© 1997 American Heart Association, Inc.
Articles |
Prevalence of Double Pre-Beta Lipoproteinemia in Hyperlipidemic Patients Is Influenced by Gender, Menopausal Status, and ApoE Phenotype
From the Hyperlipidemia and Atherosclerosis Research Group, Clinical Research Institute of Montreal, Montreal, Quebec, Canada.
Correspondence to Dr J.S. Cohn, Hyperlipidemia and Atherosclerosis Research Group, Clinical Research Institute of Montreal, 110 Pine Ave W, Quebec, Canada, H2W 1R7.
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Abstract |
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Abstract Double pre-beta lipoproteinemia (DPBL) is a plasma lipoprotein phenotype characterized by the presence of two agarose gel electrophoretic populations of very low density lipoproteins (VLDLs, d<1.006 g/mL), ie, normal pre–ß-migrating VLDL and slow pre–ß VLDL. Slow pre–ß VLDL represents remnant lipoproteins derived from the hydrolysis of triglyceride (TG)-rich lipoproteins (TRLs), and thus DPBL is a characteristic of plasma remnant lipoprotein accumulation. To determine the prevalence of DPBL in our lipid clinic population, patients (n=2501) were selected who (1) had an unambiguous VLDL electrophoretic phenotype and could be classified as having either DPBL (DPBL+), ß-migrating VLDL (ß-VLDL+), or an absence of both (DPBL/ß-VLDL -/-) and (2) had hypercholesterolemia (HC: plasma cholesterol
6.2 mmol/L, n=1017),
hypertriglyceridemia (HTG: plasma TG
2.3 mmol/L but <15 mmol/L, n=554) or combined
hyperlipidemia (HC+HTG, n=930). Patients with TG
<2.3 mmol/L and cholesterol <5.2 mmol/L acted
as control subjects (n=343). Using a commercially available agarose gel
electrophoresis system, we identified 220
hyperlipidemic patients (8.8%) with DPBL (versus <1%
of control). The prevalence of DPBL was higher in (1) male than in
female patients (10.7% versus 6.7%), (2) postmenopausal than in
premenopausal females (7.3% versus 4.1%), and (3) patients with
HC+HTG than in those with HTG or HC alone (15.8% versus 8.3% versus
2.7%, respectively). Patients with an 
2 allele had a higher
prevalence of DPBL; ie, 26.9% of apoE 3/2 and 26.2% of apoE 4/2
patients had DPBL compared with 6.5%, 6.8%, and 7.4% of apoE 3/3,
4/3, and 4/4 patients, respectively. DPBL patients consistently
had increased levels of VLDL-C and (LDL+HDL)-TG and decreased levels of
LDL-C, and their plasma lipid profiles were intermediate between those
of ß-VLDL+ and DPBL/ß-VLDL-/- patients. These results
demonstrate that male sex, postmenopausal status in women, and the
presence of an apoE 3/2 or apoE 4/2 phenotype are associated
with an increased incidence of DPBL in hyperlipidemic
patients.
