© 1995 American Heart Association, Inc.
Articles |
LDL Physical and Chemical Properties in Familial Combined Hyperlipidemia
Presented in part at the annual meeting of the American Society for Clinical Investigation, Washington, DC, May 4, 1984.
From the Division of Metabolism, Endocrinology, and Nutrition, Department of Medicine (J.E.H., J.J.A., J.D.B.), and the Department of Epidemiology, School of Public Health and Community Medicine (J.E.H., M.A.A.), University of Washington, Seattle, and the Donner Laboratory, Lawrence Berkeley Laboratory, University of California (R.M.K.), Berkeley.
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Abstract |
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Abstract Familial combined hyperlipidemia (FCHL) is characterized by elevations of triglyceride and/or cholesterol within families and an elevation in apoB. Although small dense LDL has been consistently associated with hypertriglyceridemia, small dense LDL persists despite reductions in triglyceride after treatment with gemfibrozil in FCHL. The current study evaluated potential differences in the distribution and chemical composition of LDL species in patients with FCHL and normolipidemic control subjects. LDL from FCHL patients was characterized by a relative abundance of a discrete LDL species with a mean peak analytic ultracentrifuge flotation rate (S°f) of 4.7±0.5 (SEM), a density of 1.041±0.001 g/mL, and a particle diameter of 250±1 Å as assessed by gradient gel electrophoresis. The major LDL species in the control subjects had a higher mean S°f rate (6.3±0.4), was more buoyant (density, 1.037±0.001 g/mL), and was larger (diameter, 262±2 Å). In addition, in a series of six LDL fractions separated by equilibrium density gradient ultracentrifugation, particle diameters were significantly smaller in all fractions from FCHL patients compared with those from control subjects. LDL particles from patients contained less free cholesterol, cholesteryl ester, and phospholipid than LDL from control subjects. The amount of triglyceride per LDL particle, however, did not differ between FCHL patients and control subjects. Differences in flotation rate and mass of the major LDL species between patients and control subjects could not be fully accounted for by differences in plasma triglyceride levels. Thus, LDL particles from FCHL patients are smaller and more dense with less cholesterol and phospholipid. Many of these differences appear to be independent of plasma triglyceride. Differences in LDL physical and chemical properties may contribute to the increase in premature coronary disease in FCHL.
Key Words: LDL • apoB • gradient gel electrophoresis • triglyceride • density gradient ultracentrifugation • familial combined hyperlipidemia
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Introduction |
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Familial combined hyperlipidemia (FCHL) is a genetic disorder of lipoprotein metabolism characterized by variable elevations in plasma lipid levels,1 VLDL, and LDL.2 Patients with FCHL have increased production of apoB, the major protein component of VLDL and LDL.3 4 Elevated apoB levels are independently associated with an increased risk of coronary disease.5 6 7 Also, families with FCHL have a high prevalence of premature coronary disease.1 8
Previous studies have shown that LDL comprises multiple distinct subspecies.9 10 11 Density subspecies have been identified by analytic and density gradient ultracentrifugation (DGUC)9 10 11 12 ; size subspecies, by nondenaturing gradient gel electrophoresis (GGE).9 13 In both healthy14 15 and FCHL16 families, small dense LDL segregates in a manner consistent with its being a mendelian trait. On the basis of complex segregation analysis, the frequency of the proposed allele for small dense LDL in FCHL families is only slightly higher than that in healthy families, 0.32 versus 0.25.14 16
Studies have consistently associated small dense LDL with elevated triglyceride levels13 16 17 18 19 20 21 and low levels of HDL,13 16 18 19 20 21 a lipid phenotype consistent with FCHL.1 2 In FCHL patients, overall LDL flotation rate is increased with decreasing plasma triglyceride concentrations.17 22 23 However, small dense LDL persisted despite substantial reductions in plasma triglyceride after treatment with gemfibrozil.23
The present study was undertaken to determine whether (1) small dense LDL is a characteristic of FCHL, (2) there are chemical differences in LDL that relate to these physical differences, and (3) plasma triglyceride concentrations alone account for the physical differences of LDL in FCHL.
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Methods |
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Subjects
The study subjects included 10 male and 4 female members of family number 41 previously reported to be affected with FCHL1 (Fig 1
). Additional affected
patients, subject I-7 from family number 6508 and
subject IV-2 from family number 448,1 also were studied.
