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3 Departamento de Nutrición y Bromatología I (Nutrición), Facultad de Farmacia, Universidad Complutense de Madrid, 28040 Madrid, Spain; 4 Provident Clinical Research (formerly with the Chicago Center for Clinical Research), Chicago, IL; 5 Lipid Metabolism Laboratory, and 6 Nutrition and Genomics Laboratory, Jean Mayer USDA Human Nutrition Research Center on Aging, Tufts University, Boston, MA
* To whom correspondence should be addressed. E-mail: frasan{at}farm.ucm.es.
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ABSTRACT |
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 TOP ABSTRACT IntroductionSubjects and MethodsResultsDiscussionLITERATURE CITED |
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-tocopherol was lower in E4 subjects than in E2 individuals, and LDL-C was lower in E2 carriers than in E3 and E4 carriers (P < 0.05 for all). During sterol consumption, TC, LDL-C, and ApoB concentrations and the TC:LDL-C and LDL-C:HDL-C ratios decreased in only E2 and E3 subjects and TAG decreased in only E2 subjects (all P < 0.05 vs. control). Significant reductions in serum carotenoids (P < 0.05 vs. control) were demonstrated for some alleles: β-carotene and lycopene in E2 and E4; -carotene in E3; cryptoxanthin in E3 and E4; zeaxanthin in E4; lycopene in E2 and E4; and lutein in E2 carriers. Thus, responses to plant sterols vary by ApoE genotype and may be of little value in ApoE4 carriers, who had reductions in serum carotenoid concentrations but not in TC, LDL-C, or ApoB.
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Introduction |
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TOPABSTRACTIntroductionSubjects and MethodsResultsDiscussionLITERATURE CITED |
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Apolipoprotein (Apo) E plays an essential role in the metabolism of cholesterol and triacylglycerols (TAG). ApoE is a major protein constituent of TAG-rich lipoprotein (chylomicron and VLDL) particles and their remnants, as well as HDL. It serves as ligand for the uptake of these lipoprotein particles by their receptors (10,11). The APOE gene is located in the long arm of chromosome 19 (12). The human APOE gene locus is highly polymorphic, but 3 common alleles (
2, 3, and 4) coding for 3 major ApoE isoforms, designated E2, E3, and E4 (12), have received the most attention. The ApoE variants differ in binding affinity for several receptors and lipoproteins (13), which translates into alterations in lipoprotein metabolism as well as plasma lipid phenotypes (14). In addition, subjects with the various APOE alleles differ in their ability to absorb cholesterol from the intestine, synthesize cholesterol and bile acids, and convert VLDL-ApoB to LDL-ApoB (15–17).
Data from population studies indicate that E2 allele carriers have lower LDL-C levels than E3 homozygotes, whereas the E4 allele tends to be associated with higher LDL-C concentrations (18). There is also some evidence supporting the hypothesis that E4 carriers may respond better to a low-fat diet intervention, in terms of LDL-C reduction, than E2 and E3 carriers (9,18).
Several studies have investigated the plasma lipid responses to plant stanol supplementation in the context of the APOE locus (17,19–24). In most studies, sterols and stanols have been shown to lower LDL-C irrespective of the APOE alleles. The specific role of ApoE as a determinant of plasma lipid response to plant sterols remains controversial, due in part to considerable differences in study design and the relatively low numbers of participants in most studies (3,4,6).
The present study evaluated the potential modulation of the APOE locus on the serum lipid responses to reduced-fat spreads containing plant sterol esters consumed as part of the National Cholesterol Education Program Step-I (NCEP-I) diet among individuals with mild-to-moderate primary hypercholesterolemia. In addition, due to the effects of plant sterols on fat absorption (3,4,6,25,26), we hypothesized that the absorption and plasma levels of fat-soluble vitamins and carotenoids may be modulated by the APOE locus.
