© 1998 American Heart Association, Inc.
Original Contributions |
Charge Heterogeneity of LDL in Asymptomatic Hypercholesterolemic Men Is Related to Lipid Parameters and Variations in the ApoB and CIII Genes
From the Laboratoire de Biochimie Appliquée, Faculté des Sciences Pharmaceutiques et Biologiques, Châtenay-Malabry (B.V., I.M., H.T., N.M.), and the Laboratoires de Biochimie (B.V., I.M., N.M.), Biologie Moléculaire (X.J.), and the Centre de Medecine Préventive Cardiovasculaire and INSERM U28 (J.L.M., A.S.), Hôpital Broussais, Paris, France.
Correspondence to Dr Benoit Védie, Laboratoire de Biochimie, Hôpital Broussais, 96 rue Didot, 75674 Paris Cedex 14, France. E-mail benoit.vedie{at}brs.ap-hop-paris.fr
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Abstract—This study was carried out to examine the relationship between the charge on low density lipoproteins (LDLs) and lipid and clinical parameters in 104 asymptomatic dyslipidemic men and to identify biochemical and genetic factors that could contribute to the charge variability of LDL. LDL charge heterogeneity was evaluated by relative electrophoretic mobility (REM) on preformed 0.5% agarose gels and by chromatographic quantification of a minor electronegative LDL subfraction designated LDL(-). The mean REM value for LDL was 0.147±0.016 and the mean LDL(-) subfraction percentage was 5.6±2.8%. Both were positively correlated with common atherosclerotic risk factors, especially total cholesterol [for REM, r=0.27, P<0.005; for LDL(-), r=0.28, P=0.008] and LDL cholesterol [for REM, r=0.27, P=0.007; for LDL(-), r=0.26, P=0.01)] levels, and REM was positively correlated with triglycerides (r=0.27, P<0.005) and negatively with apoAI levels (r=-0.30, P<0.002). The variations in LDL charge were not due to oxidation, as measured by the lag phase and binding to the LDL receptor. The results of the 2 methods used to measure LDL charge were significantly correlated and had some identical characteristics (eg, association with LDL apoCIII content and plasma triglyceride levels in borderline and IIb dyslipidemic subjects); these methods reflect different specific features of LDL charge. The percentage of LDL(-) was correlated positively with the LDL sialic acid content (P<0.0001), whereas the REM was related to at least 2 distinct chromosomal loci. Multiple logistic analysis showed that individuals carrying minor alleles of BsrDI (P<0.05), apoCIII/SacI (P<0.01), as well as the frequent allele of XbaI (P<0.05) at the apoB and CIII gene loci had high REMs. This result suggests that LDL charge heterogeneity, which is positively correlated with the atherogenic lipid profile, is influenced by both genetic and biochemical factors.
Key Words: LDL • charge heterogeneity • apolipoprotein polymorphisms • hypercholesterolemia
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Elevated serum concentrations of LDL cholesterol are associated with an increased risk of coronary heart disease.1 2 3 Several studies have demonstrated that LDL particles vary in size,4 density,5 chemical composition,6 and electric charge.7 Hypercholesterolemic subjects may also have an altered electrophoretic mobility of their LDL on agarose.7 8 An electronegative subfraction of LDL can be isolated by ion-exchange chromatography.9 10 This subfraction may be mildly oxidized.9 We have used fast protein-liquid chromatography (FPLC) to describe a procedure based on multistep NaCl gradient elution11 to separate a minor electronegative LDL subfraction designated LDL(-).12 Although LDL(-) is not oxidized, it differs markedly from the bulk of plasma LDL and is cytotoxic to endothelial cells.12 FPLC was also used by Holvoet et al13 to characterize malondialdehyde-modified LDL isolated from acute myocardial infarction patients and seems particularly suitable for investigations of LDL charge heterogeneity.
