© 1997 American Heart Association, Inc.
Articles |
Role of Lipoprotein-Bound NEFAs in Enhancing the Specific Activity of Plasma CETP in the Nephrotic Syndrome
From Service de Médecine V, Hôpital Henri Mondor, Créteil (S.B., C.M., B.J.); Laboratoire de Biochimie des Lipoprotéines, INSERM CJF 93-10, Faculté de Médecine, Dijon (D.M., E.F., A.A., P.G., C.L., L.L.); and Service de Néphrologie, Hôpital Henri Mondor, Créteil (G.R.), France.
Correspondence to Laurent Lagrost, Laboratoire de Biochimie Médicale, Hôpital du Bocage, BP 1542, 21034 Dijon, France.
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Abstract |
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Abstract Plasma cholesteryl ester transfer protein (CETP) activity, evaluated by the transfer of radiolabeled cholesteryl esters from a tracer dose of tritiated HDL to the plasma apolipoprotein B–containing lipoproteins, was significantly higher in patients with untreated idiopathic nephrotic syndrome (n=15) than in normolipidemic control subjects (n=22) (81.5±8.4 versus 43.1±3.1 µg CE · mL-1 · h-1, respectively; P<.001). The increased CETP activity in nephrotic plasma was explained by a significant rise in both the CETP mass concentration (3.2±0.2 versus 2.1±0.1 mg/L; P<.001), and the specific CETP activity, calculated as the ratio of CETP activity to CETP mass (25.3±1.7 versus 20.4±1.6 µg CE · mg-1 · h-1; P<.05). Elevated CETP activity in nephrotic patients was shown to be associated with a significant decrease in the mean size of LDL (24.4±0.5 versus 26.3±0.5 nm; P<.0001) as well as in the relative abundance of HDL2a (29.6±1.6% versus 34.8±1.1%; P<.05). The nephrotic syndrome was characterized by a significant increase in the relative proportion of lipoprotein-bound nonesterified fatty acids (NEFAs) (35.4±7.7% versus 7.6±3.0% of total; P<.01), leading to a significant increase in the electronegative charge of LDL (-4.3±0.1 versus -3.9±0.1 mV; P<.05) and HDL (-11.5±0.1 versus -11.1±0.2 mV; P<.05). Compared with native, nonsupplemented plasma, removal of lipoprotein-bound NEFAs by addition of fatty acid–poor albumin to total plasma from nephrotic patients or control subjects significantly decreased CETP activity and specific CETP activity. Specific CETP activity no longer differed between nephrotic and control groups after albumin supplementation (19.7±1.5 versus 17.7±1.5 µg CE · mg-1 · h-1; NS). It is concluded that, in addition to elevated CETP mass concentration, lipoprotein-bound NEFAs, by increasing the negative electrostatic charge of nephrotic lipoproteins, can facilitate the CETP-mediated neutral-lipid transfer reaction in total plasma from nephrotic patients.
Key Words: lipids • nephrosis • hypoalbuminemia • kidney • triglycerides
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Introduction |
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It is now clearly established that the nephrotic syndrome is associated with hyperlipidemia, which is mainly characterized by marked increases in plasma total cholesterol, apo B, and LDL cholesterol levels.1 2 3 Moreover, patients with severe proteinuria display substantial increases in plasma TG and VLDL levels.4 5 Hypercholesterolemia in the nephrotic syndrome has also been shown to be significantly related to both hypoalbuminemia2 3 and proteinuria,6 7 8 thus indicating a direct link between the pathophysiology of the nephrotic syndrome and alterations in cholesterol metabolism. However, the reasons that may account for the observed abnormalities in plasma lipid and lipoprotein parameters still remain unclear. Earlier studies conducted in patients and animals suggested that the hyperlipidemic state associated with the nephrotic syndrome might result from either reduced catabolism of apo B–containing lipoproteins, ie, VLDL and LDL,4 5 9 10 11 12 13 or hepatic oversynthesis of apo B–containing lipoproteins in response to low oncotic pressure.1 4 14 15 However, the latter hypothesis was challenged by recent studies that revealed that the overproduction of apo B–containing lipoproteins does not constitute a constant feature in nephrotic patients.16 Furthermore, the nephrotic syndrome has recently been shown to be associated with a significant increase in plasma CETP activity,17 18 19 which in vivo catalyzes the net mass transfer of CEs from LDL and HDL to the TG-rich lipoproteins, ie, VLDL and IDL, together with the reciprocal net mass transfer of TGs from the TG-rich lipoproteins to LDL and HDL.20 21 Increased CE transfer activity in the plasma from nephrotic patients was shown to be related to both increased concentrations of CETP17 and some dysfunction of the donor and acceptor lipoprotein substrates.18 However, the abnormalities in lipoprotein particles that might account for alterations in specific CETP activity in nephrotic patients remain unknown.