Control subjects included 6 unrelated men and 3 unaffected women
from family 41,1 all with plasma levels of cholesterol,
triglyceride, and HDL cholesterol between the 33rd and 67th percentile
of normal, as determined by the Lipid Research Clinics Prevalence
Study,24 and normal plasma levels of apoB.25
All subjects were nondiabetic. No subjects were taking any medication
known to alter lipid metabolism, including lipid-lowering medications
or steroidal hormones. This study was approved by the human subjects
review committee. Informed consent was obtained from all subjects.
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Lipoprotein Separation
Blood was collected from subjects who had fasted at least
12 hours overnight into tubes that contained 1.4 mg/mL
Na2EDTA that were kept at 4°C throughout processing.
Plasma was separated in a Sorvall JVC centrifuge at 1500 rpm for 20
minutes. Fractions of d<1.019 g/mL and d=1.019
to 1.063 g/mL were prepared by ultracentrifugation, and the
d=1.019 to 1.063 g/mL fraction was subjected to equilibrium
DGUC as described previously.11 The d=1.019 to
1.063 g/mL fraction was dialyzed to d=1.040 g/mL in NaBr,
and 2 mL was layered in a 7-mL cellulose nitrate tube above 2.5 mL NaBr
solution at 1.054 g/mL and below 2.5 mL NaBr solution at 1.027 g/mL.
The tube was subjected to ultracentrifugation to equilibrium in a
Beckman SW45 rotor at 40 000 rpm at 17°C for 40 hours. Tube contents
were then scanned vertically for optical density at a wavelength of 455
nm for carotene with a Transidyne RFT Densitometer. Fractions (1 mL)
were collected by pipette for subsequent analysis, except for the
first and last 0.5 mL, which were discarded. There is uniform recovery
of LDL subfractions by this method.12 Background density
of each fraction was determined by refractometry from a tube containing
only the NaBr gradient. Buoyant densities of the major lipoprotein
components were determined on the basis of the positions of the most
abundant densitometric peaks along the density gradient.
Analytic Procedures
Analytic ultracentrifugation, with quantification of mass as a
function of flotation rate (S°f), was performed on
lipoproteins of d<1.063 g/mL by the procedures of Lindgren
et al.26 Mean peak analytic ultracentrifuge flotation rate
values were corrected for lipoprotein concentration and the
Johnson-Ogston effect. Nondenaturing GGE of whole plasma and of LDL
density fractions was performed with Pharmacia 2% to 16% gels as
described previously.9 For electrophoresis of plasma
samples, gels were stained for lipid with oil red O; for LDL density
fractions, gels were stained for protein with Coomassie blue R250. Gels
were scanned in a Transidyne RFT densitometer, and particle diameters
of the major peaks were estimated from a quadratic calibration curve
based on established size markers.
Chemical Determinations
Plasma total cholesterol, triglyceride, and HDL cholesterol were
determined by the methods of the Lipid Research Clinics.27
Free and unesterified cholesterol, triglyceride, and phospholipid
content of DGUC fractions were measured as described
previously,28 with adjustment of cholesterol and
triglyceride values to Lipid Research Clinics standards.27
ApoB concentration was determined by
radioimmunoassay.25
Statistical Methods
Two-tailed Student's t tests were used to compare
mean values of variables between patients and control subjects, and
probability values were corrected when multiple comparisons were
made.29 Two-way ANOVA was performed for comparison of DGUC
fractions between patients and control subjects.29 Pearson
product-moment correlation coefficients were calculated to assess the
relation of LDL measurements to triglyceride levels in FCHL patients
and control subjects.30 Linear regression was also
performed, and 95% confidence intervals (CIs) for expected value of
the mean were determined.29 Logarithms of triglyceride
values were used because of skewness in the triglyceride
distribution.
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Results |
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Table 1
gives age, sex, body mass index (BMI), and
plasma lipid values of the FCHL patients and control subjects. Because
no consistent significant differences were found between men and women
within each group, results for both sexes were pooled for statistical
analyses. The patients had a mean BMI 12% higher than control subjects
and higher mean levels of plasma cholesterol (P<.05),
triglyceride (P<.01), and apoB (P<.01), as well
as slightly lower levels of HDL cholesterol (P<.05).
