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INSERM (B.H., S.G., G.S., S.V.S.)
Université Henri Poincaré, Faculté de Pharmacie (B.H., G.S., S.V.S., P.L.), Nancy, FRANCE
Address reprint requests to: Bernard Herbeth, INSERM U525, Faculté de Pharmacie, 30 rue Lionnois, F54000 Nancy, FRANCE. E-mail: Bernard.Herbeth{at}nancy.inserm.fr
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ABSTRACT |
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ABSTRACT
INTRODUCTION
METHODS
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Objective: We aimed to analyze whether serum concentrations of 5 carotenoids (lutein-zeaxanthin, β-cryptoxanthin, lycopene, 
-carotene, β-carotene) are associated with common polymorphisms of Apo E, Apo B, Apo CIII, CETP, and LPL.
Methods: Serum concentrations of lutein-zeaxanthin, β-cryptoxanthin, lycopene, -carotene, and β-carotene were measured and polymorphisms of Apo E (cys112arg and arg158cys), Apo B (thr71ile), Apo CIII [C(–482)T, Apo CIII T(–455)C, Apo CIII C1100T, Apo CIII C3175G, Apo CIII T3206G], CETP (ile405val), and LPL (S447X) were determined in a sample of 447 children and adults drawn from the Stanislas Study.
Results: After adjustment for age, sex, smoking, physical activity, oral contraceptive use, BMI, serum cholesterol and triglyceride concentrations, and fruit and vegetable intakes, carriers vs. non carriers of the lipoprotein lipase X447 allele had significant lower concentrations of lutein-zeaxanthin, β-cryptoxanthin, -carotene and β-carotene; differences vs. S447S genotype being the largest for X447X: –18.8%, –50.5%, –54.8% and –47.1%, for the four carotenoid fractions, respectively. No significant association was noticed for lycopene concentration. None of the other tested polymorphisms was significantly related to the serum carotenoid concentrations.
Conclusions: Our investigation for the first time demonstrates that LPL S447X polymorphism could alter serum concentrations of carotenoids in healthy individuals, independently of serum cholesterol and triglyceride concentration. These data indicate that genetic factors could be involved in the variability of carotenoid bioavailability and bioconversion.
Key words: lutein-zeaxanthin, β-cryptoxanthin, lycopene, carotene, lipoprotein lipase S447X polymorphism
Abbreviations: apo = apolipoprotein • CETP = cholesterol ester transfer protein • LPL = lipoprotein lipase • BMI = body mass index • HDL = high density lipoproteins • VLDL = very low density lipoproteins • LDL = low density lipoproteins • TG = triglyceride
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INTRODUCTION |
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INTRODUCTION
METHODS
RESULTS
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The absorption and effective use of β-carotene and other carotenoids may not be uniform among individuals and populations [6–11]. Some people show little or no increase in blood β-carotene after an oral dose of β-carotene or a β-carotene-rich diet [10–12]. Possible explanations for this low response are impaired intestinal absorption of β-carotene, exaggerated conversion to vitamin A, inefficient incorporation into chylomicrons, or accelerated clearance due to atypical lipoprotein metabolism. As the low-responder trait seems to be a stable characteristic, genetic factors included in the G class of the mnemonic term SLAMENGHI [13] could be implicated but have not been quantified in a systematic way [2]. Genes whose products affect lipoprotein metabolism, e.g., apolipoproteins, enzymes and receptors, particularly in response to dietary change, should be potential candidates [14,15], however, no study has investigated this possibility. The present study was designed to investigate the associations of serum concentrations of 5 carotenoid fractions (lutein-zeaxanthin, β-cryptoxanthin, lycopene, -carotene and β-carotene) with common polymorphisms of apolipoproteins E, CIII and B, cholesteryl ester transfer protein (CETP) and lipoprotein lipase (LPL) known to affect lipid and lipoprotein metabolism, taking into account the effect of covariates such as lipid carriers.
