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(Arteriosclerosis, Thrombosis, and Vascular Biology. 1995;15:208-213.)
© 1995 American Heart Association, Inc.

Articles

Cholesterol Absorption and Metabolism and LDL Kinetics in Healthy Men With Different Apoprotein E Phenotypes and Apoprotein B Xba I and LDL Receptor Pvu II Genotypes

Helena Gylling; Kimmo Kontula; Tatu A. Miettinen

From the Second Department of Medicine, University of Helsinki, Finland.

Correspondence to Tatu A. Miettinen, MD, Second Department of Medicine, University of Helsinki, Haartmaninkatu 4, FIN-00290 Helsinki, Finland.

	Abstract

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Abstract Apoprotein (apo) E, apoB Xba I, and LDL receptor gene Pvu II polymorphisms are associated with LDL cholesterol level, but little is known about cholesterol and LDL metabolism in subjects with the latter two genetic polymorphisms alone or in combination with different apoE phenotypes. We studied cholesterol absorption efficiency, cholesterol and bile acid synthesis, and LDL apoB kinetics in 52 healthy men and related the metabolic results to the apoB Xba I and LDL receptor Pvu II restriction fragment length polymorphism (RFLP) and apoE phenotypes. New findings were as follows. ApoB Xba I polymorphism was not associated with the metabolic variables of cholesterol, but LDL receptor Pvu II RFLP was associated with fractional catabolic rate for LDL apoB, cholesterol absorption, and cholesterol and bile acid synthesis. ApoE polymorphism exerted the most powerful effect on the LDL cholesterol concentration, so that the apoE2 subjects had the lowest LDL cholesterol and apoB levels and cholesterol absorption, and the highest fractional catabolic rate and bile acid and cholesterol synthesis compared with the apoE3 or especially apoE4 phenotypes in different genetic combinations. In multiple stepwise regression analysis with LDL cholesterol as the dependent and the genetic and metabolic parameters as the independent variables, 47.0% (n=35, P<.001) of the variability of LDL cholesterol was explained by the apoE polymorphism, 7.1% (P<.05) by the LDL receptor Pvu II RFLP, and 11.3% (P<.01) by bile acid synthesis, while the contribution of the apoB Xba I RFLP was nonsignificant. In conclusion, LDL cholesterol level was lowest in subjects with the 2 allele, irrespective of their Xba I or Pvu II genotypes; this was due to lower cholesterol absorption efficiency, more effective cholesterol and bile acid synthesis, and more efficient fractional catabolic rate in carriers of the 2 allele when compared with the other genetic subgroups.

Key Words: apoprotein B • apoprotein E • cholesterol absorption • cholesterol synthesis • LDL kinetics

	Introduction

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Both genetic and dietary factors contribute to the regulation of serum total and low-density lipoprotein (LDL) cholesterol levels. The association of apoprotein (apo) E phenotypes with the variability of serum cholesterol concentration, corresponding to 4% to 12% of the variability of LDL cholesterol level,^{1 2 3} is well documented in several populations.^{1 4} Genetic polymorphisms of the apoB gene locus revealed with the restriction enzymes Xba I, EcoRI, and Msp I as well as the LDL receptor gene Pvu II polymorphism are associated with serum total and LDL cholesterol levels according to many^{3 5 6 7 8 9 10 11 12} though not all^{13 14 15 16} studies. Different combinations of apoE and apoB polymorphisms may contribute to variable modifications of serum cholesterol concentration. Indeed, there is some evidence that the combined effects of apoE and apoB polymorphisms result in a very low LDL cholesterol level in subjects with the 2 allele along with the apoB X1 allele (absence of the polymorphic Xba I restriction site in exon 26).^{17 18} In addition, the presence of the intron 15 Pvu II restriction site of the LDL receptor gene was shown to eliminate the cholesterol-elevating effect of the 4 allele.^{19 20}

It is known that the 4 allele is associated with a decreased LDL receptor binding capacity in in vitro assays^{21 22} and with a decreased fractional catabolic rate (FCR) for LDL apoB in vivo compared with the 2 allele.^{23 24} In addition, the subjects with the 4 allele have a markedly high intestinal cholesterol absorption efficiency.²⁵ However, nothing is known about cholesterol metabolism in subjects with different apoB Xba I and LDL receptor gene Pvu II polymorphisms, alone or in combination with different apoE phenotypes. Thus, to evaluate the variation of the different apoprotein gene loci on cholesterol and LDL metabolism separately and in concert, we studied the absorption efficiency, synthesis, and excretion of cholesterol and the LDL apoB kinetics and related them to the apoB Xba I and LDL receptor Pvu II restriction site polymorphisms and apoE phenotypes in a healthy male population.