In addition to the putative oversynthesis of some hepatic proteins in response to low oncotic pressure,1 4 14 15 the marked increase in lipoprotein-bound NEFAs 22 might constitute another important factor in accounting for the dyslipidemic state associated with the nephrotic syndrome. Indeed, it is now well known that marked increases in lipoprotein-bound NEFAs constitute one common feature of hypoalbuminemic states, as observed in the nephrotic syndrome and congenital analbuminemia.22 23 24 During the last decade, several studies conducted in reconstituted experimental mixtures containing isolated lipoproteins and purified CETP25 26 27 28 and in total human plasma24 29 have demonstrated that lipoprotein-bound NEFAs can increase the interaction of CETP at the lipoprotein surface. The resulting increase in CETP-mediated transfer of neutral lipids between lipoprotein fractions has been explained in terms of the ability of NEFAs to increase the electronegativity of lipoproteins, thereby raising the electrostatic interaction of CETP at the lipoprotein surface, one key step of the neutral-lipid transfer reaction.21 Therefore, in addition to a significant increase in CETP levels in nephrotic plasma,17 lipoprotein-bound NEFAs might act as activators of CETP activity by enhancing the electrostatic interaction of CETP with nephrotic lipoproteins.
The aim of the present study was to investigate the putative role of lipoprotein-bound NEFAs in enhancing CETP activity in nephrotic patients and to evaluate the relevance of such a mechanism in accounting for some of the lipoprotein disorders associated with the nephrotic syndrome. To this end, both the mass concentration and the activity of CETP were measured in total plasma from 15 patients with untreated idiopathic nephrotic syndrome and from 22 normolipidemic control subjects. The amount of lipoprotein-bound NEFAs, as well as their ability to influence the lipoprotein electronegativity, were studied in parallel.
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Methods |
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Subjects
Fifteen consecutive proteinuric patients (6 women and 9 men; mean age±SEM, 44±3 years) from the Nephrology Department of Henri Mondor Hospital, Créteil, France, were studied. All of the patients had untreated nephrotic syndrome, as defined by severe proteinuria (>3 g/d) and hypoalbuminemia (<35 g/L). Patients with diabetes mellitus, systemic lupus erythematosus, hypothyroidism, chronic hepatic disease, dyslipidemia preexisting the kidney disease, a hematocrit value <.35, or a history of familial dyslipidemia were excluded. The study patients were prescribed diuretics (furosemide in 7), hypotensive drugs (prazosin in 2, calcium channel blockers in 2, and angiotensin-converting enzyme inhibitors in 5), allopurinol in 1, and colchicine in 2. All of the patients were consuming a low-salt (<2 g/d sodium) hypocholesterolemic phase I diet as indicated in the National Cholesterol Education Program.30 Histopathological diagnoses were conducted in all nephrotic patients and were as follows: membranous glomerulonephritis in 7; focal segmental sclerosis in 4; glomerular minimal change in 2; and amyloidosis in 2. Creatinine clearance was calculated by the reciprocal of serum creatinine by the formula of Cockroft and Gault.31 The control group consisted of 22 healthy, normolipidemic subjects (8 women and 14 men) who had been recruited from the medical staff. All of the study subjects (patients and control subjects) were nonsmokers and consumed <10 g of alcohol per day. All of them had a sedentary profession. They did not practice any physical activity, except for 1 nephrotic male and 1 control male who practiced jogging for 3 to 5 h/wk and 1 nephrotic female and 1 control female who practiced aerobics for 1 to 2 h/wk. The study was approved by the ethics committee of Henri Mondor Hospital, and informed consent was obtained from all of the study subjects.
Blood Sampling
Venous blood was collected after an overnight fast into tubes
containing 0.1 mg/mL of disodium EDTA and placed immediately on
ice. Plasma was promptly separated by a 15-minute
centrifugation at 3000 rpm and maintained at 4°C
before lipoprotein analysis. Plasma aliquots for CE transfer
activity measurements were kept at -80°C until analysis.