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INTRODUCTION
METHODS
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Dietary Record
Dietary intake was assessed by a 3-day dietary record [17], which was completed during 2 weekdays and a weekend day assigned at random for each family. All subjects received guidelines from a dietician on the procedures to complete the dietary record and to measure food portion. For young children, the 3-day diary was filled in by the mother and the child together. One week later, the 3-day record was checked and completed by the dietician by using colored photographs of foods, each with three different portion sizes. Macronutrient and micronutrient intakes were estimated with a updated computerized version of the "Répertoire Général des Aliments" [18]. β-carotene equivalents were calculated as the sum of β-carotene and of half of the amounts of -carotene, 
-carotene and cryptoxanthin [19]. Due to the design of the Stanislas study, the 3-day food consumption diary was filled during the week following the blood sampling. The three-day diary used in the Stanislas study is considered as a dietary instrument that estimates habitual diet with a relatively good repeatability, this several-day gap should not induce imprecision in the estimation of the relationships between food intakes and carotenoid concentrations in serum. Within this short period of time, changes in dietary habits are minimized.
Biological Measurements
Blood samples were collected after an overnight fast. Fresh serum was used for lipid profile determination. Serum total cholesterol (TC) and triglyceride (TG) concentrations were measured by using commercially available kits (Merck, Darmstadt, Germany) on AU5021 apparatus (Olympus, Merck). Serum HDL cholesterol (HDL-C) was determined by using phosphotungstate precipitation on Cobas-mira apparatus (Roche) [16] and LDL cholesterol (LDL-C) calculated according to Fridewald formula [LDL-C (mmol/L) = CT (mmol/L) – HDL-C (mmol/L) – TG/2.2 (mmol/L), when TG < 4.6 mmol/L]. None of the 447 individuals of this study had TG concentration higher than 4 mmol/L. Inter-assay precisions were between 1.7% (cholesterol) and 5.2% (HDL cholesterol).
Serum carotenoid concentrations were measured in samples stored in liquid nitrogen in the bio bank of the Centre de Médecine Préventive of Vandoeuvre les Nancy, France. An isocratic high-performance liquid chromatography (HPLC) method adapted from Talwar et al. [20] was used for the simultaneous determination of the five carotenoids (lutein-zeaxanthin, β-cryptoxanthin, lycopene, and - and β-carotene) [21]. Serum samples were deproteinized with ethanol and extracted once with n-hexane. Resulting extracts were injected onto a C18 reversed-phase column eluted with methanol-acetonitrile-tetrahydrofuran (75:20:5, v/v/v), and full elution of all the analytes was realized isocratically within 20 min. The detection was operated by using a diode-array spectrophotometer at channel 450 nm. Echinenone was used as an internal standard. Inter-assay precision ranged from 4.5% (lutein-zeaxanthine) to 13.7% (β-cryptoxanthin).
DNA Polymorphism Determination
Genomic DNA was extracted from peripheral blood samples by the salting out method [22]. The genotypes of apo E cys112arg and arg158cys, Apo B thr7lile, Apo CIII C(–482)T, Apo CIII T(–455)C, Apo CIII C1100T, Apo CIII C3175G, Apo CIII T3206G, CETP ile405val and LPL S447X were determined by a multiplex assay previously described by Cheng et al. [23].
Statistical Analysis
Statistical analyses were performed by using the SAS software package version 8.01 (SAS Institute, Inc., Cary, NC). Triglyceride (TG), lutein-zeaxanthin, β-cryptoxanthin, lycopene, -carotene and β-carotene concentrations were log10-transformed in the analyses in order to improve normality. A chi-square test was performed to determine whether genotype frequencies were in Hardy-Weinberg equilibrium.
For continuous variables, an analysis of variance was performed for characteristic differences among the 4 sex-by-generation groups. When the analysis of variance was significant, a Tukey-Kramer test was used to detect which groups were statistically different from each other. The significance of differences among the groups for the categorical variables was analyzed by using the chi-square test or the Fisher's exact test when cells had expected counts less than 5.
In the overall sample, stepwise multiple regression analysis was carried out to select significant covariates (p 
0.05) among style life factors, drug use, diet intake and related biological analytes. Then, multiple regression analysis was used to test relationships between lipid fractions and/or carotenoid levels and gene polymorphisms accounting for age, sex and the significant covariates selected in the previous step. The polymorphisms were tested in allelic dose effect model. To calculate P for trend, the three genotypes were coded as a single variable and were assigned the following values: AA = 0, Aa = 0.5, aa = 1, "a" being the minor allele.
