© 1996 American Heart Association, Inc.
Articles |
Increased Cholesteryl Ester Transfer Activity in Plasma From Analbuminemic Patients
From the Service de Médecine V and INSERM U391, Hôpital Henri Mondor (S.B., C.M., B.J.), Créteil; the Laboratoire de Biochimie des Lipoprotéines, INSERM CJF 93-10, Faculté de Médecine (L.L., E.F., A.A., P.G., C.L.), Dijon; and the Service d'Endocrinologie, Hôtel Dieu (M.K.), Nantes, France.
Correspondence to Laurent Lagrost, Laboratoire de Biochimie Médicale, Hôpital du Bocage, 21034 Dijon, France.
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Abstract |
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Abstract Hypercholesterolemia associated with analbuminemia, an inherited disease manifesting low plasma albumin concentration, is characterized by enhanced LDL cholesterol levels and reduced HDL cholesterol levels. In addition, compared with normal counterparts, the esterified cholesterol:triglyceride ratio tends to be higher in analbuminemic apoB-containing lipoproteins and lower in analbuminemic HDL. The aim of the present study was to investigate the mechanism that may account for the association of a hypoalbuminemic state with alterations in the concentration and composition of plasma lipoprotein fractions. To this end, endogenous cholesterol esterification activity, phospholipid transfer activity, and cholesteryl ester transfer activity were measured in total plasma from three analbuminemic patients and five control subjects. Whereas endogenous cholesterol esterification and phospholipid transfer rates were not significantly affected in analbuminemia, the transfer of radiolabeled cholesteryl esters from HDL toward apoB-containing lipoproteins was constantly higher in analbuminemic plasmas than in normal control plasma (473.6±107.3%·h-1·mL-1 versus 227.5±84.0%·h-1·mL-1, respectively; P=.036). The rise in cholesteryl ester transfer protein (CETP) activity in analbuminemic plasma was due to a significant increase in the transfer of radiolabeled cholesteryl esters toward LDL but not toward the triglyceride-rich lipoproteins. The CETP mass was higher in analbuminemic patients than in control subjects, but the difference did not reach the significance level (5.18±0.82 mg/L versus 3.13±1.19 mg/L, respectively; P=.07). Since abnormally elevated nonesterified fatty acid (NEFA) levels were shown to be associated with analbuminemic lipoproteins, mostly LDL, the direct role of lipoprotein-bound NEFA in enhancing CETP activity was suspected. In support of this view, supplementation of total plasmas with fatty acid–poor albumin was shown to reduce CETP activity to a significantly greater extent in analbuminemic plasmas than in normal control plasma. It is concluded that hyperlipidemia associated with the hypoalbuminemic state can relate, at least in part, to the combined effect of CETP and NEFA in promoting the transfer of cholesteryl esters from the antiatherogenic HDL toward the proatherogenic apoB-containing lipoproteins.
Key Words: lipoprotein • cholesteryl ester transfer protein • hypoalbuminemia • triglyceride • cholesteryl ester
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Introduction |
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Analbuminemia, or better called congenital hypoalbuminemia, is a rare, inherited disease characterized by an abnormally low plasma albumin concentration.1 2 3 4 5 6 7 8 9 10 11 12 13 In spite of the relatively small number of patients identified so far,1 2 3 4 5 6 7 8 9 10 11 12 13 inherited analbuminemia has been shown to be associated with several well-defined clinical symptoms, among them mild edema, low blood pressure, increased concentration of several plasma proteins, and hypercholesterolemia.6 7 9 10 11 13 14 15 The latter point is of particular interest and might account for the premature cardiovascular events that have been reported in several cases.3 5 6 13 Consequently, inherited analbuminemia may constitute an interesting model to elucidate the biochemical process that may account for the reported positive relationship between hypoalbuminemia and coronary artery disease.16 17 18
Whereas NEFAs are normally mostly bound to albumin, abnormally low albumin concentrations, such as those reported in analbuminemia15 and nephrotic syndrome,19 have been shown to be associated with the redistribution of NEFA toward the plasma lipoprotein fraction.19 These observations demonstrated clearly that in vivo lipoprotein particles can stand for alternative carriers of plasma NEFA.15 19 Moulin and coworkers20 reported increased CETP mass concentrations in dyslipidemic patients with nephrotic syndrome. In vitro studies achieved in the absence of albumin demonstrated that NEFA, when located at the surface of lipoproteins, can stimulate the ability of CETP to promote the redistribution of cholesteryl esters from HDL toward VLDL and LDL, together with the reciprocal transfer of triglycerides from VLDL+LDL toward HDL.21 22 Considered together, previously published data suggest, therefore, that the stimulation of plasma CETP by the lipoprotein-bound NEFA might account for the rise in LDL:HDL cholesterol ratio observed under pathological conditions that is associated with abnormally low plasma albumin levels.15 20
The latter hypothesis was investigated in the present study by measuring the CETP mass concentration and the isotopic exchange of cholesteryl esters between HDL and apoB-containing lipoproteins in three analbuminemic patients and five normolipidemic control subjects.