Charge heterogeneity of LDL may also affect the structure of the apoB protein, through substitutions of differently charged amino acids or the relative orientation of charged groups. Many forms of the apoB gene are known,14 15 16 but no causal relationship between amino acid changes in apoB and the LDL charge heterogeneity has yet been found.17 Genetic variation may directly cause the charge change, as with the MspI polymorphism that reflects the replacement of arginine by glutamine at position 3611. The 3 apolipoproteins encoded by the apoAI-CIII-AIV gene cluster include apoCIII, a component of VLDL and HDL, which is the most likely to influence the metabolism of apoB-containing lipoproteins. ApoCIII inhibits lipoprotein lipase activity in vitro18 and therefore reduces hydrolysis of triglyceride-rich particles, whereas plasma apoCIII levels are correlated with elevated triglycerides.19 Our previous studies12 20 have shown that LDL electronegativity is associated with the apoCIII content, so that changes within and around the apoCIII gene may influence LDL charge.
This study was carried out to evaluate LDL charge heterogeneity in asymptomatic dyslipidemic men, to investigate any correlations with their lipid and clinical parameters, and to look for changes in LDL that might account for the charge heterogeneity, such as those due to oxidation. Last, we have examined the biochemical and genetic factors that could influence LDL charge by determining the LDL apoE, CIII, and sialic acid contents and identifying changes in the apoB and CIII gene sequences.
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Selection of Subjects
The 104 hypercholesterolemic subjects included in this study were selected by a cholesterol screening program conducted at the workplaces of several Paris companies by a group of occupational health physicians (the PCVMETRA Group: Prévention Cardiovasculaire en Médecine du Travail). The subjects selected were nondiabetic men who had total cholesterol levels >5.2 mmol/L and triglycerides <4.5 mmol/L and who had never been treated with lipid-lowering drugs. Their dyslipidemia was divided into 3 subtypes21 : borderline hypercholesterolemia (n=33), with total cholesterol levels of 5.2 to 6.19 mmol/L and triglycerides <2 mmol/L; type IIa hypercholesterolemia (n=46), with total cholesterol levels of 6.2 mmol/L or above and triglycerides <2 mmol/L; and type IIb hypercholesterolemia (n=25), with total cholesterol levels of 6.2 mmol/L or above and triglycerides of 2 mmol/L or above. Each subject was assessed for a history or symptoms of cardiovascular disease by a complete clinical examination and careful questioning. Those with cardiovascular disease were excluded from the study.22 None of the included subjects had ever undergone any coronary investigation.
Isolation of LDL
Blood was collected after a fast of at least 14 hours into
EDTA-containing Vacutainers and was immediately centrifuged for
15 minutes at 2000g at 4°C to separate the plasma. LDLs
(d=1.019 to 1.063 g/mL) were separated from fresh plasma (8
mL) by sequential ultracentrifugation in a Beckman L90
ultracentrifuge (Beckman Instruments) equipped with a
Beckman NTV 90 rotor. LDLs were dialyzed for 24 hours at 4°C against
4 changes of 100 volumes of 10 mmol/L Tris-HCl buffer, pH 7.4,
containing 1 mmol/L EDTA and were stored at 4°C in the dark.
Lipid Measurements
Serum lipids (total cholesterol,
triglycerides, HDL cholesterol, and LDL
cholesterol) were determined by standard enzymatic
methods.23 24 ApoAI and B levels were measured by
immunonephelometry using a BNA analyzer and nonspecific
antibodies (Behring). Lipoprotein(a) [Lp(a)] in serum and apoCIII and
E in LDL were determined by noncompetitive ELISA on a Biomek 1000
analyzer (Beckman Instruments).20 25
Agarose Electrophoresis
The mobility of LDL was determined by electrophoresis on
preformed 0.5% agarose gels (Beckman, Paragon Lipo kit), as described
by Sparks and Phillips.26 The LDL concentration
was normalized to a protein concentration of 1 mg/mL in 10 mmol/L
Tris-HCl buffer, pH 7.4, containing 1 mmol/L EDTA, and fatty
acid–free bovine albumin (final albumin concentration,
4 g/L) was added as a migration reference. Samples (5 µL) were
applied to the gel wells and electrophoresed (100 V, Paragon power
supply) for 30 minutes in Tris-barbital buffer, pH 9.2. The gels were
then fixed for 5 minutes in ethanol/acetic acid/water (60:10:30,
vol/vol/vol), dried, stained for 5 minutes with 0.07% Coomassie
blue in ethanol/water, destained for 10 minutes in ethanol/water
(45:55, vol/vol), and scanned with a densitometer (Preference, Sebia).
