Common sequence variations in ABCG8 are related to plant sterol metabolism in healthy volunteers
2004; Elsevier BV; Volume: 46; Issue: 1 Linguagem: Inglês
10.1194/jlr.m400210-jlr200
ISSN1539-7262
AutoresJogchum Plat, Marjolijn C. E. Bragt, Ronald P. Mensink,
Tópico(s)Lipid metabolism and biosynthesis
ResumoPolymorphisms in the ATP binding cassette (ABC) transporters ABCG5 and ABCG8 are related to plasma plant sterol concentrations. It is not known whether these polymorphisms are also associated with variations in serum plant sterol concentrations during interventions affecting plant sterol metabolism. We therefore decided to study changes in serum plant sterol concentrations with ABCG5/G8 polymorphisms after consumption of plant stanol esters, which decrease plasma plant sterol concentrations. Cholesterol-standardized serum campesterol and sitosterol concentrations were significantly associated with the ABCG8 T400K genotype, as were changes in serum plant sterol concentrations after consumption of plant stanols. The reduction of −57.1 ± 38.3 102 × μmol/mmol cholesterol for sitosterol in TT subjects was significantly greater compared with the −36.0 ± 18.7 reduction in subjects with the TK genotype (P = 0.021) and the −16.9 ± 13.0 reduction in subjects with the KK genotype (P = 0.047). Changes in serum campesterol concentrations showed a comparable association. No association with serum LDL cholesterol was found.Genetic variation in ABCG8 not only explains cross-sectional differences in serum plant sterol concentrations but also determines a subject's responsiveness to changes in serum plant sterols during interventions known to affect plant sterol metabolism. Polymorphisms in the ATP binding cassette (ABC) transporters ABCG5 and ABCG8 are related to plasma plant sterol concentrations. It is not known whether these polymorphisms are also associated with variations in serum plant sterol concentrations during interventions affecting plant sterol metabolism. We therefore decided to study changes in serum plant sterol concentrations with ABCG5/G8 polymorphisms after consumption of plant stanol esters, which decrease plasma plant sterol concentrations. Cholesterol-standardized serum campesterol and sitosterol concentrations were significantly associated with the ABCG8 T400K genotype, as were changes in serum plant sterol concentrations after consumption of plant stanols. The reduction of −57.1 ± 38.3 102 × μmol/mmol cholesterol for sitosterol in TT subjects was significantly greater compared with the −36.0 ± 18.7 reduction in subjects with the TK genotype (P = 0.021) and the −16.9 ± 13.0 reduction in subjects with the KK genotype (P = 0.047). Changes in serum campesterol concentrations showed a comparable association. No association with serum LDL cholesterol was found. Genetic variation in ABCG8 not only explains cross-sectional differences in serum plant sterol concentrations but also determines a subject's responsiveness to changes in serum plant sterols during interventions known to affect plant sterol metabolism. Despite the fact that Western diets provide approximately the same amounts of plant sterols and cholesterol, plasma concentrations of cholesterol are much higher. This is partly attributable to the very low absorption rates of plant sterols, which are less than 2% for campesterol and less than 1% for sitosterol, the two most abundant plant sterols in nature (1Ostlund R.E. McGill J.B. Zeng C.M. Covey D.F. Stearns J. Stenson W.F. Spilburg C.A. Gastrointestinal absorption and plasma kinetics of soy Delta(5)-phytosterols and phytostanols in humans.Am. J. Physiol. Endocrinol. Metab. 