	Methods

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Study Group
The study group consisted of 52 healthy men, 54.5±1.0 (mean±SEM) years old. They were volunteers from a population-based, random-age cohort of the inhabitants of the Helsinki area.²⁶ In addition, individuals with 2 or 4 alleles detected in a cholesterol screening program among the personnel of two large companies were recruited to the study. The mean weight, height, and body mass index (BMI) of the study group are shown in Table 1. None had gastrointestinal, thyroid, liver, or renal disease or diabetes mellitus; none used lipid-lowering medication. All volunteered for the study, which had been accepted by the ethics committee of our hospital.

View this table: [in this window] [in a new window]   Table 1. Weight, Height, Body Mass Index, and Serum Lipids in Different Apoprotein E Phenotypes and Apoprotein B Xba I and LDL Receptor Gene Pvu II Genotypes

Study Design
Calculation of the dietary constituents was based on a food record that the subjects kept for 7 days.²⁷ During this week, they consumed a capsule containing 4-[¹⁴C]cholesterol, 22,23-[³H]sitosterol, and 200 mg Cr₂O₃ three times a day with their regular meals. At the end of this week, a 3-day stool collection was performed for cholesterol absorption and fecal steroid measurements. After stool collection, LDL turnover studies were performed, during which serum lipids, lipoproteins, and LDL apoB were analyzed four times from samples taken after a 12-hour fast.

Serum Lipids, Lipoproteins, and ApoB
Cholesterol and triglycerides were analyzed enzymatically (Boehringer Diagnostica) and apoB immunoturbidimetrically (Orion Diagnostica) with commercial kits. Lipoproteins were separated with density gradient ultracentrifugation in fixed-angle rotors (Beckman) into LDL and HDL as described in the Manual of Laboratory Operations of the Lipid Research Clinics Program.²⁸

Cholesterol Absorption and Metabolism
Cholesterol absorption was measured by the peroral double-isotope feeding method.²⁹ Chromic oxide was analyzed from the 3-day fecal specimens,³⁰ and fecal sterols were determined with gas-liquid chromatography using a 35-m-long SE-30 capillary column.^{31 32 33} Cholesterol synthesis was calculated as the difference between fecal sterols (neutral and acidic) and dietary cholesterol. The latter was quantified from the dietary records.²⁷

LDL ApoB Kinetics
Total LDL (1.019 to 1.063 g/mL) was separated from 50 mL of fasted EDTA-plasma by serial preparative ultracentrifugations, reultracentrifuged to remove contaminants, dialyzed extensively, and iodinated with ¹²⁵I by a modification of the iodine-monochloride method.^{34 35} Three days before injection of the autologous tracer, the subjects started to take peroral potassium iodide. Approximately 1 mg of labeled LDL was mixed with human serum albumin, filtered through a 0.22-µm Millipore filter, and injected intravenously. The total amount of radioactivity did not exceed 30 µCi.

After the injection, blood samples were collected for 14 days and counted for radioactivity. The die-away curves were constructed in whole plasma for ¹²⁵I-LDL. The FCR for LDL was determined by a two-pool model.³⁶ Transport rate (TR) was calculated by multiplying FCR by pool size, which was calculated to be 4.5% of body weight.

LDL kinetics were completed in 33 randomly selected subjects out of 52, who did not differ from the remaining subjects with respect to apoE, Xba I and Pvu II polymorphisms, lipid and lipoprotein profiles, or cholesterol absorption and metabolism.

ApoE phenotyping in serum samples was performed by isoelectric focusing.³⁷ Those either homozygous or heterozygous for 2 or 4 alleles were categorized as apoE2 or apoE4 phenotypes, respectively, and those subjects homozygous for the 3 allele were called apoE3.

DNA Analysis
DNA was isolated from frozen whole-blood samples with the technique described by Bell et al.³⁸ Determination of apoB and LDL receptor gene polymorphisms was carried out with Southern blot techniques described previously.^{8 39} The apoB allele lacking the polymorphic Xba I restriction site in exon 26 is designated as X1 and the allele with the restriction site present as X2. The P+ allele denotes the presence of the polymorphic Pvu II restriction site in intron 15 of the LDL receptor gene while its P- counterpart is the allele lacking this site.