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The mean analytic ultracentrifuge schlieren profiles of lipoproteins of
d<1.063 g/mL demonstrated differences between patients and
control subjects (Fig 2). The mean total mass
concentrations of the major lipoprotein categories according to
S°f are also shown for each group. Mean VLDL mass
(S°f=20 to 400) was significantly increased in the FCHL
patients (P<.05), consistent with their higher mean plasma
triglyceride concentration. IDL of S°f=12 to 20 was also
significantly higher in the patients (P<.05). Mass of LDL
of S°f=0 to 12 was also higher in the patients, with a
major component of mean peak corrected S°f of 4.7±0.5,
whereas the major component in control subjects had a mean peak
S°f of 6.3±0.4. Fig 3 shows the
differences in LDL mass between patients and control subjects across
the LDL S°f range. Mass of LDL of S°f=2 to
6 was significantly greater in patients; mass of LDL of
S°f=7 to 9 was lower.
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Because both size and density of lipoprotein particles contribute to
their flotation rates in the analytic ultracentrifuge, techniques that
separate LDL species on the basis of these characteristics were used to
further investigate the differences in LDL between FCHL patients and
control subjects. DGUC revealed consistent differences in the density
banding patterns in the patients compared with control subjects. Fig 4 shows four representative profiles (two FCHL
patients and two control subjects). In all FCHL patients, the major LDL
band was in fraction 4, with a mean density of 1.042±0.002 g/mL, and
most patients had a smaller peak in fraction 3 (d=1.035 to
1.040 g/mL) of varying height. In the control subjects, the peak of the
most abundant LDL species was in fraction 3, with a mean density of
1.037±0.001 g/mL, and there were minor peaks or shoulders in fraction
2 or 4 in some individuals. Mean apoB concentrations in 1-mL fractions
across the LDL density gradient (Table 2) showed highly
significant increases in apoB in fractions 4 through 6 in patients
compared with control subjects, accounting for most of the increase in
plasma and LDL apoB levels in these FCHL patients.
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The size distribution of LDL subspecies was determined with GGE.
Representative densitometric scans for the same two patients and
two control subjects (Fig 5) show a characteristic
increase in LDL of approximately 250 Å in the patients, whereas the
major species in control subjects had a larger diameter. Mean peak
particle diameters of the major peak of LDL in plasma and from DGUC
fractions show consistently smaller LDL among FCHL patients compared
with control subjects (Table 3). Mean peak particle
diameters of LDL (from plasma or isolated whole LDL) show a
statistically significant difference between FCHL subjects and control
subjects (P<.01). Furthermore, the diameters of LDL
particles isolated in each of the density fractions are significantly
smaller among the FCHL patients compared with control subjects
(P<.001 by ANOVA). Among the fractions, those having
particle diameters most similar to the major species in total LDL are
fraction 4 for patients and fraction 3 for control subjects (Table 2
).
These results parallel the measurements of distributions of total mass
and protein among the density fractions (Fig 3 and Table 2).
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Lipoprotein mass and chemical composition were analyzed in the four
DGUC fractions (2 through 5) that contain the majority of the LDL
(Table 4). As expected, percent apoB increased with
increasing density of the fractions in both groups of subjects. The
values for the FCHL patients, however, were consistently higher than
those for control subjects (P<.0005 by ANOVA). The relative
contents of both free cholesterol and cholesteryl ester were
significantly reduced in all LDL fractions from FCHL patients. Percent
phospholipid was also slightly but not significantly reduced. In FCHL
patients, LDL in fractions 2 and 3 was relatively enriched in
triglyceride (although not significantly at P<.05), while
there was a statistically significant reduction in triglyceride content
in fraction 5.
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The mass ratios of the various lipid components to apoB in DGUC density
fractions (Table 5) indicate compositional differences
in LDL particles between FCHL patients and control subjects. LDL
particles among all density fractions from FCHL patients had less free
cholesterol, cholesteryl ester, and phospholipid (P<.05 by
ANOVA), while not differing in triglyceride content. No mass ratios
were significantly correlated with plasma triglyceride levels in
patients or control subjects, except for a positive correlation of
triglyceride/ apoB ratio in fraction 3 in patients (P<.01)
and a negative correlation of this ratio in fraction 5 in control
subjects (P<.05).