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Subjects and Methods |
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This was a randomized, double-blind, controlled clinical trial conducted at a single research center (Chicago Center for Clinical Research, Chicago, IL). Participants were recruited via advertising and included men and women (21–75 y of age) with LDL-C concentrations between 3.4 and 5.2 mmol/L and TAG concentrations < 4.0 mmol/L who had abstained from all hypolipidemic therapy (including lipid-lowering medications and supplements thought to alter lipid metabolism) during the 4 wk prior to screening. Use of lipid-altering drugs and supplements was also excluded throughout the entire study. Details regarding additional eligibility requirements such as BMI, previous metabolic or related diseases, high blood pressure, and medication use have been previously described (27).
Following a 4-wk lead-in period for diet stabilization during which participants consumed a 50% fat spread as part of a NCEP-I diet (as instructed by a registered dietitian), subjects were randomly assigned to receive a control spread without added plant sterol-esters (control group, n = 87) or 1 of 2 plant sterol-ester enriched spreads (n = 120) designed to provide intakes of 1.1 or 2.2 g/d plant sterols for 5 wk. Subjects and investigators were unaware of subject randomization status. The protocol was approved by Schulman Associates institutional review board. Procedures were conducted according to good clinical practice, the Declaration of Helsinki (1996), and United States 21 Code of Federal Regulations Part 50, Protection of Human Subjects, and Part 56, Institutional Review Boards. Signed, written informed consent was obtained from all subjects before protocol-specific procedures were conducted.
Study products
Nutrient compositions and ingredients of the control and plant sterol-enriched, reduced-fat spreads (Lipton) are shown (Table 1). The plant sterols (Archer Daniels Midland) were blended into the oil phase of the spreads and processed into the test products according to standard procedure on a full-scale margarine processing line. The major plant sterols in the products were β-sitosterol (
50%), campesterol (25%), and stigmasterol (20%). Concentrations of plant sterols in 100 g of the low plant sterol-ester–enriched spread were 4 g β-sitosterol, 2 g campesterol, and 1.6 g stigmasterol. The high plant sterol-ester–enriched spread contained 8 g β-sitosterol, 4 g campesterol, and 3.2 g stigmasterol per 100 g.
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Subjects visited the clinic at wk –2 and –1 (screening), wk 0 (baseline), and wk 2, 3.5, and 5 (treatment) for assessment of vital signs, body weight, and serum lipid profile. Additionally, ApoA-I and B were measured at wk –1, 0, 3.5, and 5. Serum concentrations of carotenoids were measured at wk –1, 0, 3.5, and 5. In a subgroup of subjects (n = 71), serum fat-soluble vitamins and sterol concentrations were assessed at wk 0 and 5. To evaluate the effects of APOE on responses, ApoE isoforms were determined and subjects were categorized into 3 groups: E3, 3/3; E2, 2/3 plus 2/2; and E4, 3/4 plus 4/4. Ten subjects with the 2/4 alleles were excluded from the study.
Analyses
DNA extraction and genotyping. Leukocyte DNA was extracted from 100–200 µL buffy coats by a salting-out procedure as previously described (28) using a capture column kit (Gentra System). A 244 bp of the APOE including the 2 polymorphic sites was amplified by PCR in a DNA Thermal Cycler (PTC-100 MJ Research) using standard protocols of Lipid Metabolism Laboratory at the USDA Human Nutrition Research Center on Aging at Tufts University.
Each amplification was performed by using 25 ng of genomic DNA in a volume of 12 µL containing 7.5 pmol of each oligonucleotide: forward 5'-ACAGAATTCGCCCCGGCCTGGTACAC-3', reverse 5'-TAAGCTTGGCACGGCTGTCCAAGGA-3', 2.5 µmol/L nucleotides, 50 mmol/L magnesium chloride, 10 mmol/L Tris, pH 8.4 buffer, and 5 U in 1 µL of Taq polymerase (Gibco BRL, Life Technologies).
Each reaction mixture was heated at 94°C for 30 s, 64°C for 30 s, and 72°C for 35 s. Forty cycles were used. A final extension was performed at 72°C for 5 min. The 12-µL PCR products were subjected to restriction enzyme analysis by digestion with 0.4 U of the restriction endonuclease Hha I and the fragment products were separated by gel electrophoresis on a 4% methaphore gel. After electrophoresis, the gel was treated with ethidium bromide for 20 min and DNA fragments were visualized by UV illumination.