Statistical Methods
Statistical significance was tested with two-tailed Student's t test, and correlation coefficients were calculated with the Pearson product-moment correlation or Spearman's rank correlation test. The genetic variables were semiquantified as subscripts in the analyses, ie, apoE2/2=1, E2/3=2, E2/4=3, E3/3=4, E4/3=5, E4/4=6, Xba I X1X1=1, X1X2=2, X2X2=3, P-P-=1, P-P+=2, and P+P+=3, respectively. One-way ANOVA was used to test the null hypothesis that genotypic variance of apoE, apoB, and the LDL receptor gene was not associated with lipid values, cholesterol absorption, and cholesterol and LDL metabolism. Multiple stepwise regression analysis was used to assess associations between LDL cholesterol (dependent variable) and the genetic polymorphisms and cholesterol metabolic variables (independent variables). The lipid values were adjusted for weight and BMI, but because the differences were minor and did not change the interpretation of results, the BMI-unadjusted values are given in the tables. A value of P<.05 was considered statistically significant.

	Results

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Of the 52 subjects, 15% had the 2 allele (apoE2 group; apoE2/2, E2/3, or E2/4), 54% had the apoE3/3 phenotype (apoE3 group), and 31% had the apoE3/4 or apoE4/4 phenotype (apoE4 group), respectively (Table 1). Of the subjects, 25% were homozygous for the absence of the apoB Xba I restriction site (X1 group; X1X1), 60% were heterozygous (X1X2), and 15% were homozygous for the presence of the restriction site (X2X2). LDL receptor gene Pvu II polymorphism was available for 43 subjects, of whom 25 (58%) were homozygous for the absence of the restriction site (P- group; P-P-), 15 (35%) were heterozygous (P-P+), and 3 (7%) subjects were homozygous for the presence of the restriction site (P+P+).

Body characteristics of the study population, lipid levels, and metabolic aspects for separate genetic polymorphisms are shown in Tables 1 and 2. Weight, height, and BMI were similar in the three apoE groups, but the serum total and LDL cholesterol and LDL apoB levels were significantly lower in the apoE2 group than in the E3 and E4 subjects. The lower cholesterol absorption efficiency and higher cholesterol and bile acid synthesis and FCR for LDL apoB in the apoE2 group (Table 2) are consistent with previous results in smaller study groups.^{23 25 40} The subscript of the apoE phenotypes was significantly related to the serum levels of total (r=.347, P<.05) and LDL (r=.452, P<.001) cholesterol, LDL apoB (r=.395, P<.01) and cholesterol absorption (r=.415, P<.01) (Figure) and negatively to FCR for LDL apoB (r=-.660, P<.001). Thus, the low LDL cholesterol and apoB levels in the apoE2 subjects were characterized by low absorption and high fecal elimination and endogenous synthesis of cholesterol and enhanced FCR for LDL apoB.

View this table: [in this window] [in a new window]   Table 2. LDL Apoprotein B Kinetics, Cholesterol Absorption Efficiency, and Cholesterol and Bile Acid Synthesis in Different Apoprotein E Phenotypes and Apoprotein B Xba I and LDL Receptor Gene Pvu II Genotypes

View larger version (19K): [in this window] [in a new window]   Figure 1. Plot shows apoprotein (apo) E phenotypes, LDL receptor gene Pvu II genotypes [closed circles=P-P- (n=24); open circles=P-P+ (n=13); shaded circles=P+P+ (n=3)], and cholesterol absorption efficiency in a random male population. Correlation coefficient of cholesterol absorption to apoE phenotypes (apoE subscript from 1 to 6) was r=.415 (P<.01) and to Pvu II phenotypes (Pvu II subscript from 1 to 3) was r=.281 (P<.05).

Serum total and LDL cholesterol and triglyceride levels and cholesterol absorption and synthesis were similar in the three apoB Xba I groups even after adjustment for weight and BMI, but LDL apoB, TR for LDL apoB, and BMI were different. The subscript of the X polymorphism was positively related to serum HDL cholesterol level (r=.280, P<.05) even after BMI adjustment and to FCR for LDL apoB (r=.290, P<.05) and negatively to BMI (r=-.342, P<.01) and TR for LDL apoB (r=-.479, P<.001). Accordingly, the high FCR and low TR for LDL apoB allowed LDL cholesterol to increase only insignificantly from 2.9 mmol/L in X2X2 to 3.6 mmol/L in X1X1.

