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Biochemical, Molecular, and Genetic Mechanisms

The ABCG5 Polymorphism Contributes to Individual Responses to Dietary Cholesterol and Carotenoids in Eggs¹

Kristin L. Herron^*, Mary M. McGrane^*, David Waters^*, Ingrid E. Lofgren^*, Richard M. Clark^*, Jose M. Ordovas and Maria Luz Fernandez^*,2

^* Department of Nutritional Sciences, University of Connecticut, Storrs, CT and USDA-HNRCA, Tufts University, Boston, MA

² To whom correspondence should be addressed. E-mail: maria-luz.fernandez{at}uconn.edu.

	ABSTRACT

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
The ATP binding cassette G5 (ABCG5) polymorphisms have been postulated to play a role in the response to dietary cholesterol. The objective of this study was to examine the contribution of the ABCG5 polymorphism on the plasma response to consumption of cholesterol and carotenoids from eggs. For this purpose, genotyping was conducted for 40 men and 51 premenopausal women who were randomly assigned to consume an egg (EGG, 640 mg/d additional dietary cholesterol and 600 µg lutein+ zeaxanthin) or placebo (SUB, 0 mg/d cholesterol, 0 µg lutein + zeaxanthin) diet for 30 d. The two arms of the dietary intervention were separated by a 3-wk washout period. Plasma concentrations of total cholesterol, LDL cholesterol (LDL-C), and HDL cholesterol were determined. Because eggs are an excellent source of lutein and zeaxanthin, the plasma levels of these carotenoids were also measured in a subset of subjects to determine whether the response to carotenoid intake was similar to that seen for dietary cholesterol and to evaluate the contribution of ABCG5 polymorphism to both responses. Individuals possessing the C/C genotype experienced a greater increase in both LDL-C (P < 0.05) and a trend for lutein (P = 0.08) during the EGG period compared with those individuals with the C/G (heterozygote) or G/G genotypes (homozygotes). These results, although obtained from a small number of subjects, suggest that the ABCG5 polymorphism may play a role in the plasma response to dietary cholesterol and carotenoids.

KEY WORDS: • eggs • lutein • dietary cholesterol • ABCG5 polymorphism • LDL cholesterol

Epidemiological and intervention studies have shown that dietary factors affect the concentration, composition, and metabolism of lipoproteins (1). On the basis of this evidence, the nutrition scientific community has been providing general dietary guidelines as a method by which individuals may decrease their risk for coronary heart disease (CHD)³ through normalizing plasma lipoprotein concentrations (2). One of the recommendations has been to limit the intake of high cholesterol foods, such as eggs, in an attempt to reduce atherogenic concentrations of plasma total cholesterol (TC) and LDL cholesterol (LDL-C). Early studies (3,4) provided evidence, which remains consistent today, that increased consumption of dietary cholesterol can elevate TC values to some extent in certain individuals. However, extensive research does not support a consistent relation between egg intake and increased CHD incidence (5–7) This could be due to the fact that individuals do not experience a homogeneous response to cholesterol consumption (6). Therefore, it is difficult to predict the effect of cholesterol intake on plasma lipoproteins and cardiovascular risk at the individual level.

Interindividual variation in the response to cholesterol and other dietary components can be attributed in part to factors such as ethnicity, hormonal status, and obesity (8). Polymorphisms in genes that encode key proteins in lipoprotein metabolism may also explain some of the variance. Several proteins were identified that may function to regulate sterol absorption in the intestine. Sterols that enter the mucosal cell are either directed toward chylomicron synthesis or may be excreted back into the intestinal lumen. This latter fate of dietary sterols may be regulated by the activity of ATP-binding cassette (ABC) transporters, found both in the intestine and the liver, which utilize ATP as an energy source to drive the transport of lipids and other metabolites across cell membranes (9). There is evidence that ABCG5 may play an important role in limiting dietary cholesterol absorption. The response of this possible transport mechanism to dietary intake was examined in healthy mice, which experienced an increase in ABCG5 expression after intake of a high-cholesterol diet (10).