Analysis of Plasma Lipid and Lipoprotein
Components
Total cholesterol, free cholesterol, TG,
and phospholipid concentrations were determined in total plasma and
isolated lipoprotein fractions with enzymatic reagents
(Boehringer Mannheim) and an Abbott diagnostic VP
analyzer. Plasma concentrations of HDL cholesterol
were determined after precipitation of plasma apo B-containing
lipoproteins with phosphotungstic acid and MgCl2
(Boehringer Mannheim). LDL cholesterol levels were
calculated by using the formula of Friedewald et al.32 The
CE mass content of each lipoprotein fraction was calculated as the
difference between total cholesterol and free
cholesterol and then multiplied by 1.67, thus
representing the sum of esterified cholesterol
and fatty acid moieties. Protein concentrations were measured by the
method of Lowry et al33 with serum albumin as the
standard. NEFA concentrations were determined in triplicate by using an
enzymatic kit (Wako Pure Chemicals Industries).
Preparation of Lipoprotein Fractions by Sequential
Ultracentrifugation
Lipoprotein fractions were separated by sequential
ultracentrifugation in an L8-65 ultracentrifuge
(Beckman) according to a modification of the procedure of Havel et
al.34 Densities were adjusted by adding 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.
To avoid contamination with plasma albumin, HDL2
and HDL3 fractions were washed by a second run at
d=1.125 g/mL and d=1.250 g/mL,
respectively.
Native Polyacrylamide Gel Electrophoresis
The size distribution of LDL and HDL was determined by
electrophoresis of the d<1.21 g/mL plasma fraction
on nondenaturing gradient gels ranging from 15 to 250 g/L
polyacrylamide according to the general procedure previously
described.35 Densitometric profiles of LDL and HDL were
obtained by analyzing Coomassie Brilliant Blue G–stained gels on a
GS-670 imaging densitometer (Bio-Rad). The mean apparent diameter of
LDL and HDL subfractions was determined by comparison with protein
standards (Pharmacia High Molecular Weight Protein Calibration kit) and
carboxylated latex beads (Duke Scientific).
Agarose Gel Electrophoresis and Determination of Lipoprotein
Charge
The electrophoretic mobility of LDL and HDL particles was
determined by electrophoresis of total plasma on 0.5% agarose gels
(Paragon Lipo Kit, Beckman) according to the method described by Sparks
and Phillips.36 Mean migration distances of LDL and HDL
fractions were obtained by analyzing the gel on a Bio-Rad GS-670
imaging densitometer. The surface charge of LDL and HDL was estimated
by using the equations given by Sparks and Phillips.36
Electrophoretic mobilities (U) were calculated by dividing the
electrophoretic velocity (mean migration distance in millimeters
divided by the time in seconds) by the electrophoretic potential
(voltage per gel distance in centimeters). To correct the isoelectric
point–dependent retardation effects, the following equation was
applied:
Ucorrected=(Uagarose-0.136)/1.211.
The surface potentials of lipoproteins were calculated by using the
Henry equation37 : S=Ux6
n/D, where n is the coefficient
of viscosity (0.0089 poise) and D the solvent dielectric constant.
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).38 CETP mass concentration values
were determined in quadruplicate from a calibration curve obtained with
a frozen plasma standard. Intra-assay and interassay coefficients of
variation were 4% and 6%, respectively.38 Results are
expressed in milligrams of CETP per liter of plasma.
Isotopic Assay of Plasma CETP
Plasma CE transfer activity was determined as the capacity of a
plasma sample to induce the transfer of tritiated CEs from a tracer
dose of exogenous [3H]CE-HDL3 to plasma apo
B–containing lipoproteins. In brief, total plasma (25 µL),
[3H]CE-HDL3 (2.5 nmol
cholesterol), and iodoacetate (75 nmol) were incubated for
3 hours at 37°C in a final volume of 50 µL. At the end of the
incubation period, incubation mixtures were adjusted to
d=1.068 g/mL and ultracentrifuged for 7 hours
at 50 000 rpm (269 000g) in a 50.4 Ti rotor on an L7
ultracentrifuge (Beckman). The recovered d<1.068
and d>1.068 g/mL fractions were mixed with
scintillation fluid (OptiPhase Hisafe 3, Pharmacia), and radioactivity
was assayed for 5 minutes in a Wallac 1410 liquid scintillation counter
(Pharmacia). Results were calculated as net percentages of total
radiolabeled CEs transferred from exogenous
[3H]CE-HDL3 to the plasma apo B–containing
lipoproteins.35 Variations in CE transfer rates among
various plasma samples were previously shown to be unrelated to the
relative dilution of exogenous [3H]CE-HDL3 in
the plasma HDL pool.35 CETP activity was expressed in
micrograms of plasma HDL CEs transferred per hour and per milliliter of
plasma (µg CE · mL-1 ·
h-1). The intra-assay coefficient of variation
of CE transfer assay was 5%. Plasma-specific CETP activity was
calculated as the ratio of the plasma CE transfer activity to the
plasma CETP mass concentration and expressed in micrograms of HDL CEs
transferred per hour and per milligram of CETP (µg CE ·
mg-1 ·
h-1).