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Case Reports |
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TopAbstractIntroductionCase Reports Methods Results Discussion References |
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Case 1
ZA, a 32-year-old woman, was first hospitalized in 1981 for a severe myopathy revealing a vitamin-sensitive osteomalacia.23 Laboratory examination at that time showed a combined hyperlipidemia together with a hypoalbuminemia without proteinuria. Study of the family revealed a consanguineous union of the parents.7 Analbuminemia was detected in plasma from two of ZA's brothers, the older one suffering slight generalized edema and the other being asymptomatic (Fig 1a
). To
date, ZA still exhibits a strong dyslipidemia (plasma total
cholesterol level >400 mg/dL; plasma
triglyceride levels >200 mg/dL), with no beneficial
effects derived from a low-fat diet and hypolipidemic drug
treatment. At the present time, the physical examination of ZA is
normal, with blood pressure of 120/70 mm Hg and normal weight (BMI,
24.5 kg/m2). The only associated clinical abnormality is a
bilateral corneal arcus, with no symptom of
cardiovascular disease.
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Cases 2 and 3
BR and RR, two sisters, aged 25 and 32 years,
respectively, were
also investigated for a severe and persistent dyslipidemia
associated with inherited hypoalbuminemia. BR was first
admitted in the hospital in 1987 for a transient oligoarthritis, which
remained unexplained, and edema of the legs. RR was hospitalized in
1990 and 1992 for superficial thrombophlebitis. Review of family
history revealed a consanguineous union of the parents and maternal
grandparents (Fig 1b
). One of the maternal aunts had a
myocardial
infarction at 54 years old.13 Laboratory examination
immediately after the infarction revealed
hypercholesterolemia associated with
hypoalbuminemia (plasma cholesterol, 348 mg/dL;
plasma triglycerides, 78 mg/dL; plasma albumin, 2.7
g/L). The father of BR and RR was
hypercholesterolemic but had normal plasma
albumin concentration (plasma cholesterol, 290
mg/dL; plasma triglycerides, 180 mg/dL). He died at the age
of 63 of hepatic metastasis of a colic carcinoma. The brother of BR and
RR is also hypercholesterolemic without
hypoalbuminemia (plasma cholesterol, 324 mg/dL;
plasma triglycerides, 65 mg/dL).
At present, the physical examinations of BR and RR are normal, except that BR has edema in both legs and low blood pressure (100/70 mm Hg) and RR is overweight (BMI=30.4 kg/m2). BMI of BR is 23.3 kg/m2.
Control Subjects
The control group consisted of five healthy,
normolipidemic women
recruited from among the hospital staff (38.6±6.9 years old; BMI,
20.7±1.0 kg/m2; plasma cholesterol <230
mg/dL; plasma triglycerides <200 mg/dL). Informed consent
from patients and control subjects was obtained. They took
neither drugs nor oral contraceptives.
Control subjects did not differ
significantly from
analbuminemic patients in regard to their physical
activity, smoking habits, and diet. Only one control subject was a
smoker (10 cigarettes per day). Information on usual food intake was
obtained through a 3-day dietary record. The average energy
consumption, as well as the protein, carbohydrate, and fat contents of
the diets in analbuminemic patients and control subjects
did not differ significantly. Saturated fatty acids accounted for
50% of the total fat intake in all the subjects studied.
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Methods |
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Blood Sampling
Venous blood was collected after an overnight fast into tubes containing 0.1 mg/mL Na2-EDTA and placed immediately on ice. Plasma was promptly separated by a 15-minute centrifugation at 3000 rpm and maintained at 4°C before lipid and lipoprotein analysis. Plasma aliquots for cholesteryl ester transfer, phospholipid transfer, and cholesterol esterification assays were kept at -20°C.
Analysis of Lipoprotein Components
Total cholesterol, free
cholesterol,
triglyceride, and phospholipid concentrations were
determined in total plasma and lipoprotein fractions by using an Abbott
diagnostic VP analyzer with enzymatic reagents
(Boehringer Mannheim). Cholesteryl ester mass content of
lipoprotein fractions was calculated as the difference between total
cholesterol and free cholesterol multiplied by
1.67, representing the sum of the esterified
cholesterol and fatty acid moieties. Protein concentrations
were measured by using the method of Lowry et al,24 with
serum albumin as the standard. HDL cholesterol
levels were determined after precipitation of plasma apoB-containing
lipoproteins with phosphotungstic acid and magnesium chloride
(Boehringer Mannheim). LDL cholesterol levels were
calculated using the formula of Friedewald et al.25
Nonesterified fatty acid concentrations were determined by using an
enzymatic kit (Wako Pure Chemicals Industries).
