Dietary Assessment in the MetaCardis Study: Development and Relative Validity of an Online Food Frequency Questionnaire.

BACKGROUND: The European study MetaCardis aims to investigate the role of the gut microbiota in health and cardiometabolic diseases in France, Germany, and Denmark. To evaluate long-term diet-disease relationships, a food frequency questionnaire (FFQ) was found to be the most relevant dietary assessment method for the MetaCardis study. OBJECTIVE: The objectives of this study were to describe the development of three semiquantitative online FFQs used in the MetaCardis study-one FFQ per country-and to assess the relative validity of the French MetaCardis FFQ. DESIGN: The layout and format of the MetaCardis FFQ was based on the European Prospective Investigation of Cancer (EPIC)-Norfolk FFQ and the content was based on relevant European FFQs. Portion size and nutrient composition were derived from national food consumption surveys and food composition databases. To assess the validity of the French MetaCardis FFQ, a cross-sectional study design was utilized. PARTICIPANTS/SETTING: The validation study included 324 adults recruited between September 2013 and June 2015 from different hospitals in Paris, France. MAIN OUTCOME MEASURES: Food intakes were measured with both the French MetaCardis FFQ and 3 consecutive self-administered web-based 24-hour dietary recalls (DRs). STATISTICAL ANALYSES PERFORMED: Several measures of validity of the French MetaCardis FFQ were evaluated: estimations of food groups, energy, and nutrient intakes from the DRs and the FFQ, Spearman and Pearson correlations, cross-classification, and Bland-Altman analyses. RESULTS: The French MetaCardis FFQ tended to report higher food, energy, and nutrient intakes compared with the DRs. Mean correlation coefficient was 0.429 for food, 0.460 for energy, 0.544 for macronutrients, 0.640 for alcohol, and 0.503 for micronutrient intakes. Almost half of participants (44.4%) were correctly classified within tertiles of consumption, whereas 12.9% were misclassified in the opposite tertile. Performance of the FFQ was relatively similar after stratification by sex. CONCLUSIONS: The French MetaCardis FFQ was found to have an acceptable level of validity and may be a useful instrument to rank individuals based on their food and nutrient intakes.

Habitual diets rich in dark-green vegetables are associated with an increased response to ω-3 fatty acid supplementation in Americans of African ancestry.

Although substantial variation exists in individual responses to omega-3 (ω-3) (n-3) fatty acid supplementation, the causes for differences in response are largely unknown. Here we investigated the associations between the efficacy of ω-3 fatty acid supplementation and a broad range of nutritional and clinical factors collected during a double-blind, placebo-controlled trial in participants of African ancestry, randomly assigned to receive either 2 g eicosapentaenoic acid (EPA) + 1 g docosahexaenoic acid (n = 41) or corn/soybean oil placebo (n = 42) supplements for 6 wk. Food-frequency questionnaires were administered, and changes in erythrocyte lipids, lipoproteins, and monocyte 5-lipoxygenase-dependent metabolism were measured before and after supplementation. Mixed-mode linear regression modeling identified high (n = 28) and low (n = 13) ω-3 fatty acid response groups on the basis of changes in erythrocyte EPA abundance (P < 0.001). Compliance was equivalent (∼88%), whereas decreases in plasma triglycerides and VLDL particle sizes and reductions in stimulated monocyte leukotriene B4 production were larger in the high-response group. Although total diet quality scores were similar, the low-response group showed lower estimated 2005 Healthy Eating Index subscores for dark-green and orange vegetables and legumes (P = 0.01) and a lower intake of vegetables (P = 0.02), particularly dark-green vegetables (P = 0.002). Because the findings reported here are associative in nature, prospective studies are needed to determine if dietary dark-green vegetables or nutrients contained in these foods can enhance the efficacy of ω-3 fatty acid supplements. This trial was registered at clinicaltrials.gov as NCT00536185.

Arachidonate 5-lipoxygenase gene variants affect response to fish oil supplementation by healthy African Americans.