Key Words: triglyceride-rich remnant lipoproteins • double pre-beta lipoproteinemia • agarose gel electrophoresis
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Introduction |
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The ability of TRLs to promote atherosclerosis and thrombosis is generally believed to be dependent on their conversion by lipoprotein lipase into smaller and more dense TRL remnants.1 2 3 The potential atherogenicity of remnant lipoproteins is best exemplified by patients with familial dysbetalipoproteinemia (ie, type III hyperlipoproteinemia), who have a marked increase in circulating remnant lipoproteins and an increased risk of peripheral vascular disease and CAD.4 They possess genetically defective forms of apoE, which cause impaired receptor-mediated recognition and uptake of remnants by the liver.5 Plasma accumulation of remnant lipoproteins in these patients is evidenced by the appearance of VLDL (isolated by ultracentrifugation at d<1.006 g/mL), which are less negatively charged, and have ß-mobility when separated by agarose gel electrophoresis. These ß-VLDLs are enriched in apoE and cholesterol and are present in the plasma together with normal pre–ß-migrating VLDL.4
A number of studies have reported the presence of two electrophoretic populations of VLDLs in human subjects not afflicted by type III hyperlipoproteinemia.6 7 8 9 One VLDL population has normal pre–ß-mobility, while the second population of particles has slow pre–ß-mobility and is distinct from ß-VLDL. Pagnan et al have coined the term "double pre-beta lipoproteinemia" (DPBL) to describe this plasma lipoprotein phenotype.8 10 Slow pre–ß-VLDLs contain apoB-100 and are thus considered to be of hepatic rather than intestinal origin. They are enriched in cholesteryl ester, apoE, and apoC-III and are poor in TGs compared with normal VLDL; they thus resemble remnant lipoproteins.9
In the present study, we reviewed the agarose gel electrophoretic
results of patients who have been seen in our lipid clinic during the
last 11 years to investigate the prevalence of DPBL in male and female
patients with hyperlipidemia. Our principle aim was to
study the relationship between DPBL and different apoE
phenotypes. The human apoE gene is polymorphic, and three
apoE alleles (2, 3, 4) code for three common apoE isoforms
(E2, E3, E4).11 Apo E3 is the most prevalent isoform in
normolipidemic individuals and is considered to be the normal
allele. In vitro studies have demonstrated that apoE2 has <1% of
the receptor-binding capacity of apoE3 or apoE4,12 and
human kinetic studies have shown that apoE2 is cleared from the
circulation at a slower rate than either apoE3 or
apoE4.13 14 Evidence has also been presented
demonstrating that apoE2 in the VLDL of dysbetalipoproteinemic subjects
leads to impairment of the in vitro lipolytic conversion of VLDL to
LDL.15 16 Consequently, the presence of an 2 allele
has generally, though not always, been associated with an increase in
the plasma concentration of TRLs and/or their remnants after an oral
fat load.17 18 19 20 21 22
These data suggest that remnant lipoprotein accumulation in the plasma is more likely to occur in individuals homozygous or heterozygous for apoE2. Pagnan et al10 have reported, however, that in Italian and Finnish subjects, the prevalence of DPBL was significantly higher in individuals having an apo E4/4 or apo E4/3 phenotype. We have readdressed this issue in the present study by investigating the prevalence of DPBL in a much larger number of male and female patients who had well-defined VLDL electrophoretic phenotypes and who could clearly be classified as being hypercholesterolemic, hypertriglyceridemic, or both.
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Methods |
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Human Subjects
Plasma lipid records were reviewed for all patients referred to our lipid clinic between 1986 and 1996 and for whom an agarose gel electrophoretic analysis had been performed (N=3768). Patients were selected if they had an unambiguous VLDL (d<1.006 g/mL) electrophoretic profile. They were classified as having (1) DPBL, if two pre–ß-migrating VLDL bands were evident (DPBL+); (2) ß-VLDL, if a ß-migrating band was evident in the d<1.006 g/mL fraction (ß-VLDL+); or (3) normal pre–ß-VLDL, if DPBL or ß-VLDL was absent (DPBL/ß-VLDL-/-). Two hundred fifty-nine patients were excluded because they did not have a clearly defined VLDL phenotype. Of these individuals, 120 had trace amounts of slow pre–ß-migrating VLDL, 129 had trace amounts of ß-VLDL, 3 had a trace of both, and 7 had a pre–ß-VLDL, a ß-VLDL, and a slow pre–ß-migrating VLDL band. Patients were then selected according to their plasma lipid concentrations. Patients were selected if they were (1) HC, with a total plasma cholesterol concentration
6.2 mmol/L (240 mg/dL); (2) HTG,
with a plasma TG 2.3 mmol/L (200 mg/dL) but
<15 mmol/L (1300 mg/dL); or (3) both HC and HTG and
were thus classified as having combined hyperlipidemia.
Data from a total of 2501 patients were thus analyzed,
including 1207 females and 1294 males, having a mean±SD age of
50.5±14.4 years (range, 12 to 91) and 45.1±11.6 years (range, 7 to
84), respectively. Patients who were not classified as being
hyperlipidemic (ie, cholesterol <5.2
mmol/L TG <2.3 mmol/L) and who had an unambiguous
VLDL electrophoretic profile acted as the control group (n=343). This
group consisted of 144 females and 199 males with a mean±SD age of
39.1±15.0 years (range, 10 to 95); cholesterol,
4.56±0.52 mmol/L; TG; 1.28±0.49 mmol/L; and
HDL-C; 1.04±0.29 mmol/L.