2002; 282: E911-E916Crossref PubMed Scopus (220) Google Scholar). In contrast, cholesterol absorption varies between 30% and 80% (2Bosner M.S. Lange L.G. Stenson W.F. Ostlund Jr, R.E. Percent cholesterol absorption in normal women and men quantified with dual stable isotopic tracers and negative ion mass spectrometry.J. Lipid Res. 1999; 40: 302-308Abstract Full Text Full Text PDF PubMed Google Scholar). Two recently discovered ATP binding cassette (ABC) transporters, ABCG5 and ABCG8, play an important role in the regulation of intestinal plant sterol absorption by excreting plant sterols that have already been taken up from the enterocyte back into the intestinal lumen (3Yu L. Hammer R.E. Li-Hawkins J. Bergmann K. von Lutjohann D. Cohen J.C. Hobbs H.H. Disruption of ABCG5 and ABCG8 in mice reveals their crucial role in biliary cholesterol secretion.Proc. Natl. Acad. Sci. USA. 2002; 99: 16237-16242Crossref PubMed Scopus (595) Google Scholar). ABCG5 and ABCG8 are half-transporters that function together as a heterodimer. Formation of a heterodimer is an absolute necessity to direct the ABCG5/G8 heterodimer from the endoplasmic reticulum to the apical membrane (4Graf G.A. Li W.P. Gerard R.D. Gelissen I. White A. Cohen J.C. Hobbs H.H. Coexpression of ATP-binding cassette proteins ABCG5 and ABCG8 permits their transport to the apical surface.J. Clin. Invest. 2002; 110: 659-669Crossref PubMed Scopus (294) Google Scholar). This feature explains earlier observations that mutations in only one of the half-transporters already causes the rare inheritable autosomal recessive disease sitosterolemia (5Lee M. Lu K. Patel S. Genetic basis of sitosterolemia.Curr. Opin. Lipidol. 2001; 12: 141-149Crossref PubMed Scopus (166) Google Scholar, 6Berge K.E. Tian H. Graf G.A. Yu L. Grishin N.V. Schultz J. Kwiterovich P. Shan B. Barnes R. Hobbs H.H. Accumulation of dietary cholesterol in sitosterolemia caused by mutations in adjacent ABC transporters.Science. 2000; 290: 1771-1775Crossref PubMed Scopus (1328) Google Scholar, 7Lee M.H. Lu K. Hazard S. Yu H. Shulenin S. Hidaka H. Kojima H. Allikmets R. Sakuma N. Pegoraro R. Srivastava A.K. Salen G. Dean M. Patel S.B. Identification of a gene, ABCG5, important in the regulation of dietary cholesterol absorption.Nat. Genet. 2001; 27: 79-83Crossref PubMed Scopus (0) Google Scholar). Sitosterolemic patients are characterized by severely increased serum plant sterol concentrations, normal to moderately increased serum cholesterol concentrations, and a high risk to develop coronary heart disease at a very young age (8Bhattacharyya A.K. Connor W. β-Sitosterolemia and xanthomatosis.J. Clin. Invest. 1974; 53: 1033-1043Crossref PubMed Scopus (452) Google Scholar, 9Björkhem I. Boberg K.M. Inborn errors in bile acid biosynthesis and storage of sterols other than cholesterol.in: Scriver C.R. Beaudet A.L. Sly W.S. Valle D. The Metabolic Basis of Inherited Diseases. McGraw Hill, New York1994: 2073-2100Google Scholar). Although not generally accepted, several studies have suggested that increased concentrations of plant sterols are a risk factor for premature atherosclerosis in sitosterolemic patients (10Miettinen T.A. Strandberg T.E. Gylling H. Noncholesterol sterols and cholesterol lowering by long-term simvastatin treatment in coronary patients: relation to basal serum cholestanol.Arterioscler. Thromb. Vasc. Biol. 2000; 20: 1340-1346Crossref PubMed Scopus (196) Google Scholar, 11Miettinen T.A. Gylling H. Lindbohm N. Miettinen T.E. Radhakrishnan A. Rajaratnam A. Relas H. Serum noncholesterol sterols during inhibition of cholesterol synthesis by statins.J. Lab. Clin. Med. 2003; 141: 131-137Abstract Full Text Full Text PDF PubMed Scopus (106) Google Scholar, 12Miettinen T.A. Gylling H. Strandberg T. Sarna S. Baseline serum cholestanol as predictor of recurrent coronary events in subgroup of Scandinavian Simvastatin Survival Trial.BMJ. 1998; 316: 1127-1130Crossref PubMed Scopus (214) Google Scholar) and even in nonsitosterolemic subjects (13Sudhop T. Gottwald B.M. Bergmann K. Von Serum plant sterols as a potential risk factor for coronary heart disease.Metabolism. 