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The relationships between plasma triglyceride levels and LDL physical
properties among FCHL patients and control subjects were also examined.
LDL S°f rate and log plasma triglyceride concentration
were inversely correlated among patients (Fig 6)
(r=-.66, P<.01). Among control subjects, the
same trend was observed (r=-.59, P=.10). Peak
LDL S°f rate was strongly correlated with particle
diameter and buoyant density of the most abundant LDL species in the
patients (r=.80 and .75, respectively, P<.01).
Consequently, relationships of size and density of the major LDL
species to triglyceride levels in patients and control subjects
parallel those shown for S°f in Fig 6 (data not shown).
These correlations suggest that at least some of the reduction of
flotation rate, buoyancy, and size of the major LDL species in FCHL
patients may be related to higher plasma triglyceride levels. However,
it is apparent from Fig 6 that peak LDL S°f rates of the
eight FCHL patients with triglyceride levels overlapping those of the
control group (101 to 168 mg/dL) were all lower than those of the
control subjects. In addition, the values for all of the control
subjects fall above the upper 95% confidence interval of the
regression line for the FCHL patients. Also, the mass of LDL in the
S°f interval of 4 to 5, which showed the greatest
difference in concentration between FCHL patients and control subjects
(Fig 3), was not related to triglyceride level in FCHL patients (Fig 7). In contrast, there was a statistically significant
positive correlation between S°f=4 to 5 LDL mass and
plasma triglyceride level among the control subjects (r=.76,
P<.02).
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Discussion |
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In the present report, we have identified differences in LDL physical properties and chemical composition from patients with FCHL compared with LDL from normolipidemic subjects. The most abundant LDL species in the FCHL patients had a mean peak S°f of 4.7 (Fig 2
), a mean density of 1.042 g/mL (Table 2 and Fig 4), and a mean
diameter of 250 Å (Table 3 and Fig 5). In contrast, the major LDL
species in the control subjects has a mean peak S°f of
6.3, a mean density of 1.037 g/mL, and a mean diameter of 262 Å. The
characteristics of the major LDL species in the control subjects
conform to those used to define LDL-II and LDL subclass phenotype A,
while the dominant LDL species in the FCHL patients fall within the
ranges reported for LDL-III and LDL subclass phenotype
B.9 11 18
Plasma triglyceride levels have been reported to correlate inversely
with LDL S°f rate19 31 and other indices of
LDL density and size distribution.13 16 17 18 19 20 22 32 33
However, despite the higher mean plasma triglyceride concentrations
among FCHL patients compared with control subjects, this triglyceride
difference does not fully account for the lower flotation rates,
smaller size, and greater densities of the major LDL species among the
FCHL subjects in this study. Among FCHL patients with plasma
triglyceride levels within the range of the control subjects, the major
species of LDL had a lower flotation rate (Fig 6). Furthermore, the
mass of LDL of S°f=4 to 5 did not increase as a function
of plasma triglyceride concentration in the FCHL patients as it did
among the control subjects (Fig 7). The lack of such a relation in the
FCHL patients indicates that their increased levels of
S°f=4 to 5 cannot be accounted for by increased plasma
triglyceride levels. Thus, consistent with the results from treatment
of hypertriglyceridemia in FCHL patients,23 the presence
of smaller dense LDL in these patients cannot be entirely explained by
elevated plasma triglyceride levels.
Isolated density fractions of LDL reveal differences in the chemical
composition of LDL between FCHL patients and control subjects. The
absolute amount of apoB was higher among FCHL patients in all fractions
of LDL (Table 4). Ratios of cholesteryl ester, free cholesterol, and
phospholipid to apoB in LDL fractions were reduced among FCHL patients
compared with control subjects. However, the ratio of triglyceride to
apoB was not different between the two groups. Because there is one
apoB molecule per LDL particle,34 these ratios
represent the lipid composition per LDL particle. Thus, the
smaller size of LDL particles in FCHL patients is a reflection of less
cholesteryl ester in the core and less surface free cholesterol and
phospholipid.