Lipid and Lipoprotein Analyses
Blood was obtained after a 12-hour overnight fast and was drawn
into Vacutainer tubes (Becton Dickinson Vacutainer Systems) containing
EDTA (1.5 mg/mL). Samples were centrifuged (3000 rpm, 15
minutes, 4°C), and plasma was separated and stored at 4°C until the
time of analysis. Blood was routinely obtained from patients at
their first clinic visit, at which time they were either not taking
lipid-lowering medications or had stopped taking lipid-lowering
medications for at least 4 weeks prior to their clinic visit.
Plasma lipoproteins were separated by ultracentrifugation according to the protocol of the Lipid Research Clinics.23 Plasma samples (5 mL) were spun at d=1.006 g/mL to obtain VLDL. HDLs were separated from the d>1.006 g/mL fraction by precipitation of apoB-containing lipoproteins with heparin-manganese. Cholesterol and TG concentrations in plasma and lipoprotein fractions were measured enzymatically with an automated analyzer (ABA-100 bichromatic analyzer, Abbott Laboratories; a Cobas Mira S chemistry system, Roche Diagnostic Systems; or a Hitachi 717). All instruments had been standardized by the Centers for disease Control and Prevention and were adjusted to give similar results. Cholesterol and TG contents in LDL and HDL fractions were measured by assaying lipid concentrations in the d>1.006 g/mL fraction. Agarose gel electrophoresis24 of total plasma, d<1.006 g/mL (VLDL), and d>1.006 g/mL (LDL+HDL) fractions was carried out with a Beckman Paragon Electrophoresis System (Beckman Instruments) according to the manufacturer's instructions. In brief, samples (stored for no longer than 4 days at 4°C) were applied (5 µL) to Lipo Gels containing 0.5% agarose and 1.0% barbital buffer. Samples were separated by electrophoresis for 30 minutes at 100 V. Gels were fixed by incubation for 5 minutes in buffer containing ethanol, glacial acetic acid, and water (6:1:3, vol/vol/vol) and were stained (5 minutes with 0.07% Sudan black B stain in ethanol and water. After destaining with 45% ethanol, the gels were dried to a plastic film and stored at room temperature. ApoE phenotypes were determined after isoelectric focusing of delipidated VLDL25 or by immunoblotting of plasma separated by minigel electrophoresis.26
Statistical Analysis
Statistical analyses were performed with
SIGMASTAT statistical software (Jandel Corp). Data were
expressed as mean±SD. Student's unpaired t test was used
for comparisons between two groups. Mann-Whitney rank sum tests were
performed if data sets were not normally distributed.
Kolmogorov-Smirnov tests were used to test normality and equal variance
of data sets. The 
2 test was used to assess differences
in the distributions of categorical traits. When five or fewer
observations occurred for a particular trait, a Fisher exact test was
used to compare data in a 2x2 format. Differences with a value of
P<0.05 were considered to be statistically significant.
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Results |
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Different VLDL electrophoretic phenotypes are shown in the Figure
. Patients identified as being
DPBL/ß-VLDL-/- had a single band of pre–ß-migrating VLDL in
their d<1.006 g/mL fraction. DPBL+ patients had two
bands of pre–ß-migrating VLDL: normal pre–ß- and slow
pre–ß-VLDL. In most cases, the slow pre–ß-migrating band was less
intense than the pre-ß band; however, in a few patients they were of
equal intensity. ß-VLDL+ patients also had two electrophoretic bands
of VLDL: normal pre–ß- and ß-VLDL. The slow pre–ß-VLDL of DPBL+
patients was distinguishable from the ß-VLDL of ß-VLDL+ patients
because the former migrated farther than ß-migrating LDL in the
d>1.006 g/mL fraction, which was always run in an
adjacent lane. When a slow pre–ß-migrating band was present in
the d<1.006 g/mL fraction, a band with similar
electrophoretic migration was always observed in the corresponding
plasma sample (arrowed in the Figure). However, a slow pre-ß band in
total plasma was not always indicative of the presence of slow
pre–ß-VLDL. In these cases, the slow pre–ß-migrating band in
total plasma represented Lp(a), often referred to as
"sinking pre-ß lipoprotein,"27 which was isolated in
the d>1.006 g/mL fraction. A very faint slow pre-ß
Lp(a) band is evident in the d>1.006 g/mL fraction
of the ß-VLDL+ patient in the Figure.