Because it produces an important transporter of cholesterol in the intestine and liver, polymorphisms of the ABCG5 gene may have an effect on the absorption and subsequent appearance of cholesterol in the circulation. A single nucleotide polymorphism (SNP; C G) at nucleotide 1950 results in the substitution of a glutamic acid for a glutamine at residue 640 (Q640E). Individuals classified as homozygous for the variant ABCG5 allele (G/G) were identified by a previous study to have greater plasma total cholesterol response to dietary cholesterol intake (11), which may be due to an increased efficiency of dietary cholesterol absorption in the intestine.

The primary objective of the present study was to determine whether genetic polymorphism could be identified within a population of healthy men and premenopausal women that would explain their plasma response to an egg diet. Because it was suggested that ABCG5 may play a role in the plasma lipid response to dietary cholesterol intake, we focused on this locus in the study population. Because eggs are also rich in both lutein and zeaxanthin, the parameters of the study provided a unique opportunity to examine the role that ABCG5 may play in carotenoid absorption as well. Individual variation in the plasma response to dietary cholesterol has been investigated for many years and it was suggested that a similar hypo- and hyperresponse to dietary carotenoid consumption may exist. Data from our laboratory showed that carotenoids and cholesterol have similar plasma responses in subjects challenged with eggs, which contain both dietary cholesterol and lutein and zeaxanthin (12,13). Because of this association of carotenoids with lipoproteins during absorption and in the circulation, we hypothesized that an examination of the ABCG5 polymorphisms may provide insight into the plasma response to both dietary cholesterol and carotenoids.

	SUBJECTS AND METHODS

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Liquid pasteurized whole eggs and cholesterol-free/fat-free eggs (placebo) were purchased from Better Brands. Enzymatic TC and triglyceride (TG) kits were obtained from Roche-Diagnostics; aprotinin, sodium azide, and phenylmethylsulfonyl fluoride (PMSF) were obtained from Sigma Chemical.

    Experimental design. The experimental protocol was approved by the University of Connecticut's Institutional Review Board, and written informed consent was obtained from each subject. A total of 40 men and 51 premenopausal women recruited from the University community participated in the dietary intervention (14,15). The exclusion criteria included the presence of hypercholesterolemia [cholesterol >6.2 mmol/L (240 mg/dL)], hypertriglyceridemia [TG >3.7 mmol/L (300 mg/dL)], hypertension, and diabetes. Furthermore, those receiving lipid-lowering drugs were also excluded.

The study utilized a randomized crossover design, with subjects initially assigned to an egg (EGG) or placebo (SUB) group for 30 d, followed by a 3-wk washout period, after which the second dietary period began. Subjects assigned to the EGG group were expected to consume the liquid equivalent of 3 whole eggs/d (adding 640 mg/d cholesterol to the diet and 600 µg of lutein + zeaxanthin). Participants assigned to the SUB consumed an identical weight of cholesterol-free, fat-free egg substitute (0 mg/d dietary cholesterol + 0 mg of lutein + zeaxanthin).

Subjects were expected to follow guidelines of the NCEP Step I diet during the 2 treatment periods, and detailed instructions were provided for their self-selected diets. To ensure compliance, subjects completed 7-d dietary records during each treatment period. Nutrient intake was determined using the NDS-R software version 4.0, developed by the Nutrition Coordinating Center, University of Minnesota, Minneapolis, MN.

Two fasting (12-h) blood samples were collected, on different days within the same week into tubes containing 0.15 g/100 g EDTA. Plasma was separated by centrifugation at 1500 x g for 20 min at 4°C, and placed into vials containing PMSF (0.05 g/100 g), sodium azide (0.01 g/100 g), and aprotinin (0.01 g/100 g). Two additional blood samples were collected and processed in the same manner at the end of each diet period. The variables of weight, blood pressure, level of activity, smoking, and alcohol intake were also measured at baseline and after each dietary period to account for the possible influence of these factors on plasma lipid levels.