Isotopic Assay of Plasma PLTP 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 as previously described.24 The experimental
system is specific for PLTP activity, since CETP does not transfer
phosphatidylcholine from liposomes to HDL. The results were expressed
as percentages of radiolabeled phosphatidylcholine transferred to the
plasma HDL fraction.
Statistical Analysis
Data medians were compared with the nonparametric
Mann-Whitney U and Wilcoxon signed rank statistic
tests, as indicated. Correlation coefficients were calculated by linear
regression analysis.
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Results |
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Nephrologic Status in Nephrotic Patients and Control Subjects
Plasma albumin concentrations were significantly decreased in nephrotic patients compared with control subjects (mean±SEM, 25±1 and 45±1 g/L, respectively; P<.0001). None of the nephrotic patients showed significant renal failure, and mean plasma creatinine concentrations were not significantly different in nephrotic patients and control subjects. Among all study subjects, creatinine clearance was constantly >35 mL/min (range, 35 to 173 mL/min), and mean values did not differ between nephrotic patients and control subjects. Unlike control subjects, all nephrotic patients showed severe proteinuria, ranging from 3 to 12.5 g/d (mean±SEM, 6.8±0.7 g/d).
Plasma Lipid Parameters in Nephrotic Patients and
Control Subjects
As shown in Table 1
, the nephrotic
syndrome was characterized by significant increases in total
cholesterol, LDL cholesterol, and TG levels.
Conversely, HDL cholesterol levels were significantly lower
in nephrotic patients than in control subjects, resulting in a marked
3.4-fold increase in the plasma LDL to HDL cholesterol
ratio. Plasma NEFA levels were in the normal range in all study
subjects, and mean concentration values were similar in nephrotic
patients and normolipidemic control subjects (Table 1).
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In accordance with previous reports,2 3 plasma albumin concentration was correlated negatively and significantly with plasma cholesterol (r=-.73, P<.0001), plasma TG (r=-.55, P<.001), and plasma LDL cholesterol (r=-.74, P<.0001) concentrations and correlated positively with HDL cholesterol concentrations (r=.43; P<.01) among the whole population studied (n=37) but not within either the nephrotic or the control group alone. Furthermore, the severity of dyslipidemia seemed to be related to the severity of nephrosis, since the 24-hour urinary protein loss in nephrotic patients (n=15) correlated positively and significantly with plasma cholesterol levels (r=.60, P<.05).
Composition of Plasma Lipoproteins
Whereas the protein content of isolated lipoproteins did not
differ between nephrotic patients and control subjects, significant
alterations in the lipid composition of individual lipoprotein
fractions were observed. As shown in Table 2, variations were characterized by
significant increases in the CE content of the apo B–containing
lipoprotein fractions, ie, VLDL, IDL, and LDL. In contrast, the TG
content of nephrotic VLDL and IDL fractions was significantly decreased
while the TG content of nephrotic HDL2 and HDL3
was significantly increased with respect to control values. These data
have resulted in the CE-to-TG ratio in lipoproteins from nephrotic
patients to be significantly higher in the VLDL (P<.001)
and IDL (P<.001) fractions but significantly lower in the
HDL2 (P<.05) and HDL3
(P<.001) fractions (Table 2). In addition to alterations in
composition of the neutral-lipid core of plasma lipoprotein fractions,
phospholipids were significantly decreased in nephrotic
HDL2 (P<.001). In contrast, the unesterified
cholesterol content of all plasma lipoprotein fractions was
unaltered. Overall, the lipid composition at the lipoprotein surface
was not markedly affected in nephrotic patients, and the
phospholipid-to-unesterified cholesterol ratios did not
differ significantly between homologous lipoprotein fractions isolated
from nephrotic patients and control subjects (Table 2).