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-, pre–ß-,
and ß-migrating regions of the gel are also labeled. In the total
plasma of the DPBL/ß-VLDL-/- patient shown in the first lane on the
left, these bands correspond (from top to bottom) to HDL, VLDL, and
LDL, respectively. The band above
A slow pre–ß-VLDL band was detected in 220 (8.8%) of the
hyperlipidemic patients selected for analysis
(n=2501). The prevalence of DPBL was higher (P<.01) in males than
females such that 139 of 1294 male patients (10.7%) were DPBL compared
with 81 of 1207 female patients (6.7%). The prevalence of ß-VLDL was
2.9% in males and 3.2% in females. Information on menopausal status
was available for 1007 female patients (83.4%), of whom 38.4% were
premenopausal and 61.6% were postmenopausal. Sixteen of 387
premenopausal women were DPBL (4.1%), whereas 45 of 620 postmenopausal
women were DPBL (7.3%, P<.01). The prevalence of ß-VLDL was 1.3%
in premenopausal and 4.2% in postmenopausal women. Forty-one percent
of postmenopausal women were taking estrogen, of whom 6.3% had DPBL.
DPBL was found in a slightly higher proportion of postmenopausal women
not taking estrogen (8.0%), although this difference was not
statistically significant (P=.37). As shown in Table 1, the prevalence of DPBL for males and
females together was greater in patients with combined
hyperlipidemia (15.8%) than in patients with HTG alone
(8.3%) or HC alone (2.7%). DPBL was detected in 4 of 343 (<1%)
lipid clinic patients with normal plasma lipid levels (5 patients were
apoE 2/2 and had ß-VLDL). The majority of
hyperlipidemic patients with DPBL had combined
hyperlipidemia (n=147, 67%); 46 DPBL patients were HTG
(21%) and 27 were HC (12%).
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The prevalence of DPBL was determined in hyperlipidemic
patients grouped according to their apoE phenotype (Table 2). The majority of patients had an apoE
3/3 phenotype (55.9%); 26.6% were apoE 4/3 and 9.1% were
apoE 3/2. All hyperlipidemic patients with an apoE 2/2
phenotype were ß-VLDL+. Patients with an apoE 3/2 or 4/2
phenotype had a greater incidence of DPBL (26.9% and 26.2%,
respectively) than did patients with an apoE 3/3 or 4/3
phenotype (6.5% and 6.8%, respectively; Table 2). Thus, a
significant proportion (27.7%, n=61) of DPBL+ patients had an apoE 3/2
phenotype; 7.7% (n=17) had an apoE 4/2, and 41.4% (n=91) had
an apoE 3/3 phenotype (Table 3).
The apoE phenotype distribution in DPBL+ patients was thus
different from that of the other patient groups. The majority of DPBL+
patients had an apoE 3/3 phenotype; however, compared with
DPBL/ß-VLDL-/-, the DPBL+ group had a higher proportion of patients
with an apoE 3/2 or apoE 4/2 phenotype and a smaller proportion
with an apoE 3/3 or apoE 4/3 phenotype (P<.001 by
2 analysis). The majority (85.5%) of ß-VLDL+
patients had an apoE 2/2 phenotype. ApoE phenotype
distribution of the control patient group was very similar to that
observed in the general Canadian population.28 The
DPBL/ß-VLDL-/- group in comparison had a slightly higher proportion
of patients with an apoE 4/3 phenotype, reflecting the
hyperlipidemic nature of this patient group (Table 3).
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Plasma lipid and lipoprotein cholesterol and TG
concentrations of DPBL patients with combined
hyperlipidemia (HC+HTG) were compared with those of
ß-VLDL+ and DPBL/ß-VLDL-/- (HC+HTG) patients (Table 4). Mean age and BMI of (HC+HTG) patients
with different VLDL phenotypes was not significantly different.
