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 apolipo-
protein 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, HbA
1c
, 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 mgdl and low plasma
HDL-C 5 35 mgdl levels n = 12; group 2, subjects with normal plasma TG B 200 mgdl and low plasma
HDL-C 5 35 mgdl levels n = 12; and group 3, control subjects with normal plasma TG B 200 mgdl
and normal plasma HDL-C \ 35 mgdl 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
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 gday 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 mlh. 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 gl dextran sulfate MW 50 000 and 1.0 M MgCl
2
[21]. Within-run and between-day precision CV were 3.2 and 3.8, respec-
tively. HDL
3
was separated by precipitation of the supernatant containing total HDL with 40 gl dextran
sulfate MW 50 000 and 2.0 M MgCl
2
[21]. Cholesterol and triglycerides in total HDL and HDL
3
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. HDL
2
lipid components were calculated as the difference be- tween the corresponding values obtained for total HDL
and HDL
3
. LDL-C level was determined as the differ- ence between total cholesterol and the cholesterol con-
tained in the supernatant obtained after selective precipitation of LDL with 10 gl 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 le6els
Apo A-I, apo A-II, apo B
100
, 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
. Fu
5
AH 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 con-
taining 5 fetal calf serum. Approximately 25 000 Fu5AH cellsml were plated on 24-mm multiwell plates
Inbro, Polylabo using 2 mlwell. Then 2 days after plating, cellular cholesterol was labeled during a 72-h
incubation with [
3
H]cholesterol NEN, Dupont de Ne- murs 1 mCiwell. 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-phospholipidsml as study samples and in this case the incubation at 37°C
was performed during 6 h.
2
.
6
.
2
. J
774
cells J774 cells were maintained in RPMI, 10 fetal calf
serum. Approximately 150 000 cellsml were plated on 24-mm multiwell plates using 2 mlwell. 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 mCiwell 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 acti6ity 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 10 ml of the proteoliposome substrate with 100 ml 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 acti6ity 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
HDL
3 3
H-CE-HDL
3
towards serum apo B-containing lipoproteins was evaluated. Serum samples 25 ml 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 gml and then ultracentrifuged for 4 h at 4°C and 250 000 × g in 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 mean 9 S.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 regres-
sion. Differences were considered significant at P B 0.05 in the bilateral situation.
3. Results
