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Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2630-2637

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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1997;17:2630-2637.)
© 1997 American Heart Association, Inc.

Articles

Prevalence of Double Pre-Beta Lipoproteinemia in Hyperlipidemic Patients Is Influenced by Gender, Menopausal Status, and ApoE Phenotype

Jeffrey S. Cohn; Louise-Marie Giroux; Louis-Jacques Fortin; ; Jean Davignon

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.


*    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 {epsilon}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


*    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 ({epsilon}2, {epsilon}3, {epsilon}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 {epsilon}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.


*    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 {chi}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.


*    Results
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 *Results
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Different VLDL electrophoretic phenotypes are shown in the FigureDown. 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 FigureDown). 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 FigureDown.



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  		Figure 1. Agarose gel electrophoretic separation of total plasma, d<1.006 g/mL (VLDL), and d>1.006 g/mL (LDL+HDL) fractions from a patient with DPBL (DPBL+), a patient with ß-VLDL (ß-VLDL+), and a patient with neither (DPBL/ß-VLDL-/-). The DPBL/ß-VLDL-/- patient was a 42-year-old man with a plasma cholesterol concentration of 6.23 mmol/L, a plasma TG of 2.02 mmol/L, and an HDL-cholesterol of 1.11 mmol/L. The DPBL+ patient was a 58-year-old woman with a plasma cholesterol concentration of 6.49 mmol/L, a plasma TG of 5.56 mmol/L, and an HDL-cholesterol of 1.06 mmol/L. The ß-VLDL+ patient was a 43-year-old man with a plasma cholesterol concentration of 9.83 mmol/L, a plasma TG of 7.00 mmol/L, and an HDL-cholesterol of 0.78 mmol/L. The point of sample application (origin) is indicated. Lipoprotein bands were stained with Sudan black. The {alpha}-, 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 {alpha}-migrating HDL represents free fatty acids (FFA) bound to albumin. Two VLDL bands (pre-ß and "slow" pre-ß) are clearly identifiable in the d<1.006 g/mL fraction of the DPBL+ patient. The slow pre–ß-VLDL band is arrowed and is also visible in total plasma. The slow pre-ß band in the DPBL+ patient migrates further than the ß-migrating VLDL of the ß-VLDL+ patient (lane furthest to the right).

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 1Down, 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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  		Table 1. Prevalence of DPBL and ß-VLDL in Hyperlipidemic Patients (n=2501) Grouped According to Their Plasma Lipid Phenotype: Hypercholesterolemia (HC), Hypertriglyceridemia (HTG), or Combined Hyperlipidemia (HC+HTG)

The prevalence of DPBL was determined in hyperlipidemic patients grouped according to their apoE phenotype (Table 2Down). 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 2Down). 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 3Down). 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 {chi}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 3Down).


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  		Table 2. Prevalence of DPBL and ß-VLDL in Hyperlipidemic Patients (n=2501) Grouped According to Their ApoE Phenotype


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  		Table 3. Distribution of ApoE Phenotypes in Patients With (DPBL+), (ß-VLDL+), or Neither (DPBL/ß-VLDL-/-) Compared With Control Subjects

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 4Down). 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 4Down). 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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  		Table 4. Plasma and Lipoprotein Cholesterol and TG Concentrations in Combined Hyperlipidemic Patients Without DPBL or ß-VLDL, With DPBL, or With ß-VLDL

Plasma lipoprotein profiles of HTG DPBL+ patients are compared with those of HTG ß-VLDL+ and DPBL/ß-VLDL-/- patients in Table 5Down. 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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  		Table 5. Plasma and Lipoprotein Cholesterol and TG Concentrations in HTG Patients Without DPBL or ß-VLDL, With DPBL, or With ß-VLDL

Plasma lipid and lipoprotein levels for the HC patients are presented in Table 6Down. 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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  		Table 6. Plasma and Lipoprotein Cholesterol and TG Concentrations in Hypercholesterolemic Patients Without DPBL or ß-VLDL, With DPBL, or With ß-VLDL


*    Discussion
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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 {epsilon}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 {epsilon}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 {epsilon}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 {epsilon}2 allele (ie, 41.4% of DPBL+ were apoE 3/3 and 20.5% were apoE 4/3; Table 3Up). 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 4Up), 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 4Up) 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 5Up), 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 6Up).

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.


*    Selected Abbreviations and Acronyms
 
-C = cholesterol
CAD = coronary artery disease
DPBL = double pre-beta lipoproteinemia
HC = hypercholesterolemia
HTG = hypertriglyceridemia
TG = triglyceride
TRL = triglyceride-rich lipoprotein


*    Acknowledgments
 
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.


*    References
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 *References
 

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J. S. Cohn, C. Marcoux, and J. Davignon
Detection, Quantification, and Characterization of Potentially Atherogenic Triglyceride-Rich Remnant Lipoproteins
Arterioscler. Thromb. Vasc. Biol., October 1, 1999; 19(10): 2474 - 2486.
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