2002; 51: 1519-1521Abstract Full Text PDF PubMed Scopus (184) Google Scholar, 14Glueck C.J. Speirs J. Tracey T. Streicher P. Illig E. Vandegrift J. Relationships of serum plant sterols (phytosterols) and cholesterol in 595 hypercholesterolemic subjects, and familial aggregation of phytosterols, cholesterol, and premature coronary heart disease in hypercholesterolemic probands and their first degree relatives.Metabolism. 1991; 40: 842-848Abstract Full Text PDF PubMed Scopus (178) Google Scholar). Besides the various rare mutations in ABCG5 or ABCG8 as observed in sitosterolemic patients (15Hubacek J.A. Berge K.E. Cohen J.C. Hobbs H.H. Mutations in ATP-binding cassette binding proteins G5 (ABCG5) and G8 (ABCG8) causing sitosterolemia.Hum. Mutat. 2001; 452: 1-5Google Scholar), more common sequence variations in both half-transporters, without the sitosterolemic phenotype, have been described. These polymorphisms in ABCG5 and ABCG8 are related to serum plant sterol concentrations (16Berge K.E. Bergmann K. Von Lutjohann D. Guerra R. Grundy S.M. Hobbs H.H. Cohen J.C. Heritability of plasma noncholesterol sterols and relationship to DNA sequence polymorphism in ABCG5 and ABCG8.J. Lipid Res. 2002; 43: 486-494Abstract Full Text Full Text PDF PubMed Google Scholar). It is not known, however, whether these genetic variations are also associated with variations in serum plant sterol concentrations during interventions known to affect plant sterol metabolism. We therefore decided to examine the relationships between changes in serum plant sterol concentrations with ABCG5 and ABCG8 polymorphisms after consumption of plant stanol esters, which are known to decrease plasma plant sterol concentrations. Despite the clear association between ABCG5 and ABCG8 and intestinal plant sterol absorption, the role of ABCG5 and ABCG8 in cholesterol absorption is controversial (3Yu L. Hammer R.E. Li-Hawkins J. Bergmann K. von Lutjohann D. Cohen J.C. Hobbs H.H. Disruption of ABCG5 and ABCG8 in mice reveals their crucial role in biliary cholesterol secretion.Proc. Natl. Acad. Sci. USA. 2002; 99: 16237-16242Crossref PubMed Scopus (595) Google Scholar, 17Yu L. Li-Hawkins J. Hammer R.E. Berge K.E. Horton J.D. Cohen J.C. Hobbs H.H. Overexpression of ABCG5 and ABCG8 promotes biliary cholesterol secretion and reduces fractional absorption of dietary cholesterol.J. Clin. Invest. 2002; 110: 671-680Crossref PubMed Scopus (594) Google Scholar, 18Yu L. York J. Bergmann K. Von Lutjohann D. Cohen J.C. Hobbs H.H. Stimulation of cholesterol excretion by the liver X receptor agonist requires ATP-binding cassette transporters G5 and G8.J. Biol. Chem. 2003; 278: 15565-15570Abstract Full Text Full Text PDF PubMed Scopus (237) Google Scholar, 19Plosch T. Bloks V.W. Terasawa Y. Berdy S. Siegler K. van der Sluijs F. Kema I.P. Groen A.K. Shan B. Kuipers F. Schwarz M. Schwarz M. Sitosterolemia in ABC-transporter G5 deficient mice is aggravated on activation of the liver-X receptor.Gastroenterology. 2004; 126: 290-300Abstract Full Text Full Text PDF PubMed Scopus (115) Google Scholar). Therefore, a second aim of the present study was to look for associations between ABCG5 and ABCG8 polymorphisms with changes in LDL cholesterol after consumption of plant stanol esters. Details of the study have been described before (20Plat J. Mensink R.P. Vegetable oil based versus wood based stanol ester mixtures: effects on serum lipids and hemostatic factors in non-hypercholesterolemic subjects.Atherosclerosis. 