When a polymorphism was significantly associated with serum carotenoid concentrations, a Dunnett's two-tailed t test was used, testing if Aa or aa genotypes were significantly different from AA genotype. In addition, interaction terms between significant related polymorphisms and the four sex-by-generation groups (fathers, mothers, sons and daughters) were probed. When interactions did not reach significance and in order to improve statistical power, data from the overall sub-sample were analyzed together.
As individuals within a family were not independent, regression analysis were conducted by using the SAS GENMOD procedure with a family factor as repeated statement. GENMOD was based on the Generalized Estimating Equation (GEE). GEE provides a practical method with reasonable statistical efficiency to analyze correlated data such as familial data by modelling the covariance structure of the correlated measurements. Unlike to other methods the measurements must not be assumed to be multivariate normal [24]. A level of P 0.05 was accepted as significant.
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INTRODUCTION
METHODS
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-carotene and β-carotene were significantly different among the four groups. The Apo E, Apo B, Apo CIII, CETP and LPL polymorphism distribution did not significantly deviate from Hardy-Weinberg equilibrium.
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0.05). Oral contraceptive users had significantly lower β-carotene concentrations than non users (P 0.05). BMI was significantly and negatively related to serum concentrations of - and β-carotene. Serum cholesterol concentrations were positively correlated with the level of the 5 carotenoid fractions, while serum triglyceride concentrations were negatively associated with β-cryptoxanthin levels. Fruit intakes were positively related to lutein-zeaxanthin, β-cryptoxanthin, - and β-carotene concentrations, whereas, vegetable intakes were associated with lutein-zeaxanthin and -carotene levels. Accounting for fruit and vegetable intakes, β-carotene equivalent intakes did not significantly enter into multivariate model whatever the 5 carotenoid fractions.
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2 carriers (P for trend 0.001) and higher in minor allele carriers of Apo B thr71ile and Apo CIII C1100T polymorphisms (P for trend 0.05). Moreover, HDL-cholesterol levels were negatively associated with minor allele of Apo CIII C3175G (P for trend 0.05) and positively with minor allele of CETP ile405val (P for trend 0.01). Conversely, no tested polymorphism was significantly related to serum triglyceride concentrations (data not shown). Moreover, no interaction between all the tested polymorphisms and the four sex-by-generation groups was significant.
Relationships between carotenoid levels and the gene polymorphisms were tested in allelic dose effect model, by using multiple regression analysis on 1- age and sex adjusted values and 2- after accounting for the additional significant covariates listed in Table 2. Except for LPL S447X polymorphism none of the tested polymorphisms was significantly related to the serum concentrations of the 5 carotenoid fractions by using both types of adjusted values (data not shown). Since no interaction between LPL S447X polymorphism and the four sex-by-generation groups was significant data were presented together for the overall sample in Fig. 1. After adjustment for age, sex, physical activity, smoking, oral contraceptive use, BMI, serum cholesterol and triglyceride concentrations, and fruit and vegetable intakes, carriers vs. noncarriers of the lipoprotein lipase X447 allele had significant lower concentrations of lutein-zeaxanthin, β-cryptoxanthin, -carotene and β-carotene; differences vs. S447S genotype being the largest for X447X: –18.8%, –50.5%, –54.8% and – 47.1%, for the four carotenoid fractions, respectively. No significant association was noticed for lycopene concentration. Except for lycopene, polymorphism explained 0.8%, 1.4%, 2.4% and 1.4% of the variance after adjustment for covariates, for lutein-zeaxanthin, β-cryptoxanthin, -carotene and β-carotene, respectively.
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DISCUSSION |
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-carotene and β-carotene. No significant association was noticed for lycopene concentration. As the major enzyme involved in the hydrolysis of triglycerides in VLDL and chylomicrons, LPL plays an important role in postprandial lipid metabolism and in the formation of LDL and HDL [25] and could modulate metabolism of fat soluble micronutrient such as carotenoids. In addition to its catalytic function, it appears to modulate binding of lipoproteins to various receptors [26].