    Plasma lipids and apolipoproteins. Our laboratory has participated in the CDC-National Heart, Lung and Blood Institute Lipid Standardization Program since 1989 for quality control and standardization of plasma TC, HDL-C, and TG assays. CV assessed by the standardization program during the study period were 0.76–1.42 for TC, 1.71–2.72 for HDL-C and 1.64–2.47 for TG. For this study, plasma TC was determined by enzymatic methods (16). HDL-C was measured in the supernatant after precipitation of apolipoprotein B–containing lipoproteins and LDL cholesterol (LDL-C) was calculated using the Friedewald equation as previously reported (17). Plasma TG were determined by enzymatic methods and adjusting for free glycerol. Means of the 2 blood draws were used to assess differences between treatment periods.

    Plasma and product carotenoid analysis. Plasma samples from a subset of subjects (20 men and 20 women) who were found to be representative of the whole with regard to plasma lipid concentrations and response to dietary cholesterol (20 hypo and 20 hyperresponders) (12) were utilized for the carotenoid analysis. Plasma and product (EGG and SUB) (200 µL) were prepared for HPLC analysis by initially combining them with an internal standard of 50 µL ethyl-ß-apo-8-carotenoate and 200 µL methanol. The sample was then extracted 3 times using hexane. Centrifugation (1000 x g; 2 min)was utilized to facilitate phase separation. The resulting hexane layers were reconstituted with 0.1 mL of 2-propanol and placed into HPLC collection vials. A Waters HPLC system was utilized and equipped with a Varian column (100 x 4.6 mm microsorb-MN 100–3 C-18), which was preceded by an Upchurch C-18 guard column (Upchurch Scientific). The isocratic mobile phase consisted of 80% acetonitrile:15% dioxane:2.5% methanol:2.5% 2-propanol:0.01% triethylamine:0.01% ammonium acetate. The internal standard and carotenoid content of the plasma and product were detected at 450 nm. All solvents were HPLC grade and were filtered and degassed before use.

    Mononuclear cell isolation and DNA extraction. Mononuclear cells were isolated according to the method of Boyum et al. (18). Blood (15 mL) was diluted (1:1) with HSS and layered over Ficoll-Paque. Samples were centrifuged at 400 x g for 30 min, after which the mononuclear cell interface was removed, washed twice with HBSS, resuspended in 0.2 mL TRIS buffer and kept at –70°C for later DNA extraction. DNA was extracted using the FlexiGene DNA kit from Qiagen and held at 4°C for genotype determination.

    Genotype determination of ABCG5. SNP genotyping was used. Target genomic DNA regions were first amplified by PCR. The polymorphic site in codon 640 of the ABCG5 gene locus was examined. The use of sequence-specific PCR primers (ABCG5, Fwd: 5'CCTTGACAGGCACTCAAATG-3" and Rev: 5' TTTCTCAATGAATTGAATTCCTT-3') allowed for the fragment of DNA containing the polymorphism to be amplified. After PCR, mini-sequencing with fluorescent-labeled ddNTPs was conducted. The extension reaction was 5 µL volume consisting of 2.5 µL of SNaPshot ready reaction mastermix (Applied Biosystems), 0.5 µL water, 0.015 mL mixed PCR product, and 0.0005 mL of the following probe (5'37c- TTTCTCAATGAATTGAATTCCTT-3') mixture (0.02 mmol/L). The reaction conditions were 35 cycles of 96°C for 30 s, 50°C for 30 s and 60°C for 30 s. The reaction products were incubated for 60 min at 37°C with 5 U calf intestinal phosphatase to remove unincorporated primers and dNTPs, followed by incubation at 15 min at 75°C to inactivate the enzyme. Genotyping was carried with the final products on an ABI Prism 3100 genetic analyzer (Applied Biosystems) using Genotyper version 3.7.

    Data analysis. All statistical analysis was performed using SPSS 12.0 for windows. Differences with P < 0.05 were considered significant for metabolic measurements; P < 0.1 was considered significant for the interactions between genotype and diet. Unpaired t test was used to compare initial characteristics between men and women. Repeated-measures ANOVA was used to analyze changes in plasma lipids and carotenoids during the EGG and the SUB periods. All allele frequencies were analyzed using a ²-goodness-of-fit test to determine whether the observed values differed from Hardy-Weinberg equilibrium. Differences between allele groups with regard to baseline, EGG, and SUB plasma measurements were examined using one-way ANOVA. Multivariate ANOVA was performed to determine interactions between diet and genotype while controlling for potential cofounders such as gender and BMI. When necessary, post hoc testing was completed using the Least Significant Difference procedure.