Preparation of Lipoprotein Fractions by Sequential
Ultracentrifugation
Lipoprotein fractions were separated by flotation
in an L8-65
ultracentrifuge (Beckman Instruments) according to a
modification of the procedure of Havel et al.26 Densities
were adjusted by using KBr solutions. VLDL, IDL, LDL, HDL2, and HDL3
were isolated step by step as the d<1.006-g/mL,
1.006<d<1.019-g/mL, 1.019<d<1.063-g/mL,
1.063<d<1.125-g/mL, and 1.125<d<1.250-g/mL
plasma fractions, respectively. HDL2 and HDL3 fractions were washed by
a second run of ultracentrifugation in solutions of
d=1.125 g/mL and d=1.250 g/mL,
respectively, to avoid contamination with plasma albumin.
Density Gradient
Ultracentrifugation
Plasma lipoproteins were fractionated according to
a continuous
KBr gradient by using the general procedure described by Chapman and
coworkers.27 Briefly, the density of the plasma samples
was first adjusted to 1.210 g/mL by the addition of solid KBr. A
discontinuous gradient was then constructed in cellulose nitrate tubes
in a Beckman SW 41-Ti swinging-bucket rotor by using KBr solutions
in the following order: 0.5 mL of a d=1.250-g/mL NaCl-KBr
solution, 2 mL of plasma adjusted to d=1.210 g/mL, 3 mL of a
d=1.120-g/mL NaCl-KBr solution, 2.5 mL of a
d=1.063-g/mL NaCl-KBr solution, 1.5 mL of a
d=1.019-g/mL NaCl-KBr solution, 1 mL of a
d=1.006-g/mL NaCl solution, and 1 mL of distilled water.
Tubes were immediately centrifuged at 40 000 rpm for 24 hours
at 15°C in a Beckman L8-55 ultracentrifuge.
Isotopic Assay of Plasma Cholesteryl Ester Transfer
Activity
Cholesteryl ester transfer activity was determined in total
plasma according to the general procedure previously
described.28 Briefly, the capacity of a plasma sample to
promote the transfer of tritiated cholesteryl esters from a tracer
amount of biosynthetically labeled HDL3 (3H-CE-HDL3) toward plasma
apoB-containing lipoproteins was evaluated during the incubation at
37°C of total plasma (25 µL), 3H-CE-HDL3 (2.5 nmol of
cholesterol), and iodoacetate (75 nmol) in a final volume
of 50 µL. At the end of the incubation, apoB-containing lipoproteins
were separated by using either ultracentrifugation
or PAGE. In the former case, incubation mixtures were adjusted to
d=1.068 g/mL, ultracentrifuged for 7 hours at 50
000 rpm (269 000g) in a 50.4 Ti rotor on an L7
ultracentrifuge (Beckman), and the d<1.068- and
d>1.068-g/mL fractions were recovered.28 In
the latter case, plasma lipoprotein fractions were separated by
electrophoresis in 20- to 160-g/L polyacrylamide
gels.29 Subsequently, gel fragments containing the
different plasma lipoprotein fractions (VLDL+IDL, LDL, and HDL) were
cut off and dissolved with NaOCl according to the procedure of
Florentin et al.29 Lipoprotein fractions recovered by
using either the ultracentrifugation or the PAGE
method were mixed with a scintillation fluid (OptiPhase Hisafe 3,
Pharmacia), and radioactivity was assayed for 5 minutes in a Wallac
1410 liquid scintillation counter (Pharmacia). Results were expressed
as percentage of total radiolabeled cholesteryl esters recovered in
each lipoprotein fraction.
Isotopic Assay of Plasma Phospholipid Transfer
Activity
Phospholipid transfer activity was determined in total plasma
by
measuring the transfer of radiolabeled phosphatidylcholine from
phospholipid liposomes (14C-PC-liposomes) to the plasma HDL fraction
according to the method previously described30 and derived
from the general procedure of Damen et al.31 Briefly,
total plasma (30 µL), 14C-PC-liposomes (110 nmol of
phosphatidylcholine), and iodoacetate (120 nmol) were incubated for 30
minutes at 37°C in a final volume of 80 µL. Phospholipid liposomes
and apoB-containing lipoproteins were subsequently precipitated by
addition of 60 µL of a 500-mmol/L NaCl, 215-mmol/L
MnCl2, 445-U/mL heparin solution.30
Radioactivity in resulting supernatants was assayed as described above
and phospholipid transfer activity was calculated as the rate of total
radiolabeled phospholipids transferred from liposomes to plasma HDL
after deduction of nonincubated control values.