After absorption by the mucosa of the small intestine, provitamin A carotenoids (β-cryptoxanthin, -carotene and β-carotene) and nonprovitamin A carotenoids (lutein, zeaxanthin and lycopene) are incorporated into chylomicrons and secreted into the lymph for delivery to the blood stream [3,27]. Due to their polarity, it is hypothesized that xanthophylls (lutein, zeaxanthin and β-cryptoxanthin) are surface oriented, whereas non polar hydrocarbon carotenes (lycopene, -carotene and β-carotene) are localized in the core of lipoproteins [28,29]. Prior to hepatic uptake, chylomicrons in the bloodstream are rapidly degraded by lipoprotein lipase associated with tissue endothelium and transformed into chlylomicron remnants. During this process, some carotenoids may be taken up by extrahepatic tissues. However, most chylomicron remnants deliver carotenoids to the liver where they are stored or resecreted into the bloodstream in very low density lipoproteins (VLDL) [28]. Circulating VLDL are subsequently delipidated to low density lipoproteins (LDL). In the fasted state, as in our study, LDL are the main carriers of non-polar carotenoids in human serum [29]. The more polar xanthophylls are evenly distributed between high density lipoproteins (HDL) and LDL, and to a lesser extent VLDL.
The S447X polymorphism results in a truncation of the LPL protein by two amino acids (Ser-Gly) at the carboxy-terminal [30,31] and functional differences between the LPL isoforms, which could account for their effect on plasma HDL-cholesterol, triglyceride and related fat soluble micronutrients levels, have not been satisfactory elucidated. An excess of the truncated monomers might enhance binding of triglyceride-rich lipoproteins to endothelial surfaces or to some receptors [32] rather than altering lipolytic activity [33–35]. With regard to plasma lipid, the X447 allele in meta-analysis was shown to be associated with small decreases in plasma triglycerides and increases in HDL-cholesterol [36,37]. In the present study, we did not find the association of the X447 allele with more favorable HDL-cholesterol and triglyceride profiles.
There are several mechanisms by which the S447X polymorphism may alter serum carotenoid concentrations accounting for increased secretion of monomer of LPL. First, carotenoids could dissociate from the chylomicrons and then be internalized as a component of the surface lipid that is shed from the chylomicrons during lipolysis. Second, the entire chylomicrons or chylomicron remnants might associate with LPL and then be internalized either via classical receptors or along with recycling of cell surface proteoglycans. Or third, it is conceivable that the core carotenoids can be exchanged for another non-polar lipid at or near the cell surface. Of these mechanisms, the first requires the enzymatic actions of LPL to hydrolyze triglycerides within the chylomicrons. The last two mechanisms involve the non-enzymatic "bridging’ functions of LPL.
However, our study had several limitations. In most of preliminary studies such as this one, very little information about the genetic effect size is available beforehand and thus it is difficult to calculate a reasonable sample size. According to power analysis, in population with allele frequencies of 0.11, the number of the participants in the present study was adequate to evaluate 1,4% and more of heritability (percent of variance explained by the polymorphism) with an additive mode of inheritance, achieving statistical power >70% at 5% probability level (P value). X447X homozygotes were rare in our sample population (2 among fathers, 1 among mothers, 3 among sons and 2 among daughters). Since interaction terms between S447X polymorphism and the four sex-by-generation groups were not significant and in order to improve statistical power, data from the overall sub-sample were analyzed together by using the SAS GENMOD procedure with a family factor as repeated statement.
At this time, we cannot rule out the possibility that the absence of association with the other polymorphisms is caused by low power level resulting from the small sample size. In addition, reliance of the 3-day dietary record could increase the random variability of diet intake and consequently adjustment could not be optimal.
In conclusion, nevertheless the limitations listed above, our investigation for the first time demonstrates that LPL S447X polymorphism could alter serum concentrations of carotenoids in healthy individuals, independently of serum cholesterol and triglyceride concentration. These data indicate that genetic factors could be involved in the variability of carotenoid bioavailability and bioconversion.
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ACKNOWLEDGMENTS |
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TOP
FOOTNOTES
ABSTRACT
INTRODUCTION
METHODS
RESULTS
ACKNOWLEDGMENTS
REFERENCES |
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INTRODUCTION
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Received May 25, 2005. Accepted May 4, 2006.
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FOOTNOTES
ABSTRACT
INTRODUCTION
METHODS
RESULTS
ACKNOWLEDGMENTS
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