© 1997 American Heart Association, Inc.
Articles |
Polymorphisms of the Genes Encoding Apoproteins A-I, B, C-III, and E and LDL Receptor, and Cholesterol and LDL Metabolism During Increased Cholesterol Intake
Common Alleles of the Apoprotein E Gene Show the Greatest Regulatory Impact
the Department of Medicine, Division of Internal Medicine (H.G., K.K., H.E.M., T.A.M.), and the Institute of Biotechnology (U.-M.K.), University of Helsinki, Finland.
Correspondence to Tatu A. Miettinen, MD, Department of Medicine, Division of Internal Medicine, University of Helsinki, Haartmaninkatu 4, FIN-00290 Helsinki, Finland.
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Abstract |
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Genetic and dietary factors regulate serum cholesterol level, but detailed investigations into their interactions have not been established. We assessed the effects of apoprotein (apo) E phenotype and polymorphic alleles of the apo A-I, apo B, apo C-III, and LDL receptor genes, separately and together, on regulation of serum LDL cholesterol level. The study group consisted of 29 middle-aged men, and cholesterol absorption, bile acid, and cholesterol synthesis and LDL apo B kinetics were studied in these men during low- and high-cholesterol diets. The six apo B alleles were identified on the basis of Xba I, EcoRI, and Msp I restriction fragment length polymorphism (RFLP), the apo A-I alleles with the Msp I RFLP, and the apo C-III and LDL receptor alleles corresponded to the Sst I and PvuII RPLPs of these genes, respectively. During low cholesterol intake, LDL cholesterol levels were similar in all of the genetic groups except for men with apo E2 phenotype. They had significantly (P<.05) lower levels of LDL apo B and cholesterol than men without the
2 allele. The low values were caused by a significantly higher removal of LDL apo B (apo E2, 0.453±0.03 versus apo E3, 0.312±0.01 pools per day, P<.05). High cholesterol intake increased LDL cholesterol levels in all genetic categories except in the apo E2 phenotype irrespective of the combinations with other polymorphisms. Carriers of the apo B R+ allele (EcoRI site present) presented with the most prominent LDL cholesterol rise (from 2.71±0.14 to 3.37±0.29 mmol/L). In multiple stepwise regression analysis, apo B EcoRI RFLP and apo E phenotypes were the only variables that explained the variability of high cholesterol intake–induced change in LDL cholesterol levels. In summary, in any genetic combination, individuals with the 2 allele had the lowest LDL cholesterol values and were nonresponders to dietary cholesterol, whereas subjects with the apo B R+ allele had marked LDL elevations, especially in combination with the 4.
Key Words: cholesterol absorption • cholesterol synthesis • LDL kinetics • genetic polymorphism • dietary cholesterol
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Introduction |
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Genetic and dietary factors regulate serum cholesterol level, but detailed mechanisms of their interplay are insufficiently known. It is not known why increased dietary cholesterol intake raises serum cholesterol level in some but not all subjects.1 2 3 4 5 Genetic variations of apoproteins essential in LDL metabolism have been postulated to be involved, at least in part, in the regulation of serum total and LDL cholesterol levels. Thus, common apo E polymorphisms have been shown to contribute to the magnitude of cholesterol response in several4 6 7 8 but not all9 10 11 12 dietary intervention studies. In addition, the RFLPs of the apo B gene, as disclosed with the enzymes Xba I, EcoRI, or PvuII, are associated, although inconsistently, with the variability of the serum cholesterol level.12 13 14 15 Recently, a single-base variation in the apo A-I gene promoter was reported to influence the response of serum LDL cholesterol level to dietary fat.16
It is noteworthy that in all these earlier studies, genetic polymorphisms of apolipoprotein genes have been evaluated separately. We previously studied the effects of apo E, apo B gene Xba I, and LDL receptor gene PvuII polymorphisms on LDL and cholesterol metabolism in a random male population consuming their habitual home diet.17 Common polymorphism of the apo E gene exerted the most powerful effect on the variability of serum LDL cholesterol level. Some effect was contributed by the LDL receptor PvuII RFLP, whereas the apo B gene Xba I RFLP had no consistent effect. In addition, not only apo E variation4 7 18 19 but also PvuII polymorphism of the LDL receptor gene were found to be associated with variations of cholesterol absorption efficiency, cholesterol synthesis, and FCR for LDL apo B.17 It is possible that genetic polymorphisms of apos E, B, and C-III as well as the LDL receptor, all essential mediators of LDL metabolism, could alone and especially in combination influence an individual's response to dietary cholesterol. Thus, the aim of this study was to assess the effects of common polymorphisms in these gene loci, separately and in concert, on LDL cholesterol regulation by also investigating cholesterol absorption and metabolism as well as LDL kinetics during low and high cholesterol intake.