Total plasma TG, VLDL-TG and VLDL-C, TG in (LDL+HDL), and the VLDL-C/TG
ratio were significantly higher in DPBL+ than in DPBL/ß-VLDL-/-
patients (levels of statistical significance are indicated in Table 4).
LDL-C and HDL-C and the (LDL+HDL)-C/(LDL+HDL)-TG ratio were
significantly lower. Total plasma cholesterol and VLDL-C
and the VLDL-C/VLDL-TG ratio were in turn significantly higher and the
LDL-C and (LDL+HDL)-C/(LDL+HDL)-TG ratio significantly lower in the
ß-VLDL+ than in DPBL+ patients. Thus, compared with the
DPBL/ß-VLDL-/- patients, the VLDL-C concentration was, on average,
49% higher in DPBL+ and 151% higher in ß-VLDL+ patients. LDL-C
concentration, in contrast, was 14% and 30% lower, respectively.
Plasma remnant lipoprotein accumulation was associated with a relative
enrichment of VLDL with cholesterol, as reflected by higher
VLDL-C/VLDL-TG ratios in DPBL+ and ß-VLDL+ patients (21% and 91%,
respectively), compared with DPBL/ß-VLDL-/-. An increase in plasma
remnants was also associated with an enrichment of the LDL+HDL fraction
with TG, as indicated by the 27% lower (LDL+HDL)-C (LDL+HDL)-TG ratio
in DPBL+ and the 40% lower ratio in ß-VLDL+. The proportion of males
and females in the DPBL/ß-VLDL-/- and DPBL+ groups was not
significantly different, although the ß-VLDL+ group contained
proportionately more females. Significant differences in lipid and
lipoprotein levels of patients with different VLDL phenotypes
were unchanged when data for males and females were analyzed
separately (data not shown).
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Plasma lipoprotein profiles of HTG DPBL+ patients are compared with
those of HTG ß-VLDL+ and DPBL/ß-VLDL-/- patients in Table 5. Mean age, body mass index, and sex
distributions of the three groups were not significantly different.
VLDL-C, the VLDL-C to VLDL-TG ratio, and TG concentrations in LDL+HDL
were significantly higher in DPBL+ than in DPBL/ß-VLDL-/- patients.
LDL-C and the (LDL+HDL)-C/(LDL+HDL)-TG ratio were significantly lower.
In comparison with DPBL+, ß-VLDL+ patients had significantly higher
VLDL-C concentrations and higher VLDL-C/VLDL-TG ratios and
significantly lower LDL-C and (LDL+HDL)-C/(LDL+HDL)-TG ratios. VLDL-C
and the VLDL-C/VLDL-TG ratio were 21% and 23% higher in DPBL+ and
45% and 69% higher in ß-VLDL+ than in DPBL/ß-VLDL-/- patients.
In contrast, LDL-C and the cholesterol/TG ratio in
LDL+HDL were significantly lower, by 12% and 21% in DPBL+ and by 27%
and 26% in ß-VLDL patients, respectively.
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Plasma lipid and lipoprotein levels for the HC patients are
presented in Table 6. Only 4
ß-VLDL+ patients were classified as having isolated HC, and data for
this small subgroup were not analyzed statistically. The sex
distribution in the DPBL+ and DPBL/ß-VLDL-/- groups was not the
same, but separate analyses for males and females were not
feasible due to the small number of HC patients with DPBL. As found in
the combined hyperlipidemic group, total plasma TG
concentration, VLDL-TG and VLDL-C concentrations, TG concentration in
LDL+HDL, and the VLDL-C/VLDL-TG ratio were significantly higher in
DPBL+ than in DPBL/ß-VLDL-/- patients, while LDL-C and HDL-C
concentrations and the (LDL+HDL)-C/(LDL+HDL)-TG ratio were
significantly lower.