LCAT Activity
LCAT activity in total plasma was evaluated by
using two
independent methods, which measured either the cholesterol
esterification rate with an exogenous, radiolabeled substrate
("isotopic cholesterol esterification rate") or the
decrease in plasma free cholesterol mass
("endogenous cholesterol esterification
rate").
Isotopic Cholesterol Esterification Rate
LCAT activity in total plasma was evaluated by using the general
procedure previously described.28 Briefly, 5 µL of a
3H-cholesterol–albumin emulsion
(3H-cholesterol, 6.7 mCi/L; albumin, 50 g/L) and 50
µL of total plasma were mixed and preincubated for 1 hour at 4°C.
Subsequently, mixtures were incubated for 3 hours at 37°C and
nonesterified cholesterol remaining was precipitated by
digitonin.32 The isotopic cholesterol
esterification rate was calculated as the percentage of total
radiolabeled cholesterol esterified during the 3-hour
period compared with control in which plasma was replaced by
Tris-buffered saline. Isotopic LCAT activity was expressed in
percentage of radiolabeled cholesterol esterified per hour
per milliliter of plasma
(%·h-1·mL-1).
Endogenous
Cholesterol Esterification
Rate
Endogenous cholesterol esterification
was determined by the previously described nonradioactive
method,33 which measures the decrease in plasma free
cholesterol during incubation for 40 minutes at 37°C.
Endogenous cholesterol esterification rate was
expressed in percentage of decrease in unesterified
cholesterol per hour (%/h).
CETP Enzyme-Linked Immunosorbent Assay
CETP mass
concentrations were measured by using a competitive
enzyme-linked immunosorbent assay on a Biomek 1000 Biorobotic
System (Beckman Instruments).34 CETP mass concentration
values were determined in quadruplicate from a calibration curve
obtained with a frozen plasma standard.
Native Polyacrylamide Gradient Gel
Electrophoresis
The size of isolated LDL was determined by
electrophoresis on
nondenaturing 15- to 250-g/L polyacrylamide gradient gels
according to the general procedure previously described.28
Gels were fixed and stained with Coomassie brilliant blue G before
analysis on a GS-670 Gel Densitometer (Bio-Rad). The mean
apparent diameter of LDL was determined by comparison with protein
standards (Pharmacia High Molecular Weight Protein Calibration kit) and
carboxylated latex beads (Duke Scientific).28
Agarose Gel Electrophoresis
The electrophoretic mobility of
isolated LDL particles was
determined by electrophoresis on 0.5% agarose gels (Paragon Lipo kit,
Beckman), according to the method described by Sparks and
Phillips.35 Mean migration distances were obtained by
analysis of the gel on a Bio-Rad GS-670 imaging
densitometer.
Precipitation of the Total Plasma Lipoprotein
Fraction
Lipoproteins were removed from total human plasma by using a
single-step adsorption method based on the ability of FSD to remove
large particles, such as lipid emulsions and lipoprotein particles,
from aqueous media.36 Briefly, total plasmas were treated
for 10 minutes at room temperature with FSD (final concentration, 2.5
mg/mL) according to the general procedure previously
reported.37 Under those experimental conditions, FSD
allowed the removal of all plasma lipoprotein components, whereas no
more than 5% of plasma albumin was coprecipitated.
Statistical Analysis
Data means were compared by using the
Mann-Whitney U
statistic. Correlations between various parameters were
analyzed by using Spearman's rank correlation
analysis.
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Results |
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Plasma Parameters
Table 1
presents the albumin
and lipid
levels in total plasma from three analbuminemic patients
and five control subjects. Study patients presented markedly
and significantly lower albumin levels than control subjects.
Hypoalbuminemia was associated with significantly higher
total cholesterol, LDL-C, and triglyceride
concentrations compared with normolipidemic control subjects.
Conversely, HDL-C tended to be reduced in hypoalbuminemic
plasmas. Total NEFA levels in analbuminemic patients were
within the normal range (Table 1). Plasma LDL:HDL
cholesterol ratio was significantly higher in
analbuminemic patients than in control subjects (Table 1).
