agents such as KBr or NaSCN [20]. One disadvantage of gel filtration chro-matography is the possibility of loosing small HDL subfractions such as preb particles. This is a common limitation of most methods employed to separate lipo-protein fractions, including ultracentrifugation which is the reference method for lipoprotein isolation. As preb1-HDL particles play a crucial role, being the first acceptors of cholesterol effluxed from cells, it could be thought that their loss could be conditioning cholesterol efflux experiments carried out with isolated particles. Nevertheless, the magnitude of preb loss might not be considerable because in hypertriglyceridemic patients similar results were obtained with both whole serum and HDL fractions. Moreover, Okazaki et al. [40] employed a chromatographic method and were able to separate five different HDL subclasses from samples of normal males and females. The particle diameter of the smallest subclass was reported to be about 7.6390.16 nm, similar to the size of prebparticles (preb1-HDL=

5.6 nm and preb2-HDL=12 – 14 nm), thus showing the ability of this chromatographic method to isolate small lipoprotein particles [41]. Additionally, it would be important to differentiate the in vivo situation where preb1particles are being regenerated at a constant rate and the in vitro situation where preb1particles could be rapidly saturated with free cholesterol coming from cells. Therefore, in any experimental model which in-volves long incubation periods with cultured cells, the quantitative contribution of preb1 particles could be

considerably minimized, even if it were not lost due to technical manipulation.

The significantly lower capacity to induce cellular cholesterol efflux, evidenced for both groups with hy-poalphalipoproteinemia in the assays carried out with whole serum, is determined, at least in part, by the reduced concentration of HDL particles. When experi-ments were carried out with equal quantities of isolated HDL fractions, it was interesting to note that HDL particles from group 1 were still less efficient in promot-ing cholesterol efflux than controls. In the latter case, the abnormal lipid and apolipoprotein composition of

HDL particles could be responsible for an impaired removal of free cholesterol from cells. It is possible that the presence of apo C-III in HDL particles could be playing a role as a negative modulator of cholesterol efflux promotion. Hara et al. [42] suggested that free apo C-III was one of the unique apolipoproteins unable to induce lipid release from cholesteryl ester loaded macrophages. Additionally, Bielicki et al. [43] proposed that the presence of this apolipoprotein in mixtures containing apo C-I, C-II and C-III could reduce the ability of C-I and C-II to promote efflux from fibrob-lasts. Therefore, in patients from group 2 the main alteration points out to HDL particle levels but, in patients who show hypertriglyceridemia associated with hypoalphalipoproteinemia, not only HDL concentra-tion but also its chemical composiconcentra-tion seem to be determinants for an anomalous cholesterol efflux induction.

The second step of reverse cholesterol transport was evaluated following the exogenous substrate method, which mainly reflects LCAT concentration [27]. In pa-tients from group 1, LCAT activity showed a tendency towards lower values, though this difference was not statistically significant. On the other hand, subjects from group 2 showed a significant reduction in com-parison to controls. As LCAT is generally considered the driving force of reverse cholesterol transport, this abnormality adds to the dysfunction of the whole metabolic pathway. Murakami et al. [44] found that cholesterol esterification rate was higher in a group of hypertriglyceridemic patients than in normotriglyceri-demic subjects. Two remarkable factors that could ac-count for the difference between both studies are the methodologies employed and the fact that their group of hypertriglyceridemic patients showed low HDL-C levels but apo A-I plasma concentration which was not significantly different from controls.

We finally evaluated the third step of reverse choles-terol transport, which involves CETP activity. In pa-tients from group 1, we found a significant increase in CETP activity, in comparison to normotriglyceridemic subjects and this elevation was not accompanied by an increment in CETP mass. Similarly, Murakami et al. [44] and Mann et al. [45] found that the net transfer of cholesteryl esters between endogenous lipoproteins was higher in samples from hypertriglyceridemic patients than in controls. Plasma triglyceride levels showed a strong and positive correlation with CETP activity. Therefore, the increment in CETP activity could be due to an increased cholesteryl ester transfer induced by high triglyceride levels. As regards CETP mass, Mu-rakami et al. [44] and McPherson et al. [46] also found no differences between hypertriglyceridemic patients and control subjects.