	RESULTS

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

 
Participants maintained their body weight throughout the study (data not shown). Women had higher baseline plasma TC (P < 0.05) and HDL-C (P < 0.001) than men (Table 1). In contrast, men had higher plasma TG concentration than women (P < 0.05). Baseline plasma LDL-C concentrations did not differ between gender groups (Table 1). As previously reported, subjects complied with the requirements of the NCEP step I diet during both dietary periods. The mean contribution of energy derived from total (31.4 ± 5.5%) and saturated (10.9 ± 2.4%) fat during the EGG period was higher (P < 0.01) than total (26.6 ± 7.1%) and saturated (9.4 ± 2.6%) fat consumed during the SUB period for all participants. Furthermore, intakes of monounsaturated fatty acids (MUFA; 13.05 ± 2.77%) and PUFA (6.6 ± 1.97%) were also higher during the EGG period compared with those values reported for the respective nutrients (11.42 ± 3.3 and 6.07 ± 2.1%) during the SUB period. Dietary analysis also confirmed that a significantly (P < 0.0001) greater intake of dietary cholesterol was achieved by EGG consumption compared with SUB. Similarly, the intake of lutein + zeaxanthin was 1834 ± 827 µg/d during the EGG and 1505 ± 636 µg/d during the SUB period (P < 0.001).

View larger version (9K): [in this window] [in a new window]   FIGURE 1  Changes in plasma LDL-C between the EGG and the SUB periods for individuals classified with the C/C (n = 68) or G/G or C/G phenotypes (n = 23).

View larger version (8K): [in this window] [in a new window]   FIGURE 2  Changes in plasma lutein between the EGG and the SUB periods for individuals classified with the C/C (n = 27) or G/G or C/G phenotypes (n = 13).

	DISCUSSION

TOP ABSTRACT SUBJECTS AND METHODS RESULTS DISCUSSION LITERATURE CITED

The dietary variations in cholesterol, SFA, MUFA, and PUFA that occurred between EGG and SUB can be attributed primarily to the eggs consumed. One whole egg provides 313.5 kJ, 1.5 g of saturated fatty acid (SFA), 1.9 g of MUFA, and 0.682 g of PUFA(20). Epidemiologic data clearly indicate that a strong positive relation exists between the percentage of energy obtained from SFA and CHD incidence (21). It was determined that a fluctuation in LDL-C of 0.120 mmol/L (4.6 mg/dL) can be expected for every 1% change in SFA intake with relation to the percentage of total energy consumed (22). It was also determined previously that diets that replace SFA with MUFA and PUFA decrease plasma LDL-C concentrations (23). We suggest that the changes in plasma cholesterol were due to the dietary cholesterol challenge because both types of fatty acids (SFA and MUFA) increased during the EGG period and counterbalanced their effects on plasma lipids.

In contrast to the results presented here, Weggemans et al. (11) found that the plasma TC response to dietary cholesterol tended (P < 0.25) to be higher in carriers of the G allele. That study analyzed the results of 3 different interventions with various experimental designs. These designs were similar to the one used in the present study in that participants consumed both a high- and low-cholesterol diet. Participants either received egg as a supplement to their regular diet or received all of the food that they were to consume during the high-cholesterol period. For the low-cholesterol period, subjects were given dietary guidelines to follow. The study examined 99 people who participated in one (44%) or more (56%) of 8 trials for a total of 202 samples. The authors suggested that the precision of the data was improved by multiple measurements obtained for 55 of the study participants and should be considered more accurate than a single trial. However, the multiple measurements were not combined to produce one mean response for each duplicate subject, and each trial utilized a different study design with varying amounts of cholesterol intake over different time periods. Furthermore, because the reproducibility of individual differences in response was documented previously in several controlled and field trials (24), we are confident that our results would not be different if the study were repeated with the same subjects. The discrepancy that exists with the available data may indicate that a transporter other than ABCG5 has a greater influence over the regulation of cholesterol absorption after excess dietary cholesterol consumption. Using a mouse model obtained from a genetic cross between 2 strains, Sehayek et al. (25) identified loci on chromosomes 14 and 12 (independent of the ABCG5 locus) that may contain additional genes involved in the regulation of intestinal plant sterol and cholesterol absorption.