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Size Distribution of Plasma Lipoproteins
On the basis of mean size of the major plasma LDL subfraction, all
nephrotic patients presented the typical LDL pattern B (mean
diameter of the major LDL subfraction was <25.5 nm). In contrast, all
control subjects presented the typical LDL pattern A (mean
diameter of the major LDL subfraction was >25.5 nm). Comparison of the
two groups revealed that the mean size of plasma LDL was significantly
lower in nephrotic patients (n=15) than control subjects (n=9)
(24.4±0.5 versus 26.3±0.5 nm, respectively; P<.0001).
In addition to alterations in LDL size, the distribution of plasma HDL subpopulations, ie, HDL2b, HDL2a, HDL3a, HDL3b, and HDL3c, tended to be modified in the nephrotic syndrome patients. The relative proportions of individual HDL subpopulations in nephrotic patients and control subjects, expressed as percentages of total HDL, were as follows: HDL2b, 8.9±1.3% and 9.5±1.1%, respectively; HDL2a, 29.6±1.6% and 34.8±1.1%, respectively; HDL3a, 35.2±1.3% and 33.1±1.2%, respectively; HDL3b, 22.6±2.0% and 19.7±1.7%, respectively; and HDL3 c, 3.8±0.8% and 2.8±0.3%, respectively. Differences between nephrotic patients (n=15) and control subjects (n=9) reached statistical significance with HDL2a (P<.05) only.
Plasma Phospholipid Transfer Activity
Plasma phospholipid transfer rates did not differ between
nephrotic patients (n=15) and control subjects (n=9) (6.7±0.6% versus
8.6±0.3%, respectively; P=.11).
Plasma CETP Activity
As presented in Table 3, the
rate of transfer of radiolabeled CEs from a tracer dose of tritiated
HDL3 to the plasma apo B–containing lipoprotein fraction
was significantly increased by 
2.5-fold in nephrotic patients (n=15)
compared with normal subjects (n=22). Consistently, CETP
activity expressed in micrograms of plasma HDL CEs transferred per hour
per milliliter of plasma was significantly higher in nephrotic patients
(Table 3). The increase in plasma CETP activity was explained in part
by a significant rise in the mass concentration of CETP in nephrotic
patients (Table 3). However, it is noteworthy that not only CETP mass
concentration but also the ability of CETP to interact with plasma
lipoproteins was significantly enhanced in nephrotic plasmas. Indeed,
as shown in Table 3, specific CETP activity was significantly higher in
patients with the nephrotic syndrome than in normals
(P=.028).
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Correlations between neutral-lipid transfer activity and plasma lipid
and kidney parameters are presented in Table 4. Among all study subjects (n=37), CETP
activity, CETP mass, and specific CETP activity exhibited strong and
positive correlations with concentrations of plasma
cholesterol, plasma TG, and VLDL+LDL
cholesterol and with the VLDL+LDL–to-HDL
cholesterol ratio. HDL cholesterol levels alone
did not correlate significantly with CETP mass, CETP activity, or
specific CETP activity (Table 4). Plasma TG concentrations did not
correlate siginificantly with CETP activity, specific CETP activity,
and CETP mass among nephrotic and control subjects individually.
Specific CETP activity correlated significantly with plasma
cholesterol in control subjects only (P<.01),
whereas specific CETP activity correlated significantly with the
VLDL+LDL–to-HDL cholesterol ratio in nephrotic patients
only (P<.01). Finally, whereas significant correlations
were observed between CETP mass and plasma lipid levels (except HDL) in
all study subjects (Table 4), the relationships did not reach
significance among nephrotic and control groups individually.
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As indicated in Table 4
, plasma albumin concentrations
correlated negatively with CETP activity (P<.001) and CETP
mass (P<.001), while the correlation with specific CETP
activity did not reach the significance level. The correlations were no
longer statistically significant when nephrotic patients and control
subjects were considered separately.