The increased CETP activity that we found in pa-tients from group 1 is consistent with their abnormal

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HDL lipid composition. CETP interchanges cholesteryl esters and triglycerides between HDL and apo B-con-taining lipoproteins, thus resulting in the triglyceride enrichment and cholesteryl ester depletion described in HDL from hypertriglyceridemic patients. Furthermore, studies in transgenic mice [14] have proven that hyper-triglyceridemia, originated by apo C-III overproduc-tion, interacts with CETP to considerably reduce HDL plasma levels and particle size, and also to alter its metabolism.

The present study demonstrated an abnormal reverse cholesterol transport in hypertriglyceridemic patients with low HDL-C levels, a group with potential risk of developing ischemic heart disease [47]. Hypertriglyce-ridemia seems to have affected the first three steps of this metabolic pathway, which are closely linked to one another. As regards the group with normotriglyce-ridemia and low HDL-C levels, these subjects showed lower total apo C-III plasma concentrations than the hypertriglyceridemic group, a feature generally linked to the normotriglyceridemic pattern. However, they presented some of the alterations detected in hyper-triglyceridemic patients. A possible explanation could be that even though these patients had plasma triglyce-ride levels within the reference values (B200 mg/dl), they were significantly higher than control subjects. Asztalos et al. [48] studied a group of normolipidemic subjects and found that those with low HDL-C levels also had higher plasma triglyceride concentrations than those with normal HDL-C values. In this group, the authors found abnormalities in HDL subpopulations. Additionally, about 70% of our normotriglyceridemic patients with low HDL-C levels showed plasma triglyc-eride concentrations higher than 150 mg/dl, upper limit proposed by Austin et al. [49] for the appearance of atherogenic modifications normally found in manifest hypertriglyceridemia.

In summary, in patients with primary hypertriglyce-ridemia, low HDL-C levels were accompanied by de-creased apo A-I and apo A-II concentrations. These reductions were manifested by a selective decrease in LpA-I:A-II particles. In addition, apo C-III Lp non B was found to be elevated and HDL lipid percentage composition showed a triglyceride enrichment and cholesterol depletion. The capacity of serum samples from hypertriglyceridemic patients to promote cellular cholesterol efflux was reduced, as evidenced by using two different cellular models, Fu5AH and J774 cells. This impaired cholesterol efflux promotion, also cor-roborated by incubations with HDL fractions, may be attributed to the low concentration of free cholesterol acceptors and to the abnormalities found in HDL particles. LCAT activity, the driving force of reverse cholesterol transport, showed a tendency towards lower values, while CETP activity was increased in hyper-triglyceridemic patients. In conclusion, in

hypertriglyce-ridemic patients, the reduction in plasma levels of HDL-C, HDL-phospholipids, apo A-I, apo A-II, LpA-I:A-II and the increase in HDL-triglycerides and apo C-III Lp non B particles were associated with the combination of three important factors: decreased free cholesterol removal from cells, slightly low free choles-terol esterification, and high cholesteryl ester transfer from HDL to apo B-containing lipoproteins.

Acknowledgements

Carla D. Bonavita is a Research Fellow from Univer-sity of Buenos Aires. This work was supported by a grant from the same university (FA 085) and it was part of the INSERM-CONICET International Cooper-ation Program.

References

[1] The International Committee for the Evaluation of Hypertriglyc-eridemia as a Vascular Risk Factor. The hypertriglycHypertriglyc-eridemias: risk and management. Am J Cardiol 1991;68:1A – 42A. [2] Patsch JR. Triglyceride-rich lipoproteins and atherosclerosis.

Atherosclerosis 1994;110:S23 – 6.

[3] Castelli WP. Epidemiology of triglycerides: a view from Fram-ingham. Am J Cardiol 1992;70:3 – 9.

[4] Gordon DJ, Rifkind BM. High-density lipoprotein — the clini-cal implications of recent studies. New Engl J Med 1989;321:1311 – 6.

[5] Hokanson JE, Austin MA. Plasma triglyceride level is a risk factor for cardiovascular disease independent of high-density lipoprotein cholesterol level: a meta-analysis of population-based prospective studies. J Cardiovasc Risk 1996;3:213 – 9.

[6] Grundy SM. A symposium: the role of statins in patients with hypertriglyceridemia. Am J Cardiol 1998;81:1B – 73B.

[7] Brites FD, Bonavita CD, Cloe¨s M, Yael MJ, Fruchart JC, Castro GR, et al. VLDL compositional changes and plasma levels of triglycerides and high density lipoprotein. Clin Chim Acta 1998;269:107 – 24.