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Methods |
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The study group consisted of twenty-nine 50-year-old men volunteered from a population-based random-age cohort of the inhabitants of Helsinki.20 Of the original sample of 63 men, 29 volunteered for the dietary intervention study. None of them had uncontrolled heart failure; gastrointestinal, liver, thyroid, or renal disease; or diabetes mellitus, and none were taking lipid-lowering drugs. The study protocol was accepted by the Ethical Committee of our hospital.
After entering the study, the subjects started a low-fat/low-cholesterol diet compatible with the American Heart Association type 2 diet21 for 6 weeks according to individual instructions of a dietitian. After they had completed this period, three egg yolks per day were added to the diet for 6 weeks. Adherence to the diet was monitored weekly as described in detail previously.4 7
After 4 weeks from the beginning of each dietary intervention, metabolic and kinetic studies were started. The subjects kept food records for 7 days, which allowed calculation of the dietary constituents.22 In addition, they were given a capsule containing 4-14C-cholesterol, 22,23-3H-ß-sitosterol, and 200 mg chromic oxide three times a day with their regular meals during the 7-day period. Cholesterol absorption and fecal steroids were analyzed from a 3-day stool collection. During the LDL turnover studies, serum lipid, lipoprotein, and LDL apo B concentrations were analyzed four times from serum samples after a 12-hour fast, and the mean values of these four specimens are given below.
Serum Lipids and Lipoproteins
Cholesterol, triglycerides, and apo B were analyzed enzymatically with commercial kits (Boehringer Diagnostica and Orion Diagnostica). Serum lipoproteins were separated by ultracentrifugation in fixed-angle rotors (Beckman) into density classes of VLDL, IDL, LDL, and HDL as described in the Manual of Laboratory Operations of the Lipid Research Clinics Program.23
Cholesterol Absorption and Metabolism
Cholesterol absorption was measured by the peroral double-isotope continuous-feeding method.24 Chromic oxide was analyzed from the 3-day fecal specimen25 and fecal sterols by gas-liquid chromatography with a 50-m-long SE-30 capillary column.26 27 28 Cholesterol synthesis was measured by the sterol balance technique.26 27
LDL Apo B Kinetics
Fasted EDTA plasma (50 mL) was collected, and LDL (1.019 to 1.063 g/mL) was separated by serial preparative ultracentrifugations in fixed-angle Beckman rotors and iodinated with 125I by a modification of the iodine monochloride method.29 30 Three days before injection, the subjects started to take peroral potassium iodide. Labeled autologous LDL (
1 mg) was mixed with 5% human serum albumin, filtered, and injected intravenously. The total amount of radioactivity did not exceed 30 µCi.
After the injection, blood samples of 10 mL were collected and counted for 14 days. The die-away curves were constructed in whole plasma for 125I-labeled LDL. The FCR for LDL was determined with a two-pool model.31 TR was calculated by multiplying FCR by the pool size. Plasma volume was calculated to be 4.5% of body weight.