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Discussion |
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We have found that the prevalence of DPBL was 8.8% in hyperlipidemic male and female patients from our lipid clinic. The prevalence of DPBL was higher in (1) male than female patients (10.7% versus 6.7%); (2) postmenopausal than premenopausal females (7.3% versus 4.1%), and (3) patients with combined hyperlipidemia than in patients with HTG alone or HC alone. Patients with an
2 allele had a higher prevalence of DPBL
such that 26.9% of apoE 3/2 and 26.2% of apoE 4/2 patients had DPBL
compared with 6.5%, 6.8%, and 7.4% of apoE 3/3, 4/3, and 4/4
patients, respectively. Thus, 27.7% of DPBL patients had an apoE 3/2
phenotype compared with 7.4% of patients without DPBL and
11.4% of control patients.
The increased incidence of DPBL in hyperlipidemic
patients with an apoE 3/2 or apoE 4/2 phenotype is
consistent with the concept that a single 2 allele, ie,
heterozygosity for apoE2, is able to affect plasma TRL
metabolism. This is in agreement with a
meta-analysis of data from 45 population samples in 17
different countries, which showed that individuals with an apoE 3/2 or
4/2 phenotype tended to have a higher plasma TG level than
those with an apoE 3/3 phenotype.29 Impaired TRL
catabolism, with an associated increase in the plasma concentration of
remnant lipoproteins, is likely to be a result of (1) reduced binding
of apoE2 to lipoprotein receptors12 and slower plasma
clearance of apoE213 14 ; (2) reduced binding of apoE2 to
lipoprotein lipase and/or hepatic triglyceride
lipase,30 resulting in decreased lipolytic conversion of
VLDL to LDL15 16 ; and/or (3) the formation of
heterotrimeric complexes between apoA-II and apoE2
(AII–E2-AII),31 which could cause preferential
association of apoE2 with HDL and reduced availability of apoE for
exchange with TRL. The majority of apoE 3/2 and apoE 4/2 patients
(72.2% and 73.8%, respectively) did not, however, have DPBL, and
under these circumstances, the presence of functionally active apoE3 or
apoE4 (representing 50% or slightly less of total apoE)
was evidently sufficient to mediate normal catabolism of TRL. Thus,
heterozygosity for apoE2 is normally associated with decreased levels
of LDL cholesterol28 32 33 and a reduced risk
of atherosclerosis.34 When an additional
genetic or environmental factor is present (eg, hypothyroidism,
pregnancy, diabetes, estrogen withdrawal, or obesity), heterozygosity
for apoE2 predisposes individuals to HTG, in the same way that
homozygosity for apoE2 leads to overt
dysbetalipoproteinemia.4 Plasma TRL and remnant
lipoprotein metabolism is subsequently perturbed, the
incidence of DPBL is increased, and the risk of CAD is
enhanced.1 2 3 Whether DPBL itself is a risk factor for CAD,
however, remains to be established. DPBL has been associated in a
preliminary study with a higher prevalence of peripheral
vascular disease,35 uremia,36 and
hypothyroidism,37 the latter two of which are conditions
associated with an increased prevalence of coronary
atherosclerosis.
The present finding of increased incidence of DPBL in patients with
an 2 allele is in direct contrast to the results of Pagnan et
al,10 who showed a significantly higher prevalence of DPBL
in Italian and Finnish subjects with an apoE 4/3 or 4/4
phenotype. A possible reason might be that considerably more
subjects were investigated in the present study. Furthermore, these
individuals (predominantly French Canadians) were of different
ethnicity compared with those studied previously. A more likely
explanation, however, is that both normolipidemic and
hyperlipidemic individuals were investigated by Pagnan
et al,10 whereas only hyperlipidemic
patients were investigated in this study. This is an important
difference, since in our experience, DPBL occurs more frequently in
hyperlipidemic than in normolipidemic individuals
(8.8% versus <1% prevalence). A selection bias leading to more
hyperlipidemic subjects with a certain
phenotype (particularly those with combined
hyperlipidemia) would tend to lead to a higher
incidence of DPBL in that group. This, in fact, was the case for the
Italian sample (n=167), of whom 8 of the 39 apoE 3/2 subjects (21%)
had increased lipid levels, whereas 15 of 31 (48%) of apoE 4/3 and 4/4
subjects were hyperlipidemic. In the normolipidemic
Finnish sample (n=56), total TG, cholesterol, and apoB
levels were also higher in subjects with an apoE 4/4 phenotype,
although this difference did not reach statistical significance.