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Plasma Lipoprotein Composition
Distinct lipoprotein fractions
(VLDL, IDL, LDL, HDL2, and
HDL3) were isolated from total plasma by sequential
ultracentrifugation and were assayed for protein,
unesterified cholesterol, total cholesterol,
phospholipid, and triglyceride as described in
"Methods." As shown in Table 2, lipid:protein
ratios were significantly increased only in LDL fractions of
analbuminemic patients (3.23±0.20 versus 1.87±0.95,
P=.036). Whereas the mass percentage of surface lipid
components (phospholipid, unesterified cholesterol) in
individual lipoprotein fractions was quite similar in the two groups,
some differences appeared when comparing the neutral lipid (cholesteryl
ester and triglyceride) contents. Indeed, cholesteryl ester
content tended to be higher in lipoproteins from
analbuminemic patients compared with normal control
subjects, and the difference was statistically significant in the VLDL
and LDL fractions. Triglycerides tended to be lower in
analbuminemic VLDL and IDL but higher in
analbuminemic HDL. However, differences did not reach the
significance level. Overall, the esterified
cholesterol:triglyceride ratio tended to be
higher in analbuminemic apoB-containing lipoproteins, ie,
VLDL, IDL, and LDL, but lower in analbuminemic HDL (Table 2).
Again, because of the small number of subjects studied, the
difference in the esterified
cholesterol:triglyceride ratio between
analbuminemic patients and normal subjects reached
statistical significance only for the VLDL fraction (Table 2).
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Size and Density of LDL
To determine whether the significant
increase in the
lipid:protein ratio of analbuminemic LDL was associated
with alterations in particle size and density, LDLs were separated on
native polyacrylamide gradient gel and isolated from total
plasma by density gradient ultracentrifugation (see
"Methods"). Compared with LDL from control individuals, LDL
particles from analbuminemic patients tended to be of
larger size (26.6±0.2 versus 27.0±0.5 nm, respectively; NS). In
addition, the mean density of LDL from analbuminemic
patients was significantly lower than the mean density of LDL from
control subjects (1.041±0.041 versus 1.045±0.001 g/mL,
respectively;
P=.036).
Distribution of NEFA Among Plasma Lipoprotein
Fractions
To investigate whether hypoalbuminemia was associated
with alterations in the plasma distribution of NEFA, plasma
lipoproteins were precipitated by using FSD, and NEFA contents of
lipoprotein and nonlipoprotein plasma fractions were determined as
described under "Methods." Interestingly, in spite of similar
total NEFA levels in plasmas of both analbuminemic patients
and control subjects, strong differences appeared when comparing the
plasma NEFA distribution in the two groups. Indeed, the bulk of plasma
NEFA, representing 59±3% (mean±SD of three
determinations), 68±3%, and 88±20% of the total, was located in
the
lipoprotein fraction of BR, RR, and ZA, respectively. In contrast, only
a minor proportion of total NEFA, not exceeding 10% of the total, was
located in lipoproteins from control subjects.
The precise distribution
of plasma NEFA was further studied after
separation of lipoprotein fractions by sequential
ultracentrifugation (see "Methods"). In
agreement with data obtained after precipitation of plasma lipoproteins
with FSD, most of the NEFA in analbuminemic plasmas was
bound to the ultracentrifugally isolated LDL, HDL2, and HDL3 fractions.
In contrast, only a small percentage of total NEFA was recovered in the
triglyceride-rich lipoprotein fractions, VLDL and IDL,
from both analbuminemic and control plasmas (Fig 2). In
accordance with a higher NEFA content of
analbuminemic LDL compared with normolipidemic counterparts
(33.9±4.3 versus 3.2±2.3% of total plasma NEFA, respectively;
P=.025), the mean distance of migration of LDL particles
toward the anodic end of agarose gel was significantly higher when
isolated from anabuminemic than normolipidemic plasma (7.1±0.3 versus
5.4±0.4 mm, respectively; P=.036) (Fig
3).
The distance of migration of plasma LDL in agarose gel correlated
positively with its NEFA content (P=.78;
P=.04).
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LCAT Activity in Analbuminemic and Control
Plasmas
LCAT activity was determined by two independent methods,
which measured either the esterification of exogenous radiolabeled
unesterified cholesterol (isotopic cholesterol
esterification rate) or the esterification of endogenous
unesterified cholesterol (endogenous
cholesterol esterification rate; see "Methods").
Whereas the isotopic cholesterol esterification rate was
markedly decreased in the three analbuminemic patients
(38.1±12.8%·h-1· mL-1
versus
117.2±19.9%·h-1 · mL-1
in control subjects, P=.036), the endogenous
cholesterol esterification rate was normal
(8.75±1.96%/h versus 7.27±2.62%/h in control subjects,
NS).