Serum lipids and Apo. Serum TC, HDL-C, TAG, and ApoA-I and B were measured by the methods of Myers et al. (29) in accordance with the CDC lipid measurement standardization program (Covance Central Laboratory Services). LDL-C was calculated by using the Friedewald et al. (30) equation.
Serum vitamin, sterol, and carotenoid profiles.
Samples for vitamin, carotenoid, and sterol analyses were frozen at –80°C and all measures for each subject were completed in the same analysis (Lipid Metabolism Laboratory, Tufts University, Boston, MA). Analyses of retinol, tocopherol, dihydroxyvitamin D, and phylloquinone were conducted according to previously described HPLC procedures (31–33). After lipid extraction, saponification, and reextraction, serum total sterols, total plant sterols, β-sitosterol, and campesterol were separated from fatty acids and quantified by HPLC (34). Serum concentrations of carotenoids (-carotene, trans-β-carotene, lycopene, lutein, zeaxanthin, and cryptoxanthin) were determined by HPLC (35) using a C30 carotenoid column (Gastrointestinal Nutrition Laboratory, Tufts University). To prevent photodegradation of carotenoids, all serum handling, standard preparation, and HPLC procedures were performed under dim red light.
Statistical analyses. Statistical analyses were carried out using SPSS (version 15 for the personal computer). Lipid, lipoprotein, and vitamin responses were similar in the 1.1 and 2.2 g/d plant sterol treatment groups (data not shown). Therefore, to increase the statistical power of the current analyses, data from both groups receiving plant sterols were combined into a single plant sterol group.
For lipid variables, baseline was defined as the mean of values obtained at wk –2, –1, and 0 and end-of-study was the mean of values obtained at wk 3.5 and 5. For Apo, carotene, and carotenoids, baseline was defined as the mean of values obtained at wk –1 and 0 and end-of-study was the mean of values obtained at wk 3.5 and 5. This study was designed to have a power of 85% to detect a 5% difference between control and plant sterol groups in LDL-C response. A pooled SD of 10% for the change from baseline LDL-C was assumed for this calculation. Serum concentrations of TAG, the TC:HDL-C, LDL-C:HDL-C, and ApoA-I:ApoB ratios, plant sterols, carotenoids, and fat-soluble vitamins were not normally distributed; therefore, logarithmic transformations were applied. Baseline values were compared in different ApoE carriers using 1-way ANOVA. Post hoc multiple comparisons between groups were carried out using the Scheffé method. Repeated-measures ANOVA was used to compare the effect (absolute changes in mmol/L, g/L, or µmol/L) of plant sterol to control on the studied variables in each ApoE carrier. Gender, age, BMI, tobacco, and alcohol consumption were included as covariates in some statistical comparisons. Spearman correlations between changes in LDL-C and fat-soluble vitamin, carotene, and carotenoid responses were performed.
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Results |
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TOPABSTRACTIntroductionSubjects and MethodsResultsDiscussionLITERATURE CITED |
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-tocopherol was lower (P < 0.05) in the E4 group (36.0 ± 10.1 µmol/L) than in the E2 group (53.9 ± 19.7 µmol/L) and phylloquinone was higher (P < 0.05) among E2 subjects (0.064 ± 0.086 nmol/L) than E3 (0.026 ± 0.020 nmol/L) and E4 (0.024 ± 0.012 nmol/L) subjects.
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-carotene was lower in E3 (P < 0.05), cryptoxanthin was lower in E3 (P < 0.01) and E4 (P < 0.05), zeaxanthin was lower in E4 (P < 0.01), lycopene was lower in E2 (P < 0.05) and E4 (P < 0.05), and lutein was lower in E2 carriers (P < 0.05) (Table 4). After adjusting data for TC, zeaxanthin decreased (P < 0.05) in E4 and β-carotenes and cryptoxanthin were decreased (P < 0.001 and P < 0.05, respectively) in E3, whereas lycopene was reduced in E2 and E4 carriers (both P < 0.05) (data not shown). Plant sterols and campesterol increased in E3 (P < 0.001) and E4 (P < 0.01) and β-sitosterol increased in only E3 subjects (P < 0.01) (Fig. 1). vitamin K, cholecalciferol, and tocopherols were not significantly affected in any E carriers (data not shown).