Arachidonate 5-lipoxygenase (ALOX5) gene variants that are common in people of African ancestry are associated with a differential cardiovascular disease (CVD) risk that may be ameliorated by intake of (n-3) PUFA, such as EPA or DHA. We conducted a double-masked, placebo (PL)-controlled trial of fish oil (FO) supplements to determine if changes in erythrocyte (n-3) PUFA composition, heart rate, blood pressure, and plasma lipid and lipoprotein concentrations are modified by genotype. Participants received 5 g/d FO (2 g EPA, 1 g DHA) or 5 g/d corn/soy oil (PL). A total of 116 healthy adults of African ancestry with selected genotypes (genotypes = "dd," "d5," and "55" with "d" representing the deletion of 1 or 2 Sp1 binding sites in the ALOX5 promoter and "5" indicating the common allele with 5 sites) were enrolled and 98 completed the study. FO caused significant increases (relative to PL) in erythrocyte EPA, DHA, and total (n-3) PUFA and a decrease in the (n-6) PUFA:(n-3) PUFA ratio in the low-CVD risk "d5" and "55" genotypes but not in the high-risk "dd" genotype. Similarly, HDL particle concentration decreased with FO relative to PL in the "d5" and "55" but not "dd" genotypes. The plasma TG concentration decreased significantly with FO relative to PL in the "d5" but not "dd" and "55" genotypes. No changes were seen in LDL particle or cholesterol concentrations, heart rate, or blood pressure. These findings indicate that the efficacy of FO supplements vary by ALOX5 genotype.

ALOX5 gene variants affect eicosanoid production and response to fish oil supplementation.

The objective of this study was to determine whether 5-lipoxygenase (ALOX5) gene variants associated with cardiovascular disease affect eicosanoid production by monocytes. The study was a randomized, double-masked, parallel intervention trial with fish oil (5.0 g of fish oil daily, containing 2.0 g of eicosapentaenoic acid [EPA] and 1.0 g of docosahexaenoic acid [DHA]) or placebo oil (5.0 g of corn/soy mixture). A total of 116 subjects (68% female, 20-59 years old) of African American ancestry enrolled, and 98 subjects completed the study. Neither ALOX5 protein nor arachidonic acid-derived LTB4, LTD4, and LTE4 varied by genotype, but 5-hydroxyeicosatetraenoate (5-HETE), 6-trans-LTB4, 5-oxo-ETE, 15-HETE, and 5,15-diHETE levels were higher in subjects homozygous for the ALOX5 promoter allele containing five Sp1 element tandem repeats ("55" genotype) than in subjects with one deletion (d) (three or four repeats) and one common ("d5" genotype) allele or with two deletion ("dd") alleles. The EPA-derived metabolites 5-HEPE and 15-HEPE and the DHA-derived metabolite 17-HDoHE had similar associations with genotype and increased with supplementation; 5-HEPE and 15-HEPE increased, and 5-oxo-ETE decreased to a greater degree in the 55 than in the other genotypes. This differential eicosanoid response is consistent with the previously observed interaction of these variants with dietary intake of omega-3 fatty acids in predicting cardiovascular disease risk.

Genetic contribution of the leukotriene pathway to coronary artery disease.

We evaluated the genetic contribution of the leukotriene (LT) pathway to risk of coronary artery disease (CAD) in 4,512 Caucasian and African American subjects ascertained through elective cardiac evaluation. Of the three previously associated variants, the shorter "3" and "4" alleles of a promoter repeat polymorphism in ALOX5 increased risk of CAD in African Americans (OR = 1.4, 95% CI 1.0-1.9; p = 0.04), whereas a haplotype of LTA4H (HapK) was associated with CAD in Caucasians (OR = 1.2, 95% CI 1.01-1.4; p = 0.03). In Caucasians, first-stage analysis of 254 haplotype-tagging SNPs in 15 LT pathway genes with follow-up of 19 variants in stage 2 revealed an LTA4H SNP (rs2540477) that increased risk of CAD (OR = 1.2, 95% CI 1.1-1.5; p = 0.003) and a PLA2G4A SNP (rs12746200) that decreased risk of CAD (OR = 0.7, 95% CI 0.6-0.9; p = 0.0007). The PLA2G4A rs12746200 variant also decreased risk of experiencing a major adverse cardiac event (MACE = myocardial infarction, stroke, or death) over 3 years of follow-up (HR = 0.7, 95% CI 0.5-0.9; p = 0.01), consistent with its cardioprotective effect. Functional experiments demonstrated that stimulated monocytes from carriers of LTA4H variants HapK or rs2540477 had 50% (p = 0.002) and 33% (p = 0.03) higher LTB(4) production, respectively, compared to non-carriers. These ex vivo results are consistent with LTB(4) being the direct product of the reaction catalyzed by LTA4H and its role in promoting monocyte chemotaxis to sites of inflammation, including the artery wall of atherosclerotic lesions. Taken together, this study provides additional evidence that functional genetic variation of the LT pathway can mediate atherogenic processes and the risk of CAD in humans.