Phospholipid Transfer Activity in Analbuminemic and
Control Plasmas
The phospholipid transfer activity was determined by
measuring the transfer of radiolabeled phosphatidylcholine from
phospholipid liposomes to the plasma HDL fraction (see
"Methods"). The difference in plasma phospholipid transfer
activity between analbuminemic patients and control
subjects was not statistically significant
(358±55%·h-1·mL-1
versus
464±35%·h-1·mL-1,
NS).
Isotopic Transfer of Cholesteryl Esters in
Analbuminemic and Control Plasmas
Fig 4 shows the
time-dependent transfer of
radiolabeled cholesteryl esters from HDL3 to apoB-containing
lipoproteins measured in analbuminemic patients and control
subjects. The rate of cholesteryl esters transferred toward the
d<1.068-g/mL plasma fraction increased progressively during
the first 3 hours of incubation and tended to reach a plateau for
incubation times exceeding 4.5 hours (Fig 4). During the period
studied, cholesteryl ester transfer activity in
analbuminemic patients was constantly and significantly
higher than in normolipidemic control subjects (P=.036). As
measured after 3 hours of incubation, mean cholesteryl ester transfer
activity, expressed in percentage of radiolabeled cholesteryl esters
transferred from HDL3 to apoB-containing lipoproteins per hour per
milliliter of plasma, was significantly higher in
analbuminemic plasmas than in normal control plasma
(473.6±107.3 versus
227.5±84.0%·h-1·mL-1,
respectively; P=.036).
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As observed by using PAGE,
significant increase in the transfer of
radiolabeled cholesteryl esters toward LDL but not toward the VLDL+IDL
plasma fraction accounted for the rise in cholesteryl ester transfer
activity in analbuminemic plasmas (Table 3).
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CETP Mass
To determine whether increased CETP mass might also
account for
the increased transfer of neutral lipids between lipoproteins, plasma
CETP concentrations were measured by using a specific enzyme-linked
immunosorbent assay. CETP mass was higher in analbuminemic
patients than in control subjects, but the difference did not reach the
significance level (5.18±0.82 mg/L versus 3.13±1.19 mg/L,
P=.07).
Effect of Albumin Supplementation on the Isotopic Transfer
of Cholesteryl Esters From HDL3 Toward the d<1.068 g/mL
Plasma Fraction
In vitro albumin is able to remove NEFA from
lipoprotein
substrates when added to normal or hypoalbuminemic
plasmas.19 In the present study, we made use of the
high binding capacity of albumin for NEFA to confirm that
increased cholesteryl ester transfer activity was, at least in part,
due to high levels of lipoprotein-bound NEFA. To this end,
analbuminemic and control plasmas were supplemented with
increasing amounts of fatty acid–poor albumin. Compared
with nonsupplemented plasma counterparts, changes in cholesteryl ester
transfer activity after supplementation of total plasma with 5, 10, 15,
and 20 g/L of fatty acid–poor albumin were
-28.3±16.4, -42.2±20.7, -47.1±25.6, and
-50.1±23.7%·h-1·mL-1
in control plasmas, respectively. In analbuminemic plasmas,
changes in cholesteryl ester transfer activity after supplementation of
total plasma with 5, 10, 15, and 20 g/L of fatty acid–poor
albumin were -54.4±46.6, -92.4±46.6,
-122.2±31.1, and
-144.6±66.2%·h-1·mL-1,
respectively. Differences in the reduction of cholesteryl ester
transfer activity between control and analbuminemic plasmas
were statistically significant for the two highest albumin
concentrations studied, 15 and 20 g/L (P=.05 and
P=.027, respectively).
Specific activity of CETP in
albumin-supplemented plasmas
was calculated as the ratio of plasma cholesteryl ester transfer
activity to plasma CETP mass concentration and expressed in percentage
of total radiolabeled cholesteryl esters transferred per hour per
microgram of CETP
(%·h-1·µg-1).
Specific CETP activity in analbuminemic plasmas was higher
than in control plasmas (93.4±10.1 versus
78.1±15.9%·h-1·µg-1,
respectively), but the difference did not reach the significance level.
When analbuminemic plasmas were supplemented with
increasing concentrations of fatty acid–poor albumin, mean
specific CETP activity decreased markedly, with an approximately linear
progression along the albumin concentration range studied (Fig
5). In contrast, mean specific CETP activity in normal
plasmas was substantially reduced with low levels of fatty
acid–poor albumin (5 and 10 g/L) but subsequently did not
decrease further (Fig 5). For the highest fatty acid–poor
albumin concentration studied (20 g/L), specific CETP
activities measured in analbuminemic and control plasmas
were remarkably similar (66.1±7.7 versus
61.7±15.1%·h-1·µg-1,
respectively). When plotted against the added fatty acid–poor
albumin concentration, the difference between specific CETP
activity measured in both analbuminemic and normal plasmas
decreased progressively, reaching virtually the zero value when 28 g/L
of fatty acid–poor albumin was added to total plasmas (see
Fig 5).