DNA Analysis and Apo E Phenotyping
DNA was isolated from EDTA-anticoagulated blood samples by the technique of Bell et al.32 Determination of the apo B gene Xba I, Msp I, and EcoRI RFLPs was carried out with the Southern blot technique as described previously.33 The apo B cDNA probes (pB23 and pB8) used in the hybridizations were gifts of Dr Jan L. Breslow (Rockefeller University, New York, NY). The positions of the different RFLPs of the apo B gene are as follows: Xba I, codon 2488 in exon 2634 ; Msp I, codon 3611 in exon 2635 ; and EcoRI, codon 4154 in exon 29.34 The apo B alleles with the restriction sites present for the enzymes Xba I, Msp I, and EcoRI are designated as X+, M+, and R+ and those alleles with the restriction site absent as X-, M-, and R-, respectively. The alleles X+ and X- correspond to the designations X2 and X1, respectively, that we used previously.36 The PvuII RFLP of intron 15 of the LDL receptor gene was assayed by Southern blotting using a 3'-end LDL receptor cDNA probe (a gift of Drs D. Russell, M.S. Brown, and J.L. Goldstein, Dallas, Tex) as described previously.37 The allele with the extra PvuII cleavage site is designated as P+ and that without the cleavage site as P-. The Sst I RFLP of the 3'-nontranslated region of the apo C-III gene was likewise analyzed by a Southern blot technique38 using an apo A-I genomic DNA probe, pSV2 2.2 kb apo A-I, a gift of Dr Jan L. Breslow. The apo C-III allele with the Sst I restriction site present is designated as S+ and that lacking this site as S-. Apo A-I gene Msp I promoter polymorphism was assayed by the polymerase chain reaction technique as described before.39 The allele with the restriction site present is designated as M+ and the allele without restriction site as M-.
Apo E phenotyping was performed from serum by isoelectric focusing.40 In the following, subjects either homozygotic or heterozygotic for the 2 or 4 allele were categorized to possess apo E2 or apo E4 phenotypes, respectively, and subjects homozygotic for the 3 allele were categorized as the apo E3 group.
Statistical Methods
Statistical significances were tested with the two-tailed Student's t test and paired t test, and correlation coefficients were calculated with either Pearson's product-moment correlation or Spearman's rank correlation test. One-way ANOVA was used to test the null hypothesis that the genotypic variances of apos A-I, E, B, and C-III and the LDL receptor gene were not associated with the lipid or metabolic values or the response to diet. Multiple stepwise regression analysis was used as the multivariate method to explain the variability of the change in serum LDL cholesterol level during high cholesterol intake. The skewness of distributions was checked, and logarithmic transformations were used in the calculations when appropriate. In addition, the variables were adjusted for BMI, in kilograms per square meter. To simplify the calculations, the genetic variables were semiquantified. Numerical values for the apo E phenotypes were given as follows: apo E2/2, 1; E3/2, 2; E4/2, 3; E3/3, 4; E4/3, 5; and E4/4, 6. For Apo B Xba I: X-X-, 1; X-X+, 2; and X+X+, 3; and LDL receptor gene PvuII: P-P-, 1 and P-P+, 2. In addition, the homozygotes R+R+ (n=1), S+S+ (n=1), and apo A-I Msp I M-M- (n=1) were combined with the heterozygotes R-R+, S-S+, and M-M+, respectively, and the respective combined groups were denoted as R+, S+, and M-. A value of P<.05 was considered statistically significant.
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Results |
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During the low-fat/low-cholesterol period, the mean daily cholesterol intake was 208±13 mg/d (mean±SEM) and fat intake was 53±3 g/d, whereas during the high-cholesterol/low-fat period, the respective figures were 878±38 mg/d and 69±3 g/d. The amounts of dietary cholesterol, fat, calories, and the type of ingested fat did not differ between the various genetic groups.
BMI and serum lipid levels are summarized in Table 1
. Subjects with the apo B R+ allele had lower BMIs than those homozygous for the R- allele. During low cholesterol intake, serum total cholesterol (Table 1), triglyceride (Table 1), and LDL cholesterol levels (Fig 1) were similar in the different genetic subgroups, with the exception of the apo E2 subjects, in whom total and LDL cholesterol levels were lower than in the apo E3 and E4 subjects. Although the mean HDL cholesterol level was higher in the apo B X+X+ group than in the X+X- and X-X- groups, there was no apparent gene-dosage effect dependent on this RFLP (Table 1).