Relative electrophoretic mobilities (REMs) were determined as the LDL
to albumin migration distance ratio.
FPLC
Chromatography was
performed11 12 on an anion-exchange column (mono
QHR 5/5) and an LCC-500 programmer controlling 2 P-500 pumps. The
buffers were A, consisting of 10 mmol/L Tris-HCl, pH 7.4,
containing 1 mmol/L EDTA, and B, consisting of 1 mol/L NaCl in
buffer A. Buffers were degassed before use, and the system was operated
at 4°C. The LDL sample (0.5 g/L LDL protein) was filtered (0.2-µm
filter, Satorius), introduced into the system via a 0.5-mL loop, and
eluted at a rate of 1 mL/min by a linear gradient of 0% to 20% buffer
B for the first 20 minutes, followed by 30% buffer B for 5 minutes.
The eluate was monitored by a single-path UV monitor at 280 nm,
and the areas under the peaks were integrated with LLC-500 software.
Two LDL subfractions, designated native LDL and LDL(-), were
separated. The amount of LDL(-) was expressed as a percentage of total
LDL. The major peak of native LDL was eluted with a theoretical NaCl
concentration of 0.2 mmol/L at a retention time of 11 minutes, and
a second minor peak, LDL(-), was eluted at 17 minutes with a
theoretical NaCl concentration of 0.3 mmol/L.
LDL Sialic Acid Content
Bound sialic acid was released by mild hydrolysis (15 minutes at
80°C in 0.05 mol/L
H2SO4),27
and total LDL sialic acid content was determined according to
Warren.28
Susceptibility of LDL to Oxidation
The susceptibility of LDL to oxidation was determined as
described elsewhere.29 LDLs were dialyzed for 24
hours against 10 mmol/L phosphate buffer, pH 7.4, containing
160 mmol/L NaCl and 10 µmol/L EDTA, and LDL was diluted
10-fold with EDTA-free phosphate buffer (final EDTA concentration,
1 µmol/L). Oxidation was initiated by adding freshly prepared
CuCl2 · 2H2O
solution to a final concentration of 5 µmol/L. The kinetics of
LDL oxidation were monitored by the change in absorbance at 234 nm at
30°C in a spectrophotometer (Uvikon 930, Kontron) equipped with a
10-position automated sample changer, so that as many as 10 samples
could be monitored simultaneously. Absorbance was
recorded every 4 minutes for 4 hours. The lag phase was calculated
from the oxidation profile of each LDL preparation by drawing a tangent
to the slope of the propagation phase and extrapolating it to intercept
the initial-absorbance axis. The lag phase
represented the length of the antioxidant-protected phase
during LDL oxidation by Cu2+ in vitro.
LDL Receptor Binding Assay
LDL was labeled with
1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine
perchlorate (DiI).30 The fibroblasts were human
fetal lung fibroblasts (MRC5; Bio-Merieux, Marcy l'Etoile, France)
cultured as monolayers in Dulbecco's modified Eagle's medium (DMEM)
containing 10% FCS. Experiments were performed on confluent monolayers
of fibroblasts (2x105 cells per 60-mm dish)
plated out 5 days previously. The medium was replaced with DMEM
containing 10% lipoprotein-deficient serum 2 days before the assay.
The cell monolayer was washed twice with PBS, and 2 mL of DMEM
containing normal human DiI-labeled LDL (5 µg LDL protein per mL) was
added, together with increasing concentrations (0, 2.5, 5, 10, 20, and
50 µg/mL) of each patient's LDL for lipoprotein competition assays.