Among normolipidemic subjects, a similar relation between LDL size and LDL-particle composition was observed.35 In that study, LDL-particle free cholesterol, phospholipid, and cholesteryl ester were positively correlated with LDL size, while there was no significant relation between LDL-particle triglyceride and LDL size. Thus, LDL from FCHL patients is depleted in cholesterol and phospholipid, as is small dense LDL from normolipidemic subjects.
A recent complex segregation analysis in FCHL families suggests a threshold model for FCHL36 that is influenced by a major gene controlling the level of apoB37 38 interacting with a common gene controlling small dense LDL (LDL subclass phenotype B).14 16 Homozygotes for the putative apoB-elevating allele express FCHL. Among heterozygotes at this locus, the presence of the proposed allele for LDL phenotype B increases the probability of being affected with FCHL. Consistent with this model is the bimodal distribution of apoB levels among LDL phenotype B subjects in FCHL families.39 The similar depletion of cholesterol and phospholipid in small dense LDL among both FCHL and normolipidemic subjects implies common determinants of small dense LDL in both FCHL and non-FCHL individuals.
It has been reported that among hypertriglyceridemic patients, small LDL is enriched with triglyceride.40 41 However, the degree of hypertriglyceridemia among the subjects in the current study is much lower (triglyceride range among FCHL patients, 107 to 378 mg/dL) than severe hypertriglyceridemia (>1000 mg/dL), which can lead to triglyceride-enriched LDL. In studies of moderately hypertriglyceridemic subjects, triglyceride per LDL particle was not reported.22 32 However, both percent LDL triglyceride and percent LDL protein (apoB) were inversely correlated with LDL diameter or DGUC relative position (Rp).22 32 In addition, percent LDL free and esterified cholesterol22 32 and percent LDL phospholipid32 were positively correlated with LDL diameter or Rp. These results are entirely consistent with the LDL-particle compositions observed in the present study.
Recently, Dejager et al42 reported no differences in LDL composition between "combined hyperlipidemic" patients (defined as type IIB hyperlipidemia) and control subjects, despite differences in density of LDL between the two groups. However, their patient population appears to be heterogeneous, with three of nine subjects having tendinous xanthomas, pathognomonic of familial hypercholesterolemia or familial defective apoB-100. This etiologic heterogeneity may have obscured the differences among specific patient populations and may explain the apparent differences with the present study.
Although the association between small dense LDL and coronary disease is well established from cross-sectional studies,18 19 20 22 32 43 the mechanism has yet to be elucidated. Dense LDL fractions have been shown to be more susceptible to in vitro metal ion oxidation.44 45 There are conformational changes in apoB that affect its interaction with the LDL receptor in small LDL from hypertriglyceridemic subjects, although this may be unique to severe hypertriglyceridemia.46 Small LDL may also be a marker for high plasma triglyceride and low HDL (the atherogenic lipoprotein profile)47 or the insulin-resistance syndrome.48 49 50 It is possible that these mechanisms or others—eg, an increase in LDL particle number2 5 51 52 —may lead to the increased risk of premature coronary disease in FCHL.
In conclusion, among FCHL patients, there is a predominance of small dense LDL. As previously reported23 this is a persistent trait in FCHL, which is independent of plasma triglyceride concentrations. LDL particles from FCHL patients are depleted in cholesteryl ester from the core and of surface free cholesterol and phospholipid. LDL particle triglyceride is not different between FCHL patients and control subjects. This depletion in cholesterol and phospholipid is similar to what is seen in small dense LDL from non-FCHL subjects. Plasma triglyceride, although elevated in these FCHL patients, does not account for the differences in LDL physical properties.
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Acknowledgments |
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This study was supported by National Institutes of Health (NIH) program project grant HL-30086. This study was conducted at the Lawrence Berkeley Laboratory (US Department of Energy contract DE-AC03-76SF00098 to the University of California) and at the Clinical Research Center, University of Washington Medical Center (NIH grant RR-37). We greatly appreciate the technical assistance of Dennis Duncan, Weiling King, Maria Culala, and Janet Adolphson. We thank Dr Frank Lindgren and staff for the analytic ultracentrifugation measurements.
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Footnotes |
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Reprint requests to John D. Brunzell, MD, Division of Metabolism, Endocrinology, and Nutrition, Department of Medicine, Mail Stop RG-26, University of Washington, Seattle, WA 98195.
Received October 14, 1994; accepted January 25, 1995.
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