Despite the increased incidence of DPBL in
hyperlipidemic patients with an apoE 3/2 or apoE 4/2
phenotype, it is significant that a large proportion of
patients with DPBL did not, in fact, have an 2 allele (ie,
41.4% of DPBL+ were apoE 3/3 and 20.5% were apoE 4/3; Table 3
). A
number of different factors could be responsible for remnant
accumulation in these individuals—factors that ultimately cause an
imbalance between the rate of plasma remnant lipoprotein
production and clearance. Overproduction of
apoB-100–containing TRL remnants could result from increased hepatic
VLDL production or increased conversion of VLDL to IDL through
increased lipolysis. Reduced remnant lipoprotein clearance could
equally be caused by reduced or impaired hepatic lipase activity or to
a decreased hepatic lipoprotein receptor activity. Diet, hormones,
and/or lack of exercise could effect these parameters and
ultimately be responsible for the presence of DPBL in apoE 3/3 and apoE
4/3 individuals.
A definite, slow pre–ß-VLDL band was detected in 8.8% of our hyperlipidemic lipid clinic patients and in <1% of normolipidemic control patients. This may, however, represent an underestimate of the prevalence of DPBL, since patients having an ambiguous electrophoretic gel pattern with trace amounts of slow pre–ß-VLDL were excluded. If these patients are included, the overall prevalence of DPBL becomes 12.3% (n=321) in hyperlipidemic patients and 0.3% (n=10) in normolipidemic patients. Even with the inclusion of patients with trace DPBL patterns, these prevalence rates are lower than those published by other investigators. The prevalence of DPBL has been reported to be 35% in male and 25% in female healthy Swedish subjects,6 22% in healthy 50-year-old Swedish men,38 50% in normolipidemic and 30% in hyperlipidemic male and female Americans,8 34% in normolipidemic and hyperlipidemic Italians, and 39% in normolipidemic Finns.10 Variability in DPBL prevalence rates can in part be explained (as pointed out by Carlson and Olsson39 ) by methodological factors that affect the resolution of slow- and fast-migrating VLDL, such as (1) the concentration of agarose in gels used for lipoprotein separation, (2) the brand of agarose used—differences in the amount of charged groups in the agarose will cause differences in the electroendosmotic flow during electrophoresis, and (3) the sensitivity of the staining procedure. In this study, commercially available agarose gels were used, and the present results reflect the performance of these gels used on a routine basis over an extended period of time. Variability in reported prevalence rates is also a function of the subjective criteria used to define DPBL. As pointed out by Pagnan et al,8 it is sometimes difficult to make a distinction between the presence of one or two pre–ß-VLDL bands (due to "trailing" of the faster-migrating band), and prevalence rates are therefore dependent on the subjective criteria by which patients with this phenotype are selected in different laboratories.
