Micronutrients and Markers of Oxidative Stress and Inflammation Related to Cardiometabolic Health: Results from the EHES-LUX Study

Metabolic syndrome (MetS) characteristics include chronic inflammation and elevated oxidative stress. This study assessed associations between circulating concentrations of micronutrients/phytochemicals and inflammatory/oxidative stress markers with MetS and MetS components. Adults (N = 606) from the European Health Examination Survey in Luxembourg (2013–2015) were randomly selected. We performed a multivariable logistic regression model using the least absolute shrinkage and selection operator to identify MetS-associated variables. Participants with MetS had higher concentrations of C-reactive protein (CRP), 8-iso-prostaglandin F2α, leptin, insulin, and vitamins E/A, but lower concentrations of adiponectin, beta-carotene, and oxidized low-density lipoprotein. A one-unit increase in log-CRP was associated with 51% greater odds of MetS (OR = 1.51 (95% CI: 1.16, 1.98)). Adults with a one-unit increase in log-leptin were 3.1 times more likely to have MetS (3.10 (2.10, 4.72)). Women with a one-unit increase in vitamin A were associated with 3% increased odds of MetS (1.03 (1.01, 1.05)), while those with a one-unit increase in log-adiponectin were associated with 82% decreased odds (0.18 (0.07, 0.46)). Chronic inflammation best characterized adults with MetS, as CRP, adiponectin, and leptin were selected as the main MetS determinants. Micronutrients did not seem to affect MetS, except for vitamin A in women.


Introduction
Metabolic syndrome (MetS) is defined as a constellation of different metabolic disorders (including abdominal obesity, insulin resistance, high cholesterol, and high blood pressure) that increases the risk of cardiovascular diseases (CVD), the leading cause of mortality worldwide. MetS is a global health problem, affecting nearly 25% of the world's adult population (with regional variabilities) [1]. It is commonly associated with chronic inflammation and high levels of oxidative stress [2]. Possible causes for MetS and associated risk factors include changes in lifestyle (e.g., sedentary lifestyle or unbalanced diet), chronic Examinations included blood pressure and anthropometric measurements (height, weight, waist circumference (WC), hip circumference, and thigh size), an electrocardiogram, visual acuity examination, and a spirometry. Blood samples were collected to analyze fasting blood glucose, plasma-triglycerides, plasma-cholesterol (total cholesterol, high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C)), and thyroid hormones. Moreover, we analyzed concentrations of selected micronutrients, markers of oxidative stress, and inflammation in the 606 selected individuals. Of these, we excluded 55 individuals with values below the limit of detection for any of the outcome measures above. We also excluded from the present analysis 16 outliers (one value of CRP > 90000 mg/mL, nine values of insulin > 2000 pmol/L, four values of F2-isoprostanes > 100.000 ng/mL, two values of folic acid > 90.8 nmol/L). Thus, 508 individuals had complete biological information. From those, 504 had complete information on socioeconomic and lifestyle characteristics (280 with MetS, i.e., cases, and 224 without MetS, i.e., controls) ( Figure 1). EHES-LUX was approved by the National Ethics Committee of Luxembourg (CNER, N • 201205/07) and notified to the Commission Nationale pour la Protection des Données (CNPD). All individuals approved their participation by written informed consent.
vey, representative of the general population, conducted between February 2013 and Jan uary 2015 in Luxembourg. The target sample of EHES-LUX was residents (excluding those who were living in institutions) of the Grand Duchy of Luxembourg, aged 25 to 64 years who agreed to participate [26]. For each participant, trained nurses performed a medica examination and collected health questionnaires and biological samples (hair, blood, and urine). Examinations included blood pressure and anthropometric measurements (height weight, waist circumference (WC), hip circumference, and thigh size), an electrocardio gram, visual acuity examination, and a spirometry. Blood samples were collected to ana lyze fasting blood glucose, plasma-triglycerides, plasma-cholesterol (total cholesterol high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C)) and thyroid hormones. Moreover, we analyzed concentrations of selected micronutrients markers of oxidative stress, and inflammation in the 606 selected individuals. Of these, we excluded 55 individuals with values below the limit of detection for any of the outcome measures above. We also excluded from the present analysis 16 outliers (one value of CRP > 90000 mg/mL, nine values of insulin > 2000 pmol/L, four values of F2-isoprostanes > 100.000 ng/mL, two values of folic acid > 90.8 nmol/L). Thus, 508 individuals had complete biologica information. From those, 504 had complete information on socioeconomic and lifestyle char acteristics (280 with MetS, i.e., cases, and 224 without MetS, i.e., controls) ( Figure 1). EHES LUX was approved by the National Ethics Committee of Luxembourg (CNER, N° 201205/07 and notified to the Commission Nationale pour la Protection des Données (CNPD). All indi viduals approved their participation by written informed consent.