Functional analysis of 5-lipoxygenase promoter repeat variants.

Variants of a hexanucleotide repeat polymorphism in the promoter of the 5-lipoxygenase (5-LO) gene have been associated with cardiovascular disease traits in humans, which may be due, at least in part, to differential expression of the at-risk alleles. To more fully characterize these variants, we carried out gene expression and DNA methylation studies in primary leukocytes from healthy individuals carrying various 5-LO promoter alleles. Regardless of genotype, 5-LO and 5-LO-activating protein (FLAP) gene expression was higher in granulocytes compared with monocytes and lymphocytes, whereas leukotriene A4 hydrolase (LTA4H) expression was higher in monocytes. In all three leukocyte populations, 5-LO mRNA levels were positively correlated with those of FLAP and LTA4H, with the highest correlation observed in granulocytes. In lymphocytes, individuals homozygous for the shorter 3 and 4 repeat alleles had between 20-35% higher 5-LO, FLAP and LTA4H expression compared with homozygous carriers of the wild-type 5 repeat allele (P = 0.03-0.0001). DNA methylation analysis of four CpG islands in a 1500 bp region encompassing the 5-LO promoter and the first approximately 100 bp of intron 1 revealed relatively low overall DNA methylation across all genotypes and leukocyte populations. However, analysis of the promoter repeats themselves demonstrated that, regardless of cell population, the 4 allele was methylated approximately twice as much as the 3 allele (P < 0.0001). Our results demonstrate that, in lymphocytes, the shorter repeat alleles of the 5-LO promoter lead to higher gene expression, which may be regulated through differential DNA methylation of the CpGs located within these repeats.

Cadmium toxicity on arterioles vascular smooth muscle cells of spontaneously hypertensive rats.

Cadmium (Cd) is frequently used in various industrial applications and is a ubiquitous environmental toxicant, also present in tobacco smoke. An important route of exposure is the circulatory system whereas blood vessels are considered to be main stream organs of Cd toxicity. Our previous results indicate that cadmium chloride (CdCl2) affects mean arterial blood pressure in hypertensive rats. We hypothesized that Cd alters the intracellular calcium transient mechanism, by cadmium-induced stimulation of MAPKs (ERK 1 & 2) which is mediated partially through calcium-dependent PKC mechanism. To investigate this hypothesis, we exposed primary cultures of vascular smooth muscle cells (VSMCs) from wistar kyoto (WKY) and spontaneously hypertensive rats (SHR) to increased concentrations of CdCl2 on cell viability, expression of mitogen-activated protein kinases (MAPKs/ERK 1 & 2), and protein kinase C (PKC) which are activated by Cd in several cell types. The results from these studies indicate that CdCl2 decreased cell viability of both SHR and WKY VSMCs in a concentration dependent-manner. Viability of both cell types decreased 33+/-5.3 (SHR) and 39+/-2.3% (WKY) when exposed to 1 microM CdCl2, whereas, 8 and 16 microM reduced viability by 66+/-3.1 and 62+/-4.5% in SHR cells. CdCl2 increased ERK 1 & 2 in a biphasic manner with maximum increase occurring when cells are exposed to 1 and 4 muM in SHR VSMCs, whereas, a reduction in ERK 1 and 2 is observed when WKY cells are treated with 2 microM. The results also indicate that CdCl2 increased PKC a/Beta in both SHR and WKY VSMCs with a greater increase in expression in SHR VSMCs. In addition, the [Ca2+]i chelator, BAPTA, suppressed the CdCl2 effect, whereas, the PKC inhibitor, GF109203X, reduced the CdCl2 induced-effect on PKC expression. The present studies support the hypothesis that Cd can be a risk factor of hypertension through dysfunction of vascular smooth muscle cells under certain conditions.