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Discussion |
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TOPABSTRACTIntroductionSubjects and MethodsResultsDiscussionLITERATURE CITED |
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Lipid and lipoprotein responses to plant sterol and control consumption differed among APOE alleles. Comparisons of the effects of control and plant sterols within ApoE isoforms indicated that E2 subjects experienced greater lipid altering than E3 and E4 carriers, whereas E4 subjects had fewer changes than E2 and E3 subjects. These findings are of interest, because most previous reports focused on the comparison between the E4 and E3 alleles and the E2 alleles were rarely considered separately. The difference in results between individuals with E3 and E4 alleles is in disagreement with those reported in other studies. Plat and Mensink (19) did not find significant variations between the different APOE individuals with regard to their lipoprotein response to 4 g/d of stanol esters. Katan et al. (24) also observed that 31 E3/E4 and 57 E3/E3 healthy normocholesterolemic individuals responded similarly to 3.2 g/d of plant sterols. Vanhanen et al. (37) suggested that the decrease in TC and LDL-C concentrations due to sitostanol intake was more consistent in E4 than in E3 hypercholesterolemic subjects. Similar results were reported by Ishiwata et al. (22) using 2 g/d of plant stanols. Nonetheless, the effects of 3 g/d of stanol in the diet on LDL-C and TC levels were significant in E3, but not in E4, subjects (22). In the present study, plant sterol treatment (compared with control) was more effective for lipid lowering in E2 and E3 carriers than in E4 individuals. The different responses were apparently not related to the increased serum sterol levels. At present, we have no clear explanation for these divergent findings, although the cause may involve differences in study design, the particular plant sterol or stanol used, or the number and/or type of volunteers (normo- or hypercholesterolemic).
Baseline serum plant sterol concentrations do not appear to be related to APOE alleles in the present study. These results were probably due to the fact that Westernized diets contain very low amounts of plant sterols (25). Uusitupa et al. (16) found higher serum campesterol and sitosterol levels in E4 than in E3 subjects. In contrast, Miettinen and Vanhanen (20) found lower circulating levels of plant stanols in E4 patients consuming low amounts of sitostanol. However, because sitostanol decreases absorption of sterol esters (38), the results of studies employing stanols are not comparable to those using sterols. Serum plant sterol concentrations increased in all of the subjects combined (net difference from control, 38.0%; P < 0.001) but rose significantly only in the E3 and E4 participants (net difference from control, 46.6% and 26.8%, respectively). The lack of a significant change in E2 subjects consuming plant sterols (net difference from control, 10%; P > 0.1) may be explained by the small number of individuals in this group in whom serum plant sterols were measured and the relatively large SD of the response.
In general, baseline values of fat-soluble vitamins and carotenoids did not differ among the various ApoE alleles; -tocopherol and vitamin K levels were highest in E2 and lowest in E4 carriers. These findings are of interest, considering the higher CVD incidence described in E4 carriers than in their E2 counterparts (39). Plant sterol esters are thought to reduce serum cholesterol in part by competing with dietary and bile cholesterol for incorporation into the bile micellar phase within the intestinal milieu (3,4,6,23); thus, it is possible that this physical-chemical property may also decrease the absorption of lipophilic dietary compounds such as fat-soluble vitamins and carotenoids (3,4,6,25,26). Serum concentrations of carotenes, carotenoids, and -tocopherols were lower in all of the subjects who consumed plant sterol-enriched spreads than in their control counterparts, which has important implications for the general population consuming such spreads.