2000; 148: 101-112Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 21Plat J. Mensink R.P. Effects of diets enriched with two different plant stanol ester mixtures on plasma ubiquinol-10 and fat-soluble antioxidant concentrations.Metabolism. 2001; 50: 520-529Abstract Full Text PDF PubMed Scopus (64) Google Scholar). In brief, 112 healthy nonhypercholesterolemic volunteers (Table 1) were asked to replace during a 4 week run-in period their habitual margarines and baking fats for a low-erucic acid rapeseed oil-based margarine and shortening. For the next 8 weeks, subjects were randomly allocated, stratified for gender and age, to one of three intervention groups. The control group (N = 42) continued to use the rapeseed oil-based margarine and shortening, and the second and third groups used the same margarine and shortening to which a vegetable oil-based (N = 36) or a wood-based (N = 34) plant stanol ester mixture was added. The compositions of the experimental products have been described in detail elsewhere (22Plat J. van Onselen E.N.M. Mensink R.P. Dietary plant stanol ester mixtures: effects on safety parameters and erythrocyte membrane fatty acid composition in non hypercholesterolemic subjects.Eur. Heart J. 1999; 1: 58-63Google Scholar). The margarine was used at breakfast and lunch, and the shortening was used at dinner. During the intervention period, daily intake of plant stanols in the vegetable oil-based group was 3.8 ± 0.6 g (mean ± SD) and in the wood-based group was 4.0 ± 1.8 g. Plant stanols were esterified with fatty acids from rapeseed oil. All experimental products were prepared by the Raisio Group (Raisio, Finland). Energy intake and the proportions of energy from carbohydrates, fatty acids, and protein, as well as cholesterol and fiber intakes, did not change during the study (20Plat J. Mensink R.P. Vegetable oil based versus wood based stanol ester mixtures: effects on serum lipids and hemostatic factors in non-hypercholesterolemic subjects.Atherosclerosis. 2000; 148: 101-112Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar).TABLE 1Population characteristics before the start of the studyVariableAllControl GroupExperimental GroupNumber of males411625Number of females712645Age (years) 33 ± 16 33 ± 16 33 ± 15Body mass index (kg/m2)22.9 ± 3.623.0 ± 2.822.8 ± 4.0Total cholesterol (mmol/l)5.02 ± 0.814.97 ± 0.735.04 ± 0.86Triacylglycerol (mmol/l)0.99 ± 0.540.88 ± 0.371.05 ± 0.62Values shown are means ± SD. Open table in a new tab Values shown are means ± SD. At the start of the study (day 1), blood was sampled in a 10 ml EDTA tube (Monoject sterile; Sherwood Medical, Ballymoney, Northern Ireland), which was used for DNA isolation (23Plat J. Mensink R.P. Relationship of genetic variation in genes encoding apolipoprotein A-IV, scavenger receptor BI, HMG-CoA reductase, CETP and apolipoprotein E with cholesterol metabolism and the response to plant stanol ester consumption.Eur. J. Clin. Invest. 2002; 32: 242-250Crossref PubMed Scopus (55) Google Scholar). At the end of the run-in period (weeks 3 and 4) and at the end of the experimental period (weeks 11 and 12), blood was sampled in a 10 ml clotting tube (CORVAC integrated serum separator tube; Sherwood Medical Co., St. Louis, MO) after an overnight fast. Subjects abstained from drinking alcohol on the day preceding, and from smoking on the morning of, blood sampling. Serum was prepared by centrifugation at 2,000 g for 30 min at 4°C, minimally 1 h after venipuncture, and aliquots were stored directly at −80°C for analysis of serum lipids, lipoproteins, plant sterols, plant stanols, and cholesterol precursor concentrations at the end of the study (20Plat J. Mensink R.P. Vegetable oil based versus wood based stanol ester mixtures: effects on serum lipids and hemostatic factors in non-hypercholesterolemic subjects.Atherosclerosis. 