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High cholesterol intake increased LDL cholesterol and apo B levels (data not shown) significantly in most but not all single genotype categories (Fig 1
). The highest increment of LDL cholesterol occurred in the subjects with the apo B R+ allele, and the lowest (insignificant) in the apo E2 group.
During low cholesterol intake, the lowest FCR values for LDL apo B (0.308 to 0.318 pools per day) were found in the carriers of the apo B R+ and apo C-III S+ alleles and in the apo E3 and E4 phenotypes (Table 2). The highest removal values (0.369 to 0.453 pools per day), respectively, were observed in the R-, P-, and apo E2 groups. Cholesterol absorption efficiency was lower in the carriers of the apo C-III S+ allele than in those without it, and cholesterol synthesis was lower in the carriers of the LDL receptor P+ allele than in those lacking it (9.7±0.7 versus 12.0±0.7 mg·kg-1·d-1, P<.05), but none of these differences resulted in significant variations in circulating LDL cholesterol level. Apo E2 phenotype was associated with lower cholesterol absorption efficiency, more effective bile acid and cholesterol synthesis, and higher FCR for LDL apo B on a normal diet,4 7 but on the low-cholesterol diet these associations were less consistent (Table 2).
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) When Switched to High Cholesterol Intake
During cholesterol feeding, FCR for LDL apo B was decreased in all genetic subgroups, TRs for LDL apo B and cholesterol synthesis (data not shown) were unaffected, cholesterol absorption was decreased mostly insignificantly, and bile acid synthesis was frequently stimulated (Table 2).
The combined effects of selected genetic variables on serum LDL cholesterol levels are shown in Fig 2. The lowest LDL cholesterol levels during low cholesterol intake were observed in the apo E2 groups. During high dietary cholesterol consumption, LDL cholesterol level was increased most in the apo E4/apo B R+ group (+1.0±0.3 mmol/L, n=4, P<.05 from the change in all other groups), whereas in the apo E4/apo B R-, it was increased by +0.3±0.1 (n=6) and in the apo E3 R- and R+ groups, by +0.31±0.10 (n=5) and +0.40±0.12 mmol/L (n=4), respectively (low versus high, P<.05 for all groups). The E2 group in any combination was nonresponding, whereas the E3 groups were located midway between E2 and E4 in every combination.
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P<.05, E4R- vs E4R+. Values are mean±SEM. E2X2, n=5; E4X2, n=9; E2R-, n=7; E4R-, n=6; E4R+, n=4; E2P-, n=6; E4P-, n=5; and E4P+, n=7.
During the two dietary studies, LDL cholesterol concentration was significantly correlated with apo E phenotype and TR for LDL apo B and inversely with FCR for LDL apo B but not at all with apo B Xba I or EcoRI or LDL receptor gene PvuII RFLPs (Table 3). During high cholesterol intake, LDL cholesterol concentration was correlated with cholesterol absorption efficiency and inversely with bile acid and cholesterol synthesis. During the latter diet, apo E polymorphism was correlated with FCR for LDL apo B (r=-.775, P<.001), apo B Xba I genotypes with TR for LDL apo B (r=.468, P<.05), and apo B EcoRI genotypes with cholesterol absorption efficiency (r=.378, P=.05).
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Finally, by multiple stepwise regression analysis with the change of LDL cholesterol as the dependent variable and the genetic parameters (apo E phenotypes; apo B Xba I, EcoRI, and Msp I RFLPs; apo C-III Sst I RFLP; and LDL receptor gene PvuII RFLP) as the independent variables, apo B EcoRI RFLP (P<.01) and apo E phenotypes (P<.05) were the only parameters (as in the univariate model, Table 3) that explained the variability of LDL cholesterol change, of which 53.9% was explained with the model.