Cells were incubated at 4°C for 2 hours, washed 4x with PBS
containing 0.4% BSA at 4°C and once with PBS alone; isopropanol (1
mL) was added to each well, and the plates were gently shaken for 15
minutes. Fluorescence in the isopropanol extract was determined
with a spectrofluorometer (American Instrument Co), with excitation and
emission wavelengths of 520 and 578 nm, respectively. Cells were
dissolved in 0.2N NaOH for protein determination. The concentration of
each patient's LDL (IC50) that inhibited 50% of
the DiI-LDL binding to the cell surface was calculated as the log
concentration (µg protein per mL) versus probits. Probits were read
from a probit transformation table.
Protein Assay
Total protein was measured by Peterson's
method31 with BSA as the standard.
DNA Analysis
Genomic DNA was extracted from proteinase K–treated crude buffy
coats by a salting-out procedure.32 All forms of
polymorphism were typed by the polymerase chain reaction (PCR). DNA
was analyzed for apoB restriction fragment length
polymorphisms (RFLPs) with the endonuclease XbaI,
EcoRI, MspI, RsaI, and
BsrDI sites, all located in the coding region of the apoB
gene,14 15 and with the endonuclease
SacI, HphI, MspI, and
PstI sites in the 5' and 3' noncoding regions of the apoCIII
and apoAI genes.33 34 35 A 9-bp insertion/deletion
(I/D) variant in the signal-peptide coding region of the human apoB
gene36 and a variable number of tandem
repeats (VNTRs) at the 3' end of the apoB gene16
were also analyzed. All PCR reactions were carried out in a
final volume of 20 µL containing 100 ng genomic DNA, 0.2 U
Thermus aquaticus DNA polymerase (ATGC Biotechnologie),
200 µmol/L dNTP (Boehringer Mannheim), 10 pmol of each
primer, and 2 µL of the amplification buffer recommended by the
manufacturer (10x buffer: 500 mmol/L KCl, 15 mmol/L
MgCl2, 100 mmol/L Tris-HCl, 0.1% [wt/v]
gelatin; pH 9.0). The mixture was overlaid with 50 µL mineral oil
(Sigma). The primer sequences and conditions used to type the 7
variants of the apoB gene and the 3 variants of the apoAI-CIII gene
cluster have been reported
elsewhere.15 34 35 37 38 39 The following primers
were used to amplify the polymorphic sites at
nucleotides -625 and -482 in the apoCIII promoter region:
-625/apoCIII, 5' (sense, A): TCA TCC TGC CAT CCC TGC TG; 5'
(antisense, B): GCC CCC ACC CTG TGT GCC TC; -482/apoCIII, 5' (sense,
C): TGC TGG GAG GGG CTG TGA GA; and 5' (antisense, D): GCT GGT GAG AGG
GGA AAT GG.
Amplification was carried out with the following cycles: initial denaturation (95°C, 5 minutes), followed by 35 cycles of denaturation (95°C, 1 minute), annealing (62°C, 1 minute with primers A and B and 54°C, 1 minute with primers C and D), and primer extension (72°C, 1 minute).
PCR was done in an automated Stratagene Robocycler gradient 96. Digestion with 10 U of XbaI, EcoRI, MspI, RsaI, PstI, and SacI and 1 U of HphI restriction nucleases was performed at 37°C overnight with 20 µL of each amplification product in the buffer recommended by the supplier (Biolabs) and with 2 U of BsrDI restriction nuclease at 60°C. The restriction products were electrophoresed in 1.5% agarose gels (Eurobio) at 80 V for 1 hour. The I/D variants of the apoB gene and the restriction products of the polymorphic site at nucleotide -625 in the apoCIII promoter region were analyzed by visual inspection after electrophoresis of 15 µL of amplification product in a 3% Resophor agarose gel (Eurobio) at 80 V for 3 hours. The VNTR 3' alleles are referred to by their number of repeat units and were scored after electrophoresis of 20 µL of amplification product in a 2% agarose gel at 60 V overnight. The nomenclature used is that suggested by Boerwinkle et al.16
Statistical Analysis
Statistical analysis was performed on an Apple Macintosh
computer with JMP (SAS Institute) software. Quantitative variables
are expressed as mean±SD and range. The normal distributions of
electronegative charge parameters [REM and LDL(-)] were
checked with the Shapiro-Wilk test. All nonnormally distributed
variables were logarithmically transformed to a normal
distribution, but untransformed means are shown in the Tables. LDL
charge was analyzed according to the quartile values of age.