2000; 148: 101-112Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 21Plat J. Mensink R.P. Effects of diets enriched with two different plant stanol ester mixtures on plasma ubiquinol-10 and fat-soluble antioxidant concentrations.Metabolism. 2001; 50: 520-529Abstract Full Text PDF PubMed Scopus (64) Google Scholar). For this study, we decided to evaluate only associations between serum plant sterols, lipids, and lipoproteins with single nucleotide polymorphisms (SNPs) in ABCG5/G8 with a published population frequency of >10%. Otherwise, the number of subjects in a genotype group could become too low for meaningful statistical analyses. In addition, we have chosen to use only those SNPs that showed a significant cross-sectional association with serum plant sterol or LDL cholesterol concentrations in earlier studies. These criteria resulted in the selection of three SNPs: ABCG8 T400K, ABCG8 A632V, and ABCG5 Q604E. Genotyping of exons 8 and 13 from ABCG8 and of exon 13 from ABCG5 was performed by analysis of restriction fragment length polymorphisms, as described (15Hubacek J.A. Berge K.E. Cohen J.C. Hobbs H.H. Mutations in ATP-binding cassette binding proteins G5 (ABCG5) and G8 (ABCG8) causing sitosterolemia.Hum. Mutat. 2001; 452: 1-5Google Scholar). In brief, PCR amplifications were performed in 20 μl volumes containing 350 ng of genomic DNA, 25 pmol of each nucleotide primer (Sigma Genosys, Cambridge, UK), 1 unit of Taq polymerase (Pharmacia Biotech, Roosendaal, The Netherlands), 0.2 mM of each deoxynucleoside triphosphate (Pharmacia Biotech, Roosendaal, The Netherlands), and 1.5 mM MgCl2. Before amplification, each sample was denatured for 5 min at 95°C. For ABCG8 genotyping, each of the following 30 cycles consisted of 15 s at 96°C (denaturation), 15 s at 60°C (primer annealing), and 30 s at 72°C (extension), followed by 10 min at 72°C (elongation). For ABCG5 genotyping, the program was slightly modified in that annealing occurred for 30 s at 60°C and no elongation step was used. PCR products were digested with specific restriction enzymes, and the DNA fragments obtained were electrophoresed for 1.5 h at 125 V on a 2.5% agarose gel containing Gelstar (Sanvertech, Heerhugowaard, The Netherlands). DNA fragments were visualized by ultraviolet light at 312 nm using a Wratten gelatin filter on a VDS Imagemaster (Pharmacia Biotech, San Francisco, CA). Sequences of the primers have been described (15Hubacek J.A. Berge K.E. Cohen J.C. Hobbs H.H. Mutations in ATP-binding cassette binding proteins G5 (ABCG5) and G8 (ABCG8) causing sitosterolemia.Hum. Mutat. 2001; 452: 1-5Google Scholar). The restriction enzymes (New England Biolabs, Beverly MA) used were MseI and NcoI for ABCG8 exons 8 and 13, respectively, and XmnI for ABCG5 exon 13. Because of the limited number of subjects, and because plant sterol concentrations were not significantly different, heterozygous and homozygous carriers of the genetic variants [ABCG8 T400K (TK+KK), ABCG8 A632V (VV+VA), and ABCG5 Q604E (QE+EE)] were combined before data analysis. Excluding the homozygous carriers did not affect the conclusions. Linkage disequilibrium was evaluated according to standard procedures and reported as D′ values. At the end of the run-in period, cross-sectional differences in metabolic parameters between genotype groups were compared with an unpaired t-test. When significant, the three genotype groups were also compared using ANOVA with Bonferroni correction to determine if relations were dose allele-dependent. In view of their well-known relationships with lipid metabolism, body mass