Zeaxanthin

Biweight midcorrelation, an outlier-robust correlation measure, was used to assess marginal linear relationships between epigenetic aging measures and dietary, cardiometabolic, and socioeconomic factors. To adjust for possible socioeconomic and lifestyle confounders, we fit ethnically-stratified multivariable linear models adjusting for education, exercise, BMI, and current drinker and smoker status. We used Stouffer's method to infer the meta-analytic significance of each variable over the different ethnic strata using the square-root of the sample size as the Z-score weighting factor. Specifically for the WHI, the age acceleration measures were adjusted for differences in originating dataset and within the InCHIANTI the measures were adjusted for sex. Models including regression on biomarkers, and number of metabolic syndrome symptoms were not stratified by ethnicity due to lack of coverage for biomarker profiling. Models were designed based on common prior knowledge and in cases where there was co-linearity between confounding variables, choice for adjustment was selected based on variable commonality in order to improve comparability with other studies, e.g. BMI was chosen over WHR because BMI is more commonly measured and reported. Variables with skewness >1 were log transformed (possibly adding +1 to avoid forming the logarithm of zero). Mean carotenoids was computed as the mean across standardized measures of lycopene, log2(alpha-carotene), log2(beta-carotene), log2(lutein + zeaxanthin), and log2(beta-cryptoxanthin). Repeat measurements on the same individuals were omitted from the analysis.

Food and nutrition intake were evaluated with a brief-type self-administered diet history questionnaire (BDHQ) [21 (link),22 (link),23 (link)]. The BDHQ is a four-page self-administered questionnaire [24 (link)]. The questionnaire assesses dietary habits over the previous month and consists of five sections: (i) frequency of consumption of 46 food and non-alcoholic beverage items, (ii) daily intake of rice (including types of rice) and miso soup, (iii) frequency and amount of alcoholic beverages consumed, (iv) usual cooking methods, and (v) general dietary behavior. Many of the foods and beverages were selected from items commonly consumed in Japan, and some were added using a food list from the National Health and Nutrition Survey of Japan. Dietary intake estimates for seventy foods and beverages and ninety-nine nutrients were calculated by the developer (EBNJAPAN, Tokyo, Japan) using an ad hoc computer algorithm. The validity of BDHQ estimates of food and nutrient intakes has been validated in previous reports [24 (link),25 (link)]. Of the ninety-nine nutrients, energy, protein, fat, carbohydrate, three fatty acids, cholesterol, three dietary fibers, nine minerals, eleven vitamins and alcohol were used as representative nutrients for analysis in this study. All seventy foods were included in the statistical analysis of this study. The concentrations of serum carotenoid (lutein, zeaxanthin, β-cryptoxanthin, α-carotene, β-carotene, lycopene), serum retinol, and serum α-tocopherol were measured using high-performance liquid chromatography (HPLC) according to a previous report [26 (link)]. We used a C30 carotenoid column and photodiode array detector (Prominence LC-30AD/Nexera X2 SPPD-M30A, SHIMADZU CORPORATION, Kyoto, Japan) for HPLC analysis. Plasma vitamin C concentration was measured using a commercially available measurement kit (ROIK02, SHIMA Laboratories Co, Ltd., Tokyo, Japan) in accordance with the manufacturer’s instructions.

The ABTS (2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid)) diammonium salt, potassium peroxodisulfate, 2,4,6-tris(2-pyridyl)-s-triazine, Trolox^®, aminoguanidine hydrochloride, methylglyoxal solution, L-arginine, hexane, 5-hydroxymethyl-L-furfural, BHT (2,6-Di-tert-butyl-4-methylphenol), (+)-catechin, rutin, quercetin, D-glucose, citric, L-ascorbic, oxalic, gallic and hydroxybenzoic acid were purchased from Sigma-Aldrich (Switzerland). Acetone and trifluoroacetic acid were bought from Acros Organics (France). Methanol and acetonitrile were obtained from Fine Chemicals (Netherlands), ethyl acetate from Alfa Aesar (Germany). Acetic and sulfuric acids were from Thermo Scientific (Germany). Lycopene, β-carotene, lutein and zeaxanthin were bought from Extrasynthèse (France) and absolute ethanol from Alcosuisse (Switzerland). Fructose and saccharose were obtained from Merck (Germany).
Folin and Ciocaulteu reagent, sodium dihydrogen phosphate mono-hydrate, di-sodium hydrogen phosphate anhydrous and sodium azide were purchased from Chempur (Poland), BSA (Bovine Serum Albumin) from Pol-Aura (Poland). The pectinase Pectinex Ultra SP-L was obtained from Novozymes (Denmark).
Deionized water (Milli-Q purification system, BlancLabo, Switzerland) was used for chromatography.
All reagents used were of analytical grade or higher. All solvents were of HPLC grade.

The 2, 20-azo-bis-(2-methylpropionamidine)-dihydrochloride (AAPH), fluorescein, 2, 2-azinobis-3-ethylbenzothiazoline-6-sulphonic acid (ABTS diammonium salt), potassium sorbate, sodium carbonate, and ethylenediaminetetraacetic acid (EDTA were purchased from Sigma-Aldrich (Sintra, Portugal). Hexane, ethanol, Folin–Ciocalteu’s reagent, and potassium persulfate were purchased from Merck (Algés, Portugal). Standards of ascorbic acid, Trolox, gallic acid, rutin, p-coumaric, and 4-hydroxybenzoic acid, were purchased from Sigma-Aldrich (Sintra, Portugal), while kaempferol, β-carotene, lycopene, zeaxanthin, and lutein (Extrasynthese, France) were purchased from Extrasynthese (Lyon, France).