In the different LDL receptor Pvu II genotypes, the triglyceride and HDL cholesterol levels were similar, also after BMI adjustment, but total and LDL cholesterol, FCR for LDL apoB, cholesterol absorption, and synthesis and bile acids were different (Tables 1 and 2) and were related to the subscript of the P polymorphism (LDL cholesterol, r=.319, P<.05; cholesterol absorption, r=.281, P<.05, Figure). Cholesterol absorption efficiency, the LDL cholesterol levels, and FCR for LDL apoB were lowest in the P- group, while bile acid and cholesterol synthesis were highest.

The combined effects of the apoB Xba I and apoE polymorphisms, as well as LDL receptor Pvu II and apoE polymorphisms, are shown in Table 3. LDL cholesterol levels were lowest in the apoE2/X2 and E2/P- groups because of significantly higher FCR for LDL apoB, lower cholesterol absorption efficiency (Figure; E2/P-), and more effective cholesterol synthesis in these subjects. In addition, the presence of the P+ allele increased significantly the LDL cholesterol level compared with its absence in the apoE3 subjects without relation to cholesterol metabolism, perhaps because of low FCR for LDL apoB. ApoE and Pvu II subscripts were not significantly related. In the apoE4 subjects the presence of the P+ allele did not significantly diminish the LDL cholesterol level despite trends to increase both the removal and production of LDL apoB.

View this table: [in this window] [in a new window]   Table 3. LDL Apoprotein B Kinetics, Cholesterol Absorption Efficiency, Cholesterol Synthesis, and Fecal Bile Acids in Combined Groups According to Apoprotein E Phenotype and Apoprotein B Xba I Genotype and According to Apoprotein E Phenotype and LDL Receptor Pvu II Genotype

The combination of all three polymorphisms investigated revealed five groups large enough for further analyses, although all groups were X2 heterozygotes (Table 4). The apoE2 group had the lowest LDL cholesterol level due to low cholesterol absorption efficiency, effective cholesterol synthesis, and high FCR for LDL apoB. The E3/P- subjects had a lower LDL cholesterol level and higher FCR for LDL apoB than the E3/P+ subjects as shown in Table 3, indicating that the presence of X2 had no effect on the E3/P combination. In the E4 groups, X and P did not influence the LDL cholesterol level despite a markedly high TR for LDL apoB in the E4/X2/P+ group.

View this table: [in this window] [in a new window]   Table 4. LDL Kinetics, Cholesterol Absorption, and Cholesterol and Bile Acid Synthesis in Combined Groups According to Apoprotein E Phenotype, Apoprotein B Xba I, and LDL Receptor Gene Pvu II Genotypes

With multiple stepwise regression analysis, the variability of LDL cholesterol (dependent variable) was assessed with the apoE, apoB Xba I and LDL receptor Pvu II polymorphisms, BMI, cholesterol absorption, and bile acid and cholesterol synthesis (independent variables). In this model (all variables present in 35 cases), the apoE variation explained 47.0% (R²) of the variability of the LDL cholesterol concentration (F=29.27, P<.001); the corresponding estimates for bile acid synthesis were 11.3% (F=8.72, P<.01) and for the LDL receptor Pvu II restriction fragment length polymorphism (RFLP) 7.1% (F=6.4, P<.05), while Xba I was noncontributory. When the three genetic parameters were included into the model as the only independent variables, 32.7% (P<.001) of the LDL cholesterol variation could be explained with this model.

	Discussion

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The present results revealed that apoE polymorphism exerted the most powerful effect on the variability of LDL cholesterol level and on kinetics, absorption, and metabolism of cholesterol in a group of volunteers from a random male population. In fact, this study presents for the first time the marked role of cholesterol absorption in the regulation of serum LDL cholesterol through cholesterol metabolism and LDL apoB kinetics in different apoE phenotypes on a population level. Additional new findings were that in subjects with different LDL receptor gene Pvu II genotypes, the LDL cholesterol concentration was regulated by FCR for LDL apoB, cholesterol absorption, and bile acid and cholesterol synthesis, while the apoB Xba I polymorphism seemed to have an effect only on the LDL apoB concentration and TR. However, the regulatory role of the Xba I polymorphism was eliminated by the apoE phenotypes, which eliminated only partly those of the Pvu II genotypes. The lowest LDL cholesterol values in the apoE2 group, irrespective of the combination with different Xba I or Pvu II RFLPs, were due to low cholesterol absorption efficiency, which, according to cholesterol homeostasis, upregulated hepatic cholesterol synthesis and LDL receptor activity measured by FCR for LDL apoB.