index, gender, and age were considered as potential confounders. However, these parameters were not significantly different between the various genotype groups (data not shown) and were therefore not included into the models. In a search for gene-diet interactions, changes in metabolic parameters were calculated for each subject as differences between values of the experimental period and the run-in period. Because responses in serum lipoprotein and plant sterol and stanol concentrations were not different between the two experimental groups (20Plat J. Mensink R.P. Vegetable oil based versus wood based stanol ester mixtures: effects on serum lipids and hemostatic factors in non-hypercholesterolemic subjects.Atherosclerosis. 2000; 148: 101-112Abstract Full Text Full Text PDF PubMed Scopus (127) Google Scholar, 21Plat J. Mensink R.P. Effects of diets enriched with two different plant stanol ester mixtures on plasma ubiquinol-10 and fat-soluble antioxidant concentrations.Metabolism. 2001; 50: 520-529Abstract Full Text PDF PubMed Scopus (64) Google Scholar), results of the vegetable oil-based and the wood-based plant stanol ester groups were combined (N = 70). Effects of genotype on these responses were examined with an unpaired t-test. Again, allele dose-dependent relationships were examined by ANOVA plus Bonferroni correction. Because cross-sectional studies (16Berge K.E. Bergmann K. Von Lutjohann D. Guerra R. Grundy S.M. Hobbs H.H. Cohen J.C. Heritability of plasma noncholesterol sterols and relationship to DNA sequence polymorphism in ABCG5 and ABCG8.J. Lipid Res. 2002; 43: 486-494Abstract Full Text Full Text PDF PubMed Google Scholar) have reported that individuals with the TT genotype have higher cholesterol-standardized plasma plant sterol concentrations, we chose not to use analysis of covariance with values from the experimental period as dependent variables and genotype and baseline plant sterol concentrations as independent variables. The two latter parameters are mutually dependent, which creates the possibility of introducing collinearity into the statistical model. All statistical analyses were performed with Statview 4.5 (24Statview manual (for the Macintosh). 1992. Statview Abacus Concepts, Inc: Berkeley, CA.Google Scholar). Frequency distributions for the genotypes in exons 8 (T400K) and 13 (A632V) of ABCG8 and in exon 13 (Q604E) of ABCG5 are shown in Table 2. All genotype frequency distributions were in Hardy-Weinberg equilibrium. Significant linkage disequilibrium was found for two of three pairs of the three polymorphisms analyzed (Table 3). At the end of the 4 week run-in period, serum concentrations of LDL cholesterol, HDL cholesterol, and triacylglycerol in all subjects (N = 112) were 2.95 ± 0.78, 1.59 ± 0.38, and 0.92 ± 0.52 mmol/l, respectively. Serum concentrations of plant sterols, plant stanols, and lathosterol at the end of the run-in period are shown in Table 4.TABLE 2Frequency distribution of the different genotypes in exons 8 (T400K) and 13 (A632V) of ABCG8 and in exon 13 (Q604E) of ABCG5SubjectsAllControl GroupExperimental GroupAll1124270ABCG8 T400KTT77 (68.7)30 (71.4) 47 (67.1)TK31 (27.7)11 (26.2) 20 (28.6)KK4 (3.6)1 (2.4) 3 (4.3)ABCG8 A632VAA70 (62.5)23 (54.8) 47 (67.1)VA37 (33.0)17 (40.5) 20 (28.6)VV5 (4.5)2 (4.8) 3 (4.3)ABCG5 Q604EQQ81 (72.3)33 (78.6) 48 (68.6)QE29 (25.9)9 (21.4) 20 (28.6)EE2 (1.8)0 (0) 2 (2.9)Values shown are absolute frequencies (with relative frequencies in parentheses). Open table in a new tab TABLE 3Linkage disequilibrium between genotypes in exons 8 (T400K) and 13 (A632V) of ABCG8 and in exon 13 (Q604E) of ABCG5Locus 1Locus 2D′95% Confidence IntervalABCG8 T400KABCG8 A632V0.290.03–0.76ABCG5 Q604E0.810.06–0.97ABCG8 A632VABCG5 