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) and five normal control
plasmas (
). Differences between analbuminemic and
control samples have been calculated and plotted against the added
concentration of fatty acid–poor albumin (see inset
graph). |
Discussion |
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TopAbstractIntroductionCase Reports Methods Results Discussion References |
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The results of the present study revealed for the first time that the hyperlipidemic state associated with analbuminemia can relate at least in part to a significant increase in plasma cholesteryl ester transfer activity. Consistent observations were made by measuring the exchange of radiolabeled cholesteryl esters between HDL and VLDL+LDL fractions and the plasma CETP mass concentration. In accordance with a previous study from our laboratory in which NEFAs were shown to modulate CETP activity in normolipidemic plasma,38 the increased cholesteryl ester transfer activity in hypoalbuminemic plasmas was explained in part by the redistribution of NEFA from albumin toward the plasma lipoprotein particles.
Analbuminemia is a rare genetic disease that is associated with corneal arcus9 12 and premature atherosclerosis.3 5 6 13 It is also associated with a mild hypertriglyceridemia6 9 14 and a strong hypercholesterolemia6 7 9 10 14 15 that is mainly due to a rise in LDL cholesterol levels.15 In agreement with previous reports, the three analbuminemic patients of the present study showed abnormally high plasma cholesterol levels in association with corneal arcus in one of them.14 15 Moreover, we observed that the cholesteryl ester:triglyceride ratio in the apoB-containing lipoprotein fractions, ie, VLDL, IDL, and LDL, tended to be higher in analbuminemic patients than in normolipidemic control subjects, with only the ratio in VLDL reaching the significance level. In contrast, the cholesteryl ester:triglyceride ratio tended to be reduced in HDL from analbuminemic patients. The plasma LDL:HDL cholesterol ratio was significantly higher in analbuminemic plasmas. We demonstrated that alterations in the plasma lipoprotein profile were characterized by a marked, significant increase in the lipid:protein ratio of analbuminemic LDL, which was associated with changes in the physical properties of the particles. Indeed, we observed that analbuminemic LDL tended to be both of larger size and lower density compared with normolipidemic LDL. In addition, the electronegativity of analbuminemic LDL was significantly higher than that of normolipidemic LDL, reflecting its higher NEFA content.
It is commonly admitted that dyslipidemia associated with hypoalbuminemic states, such as congenital analbuminemia or nephrotic syndrome, is primarily induced by a hepatic oversynthesis of apoB-containing lipoproteins in response to the decreased oncotic pressure.39 40 However, recent observations suggested that the enrichment of individual LDL particles with cholesterol also contributed to elevated LDL-C levels in nephrotic syndrome.41 It is suggested, therefore, that not only an increased number of apoB-containing lipoprotein particles but also a net increase in their cholesterol content may be involved in the pathophysiology of hyperlipidemia associated with analbuminemia. In support of the latter view, we observed in the present study that LDL particles from analbuminemic patients compared with control subjects were enriched with cholesteryl esters.
Other studies achieved with analbuminemic patients,14 15 nephrotic patients,42 43 and analbuminemic rats,44 45 suggested that alteration in LCAT activity might be involved in the lipoprotein abnormalities associated with hypoalbuminemia. However, controversial observations were reported, and the hypoalbuminemic state was associated with increased,15 42 43 44 45 normal,14 42 46 or decreased42 46 plasma cholesterol esterification rate. The data presented here show that LCAT activity in analbuminemic plasmas was significantly decreased when measuring the esterification rate with an exogenous substrate but was normal when measuring the endogenous cholesterol esterification rate with the method of Albers and coworkers.33 In fact, as suspected by others,45 the decrease in exogenous cholesterol esterification rate might relate to variations in plasma lipoprotein levels, and then to differences in the ratio of added radiolabeled cholesterol to the plasma unesterified cholesterol pool. Therefore, on the basis of measurements of endogenous cholesterol esterification rates, it appears that the lipoprotein disorders associated with the hypoalbuminemic state in the present study are unlikely to result from alteration in plasma LCAT activity.
Since similar lipoprotein modifications, ie, increased cholesteryl ester:triglyceride ratio in triglyceride-rich lipoproteins and increased plasma LDL:HDL cholesterol ratio, were also observed in physiopathological conditions under which plasma CETP activity is enhanced,47 an abnormally elevated plasma CETP activity in analbuminemia was suspected. CETP activity was clearly higher in analbuminemic patients than in normal subjects in our study by measuring the isotopic transfer of cholesteryl esters from HDL to the plasma apoB-containing lipoproteins. It is noteworthy that CETP activity was measured by using radiolabeled HDL substrates that presumably did not contain the NEFA found in analbuminemic HDL. Therefore, an underestimation of the influence of lipoprotein-associated NEFA on CETP activity cannot be excluded. In contrast to neutral lipid transfer, phospholipid transfer rates were not significantly modified in analbuminemic plasma compared with normal control plasma.