Dietary cholesterol and carotenoids are metabolized in a similar manner. Carotenoids, in their ester form, are hydrolyzed in the lumen of the small intestine by various lipases and esterases for incorporation into the lipid core of the micelle. Micelles then transport these nutrients into the enterocyte where they are packaged into a chylomicron particle and released into the circulation. Plasma chylomicrons are remodeled by the action of lipoprotein lipases that hydrolyze TG to result in the formation of a smaller remnant, which can be taken up by the liver. Once in the hepatocyte, cholesterol and carotenoids are packaged into lipoproteins. Most of the lutein and zeaxanthin is directed to HDL; a smaller percentage of the total concentration is incorporated into the VLDL particle. In contrast, - and ß-carotene are carried primarily in the VLDL particle. When excess dietary cholesterol is consumed, a certain percentage of the population may experience increased intestinal absorption, which allows for more cholesterol to reach the circulating pool via lipoproteins. It is possible that these individuals may also increase absorption of carotenoids in response to elevated consumption.

Evidence suggests that increased consumption of foods rich in lutein and zeaxanthin can be directly associated with elevated serum (12,13) and adipose tissue concentrations of these carotenoids. Because of their antioxidant properties and their association with lipoproteins in the plasma, lutein and zeaxanthin may also function to protect against CHD. Circulating lutein and zeaxanthin may function to reduce arterial plaque formation by decreasing the expression of adhesion molecules, which are needed for monocyte association with the artery (26). In addition, studies showed that consumption of foods high in lutein results in an increased concentration of this carotenoid within the LDL particle (27,28). Lutein contained in LDL demonstrated antioxidant function in vivo as a scavenger of peroxynitrite, the reaction product of nitirc oxide and superoxide (29). Peroxynitrite, when in the presence of LDL, can destroy lipid-protein complexes, creating a particle that is more susceptible to uptake by the macrophage scavenger receptor. Furthermore, 2 epidemiological studies, which examined carotid intima thickness as a measure of CHD, showed that high levels of plasma lutein produced a significant reduction in disease risk (30,31).

In conclusion, although the number of individuals examined in this study was small (91 for dietary cholesterol and 40 for the carotenoids), we were able to identify a possible effect of an ABCG5 polymorphism on the absorption of dietary cholesterol and carotenoids. This information provides some insight into the mechanisms that control the plasma response to dietary components. However, it is difficult to determine what effect a single nucleotide polymorphism has on lipoprotein metabolism in isolation from other factors. Overall, it is likely influenced by variation at multiple loci, which could be further affected by various environmental factors. Therefore, we are reminded that these findings are pieces of a bigger puzzle and that further research is required to understand their effect on metabolism and disease risk.

	FOOTNOTES

 
¹ Supported in part by from a grant from the American Egg Board/Egg Nutrition Center to M.L.F. and contracts 53-K06-5-10 and 58-3148-2-083 from U.S. Department of Agriculture to J.M.O. K.L.H. is the 2002 recipient of the American Egg Board Egg Nutrition Center Dissertation Fellowship in Nutrition.

³ Abbreviations used: ABC, ATP binding cassette; CHD, coronary heart disease; HDL-C, HDL cholesterol; LDL-C, LDL cholesterol; MUFA, monounsaturated fatty acids; PMSF, phenylmethylsulfonyl fluoride; SFA, saturated fatty acids; SNP, single nucleotide polymorphism; TC, total cholesterol; TG, triglycerides.

Manuscript received 8 January 2006. Initial review completed 14 February 2006. Revision accepted 23 February 2006.

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This Article Abstract Full Text (PDF) Purchase Article View Shopping Cart Alert me when this article is cited Alert me if a correction is posted Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Herron, K. L. Articles by Fernandez, M. L. Search for Related Content PubMed PubMed Citation Articles by Herron, K. L. Articles by Fernandez, M. L.