Q604E0.0−0.01–0.22D′ and the corresponding 95% confidence intervals were calculated by using all 112 subjects included in the study. Open table in a new tab TABLE 4Relationships between genetic polymorphisms in ABCG8 and ABCG5 with absolute and cholesterol-standardized serum noncholesterol sterol concentrationsSubjectsCampesterolSitosterolLathosterolCampestanolSitostanolAbsolute concentrations (μmol/l)All15.8 ± 5.36.0 ± 2.45.2 ± 2.1 0.5 ± 0.30.4 ± 0.2ABCG8 T400KTT16.9 ± 5.56.6 ± 2.65.0 ± 2.1 0.5 ± 0.40.4 ± 0.2TK/KK13.5 ± 3.84.8 ± 1.45.7 ± 2.1 0.5 ± 0.30.4 ± 0.2P value<0.001<0.0010.0700.8870.675ABCG8 A632VAA16.3 ± 5.66.2 ± 2.65.1 ± 2.0 0.5 ± 0.30.4 ± 0.2VV/VA15.1 ± 4.85.7 ± 2.15.4 ± 2.4 0.6 ± 0.40.4 ± 0.2P value 0.280 0.2660.5160.3490.759ABCG5 Q604EQQ16.3 ± 5.46.3 ± 2.55.1 ± 2.1 0.5 ± 0.30.4 ± 0.2QE/EE14.6 ± 5.05.4 ± 2.25.4 ± 2.3 0.6 ± 0.40.4 ± 0.2P value 0.125 0.0930.5170.0430.768Cholesterol-standardized concentrations (102 × μmol/mmol cholesterol)All303.4 ± 98.0115.0 ± 44.597.3 ± 33.5 9.8 ± 6.67.6 ± 4.3ABCG8 T400KTT324.2 ± 98.5125.2 ± 45.893.2 ± 35.0 9.6 ± 6.87.5 ± 4.4TK/KK257.7 ± 80.892.4 ± 31.9106.4 ± 28.4 10.2 ± 6.17.7 ± 3.9P value<0.001<0.0010.0530.6360.862ABCG8 A632VAA305.8 ± 95.3117.3 ± 47.395.2 ± 32.2 9.3 ± 6.17.6 ± 4.2VV/VA299.3 ± 103.3111.0 ± 39.8101.0 ± 35.8 10.6 ± 7.27.6 ± 4.4P value 0.736 0.4670.3770.3320.938ABCG5 Q604EQQ310.5 ± 98.8118.3 ± 43.795.3 ± 34.8 8.9 ± 5.77.6 ± 4.3QE/EE284.8 ± 94.9106.2 ± 46.2102.7 ± 29.9 11.9 ± 8.17.6 ± 4.2P value 0.216 0.1980.2980.0290.987Values shown are means ± SD and were analyzed after a 4-week period of consumption of rapeseed oil-based margarine and shortening. Open table in a new tab Values shown are absolute frequencies (with relative frequencies in parentheses). D′ and the corresponding 95% confidence intervals were calculated by using all 112 subjects included in the study. Values shown are means ± SD and were analyzed after a 4-week period of consumption of rapeseed oil-based margarine and shortening. Next, associations between the different genotypes and serum plant sterol concentrations, both absolute (μmol/l) and cholesterol-standardized (102 × μmol/mmol cholesterol), were evaluated. Analysis of absolute and cholesterol-standardized ratios gave similar results, so in the following analysis only serum cholesterol-standardized ratios were used. Both cholesterol-standardized serum campesterol and sitosterol concentrations were associated with the ABCG8 T400K genotype (Table 4). Moreover, this relationship was allele-dependent (Fig. 1). The association with cholesterol-standardized serum lathosterol concentrations nearly reached statistical significance (P = 0.053). Differences for cholesterol-standardized serum plant stanol (sitostanol and campestanol) concentrations between the different genotypes never reached statistical significance (Table 4). No associations were found between the polymorphisms in the ABCG8 gene with serum lipids and lipoproteins. Subjects with the QQ genotype (ABCG5 Q604E) showed significantly higher serum LDL cholesterol concentrations compared with QE/EE subjects (Table 5).TABLE 5Relationships between genetic polymorphisms in ABCG8 and ABCG5 with serum lipid and lipoprotein concentrationsSubjectsLDL CholesterolHDL CholesterolTriacylglycerolmmol/lAll2.95 ± 0.781.59 ± 0.380.92 ± 0.52ABCG8 T400KTT2.97 ± 0.741.59 ± 0.370.85 ± 0.47TK/KK2.89 ± 0.861.60 ± 0.401.08 ± 0.58P value0.6220.9760.025ABCG8 A632VAA3.03 ± 0.781.58 ± 0.390.91 ± 0.48VV/VA2.81 ± 0.771.61 ± 0.360.93 ± 0.58P value0.1370.6660.883ABCG5 Q604EQQ3.04 ± 0.751.56 ± 0.350.89 ± 0.45QE/EE2.70 ± 0.811.68 ± 0.430.99 ± 0.65P value0.0390.1450.342Values shown are means ± SD and were analyzed after a 4-week period of consumption of rapeseed oil-based margarine and