Linear correlation coefficients were used to describe relationships
between electronegative charge parameters and quantitative
variables. Stepwise regression analysis with backward
selection (ie, all terms entered into the model and the least
significant terms removed until the remaining terms were significant,
with a probability to enter=0.25 and a probability to leave=0.10) was
used to identify links between electronegative charge
parameters, taken as dependent variables, and lipid and
clinical parameters, as independent variables. Gene
frequencies were determined by gene counting. The
2 test was used to test for Hardy-Weinberg
equilibrium. Differences in means were examined by ANOVA. Multiple
logistic analysis was used to identify the independent
contributions of minor alleles of the variants in the apoB and
apoCIII genes to electronegative charge parameters of LDL.
The covariates included combinations of age, body mass index,
diastolic blood pressure, LDL cholesterol,
apoAI, triglycerides, LDL apoCIII, LDL apoE, and LDL sialic
acid content. A P value <0.05 was considered statistically
significant.
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Subjects
Table 1
shows the mean
frequencies of risk factors in the study group. Thirty-two percent of
patients had borderline-high cholesterol concentrations,
44% had type IIa dyslipidemia, and 24% had type IIb
dyslipidemia.
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LDL Charge Heterogeneity and Lipid and
Clinical Parameters
Figure 1 shows the electrophoretic
mobility of LDL isolated from the plasma of dyslipidemic
patients. Within-run (n=8) and day-to-day (10 days, n=8)
reproducibilities of REM determinations were 1.95% and 2.5%,
respectively. The REM distribution was normal (Figure 2A). The mean±SD (n) REM value was
0.147±0.016 (104), with a range of 0.112 to 0.195. The distribution of
the percentage of LDL(-) was skewed (Figure 2B). The mean±SD LDL(-)
value was 5.6±2.8%, with a range of 1.5% to 17.0%. The percentage
of LDL(-) was positively correlated with the REM value
(r=0.35, P=0.007).
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Figure 3 shows LDL charge
parameters for each subtype of dyslipidemia.
REM values were significantly higher in type IIa and IIb
dyslipidemias than in the borderline type, whereas the
percentage of LDL(-) was higher in type IIa dyslipidemia
than in type IIb and the borderline type. Hence, the REM values were
positively correlated with the total cholesterol
(r=0.27, P<0.005), LDL cholesterol
(r=0.27, P<0.007), triglyceride
(r=0.27, P<0.005), and apoB (r=0.23,
P<0.02) concentrations and negatively correlated with the
apoAI concentration (r=-0.30, P<0.002) (Table 2). The only lipid
parameters that were positively correlated with the
percentage of LDL(-) were total cholesterol
(r=0.28, P=0.008) and LDL cholesterol
(r=0.26, P=0.014). The REM values were lower in
hypertensive subjects (P<0.03), and the percentage of
LDL(-) was lower in smokers (P<0.03) (data not shown).
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When the 3 dyslipidemic populations were considered
separately, the only significant associations were between REM and
apoAI (r=-0.39, P<0.008) in group IIa and REM
and triglycerides (r=0.52, P<0.007)
in group IIb (Table 2
).
The parameters influencing the electronegative charge of
LDL were identified by examining the links between charge and LDL
sialic acid, apoE, and apoCIII contents. REM was correlated weakly with
the LDL apoCIII content (r=0.20, P<0.05), and a
similar trend was observed with LDL apoE content (r=0.19,
P<0.06) for the whole population (Table 2). These relations
also held for borderline subjects for apoCIII and in IIb
dyslipidemic subects for apoE (Table 2). There was a
strong, positive correlation between the percentage of LDL(-) and the
LDL sialic acid content in borderline and type IIa
dyslipidemic subjects.