[8] Fruchart JC, Ailhaud G. Apolipoprotein A-containing lipo-protein particles: physiological role, quantification and clinical significance. Clin Chem 1992;38:793 – 7.

[9] Glomset JA. The plasma lecithin:cholesterol acyltransferase reac-tion. J Lipid Res 1968;9:155 – 67.

[10] Fielding CJ, Fielding PE. Molecular physiology of reverse cholesterol transport. J Lipid Res 1995;36:211 – 28.

[11] Castro GR, Fielding CJ. Early incorporation of cell-derived cholesterol into pre-b-migrating high density lipoprotein. Bio-chemistry 1988;27:25 – 9.

[12] Acton S, Rigotti A, Landschulz KT, Xu S, Hobbs HH, Krieger M. Identification of scavenger receptor SR-BI as a high density lipoprotein receptor. Science 1996;271:518 – 20.

[13] Manzato E, Zambon S, Zambon A, Cortella A, Sartore G, et al. Levels and physicochemical properties of lipoprotein subclasses in moderate hypertriglyceridemia. Clin Chim Acta 1993;219:57 – 65.

[14] Hayek T, Azrolan N, Verdery RB, Walsh A, Chajek-Shaul T, Agellon LB, et al. Hypertriglyceridemia and cholesteryl ester transfer protein interact to dramatically alter high density lipo-protein levels, particle sizes, and metabolism. J Clin Invest 1993;92:1143 – 52.

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Fig. 1. Cholesterol efflux from monolayer cultured cells (Fu5AH rat hepatoma cells or J774 mouse macrophage cells) induced by samples (whole serum or HDL fractions) from hypertriglyceridemic patients in comparison to normotriglyceridemic subjects with or without low HDL-C levels. HDL-C, high density lipoprotein-cholesterol; TG, triglycerides.a_P_B0.001,b_P_B0.05, c_P_{B0.01 versus normal TG, normal HDL-C group.}

(group 1) induced cholesterol efflux from Fu5AH cells to a lesser extent than HDL fractions from subjects with normal triglyceride levels.

4. Discussion

In this study, we evaluated diverse parameters in-volved in reverse cholesterol transport and the first

Table 4

Correlation coefficients of HDL related lipids, apolipoproteins and lipoprotein particles with cholesterol efflux promotion from Fu5AH rat hepatoma cells and J774 mouse macrophage cells (n=36)a

Fu5AH cells J774 cells

r PB r PB

0.001 0.62

0.001

HDL-C 0.77

HDL2-C 0.59 0.001 0.26

0.76 0.001

HDL3-C 0.66 0.001

0.001 0.52

0.001

HDL-PL 0.68

−0.14 NS

HDL-TG −0.10 NS

0.001 0.49 0.01

apo A-I 0.68

0.77 0.001

apo A-II 0.48 0.01

NS 0.27 0.01

0.42 LpA-I

0.61 0.001

LpA-I:A-II 0.45 0.01

HDL-C/A-I+A-II 0.58 0.001 0.51 0.001

a_{apo, apolipoprotein; C, cholesterol; HDL, high density}

lipo-protein; Lp, lipoprotein particle; NS, not significant (P\0.05); PL, phospholipids; TG, triglycerides.

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Table 5

LCAT activity, CETP activity and mass in serum samples from patients and control subjects (mean9S.D.)a

Normal TG, low HDL-C

High TG, low HDL-C Normal TG, normal HDL-C

(group 1,n=12) (group 2,n=12) (group 3,n=12)

8.695.3 5.793.1b _11.695.8

LCAT activity (%/h per ml)

369989c,d ₂₅₆₉₈₁

CETP activity 254959

(%/h per ml)

9679308 9149225

CETP mass (ng/ml) 9899245

a_{C, cholesterol; CETP, cholesteryl ester transfer protein; HDL, high density lipoprotein; LCAT, lecithin:cholesterol acyltransferase; TG,}

triglycerides.

b_P_{B0.05 versus group 3, by Mann–Whitney}_U_-test. c_P_{B0.005 versus group 3, by Mann–Whitney}_U_-test. d_P_{B0.01 versus group 2, by Mann–Whitney}_U_-test.