Effect of Lipoprotein-Bound NEFAs on the Electrostatic Charge of
Lipoproteins and CETP Activity in Plasma From Nephrotic Patients and
Control Subjects
The nephrotic syndrome was associated with a significant
increase in the relative proportion of lipoprotein-bound NEFAs
[35.4±7.7% of total in nephrotic patients (n=15) versus 7.6±3.0%
of total in control subjects (n=9), P<.01]. As shown in
Fig 1, while NEFA contents were increased
in all ultracentrifugally isolated nephrotic lipoproteins, the
difference between nephrotic patients and control subjects reached
significance only for the LDL fraction (14.8±3.5% of total plasma
NEFA in nephrotic LDL versus 0.4±0.4% of total plasma NEFA in control
LDL, P<.001). To assess the consequence of the higher NEFA
content of nephrotic lipoproteins in terms of electrostatic charge,
electrophoretic mobilities of plasma LDL and HDL fractions were
determined on agarose gels according to the general procedure described
in "Methods." Compared with control values (n=9), nephrotic
patients (n=15) showed significantly higher negative electrostatic
charge of lipoproteins, both in LDL (-4.3±0.1 mV versus -3.9±0.1
mV, P<.05) and HDL (-11.5±0.1 mV versus -11.1±0.2 mV,
P<.05) fractions (data not shown). In support of a direct
role for hypoalbuminemia in leading to the higher
electronegativity of plasma lipoprotein particles, plasma
albumin concentrations correlated positively with the mean
electrostatic charge of plasma LDL and HDL fractions among study
subjects (r=.77, P<.0001 and r=.52,
P<.01, respectively). However, when nephrotic (n=15) and
control (n=9) groups were studied separately, only the positive
correlation between albumin concentration and electrostatic
charge of LDL in nephrotic plasma reached significance
(r=.67, P<.01).
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Finally, the proportion of lipoprotein-bound NEFAs in plasma was positively and significantly correlated with CE transfer rates (r=.55, P<.01), whereas the correlation with plasma CETP activity (expressed in micrograms of HDL CEs transferred per hour per milliliter of plasma) did not reach significance (r=.36, P=.08) (data not shown). None of the CETP measurements correlated significantly with total plasma NEFA concentrations.
Effect of Albumin Supplementation on the Charge Properties
of Plasma Lipoproteins and CETP Activity
To demonstrate further that hypoalbuminemia in the
nephrotic syndrome can account for alterations in plasma CE transfer
activity, the high binding capacity of fatty acid–poor albumin
for NEFAs was used to remove them from plasma lipoproteins.
Subsequently, the effect of supplementation of total plasma with
albumin on specific CETP activity, as well as on the
electrostatic charge of LDL and HDL, was investigated (see
"Methods"). As expected, removal of lipoprotein-bound
NEFAs by fatty acid–poor albumin induced significant
alterations in the electrostatic charge of plasma lipoproteins. The
mean charge of LDL increased from -4.3±0.1 mV before albumin
supplementation to -4.1±0.1 mV after albumin supplementation
(P<.05) in nephrotic patients (n=15) while it remained
virtually unchanged in control subjects (n=9) (-3.9±0.1 mV in both
cases). The mean charge of HDL increased from -11.5±0.1 mV before
albumin supplementation to -10.6±0.1 mV after albumin
supplementation in nephrotic patients (P<.0001) and from
-11.1±0.1 mV before albumin supplementation to -10.3±0.1 mV
after albumin supplementation (P<.001) in control
subjects (data not shown).
Fig 2 shows the effect of
supplementation with fatty acid–poor albumin on normal and
nephrotic plasma with respect to CETP activity. Mean CETP activity
decreased significantly from 81.5±8.4 µg CE ·
mL-1 · h-1
before albumin supplementation to 65.2±8.0 µg CE ·
mL-1 · h-1
after albumin supplementation in nephrotic plasmas (n=15,
P<.01) and from 43.1±3.1 to 37.1±2.9 µg CE ·
mL-1 · h-1 in
control plasma (n=22, P<.001) (Fig 2). Mean specific CETP
activity decreased significantly from 25.3±1.7 µg CE ·
mg-1 · h-1
before albumin supplementation to 19.7±1.5 µg CE ·
mg-1 · h-1
after albumin supplementation in nephrotic plasma
(P<.001) and from 20.4±1.6 µg CE ·
mg-1 · h-1
before albumin supplementation to 17.7±1.5 µg CE ·
mg-1 · h-1
after albumin supplementation in control plasma
(P<.001) (Fig 2). The albumin-mediated reduction in
CETP activity was significantly greater in nephrotic than in control
plasma (-16.2±3.4 and -6.1±1.5 µg CE ·
mL-1 · h-1,
respectively; P<.01). In addition, the
albumin-mediated reduction in specific CETP activity was higher
in nephrotic than control plasma (-5.6±1.0 and -2.8±0.7 µg
CE · mg-1 ·
h-1, respectively; P<.01). Whereas
specific CETP activity was significantly higher in nephrotic than
control plasma (see Table 3), it is noteworthy that a significant
difference between the two groups disappeared after albumin
supplementation (19.7±1.5 and 17.7±1.5 µg CE ·
mg-1 · h-1,
respectively; NS).