In total human plasma, cholesteryl ester transfer activity values reflect the activity of the CETP protein as modulated by endogenous plasma factors, among them the concentration and composition of lipoprotein particles.47 48 Therefore, to determine the mechanism that may account for the increased cholesteryl ester transfer activity in analbuminemic patients, we searched for alterations in both CETP mass and putative CETP modulators in total plasma. As measured by using a specific enzyme-linked immunosorbent assay,34 CETP concentrations tended to be higher in analbuminemic plasmas than in normolipidemic counterparts. Due to the small number of analbuminemic patients studied, these differences did not reach the significance level. However, mean CETP concentration values in analbuminemic plasmas were clearly beyond the normal upper limit recently reported by using the same immunoassay.34 Increased plasma CETP mass is in good agreement with previous observations of Moulin and coworkers,20 who reported a significant rise in plasma CETP concentration in patients with nephrotic syndrome, another hypoalbuminemic state. The rise in CETP concentration in analbuminemic patients might relate to the well-known overproduction of a number of proteins by the liver in hypoalbuminemic states.20 43 Whether CETP is part of these overproduced proteins remains to be established.
In addition to high plasma CETP mass and elevated concentrations of triglyceride-rich lipoprotein acceptors,48 enhanced plasma cholesteryl ester transfer activity in analbuminemia might relate to significant increases in potential plasma CETP activators, among them the negatively charged lipoprotein-bound lipolytic products.21 47 49 50 51 In agreement with previous data,15 19 we observed that most NEFA is abnormally bound to analbuminemic lipoproteins, mainly LDL. In addition, Joles and coworkers45 recently demonstrated that the lysophosphatidylcholine content of plasma lipoproteins, another negatively charged lipid product, is also strongly increased in hypoalbuminemia. It results, therefore, that high amounts of lipoprotein-bound, negatively charged molecules, normally bound to plasma albumin, may facilitate the interaction of plasma CETP with analbuminemic lipoprotein substrates, resulting in a significant increase in neutral lipid transfer activity. In support of the latter view, we observed that specific activity of CETP tended to be higher in analbuminemic patients than in control subjects, and cholesteryl ester transfers were specifically and significantly directed toward LDL, the main NEFA carrier in analbuminemic plasmas.
The possibility that lipolytic products can enhance CETP activity was verified by supplementation of plasmas with fatty acid–poor albumin. Whereas in normolipidemic control plasma the CETP activity was only slightly reduced after albumin supplementation, the albumin-mediated decrease in cholesteryl ester transfers was much more pronounced in analbuminemic plasmas. In accordance with in vitro studies in which the ability of NEFA to modulate CETP activity was canceled in the presence of fatty acid–poor albumin,21 51 striking reduction of plasma cholesteryl ester transfer activity with fatty acid–poor albumin strongly suggests that lipoprotein-bound NEFA may constitute potential activators of lipid transfers in analbuminemic patients. Since plasma CETP activity tended to return to a basal, normal level when plasma albumin concentration was normalized, the elevation of CETP activity associated with analbuminemia appeared to be directly linked to increased NEFA content of plasma lipoproteins. In other words, the results of the present study strongly suggest that some of the lipoprotein disorders associated with analbuminemia might relate to enhanced plasma CETP activity, resulting itself from the hypoalbuminemic state. In support of the latter view, high plasma cholesterol values were shown to return to the normal level following albumin infusion in analbuminemic patients.4 6
In conclusion, the present study demonstrated that analbuminemia is associated with increased plasma cholesteryl ester transfer activity. It gave new insights into the mechanisms that may account for the previously reported relationship between hypoalbuminemic states and increased risk for coronary artery disease.16 17 18 By stimulating cholesteryl ester transfers from the "antiatherogenic" HDL toward the "proatherogenic" apoB-containing lipoproteins, lipoprotein-bound NEFA may represent a risk factor of atherogenicity.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This investigation was supported by the Université de Bourgogne, the Conseil Régional de Bourgogne, the Institut National de la Santé et de la Recherche Médicale (INSERM), and the Fondation pour la Recherche Médicale. Françoise Berneau and Caroline Rousseau are greatly acknowledged for their secretarial assistance.
Received June 2, 1995; accepted November 30, 1995.
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