The presence of the apoB Xba I X1 allele was associated with short stature, high BMI, and low HDL cholesterol level, ie, risk factors for coronary artery disease.^{41 42} In fact, the X1 allele has been suggested to be more frequent in patients with coronary artery disease than in control subjects,^{13 14 15} but mechanisms inducing obesity⁴³ and atheromatosis are unknown. ApoB Xba I polymorphism results from a single nucleotide change at codon 2488; this substitution is neutral in nature; ie, no amino acid alteration is occurring in apoB.⁴⁴ It is currently not known whether this RFLP is in linkage disequilibrium with another DNA alteration, which is more important in apoB function. In the present study, FCR for LDL apoB was only slightly increased with the increasing subscript of X (r=.290, P<.05), which is different from some^{45 46} but not all⁴⁷ previous studies.

The increase of the LDL cholesterol level in the P+ subjects can actually be related to the slightly increased cholesterol absorption efficiency and decreased removal of LDL apoB so that corresponding synthesis of cholesterol and bile acids were low. Previous studies have shown that the P allele may explain approximately 3% to 10% of the population variance in the serum cholesterol level,^{10 11 12} a finding roughly similar to 7% for the LDL cholesterol concentration in the present series. It has been suggested that the P+ allele may eliminate the cholesterol-elevating effect of the 4 allele.^{19 20} This observation could not be confirmed in the present study with almost identical data for the level of LDL cholesterol and absorption and metabolism of cholesterol and kinetics of LDL apoB in the E4/P+ and E4/P- groups or E4/X2/P+ and E4/X2/P- groups.

The explanatory power of the apoE polymorphism on LDL cholesterol variation, 47.0% according to the multiple stepwise regression analysis, was even higher than in previous studies.^{1 2 3} There are two caveats, however, that should be kept in mind when the present data are interpreted. First, this study group was based on volunteers of a population-based, random male age cohort enriched for 2 and 4 subjects. Thus, the frequencies of the 2, 3, and 4 alleles in the present cohort (0.125, 0.547, and 0.328, respectively) differed from those of the random Finnish population (0.041, 0.733, and 0.227, respectively).⁴⁸ In contrast, the frequencies of the X1 and X2 alleles (0.55 and 0.45, respectively) and the P- and P+ alleles (0.76 and 0.24, respectively) did not significantly differ from those in random Finnish population samples.^{8 39} It is possible that the relative enrichment of this cohort for the 2 and 4 alleles may have accentuated the contribution of the apoE gene locus on LDL cholesterol variation. This may also explain why we were not able to confirm previously reported associations between the X2 allele, or the P- allele, and elevated serum LDL.^{3 5 6 7 8 9 10 11 12} Second, relatively small groups of volunteers were subjected to comparison when combined genotypes were investigated.

The explanatory power of a combination of several genetic parameters has not been studied earlier. Additional studies of the present data showed that a combination exclusively of the three assessed genetic parameters could alone explain one third of the LDL cholesterol variation, and the other metabolic parameters (cholesterol absorption, synthesis of cholesterol and bile acids, and LDL apoB kinetics), regulating the LDL cholesterol variation, were principally related to apoE genetic polymorphism. It remains open, however, by which mechanisms apoE genotypes affect cholesterol absorption efficiency. In addition, the latter factor, a significant regulator of serum cholesterol level, may modify the LDL cholesterol responses, for example, to cholesterol feeding or hypocholesterolemic treatments with drugs causing cholesterol malabsorption, especially when different genetic polymorphisms are considered.

	Acknowledgments

 
This study was supported by grants from the Finnish Heart Research Foundation, the Medical Council of the Finnish Academy, the Juho Vainio Foundation, the Paulo Foundation, and the Sigrid Juselius Foundation. The expert technical and secretarial assistance of Eeva Gustafsson, Leena Kaipiainen, Leena Saikko, Pia Hoffström, Orvokki Ahlroos, Ritva Nissilä, Elli Kempas, Anja Salolainen, Soile Aarnio, and Raija Selivuo are greatly acknowledged.

Received June 16, 1994; accepted November 9, 1994.

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