The stepwise regression analysis with backward selection (Table 3), with LDL charge
heterogeneity as the dependent variable and lipid
and clinical parameters as independent variables,
showed that the electronegative parameters of LDL were
significantly associated with the LDL apoCIII content in borderline
subjects and significantly associated with the plasma
triglyceride levels in type IIb dyslipidemic
subjects. There was also a significant correlation between the
percentage of LDL(-) and the LDL sialic acid content in the overall
population and in each dyslipidemic group. The independent
association of LDL charge heterogeneity with LDL
cholesterol and apoAI levels within the whole population
was not consistent when each lipid group was
analyzed.
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LDL Charge Heterogeneity and Changes due to
Oxidation
We looked for a positive association between LDL charge
heterogeneity and change due to oxidation. The mean±SD
lag phase was 144±20 minutes (range, 105 to 195). There was no
correlation between the length of the lag phase and the electronegative
parameters of LDL, except in type IIa
dyslipidemics, for whom there was a trend with the
percentage of LDL(-) (r=-0.29, P=0.08). This
correlation was lost in multivariate analysis.
The electronegative parameters of LDL were then compared
with the binding affinity for the LDL receptor of MRC5 fibroblasts. LDL
binding studies were performed on samples from 23
hypercholesterolemic subjects
representative of the whole population. The result,
expressed as the IC50, was a mean±SD of
10.68±3.47 µg/mL (range, 3.22 to 16.36). There was no link between
the IC50 and REM or the percentage of LDL(-) in
univariate analysis.
LDL Charge Heterogeneity and Genetic
Factors
Table 4 shows the
estimated allele frequencies for the variants of the apoB, AI, and
CIII genes. The distribution of each diallelic genotype was in
Hardy-Weinberg equilibrium. There were 10 alleles of the 3' VNTR
region in the apoB gene, ranging from 31 to 51 repeats of the 15-bp
sequence and displaying a bimodal frequency distribution (maxima at 37
and 49 and minimum at 43 repeat units). The 3' VNTR alleles were
divided into small (S
43 repeats) and large (B>43 repeats)
alleles.16 The distributions of the small and
large alleles were in Hardy-Weinberg equilibrium (Table 4).
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We next examined the influence of selected genetic markers on the
charge heterogeneity of LDL. Table 5 shows the LDL charge
parameters for each genotype at the apoB and CIII
loci. There was a statistically significant association between
elevated REM values and the presence of MspI,
BsrDI, I/D, and 3' VNTR B/S minor alleles and the
absence of the XbaI cutting site at the apoB locus. There
was also a statistically significant association between elevated REM
levels and the minor alleles of polymorphic sites -625
(P=0.04) and SacI (P=0.0001) at the
apoCIII locus. There was a weak correlation between the percentage of
LDL(-) and the I/D polymorphism of the apoB gene. The
hypercholesterolemic men homozygous for the I
allele had higher levels of LDL(-) (P=0.04) than did
individuals homozygous or heterozygous for the D allele.
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Logistic regression analysis of variants of the apoB and CIII genes with the electronegative charge parameters of LDL was performed by using the variables that were significantly different in the univariate analysis. REM values were significantly elevated in carriers of the XbaI (-/-) allele (P=0.05) and in carriers of the minor alleles BsrDI (P=0.05) and apoCIII/SacI (P=0.01). The association between the I/D polymorphism of the apoB gene and the percentage of LDL(-) observed in univariate analysis was lost after adjustment for age, body mass index, LDL cholesterol, and LDL sialic acid content.
The relationships between XbaI, BsrDI, and apoCIII/SacI site variants and REM were further analyzed by construction of several combinations. Carriers of a combination defined by the XbaI (-/-) allele and the minor alleles BsrDI and apoCIII/SacI had significantly higher levels of REM (P<0.0001, univariate analysis) than did carriers of combinations with 1 or no rare alleles. This relation was also statistically significant in a multivariate model after adjustment for LDL cholesterol, triglyceride, apoAI, LDL apoCIII, and LDL apoE levels (P=0.005).
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LDL electronegativity has often been proposed as a feature of atherogenic LDL, but the various methods used to assess charge heterogeneity do not all provide the same type of information. We have evaluated LDL charge heterogeneity in hypercholesterolemic asymptomatic subjects by electrophoretic mobility on agarose gel and by anion-exchange chromatography based on FPLC.