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Discussion |
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In the past, hypoalbuminemia in nephrotic or analbuminemic patients has often been reported to be associated with hyperlipidemia and a high incidence of atherosclerosis.39 40 41 42 43 A recent prospective study conducted in a large group of nondiabetic patients reported a 2.8-fold increase in the relative risk of death from coronary artery disease in patients with the nephrotic syndrome compared with control subjects.44 In fact, whereas a high incidence of coronary artery disease has been demonstrated to be associated with an increased ratio of plasma NEFAs to albumin,45 the high risk for coronary artery disease in hypoalbuminemic patients is still not completely understood. A recent study from our group in patients with familial analbuminemia, a rare genetic disease, revealed that increased amounts of lipoprotein-bound NEFAs resulting directly from the hypoalbuminemic state increased the activity of plasma CETP, suggesting that the resulting increase in the transfer of CEs from plasma HDL to apo B–containing lipoproteins might account, at least in part, for alterations in the lipoprotein profile of analbuminemic patients.24 41 In the present study, we addressed the question of the ability of plasma NEFAs to substantially increase plasma lipid transfer activity in a frequent disease characterized by both hypoalbuminemia and hyperlipidemia, ie, the nephrotic syndrome.
As previously reported by others,1 2 3 we observed that the nephrotic syndrome is associated with a significant increase in plasma total and LDL cholesterol levels. Conversely, the HDL cholesterol concentration in nephrotic patients was significantly decreased compared with that in control subjects, leading to an important rise in the LDL-to-HDL cholesterol ratio. Although the mean plasma TG levels remained in the normal range in all cases, they were significantly higher in nephrotic patients compared with control subjects. In addition to alterations in the concentration of the main plasma lipid components, the composition of individual plasma lipoprotein fractions was significantly modified in nephrotic patients. In contrast to a recent study that reported a significant increase in the phospholipid percentage of nephrotic lipoproteins,3 the lipoprotein phospholipid percentage in the present study was significantly altered in nephrotic HDL2 only. Overall, the surface lipid composition of individual lipoproteins was not markedly affected in nephrotic patients, and the phospholipid-to–free cholesterol ratio in various lipoprotein fractions did not vary significantly between nephrotic patients and control subjects. Unlike the surface lipid composition, the neutral-lipid content of nephrotic lipoproteins differed significantly that of their normal counterparts. Indeed, the nephrotic TG-rich lipoproteins, ie, VLDL and IDL, showed an important and significant rise in their CE-to-TG ratio, whereas both HDL2 and HDL3 showed a significant decrease in their CE-to-TG ratio compared with control lipoproteins. The latter data are in very good agreement with previous findings in nephrotic patients.3 17 18
It is noteworthy that similar alterations in plasma lipoproteins, including significant increases in the CE-to-TG ratio in VLDL and the plasma LDL-to-HDL cholesterol ratio, were also observed in familial analbuminemia and shown to be associated with significant increases in plasma CETP activity.24 In accordance with previous studies,17 plasma CETP mass concentration was markedly and significantly increased in all nephrotic patients of the present study. Although in a previous study the plasma CETP mass concentration also tended to be raised above normal values in patients with analbuminemia, the difference from control values did not reach significance due to the small number of analbuminemic patients studied.24 Overall, it appears that the hypoalbuminemic state might result in vivo in a significant increase in plasma CETP mass concentration. Although no conclusive explanation can be provided at this stage, at least two main hypothesis might account for increased plasma CETP concentrations in nephrotic patients: (1) as previously observed for some hepatic proteins,1 4 CETP might be overproduced in response to the low oncotic pressure associated with hypoalbuminemia and (2) hypercholesterolemia per se might secondarily induce increases in plasma CETP levels.46 In support of increased plasma CETP activity in nephrotic patients, the isotopic transfer of CEs from HDL to plasma apo B–containing lipoproteins was significantly increased in nephrotic compared with normal plasma. Conversely, phospholipid transfer activity tended to be lower in nephrotic patients than control subjects, but the difference between the two groups did not reach significance. Finally, previous studies have reported significant decreases in postheparin lipoprotein lipase and/or hepatic lipase activities in nephrotic rats and nephrotic patients.47 48 49 50 Although alterations of endothelial lipases were not investigated in the present study, they may also contribute to the dyslipidemic state observed in nephrotic patients.