Carotenes and carotenoids are known to be associated primarily with LDL (40). As expected (3,6,25,26), in this study, important reductions in the lipophilic carotenoids (β-carotene, -carotene) were detected. Cryptoxanthin also had relatively high reductions in serum. Changes in all carotene and carotenoid concentrations were positively correlated to changes in LDL-C (P 
0.021 for all comparisons); β-carotene levels decreased 
20% in E2 and E3 carriers but just 3.4% in E4 individuals. According to Esterbauer et al. (41), β-carotene may play an important role in protecting LDL against peroxidation. Thus, although plant sterols resulted in lower LDL-C levels and, in turn, would be expected to reduce CVD risk, the higher relative decrease in β-carotene concentration than in LDL suggests that those LDL contained less β-carotene. Depletion of this antioxidant in certain individuals, particularly in E2 and E3 carriers after plant sterol treatment, could increase their LDL oxidative susceptibility. This hypothesis should be tested in future studies. Plant stanol esters have also been reported to significantly reduce (30%) serum β-carotene concentrations (20% after cholesterol correction), but not -carotene (42). In the present study, -carotene decreased significantly with sterol esters compared with control only in E3 carriers. Adjusting the carotene concentration for TC levels generally minimizes the effect of plant sterol therapy (3,4,6,25). Following adjustment for TC concentration, plant sterols compared with control did not significantly affect -carotene levels in E3 carriers in the present study. E4 carriers had significant changes in the concentrations of less lipophilic carotenoids (e.g. zeaxanthin and lycopene) (data not shown).
E4 carriers displayed significant changes in the concentrations of other less lipophilic carotenoids (e.g. zeaxanthin and lycopene) (data not shown). The association between blood or tissue concentrations of lycopene and other carotenoids and CVD has been studied in recent years (43). Several studies have reported an inverse association between circulating carotenoids and atherosclerosis or CVD. An L-shaped association between plasma lycopene and acute coronary event or stroke was observed in the Finnish prospective Kuopio Ischemic Disease Risk Factor study involving 1038 middle-aged men (44). The risk reduction associated with the concentration of this carotenoid remained significant during a follow-up of >7 y (43). The antioxidant activity of carotenoids in multilamellar liposomes has the following ranking: lycopene > -tocopherol > -carotene > β-cryptoxanthin > zeaxanthin > β-carotene > lutein (45). Esterbauer et al. (40) indicated that autooxidation of LDL occurs when LDL is largely depleted of its endogenous vitamin E, carotenoids, and β-carotene. The antioxidants disappearing first were - and 
-tocopherols followed by carotene and carotenoids (lycopene and cryptoxanthin first and β-carotene last). In accordance with Gylling et al. (42), although plant sterols significantly decreased β-carotene levels in the present study, serum concentrations of its metabolite, retinol, did not vary among the different APOE carriers.
In summary, the results of this study suggest that ApoE4 subjects with hypercholesterolemia, consuming plant sterols within the framework of the NCEP-I diet, experience less TC, LDL-C, and ApoB lowering than E2 and E3 carriers. The effect of plant sterols on β-carotene suggests that ApoE2 and E3 individuals may be more sensitive to peroxidative stress than other allele carriers. The European Community (46) has recently approved legislation requiring functional foods containing plant sterols to provide label information regarding target populations, including ages, concomitant medication warnings, and recommended dosages. Plant sterol therapy should be intended for hypercholesterolemic subjects and consumption of functional foods containing plant sterols (e.g. spreads, milk products) should be avoided by pregnant women or children under 5 y. Moreover, plant sterol margarines should not be consumed in the rare instances of sitosterolemia. Plant sterol supplements actually contribute to atherosclerosis (4,6,25) in sitosterolemic individuals by increasing serum plant sterol levels (3,6,47). In this study, carotenes, carotenoids, and -tocopherols decreased in the group of all subjects combined. Our findings suggest that plant sterol therapy may be of little value for E4 subjects with hypercholesterolemia due to its effect of lowering circulating carotenoid concentrations in the absence of significant benefits in TC, LDL-C, and ApoB levels.
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FOOTNOTES |
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2 Author disclosures: F. J. Sánchez-Muniz, K. C. Maki, E. J. Schaefer, and J. M. Ordovas, no conflicts of interest.
7 Abbreviations used: Apo, apolipoprotein; CVD, cardiovascular disease; LDL-C, LDL cholesterol; NCEP-I, National Cholesterol Education Program Step-I; TAG, triacylglycerol; TC, total cholesterol.
Manuscript received 1 April 2008. Initial review completed 4 May 2008. Revision accepted 15 October 2008.
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LITERATURE CITED |
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