shortening. Open table in a new tab Values shown are means ± SD and were analyzed after a 4-week period of consumption of rapeseed oil-based margarine and shortening. In the 70 subjects who consumed the plant stanol ester-enriched margarines, cholesterol-standardized serum campesterol and sitosterol concentrations decreased, whereas those of lathosterol increased, compared with the control group (Table 6). These changes indicated that plant stanol ester consumption decreased cholesterol absorption and increased cholesterol synthesis, which is in agreement with the known underlying mechanism. As a consequence, LDL cholesterol concentrations were significantly decreased. HDL and triacylglycerol concentrations were not affected (Table 7). Changes in cholesterol-standardized serum campesterol and sitosterol concentrations were significantly associated with the ABCG8 T400K polymorphism (Table 6, Fig. 1). The decrease of −116.7 ± 76.5 102 × μmol/mmol cholesterol for campesterol in TT subjects was significantly different from the change of −75.4 ± 46.9 102 × μmol/mmol in subjects with the TK/KK genotype [P < 0.05; 95% confidence interval (CI), −76.0 to −6.6 102 × μmol/mmol cholesterol]. The reduction of −57.1 ± 38.3 102 × μmol/mmol cholesterol for sitosterol in TT subjects was also larger compared with the reduction of −33.5 ± 19.0 102 × μmol/mmol cholesterol in subjects with the TK/KK genotypes (P < 0.01; 95% CI for the difference in changes, −40.5 to −6.7 102 × μmol/mmol cholesterol). Additional analysis to evaluate the association between the three different genotype groups of the ABCG8 T400K polymorphism (TT, TK, and KK) with changes in serum plant sterol and lipoprotein concentrations suggested an allele-dependent relation for both cholesterol-standardized serum campesterol and sitosterol concentrations (Fig. 1). The reduction of −57.1 ± 38.3 102 μmol/mmol cholesterol for sitosterol in TT subjects was significantly greater compared with the −36.0 ± 18.7 in subjects with the TK genotype (P = 0.021; 95% CI, −39.1 to −3.1 102 × μmol/mmol cholesterol) and the −16.9 ± 13.0 in subjects with the KK genotype (P = 0.047; 95% CI, −85.2 to −4.8 102 × μmol/mmol cholesterol). Changes in serum campesterol concentrations showed a comparable association pattern with the ABCG8 T400K genotype. No statistically significant relationships between the ABCG8 and ABCG5 polymorphisms with changes in serum plant stanol (Table 6), lipid, and lipoprotein (Table 7) concentrations were found.TABLE 6Relationships between genetic polymorphisms in ABCG8 and ABCG5 with changes in absolute and cholesterol-standardized serum noncholesterol sterol concentrations after consumption of plant stanol estersSubjectsCampesterolSitosterolLathosterolCampestanolSitostanolAbsolute concentrations (μmol/l)Experimental groupControl−0.5 ± 2.2 −0.5 ± 1.20.0 ± 1.1−0.0 ± 0.40.0 ± 0.2Stanol−6.3 ± 3.9 −3.0 ± 2.10.2 ± 1.40.3 ± 0.40.3 ± 0.2P value<0.001<0.001<0.001<0.001<0.001Genotype groupABCG8 T400KTT−7.0 ± 4.4 −3.4 ± 2.30.2 ± 1.20.2 ± 0.40.3 ± 0.2TK/KK−4.9 ± 2.4 −2.1 ± 0.90.2 ± 1.70.3 ± 0.40.3 ± 0.2P value 0.042 0.017 0.989 0.526 0.967ABCG8 A632VAA−6.5 ± 4.5 −2.9 ± 2.30.3 ± 1.10.3 ± 0.40.3 ± 0.2VV/VA−6.0 ± 2.7 −3.0 ± 1.60.2 ± 1.30.2 ± 0.40.3 ± 0.2P value 0.598 0.831 0.836 0.832 0.395ABCG5 Q604EQQ−6.6 ± 4.3 −3.1 ± 2.30.1 ± 1.50.2 ± 0.40.3 ± 0.2QE/EE−5.8 ± 3.1 −2.7 ± 1.60.6 ± 1.20.2 ± 0.40.4 ± 0.2P value 0.448 0.413 0.185 0.956 0.097Cholesterol-standardized concentrations (102 × μmol/mmol cholesterol)Experimental groupControl−5.9 ± 39.2 −7.8 ± 22.40.5 ± 19.5−0.6 ± 7.20.4 ± 4.0Stanol−103.1 ± 70.6−49.3 ± 34.917.4 ± 22.26.3 ± 8.18.2 ± 4.5P value<0.001<0.001<0.001<0.001<0.001Genotype groupABCG8 T400KTT−
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