An increased LDL electronegativity was associated with an atherogenic lipid profile. REM was positively correlated with the total cholesterol, LDL cholesterol, apoB, and triglyceride contents and negatively with apoAI in the whole population, whereas the percentage of LDL(-) was correlated with total and LDL cholesterol. As previously reported,7 there were significant differences in the charge characteristics of the LDL in different lipid groups of human subjects. The importance of triglyceride concentration for determining LDL charge was confirmed in type IIb dyslipidemia. The triglyceride enrichment might alter the lipid core and surface of LDL and affect apoB conformation.40 These alterations could then influence the net electric charge of LDL, as reported for HDL particles.41 42
Early in our analysis, we looked for changes in LDL due to oxidation that might account for LDL charge heterogeneity. We measured the susceptibility of LDL to oxidation by copper, as this modification is easy to perform and gives reproducible results. In agreement with our previous studies,12 20 oxidation does not appear to be the main change responsible for differences in LDL charge. However, the measuring of lipid peroxidation products (hydroperoxides and cholesterol oxides) may have revealed slight oxidation of LDL, which might account for some of the net electronegativity. Sevanian et al9 found that LDL(-), isolated according to Cazzolato et al,44 contained more lipid peroxides than did normal LDL from normolipidemic subjects. This apparent difference could be due to the preparation, handling, and storage of lipoproteins20 but remains to be explored. The increased negative charge of LDL particles does not appear to alter their binding to LDL receptors. However, a more detailed analysis of receptor binding affinity by Scatchard analysis for each of the dyslipidemic groups is needed, and this result must be viewed with caution.
The increased electronegativity of LDL could be due to a higher sialic acid content. In agreement with LaBelle et al,45 we found that the increased mobility of LDL on agarose gels was not influenced by the sialic acid content. However, there was a strong association between the percentage of LDL(-) and the LDL sialic acid content, both in the whole population and in each of the 3 dyslipidemic groups, strengthening our earlier observations.12
Because the apoCIII in plasma exists in several sialylated isoforms,46 47 the independent association between the LDL apoCIII content and the LDL charge may be 1 explanation for the increased sialic acid content of LDL from subjects with moderate hypercholesterolemia. The enrichment of LDL(-) in sialic acid content in other dyslipidemic groups could also be explained by the transport of different amounts of gangliosides, which contain 1 or more residues of sialic acid. Gangliosides are found in lipoproteins and predominantly in the LDL fraction in serum.48 It is unlikely that variations in the Lp(a) content contributed to the LDL charge differences, because there was no correlation between the percentage of LDL(-) and the Lp(a) content.
The charge heterogeneity of LDL could also be due to variations in the genes coding for their protein components. We have found evidence for an association between the electrophoretic behavior of LDL and changes at distinct chromosomal loci, the apoB and apoCIII genes. Because apoB is the principal structural apolipoprotein of LDL, variations in the apoB gene may result in differences in the electric charge of LDL between individuals. Many studies have investigated the contribution of variations in the apoB locus to plasma lipid levels and coronary heart disease,49 50 51 52 53 but they have given differing results.54 55 We have found that LDL REM was significantly higher in carriers of the XbaI (-/-) allele and the BsrDI minor allele. The XbaI RFLP showed a codominant relationship with REM. Because the base change that generates the XbaI RFLP occurs in the third base of codon 2488, this "silent" polymorphism per se cannot explain the change in the electrophoretic behavior of LDL.56 However, it could be in linkage disequilibrium with a causal mutation that could alter the tertiary structure and relative orientation of charged groups. The minor allele of BsrDI RFLP was also associated with an elevated REM. This polymorphism was recently described by Ilmonen et al15 and causes an AAT to AGT change in codon 1887 (resulting in the substitution of arginine for serine). The loss of a basic amino acid could lead to changes in the apoB surface, including a charge change, as shown by the impaired immunoreactivity of LDL apoB with Mab D7.2.15
The LDL REM was also associated with a variation at the apoCIII locus. There was a significant increase in LDL REM in carriers of the apoCIII SacI minor allele. Because it lies in the 3' untranslated region, the SacI site is itself unlikely to be of functional significance, but it may be in disequilibrium with an as-yet-unknown functional mutation. The promoter region, a primary site of transcriptional control, is an excellent candidate site for this mutation. Dammerman et al57 identified 5 polymorphic sites in strong disequilibrium with each other and with the SacI site. We examined polymorphic sites at nucleotides -625 and -482 in the apoCIII promoter region to determine whether any of these markers affected LDL charge heterogeneity. A significant linkage disequilibrium between these 5' sites and the 3' SacI site was found, but none of them was significantly associated with LDL charge heterogeneity in multivariate analysis. Consequently, the relationship between the electrophoretic mobility of LDL and the apoCIII SacI site is not attributable to functional variations in the 5' regulatory region of the apoCIII locus.