Plasma NEFAs are known to partition between albumin, membranes of endothelium and blood cells, and lipoprotein particles,51 and the distribution of NEFAs has been shown to be dependent on the NEFA-to-albumin ratio.52 In support of the latter view, direct analysis of the NEFA content of plasma lipoproteins revealed that hypoalbuminemic states, as observed in the nephrotic syndrome or familial analbuminemia, are accompanied by the shift of NEFAs from the albumin-containing plasma fraction toward the lipoprotein-containing plasma fraction.22 23 24 It is now clearly established that an increased proportion of lipoprotein-bound NEFAs can increase the electronegativity of plasma lipoproteins, thus increasing both the interaction of CETP at the lipoprotein surface and the transfer of CEs between lipoprotein particles.25 26 28 53 In accordance with previous studies,22 we observed that the nephrotic syndrome was associated with a redistribution of plasma NEFAs toward the lipoprotein fractions, and as a consequence, the electronegativity of LDL and HDL was significantly higher in nephrotic patients than in control subjects. In fact, significant relationships between the electronegativity of plasma lipoproteins and the severity of hypoalbuminemia were observed in nephrotic patients. These observations extend previous data from our group conducted both in normal subjects29 and in patients with familial analbuminemia.24 They indicate that the NEFA-mediated increase in plasma CETP activity may be of pathophysiological relevance.
To demonstrate further the role of lipoprotein-bound NEFAs in increasing CETP activity in plasma from nephrotic patients, we took advantage of the high binding capacity of albumin to remove NEFAs from plasma lipoprotein particles and to evaluate the effect in terms of lipoprotein electrostatic charge and CE transfer activity. As predicted, incubation of total nephrotic plasma with fatty acid–poor albumin significantly reduced the electronegativity of LDL and HDL fractions. Although fatty acid–poor albumin also reduced the electronegativity of normal HDL in normal plasma, no changes in normal LDL were observed. Finally, mean CETP activity and specific CETP activity were significantly reduced by the fatty acid–poor albumin treatment, and reductions in CE transfer rates were greater in nephrotic patients than in control subjects. After albumin treatment, the significant difference in specific CETP activity between control and nephrotic subjects no longer appeared. The latter observations might explain in part the reduction in lipoprotein abnormalities observed in vivo after infusions of albumin into patients with the nephrotic syndrome.54
Another important point of the present study was the modification in the size distribution of lipoprotein particles in nephrotic patients compared with normals. We observed that increased CETP activity in the nephrotic syndrome is associated with the predominance of small LDL particles, which are characteristic of LDL pattern B, whereas control subjects showed the typical LDL pattern A, with a predominance of large LDL particles.55 Since CETP has been shown to constitute one key factor in determining the size distribution of LDL particles56 57 and, in particular, in transforming the typical LDL pattern A into the typical LDL pattern B,57 the predominance of small LDLs in nephrotic patients might directly result from increased plasma lipid transfer activity. Not only the size distribution of plasma LDL but also the size distribution of plasma HDL subpopulations tended to be modified in the nephrotic syndrome. Indeed, compared with normal HDL, nephrotic HDL particle sizes tended to be shifted toward the smaller range. However, only the decrease in the relative abundance of HDL2a in nephrotic plasma reached statistical significance. Recent studies from our group have demonstrated that plasma CETP can specifically reduce the relative abundance of HDL2a, while plasma PLTP can specifically increase the relative abundance of HDL2b.58 In good agreement with the latter observations, it is noteworthy that in nephrotic patients the significant increase in plasma CETP activity was associated with a significant decrease in the HDL2a subpopulation, while neither plasma PLTP activity nor the relative abundance of HDL2b was significantly modified. Taken together, alterations in the size distribution of LDL and HDL particles in nephrotic patients point out the important role of CETP in reducing the size of lipoprotein particles in vivo.
In conclusion, the present study demonstrates that the nephrotic syndrome is associated with significant increases in both the mass concentration and the activity of plasma CETP. In particular, abnormally elevated proportions of lipoprotein-bound NEFAs in nephrotic patients have been shown to significantly increase the ability of CETP to transfer CEs between plasma lipoproteins. These observations might account at least in part for some of the lipoprotein abnormalities associated with the nephrotic syndrome.
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Acknowledgments |
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This work was supported by the Assistance Publique-Hôpitaux de Paris, the Groupe Lipides Nutrition, the Université de Bourgogne, the Conseil Régional de Bourgogne, and the Institut National de la Santé et de la Recherche Médicale (INSERM). Caroline Rousseau and Françoise Berneau are greatly acknowledged for their secretarial assistance.
Received October 4, 1997; accepted June 26, 1997.
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