In both groups with low HDL-C levels, a diminution in apo A-I and apo A-II plasma levels was observed. The reduction in HDL-C was due to a decrease in the

concentration of HDL2-C and in HDL3-C. In addition,

Table 6

Correlation coefficients of HDL related lipids, apolipoproteins, lipo-protein particles and different steps of reverse cholesterol transport with plasma triglyceride levels (n=36)a

r PB

HDL-C −0.56 0.001

−0.25 HDL2-C

HDL3-C −0.63 0.001

HDL-PL −0.50 0.001

−0.56 0.001

HDL-TG

apo A-I −0.07 NS

−0.32

apo A-II 0.05

apo B100 0.15 NS

NS 0.23

LpA-I

−0.20

LpA-I:A-II NS

Total apo C-III 0.89 0.001

0.001 0.79

apo C-III Lp non B

0.84

LpB:C-III 0.001

Total apo E 0.60 0.001

apo E Lp non B 0.08 NS

0.53

LpB:E 0.001

−0.32 0.05

Efflux Fu5AH

0.01

−0.43 Efflux J774

−0.10

LCAT activity NS

0.55

CETP activity 0.001

CETP mass 0.12 NS

a_{apo, apolipoprotein; C, cholesterol; CETP, cholesteryl ester}

trans-fer protein; HDL, high density lipoprotein; LCAT, lecithin:cholesterol acyltransferase; Lp, lipoprotein particle; NS, not significant (P\0.05); PL, phospholipids; TG, triglycerides.

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patients treated with b-blockers, two factors known to reduce HDL levels and its subpopulations beyond hy-pertriglyceridemia [34], unlike our study which

exclu-sively selected primary hypertriglyceridemic male

subjects. Furthermore, in a previous study [35], we evaluated HDL subfractions in a group of type II diabetic patients with moderate hypertriglyceridemia. Employing the same electroimmunoduffusion method-ology and following exactly the same conditions, in that research we were able to point out that in the diabetic patients the decrease in HDL levels was due to LpA-I subfraction and not to LpA-I:A-II particles, a com-pletely different pattern to the one observed in patients with primary hypertriglyceridemia. These data clearly show that results obtained do not depend on the analyt-ical procedure employed, and other factors could be more relevant.

In summary, analysis of HDL subfractions in hyper-triglyceridemic patients, revealed a significant decrease in HDL2-C, HDL3-C and in LpA-I:A-II particle levels.

Moreover, if the contribution of HDL2 subpopulation

signifi-cant (HDL2-C=11.1911.2% vs. 16.997.2%,

respec-tively; P\0.05). The same analysis carried out with

HDL3 subfraction also revealed no statistically

signifi-cant difference between patients and controls (HDL3

-C=88.9911.2% vs. 84.296.8%, respectively;

P\0.05). Even if different authors described a positive

correlation between HDL2 and LpA-I levels, and

be-tween HDL3 and LpA-I:A-II levels, it is interesting to

a decrease in HDL2subfraction may not always imply

a reduction of the same magnitude in LpA-I subfrac-tion. Another fact which renders the comprehension of

HDL heterogeneity more difficult is that both HDL3

and LpA-I subpopulations have been proposed to be the best cholesterol efflux promoters [36,37] and not the

pair HDL2-LpA-I or HDL3-LpA-I:A-II, as would have

been expected.

HDL-C, HDL3-C, HDL-phospholipids, apo A-I, apo

acceptors (including preb-HDL particles), LCAT

activ-ity, CETP activity and the concentration of apo B-con-taining lipoproteins. However, they provide no specific information about the role played by the different

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subfractions such as preb particles. This is a common

limitation of most methods employed to separate lipo-protein fractions, including ultracentrifugation which is the reference method for lipoprotein isolation. As preb1-HDL particles play a crucial role, being the first acceptors of cholesterol effluxed from cells, it could be thought that their loss could be conditioning cholesterol efflux experiments carried out with isolated particles.

Nevertheless, the magnitude of preb loss might not be

considerable because in hypertriglyceridemic patients similar results were obtained with both whole serum and HDL fractions. Moreover, Okazaki et al. [40] employed a chromatographic method and were able to separate five different HDL subclasses from samples of normal males and females. The particle diameter of the

smallest subclass was reported to be about 7.6390.16

nm, similar to the size of prebparticles (preb1-HDL=

5.6 nm and preb2-HDL=12 – 14 nm), thus showing the

ability of this chromatographic method to isolate small lipoprotein particles [41]. Additionally, it would be important to differentiate the in vivo situation where preb1particles are being regenerated at a constant rate and the in vitro situation where preb1particles could be rapidly saturated with free cholesterol coming from cells. Therefore, in any experimental model which in-volves long incubation periods with cultured cells, the

quantitative contribution of preb1 particles could be