We also investigated the contribution of 2 other variants in the apoAI-CIII-AIV gene cluster to the electrophoretic mobility of LDL: a guanine to adenine substitution in the promoter of the apoAI gene at position -78 bp35 and a PstI site in the intergene region of apoAI and apoCIII.34 No association was found between the electrophoretic mobility of LDL and any of the apoAI variants analyzed. Hence, the 3' region of apoCIII could influence protein synthesis, as suggested by Surguchov et al.57A The association between the SacI site and hypertriglyceridemia (P<0.04, data not shown) is widely supported.58 59 Genetic changes may influence triglyceride metabolism, as shown by studies in vitro and in transgenic mice. Increased apoCIII may also impair lipolysis of triglyceride-rich lipoproteins60 61 and interfere with receptor-mediated clearance of lipolytic remnant particles.62 ApoCIII transgenic animals have lipoproteins enriched in apoCIII, and their elimination is impaired,61 63 64 resulting in the accumulation of triglyceride-rich remnant particles and hypertriglyceridemia. The apoCIII gene could affect LDL metabolism by causing a change in lipid content. Alterations in lipid composition can alter the exposure of charged amino acid residues of apoB40 and also the diameter relative to negative charge, resulting in an increased surface charge density.45
This study thus provides evidence that the electronegative charge of LDL is associated with an atherogenic lipid profile. Although the results from the 2 methods used to measure LDL charge were correlated, these methods reflect different specific features of LDL. The REM of LDL particles is at least partly associated with several genetic loci, in particular the 3' SacI site of the apoCIII gene, whereas the percentage of LDL(-) is mainly influenced by the sialic acid content of the particles. This difference might reflect differences in the charge assessed by these 2 methods. Electrophoretic mobility on agarose gel estimates the surface charge density of LDL, whereas anion-exchange chromatography based on FPLC reflects the number of negative charges on the particle surface. The origin of the influences of biochemical and genetic factors on LDL charge remains to be established.
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Acknowledgments |
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The authors thank the staff of the PCVMETRA Group for their help: P. Segond (Chairman), D. Badet, C. Baylac-Lebot, P. Bonneau, A. de Bonnières, A. Borie, M.F. Bourillon, J. Boursier, S. Bressler, M. Bru, M. Chenet, P. Corteel, C. Coulange, C. Delmotte-Devocelle, B. Demure, M.T. Douguet, M. Dubost, T. Drumare, D. Estève, M. Fragny, O. Galamand, A.M. Giard, R. Gitel, C. Guilbert, H. Hafe, F. Kiesgen, E. Lamothe, C. Lanoiselée, M.L. Leblanc, N. Le Chevanton, I. Leprince, A. Marty, D. Miara, B. Millet, J. Oziel, A. Parini, M.C. Pasteau, M Picard, M.M. Pupponi, C. Quinio, F. Raulet, M.L. Rocca, F. Szabason, P. Taine, M.C. Tardieu, C. Tarin, A. Touati-Lumbroso, and L. Troudet. We also thank Dr V. Atger for assistance and Dr O. Parkes for checking the English text.
Received March 7, 1997; accepted May 8, 1998.
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