The higher prevalence of DPBL in male versus female patients and in postmenopausal than premenopausal females is consistent with the concept that hormonal status strongly influences the plasma metabolism of potentially atherogenic apoB-100–containing lipoproteins. Compared with premenopausal women, postmenopausal women tend to have higher levels of total and LDL cholesterol, TG, and apoB.40 Menopause results in an increase in the level of all apoB-containing lipoproteins, namely, LpB, LpB:C-III, and LpB:E.41 Hormone replacement therapy, on the other hand, lowers LDL-C and increases HDL-C levels (predominantly HDL2),42 thus considerably lowering the risk of CAD.43 The present results suggest that estrogen also tends to protect against remnant lipoprotein accumulation (reflected by a reduced presence of DPBL). Although the mechanism of this action is unclear, it is probably not mediated by hepatic lipase, since estrogen administration in fact causes a reduction in hepatic lipase activity.44
Two previous studies have compared the plasma lipid and lipoprotein
concentrations of patients with and without slow
pre–ß-VLDL.6 8 In general, we found that DPBL patients
had a plasma lipid profile intermediate between patients having normal
pre–ß-VLDL and those having ß-VLDL (ie, patients with type III
hyperlipoproteinemia). This is best exemplified
by data for the combined hyperlipidemic patient group
(Table 4), which included the majority (55 of 76) of patients with
ß-VLDL. Compared with individuals without evidence of plasma
remnants, patients with mild (DPBL+) or severe (ß-VLDL+) remnant
lipoprotein accumulations had higher levels of total plasma TGs, and
higher levels of VLDL-TG and VLDL-C. Mean VLDL-TG levels were higher by
22% and 32% and mean VLDL-C levels by 49% and 251% in the two
groups, respectively. The presence of cholesterol-enriched
remnant lipoproteins in the VLDL fraction of combined
hyperlipidemic patients (Table 4) was evidenced by a
significantly higher (19%) VLDL-C to VLDL-TG ratio for the DPBL
patients (P<.001) and an even higher (88%) ratio for the ß-VLDL
patients. Similarly, the VLDL-C to total plasma TG ratio, formerly used
as a diagnostic criterion for type III
hyperlipoproteinemia,45 was
significantly increased (P<.001) in both DPBL and ß-VLDL patients
(by 21% and 91%, respectively). Consistent with the concept
that inefficient TRL catabolism leading to plasma accumulation of
remnant lipoproteins is associated with increased levels of LDL and/or
HDL-TG, (LDL+HDL)-TG concentration was higher and the (LDL+HDL)-C to
(LDL+HDL)-TG ratio significantly lower in both DPBL and ß-VLDL
patients with combined hyperlipidemia. Enrichment of
LDL and HDL with TG has been shown to be a consistent feature
of remnant lipoprotein accumulation in patients with hepatic lipase
deficiency.46 It is significant that the
cholesterol enrichment of VLDL and the relative
TG-enrichment of LDL+HDL was also evident in the HTG DPBL patients
(Table 5), who by selection had similar mean levels of total and VLDL
TGs compared with the other two patient groups. This was also true for
the HC DPBL patients, who by selection had similar mean levels of total
plasma cholesterol compared with the other groups (Table 6).
Results of angiographic regression studies have provided recent support for the concept that TRLs play an important role in the onset and development of atherosclerosis.47 There is now greater realization that patients at increased risk of premature CAD who are likely to have impaired glucose tolerance, central obesity, and/or hypertension have an atherogenic lipoprotein profile characterized by an increase in plasma levels of TRLs and their remnants, an increase in the concentration of small, dense LDL, and a reduction in plasma HDL.48 Plasma remnant lipoprotein accumulation is not just an associated characteristic of the atherogenic lipoprotein profile, since both experimental49 50 and clinical51 evidence has shown that remnant lipoproteins can themselves cause lipid accumulation in cells of the artery wall. Detection and quantification of plasma remnant lipoproteins has, however, proved to be difficult, since remnants are normally present at very low plasma concentrations and they are difficult to distinguish from their TG-rich precursors. A number of novel approaches have recently been proposed.52 53 The use of agarose gel electrophoresis to detect the presence of slow pre–ß-VLDL is an alternative approach, and the present results provide strong support for the concept that DPBL is a marker for the presence of potentially atherogenic, cholesterol-enriched remnant lipoproteins in the TRL fraction. Unfortunately, however, this technique is of limited clinical or diagnostic value, since it has the capacity to provide only semiquantitative data. It remains an interesting and potentially useful research tool, and it is important to know more about the genetics of this trait and to what extent it enhances the risk of atherosclerosis.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This work was supported by a joint University-Industry grant (PA-14006) from the Medical Research Council of Canada and Parke-Davis and by La Succession J.A. De Sève. Dr Cohn was supported by a grant from the Heart and Stroke Foundation of Québec. The excellent technical assistance of Michel Tremblay, Claudia Rodriguez, Nancy Doyle, Chantal Lefebvre, and Ann Chamberland is gratefully acknowledged. We would especially like to thank Denise Dubreuil and the other nurses of the IRCM Lipid Clinic for their assistance in obtaining patients' blood samples. The helpful advice of Dr Charles Sing is also acknowledged, as is the statistical help of Lisa Tassoni.
Received March 5, 1997; accepted May 8, 1997.
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