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Discussion |
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The present study is the first to assess the simultaneous associations of common polymorphisms of the lipid-regulatory genes with parameters of cholesterol and LDL metabolism during changes in dietary cholesterol. Our data show that the two genetic factors, apo E phenotype distribution and apo B EcoRI RFLP, were specifically related to changes in LDL cholesterol during low and high cholesterol intake. Individuals possessing the
2 allele were nonresponders to increased cholesterol intake irrespective of apo B, A-I, or C-III or LDL receptor gene polymorphisms, whereas in the apo E3 and E4 phenotypes, LDL cholesterol was elevated in every genetic combination during high cholesterol intake. However, the presence of the polymorphic EcoRI site in the apo B gene combined with apo E4 was associated with the highest elevation of LDL cholesterol level. On the other hand, the LDL receptor P+ allele combined with apo E4 had no such inhibitory effect on the apo E4–induced rise in cholesterol that was described earlier.41 LDL cholesterol levels or their changes in response to high-cholesterol diet did not differ from each other in the different apo B, A-I, and C-III and LDL receptor RFLP groups. In many earlier studies, the allele apo B X+ has been associated with higher total and LDL cholesterol level36 42 43 44 45 46 47 48 49 and slower FCR for LDL apo B.50 51 The present study group was based on a uniform age cohort of the population of Helsinki enriched with the apo E2 phenotype. This enrichment and the small size of the cohort (n=29) might partly explain the differences. There was a trend toward a higher serum triglyceride level associated with the apo C-III Sst I S+ allele, which has been observed earlier in Finnish patients with severe primary hypertriglyceridemia,52 and in a large Finnish population sample of adolescents and young adults.38 The increases in LDL cholesterol during high cholesterol intake were similar in the apo A-I M+M+ and M+M- groups, which differs from the earlier observations by Lopez-Miranda et al16 ; in their study, however, the dietary fat intake, not cholesterol, was varied.
The apo B EcoRI polymorphism was associated with the largest change of LDL cholesterol level taking place during the switch from low to high cholesterol intake and FCR for LDL apo B (data not shown). The importance of this polymorphism for cholesterol metabolism has been difficult to conclude from previous studies, because the results are scanty and controversial, varying from no association with serum cholesterol43 53 to some association with VLDL cholesterol and triglyceride levels.48 54 This polymorphism is due to a single-base-pair change in the coding region (exon 29) of the apo B gene, resulting in a substitution of lysine for glutamic acid at position 4154.55 It is possible that this amino acid substitution could influence LDL receptor binding of the encoded apo B molecules, measured as FCR for LDL apo B in the present study, although the in vitro apo B binding to fibroblasts has been shown to be independent of this polymorphism.53 The present study also showed that the apo B EcoRI polymorphism was associated with cholesterol absorption efficiency. When cholesterol absorption efficiency is high, as is the case with the R+ subjects (Table 2), cholesterol synthesis and LDL receptor activity will be downregulated.4 7
In conclusion, apo E2 phenotype was associated with virtually no elevation of LDL cholesterol level during a high-cholesterol diet, irrespective of a variety of other apoprotein or LDL receptor gene polymorphisms that have previously been found to influence serum lipid levels. Furthermore, during high cholesterol intake, LDL cholesterol level was significantly increased in subjects with the apo E3 and E4 phenotypes independently of their apo B Xba I and Msp I, apo C-III Sst I, and LDL receptor PvuII alleles, but the increase was most conspicuous when the apo B EcoRI R+ allele was present. These findings suggest that common polymorphism of the apoprotein E and the Glu 4154 Lys polymorphism of the apoprotein B gene may result in variable responses of serum total and LDL cholesterol levels when dietary cholesterol content is markedly modified.
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Selected Abbreviations and Acronyms |
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Acknowledgments |
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This study was supported by grants from the Paulo Foundation, the Finnish Heart Research Foundation, the Juho Vainio Foundation, the Medical Council of the Finnish Academy, the University of Helsinki, and the Sigrid Juselius Foundation. The technical assistance of Leena Kaipiainen, Pia Hoffstrom, Orvokki Ahlroos, Elli Kempas, Anja Salolainen, and Antti Laine is gratefully acknowledged.
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Footnotes |
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Presented in part at the 67th Scientific Sessions of the American Heart Association, Dallas, Tex, November 14-17, 1994, and published in abstract form (Circulation. 1994;90[suppl I]:I-504.)
Received December 1, 1995;
revision received May 9, 1996;
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