Metabolic Syndrome
MetS was defined as having a WC ≥ 94 cm for men and ≥ 80 cm for women and at least the presence of two factors of the following: (1) total triglycerides ≥ 150 mg/dL or on related medication for both men and women; (2) HDL cholesterol < 40 mg/dL for men and < 50 mg/dL for women or being on related medication; (3) systolic blood pressure ≥ 130 or diastolic blood pressure ≥ 85 mm Hg or being on related medication for both men and women; and (4) fasting plasma glucose ≥ 100 mg/dL or previous diagnosis of diabetes for both men and women. We used the International Diabetes Federation definition [27].

Micronutrients, Markers of Oxidative Stress, and Inflammation
We analyzed circulating concentrations of 14 micronutrients/phytochemicals and inflammatory/oxidative stress markers. Cortisol, folic acid, and vitamin D (as 25 hydroxyvitamin D) in serum aliquots (10,25, and 20 µL, respectively) were measured by a chemiluminescent enzymatic antibody-based method (Cobas e 602, Roche) by a local commercial laboratory with an accredited method (Laboratoires Réunis, Junglinster, Luxembourg). Vitamin A (retinol) and vitamin E (alpha-tocopherol) were also measured by this laboratory from serum aliquots (Agilent 1290 Infinity II, 30 µL) based on HPLC combined with UV-Vis detection, employing the Chromosystems 34,000 kit (Chromosystems, Munich, Germany). Total carotenoids were determined following the micro-extraction procedure with heptane, similar as described by Donaldson [28]. In short, 100 µL of serum were mixed with 100 µL of methanol, including 1% butylated hydroxytoluene for protein precipitation, and then extracted with 120 and again with 70 µL of heptane. The combined fractions were measured at 450 and 470 nm spectrophotometrically (Spectra Max M2, Molecular Devices, San Jose, CA, USA) in a microcuvette (Micro Quartz Cuvette, Black, 0.7 mL, Spectrometer Cell; Science Outlet, Aliso Viejo, CA, USA). Quantification was carried out, assuming an average molecular extinction coefficient of 134.000 mol/(L * cm). Aliquots of the serum (50 µL) were measured for MDA based on the thiobarbituric acid (TBARS) assay, employing a commercial kit (10009055, Cayman Chemicals, Ann Arbout, MI, USA), based on fluorescence detection with an excitation wavelength of 525 nm and an emission wavelength of 565 nm (Spectra Max M2 spectrophotometer, Molecular Devices, San Jose, CA, USA). Total phenolics in serum aliquots were measured based on the Folin Ciocalteu assay, expressed as gallic acid equivalents. In short, 50 µL of serum and 80 µL of NaOH (2.5 M in 75% of methanol) were added to facilitate lipoprotein dissemblance. Following incubation at 37 • C for 30 min, 20 µL of 6.25 M metaphosphoric acid (MPA) was added and centrifuged (13,000× g, 5 min). The supernatant was removed and combined with a follow-up extraction of 250 µL of methanol (65%). Combined phases were quantified with 1:5 diluted Folin Ciocalteu reagent (Sigma Aldrich, St. Louis, MS, USA) at 750 nm in a wellplate reader (Polarstar, BMG Labtech, De Meern, The Netherlands); 8-iso-prostaglandin F2α in plasma was quantified by a direct enzyme immunoassay kit (ADI-901-091; Enzo Life Sciences AG, Villeurbanne, France). Ox-LDL in serum was measured by enzyme immunoassay (BI-20032; OLAB, Vienna, Austria). Leptin, adiponectin, CRP, and insulin in plasma (references DY398, DY1065, DY1707, DY8056, respectively) were measured using commercially available enzyme-linked immunosorbent assay kits purchased from Biotechne R&D systems (Lille, France). All quantifications were made according to the manufacturer's specifications, after preliminary dilution tests, and based on external calibration curves.

Covariates
We included age, sex (men/women), and country of birth (Luxembourg, Portugal, other EU countries, other non-EU countries) as sociodemographic variables. Socioeconomic status included the variables education level (categorized into no qualification, primary education, secondary education, and tertiary education) and job status (not working, working). Lifestyle characteristics included smoking (current smoking or quitted < 12 months, non-smokers or quitted > 12 months), alcohol consumption (non-alcohol consump-Nutrients 2021, 13, 5 5 of 13 tion, ≤6 drinks/week, >6 drinks/week), and physical activity (aerobic physical activity ≥ 150 min, aerobic physical activity < 150 min per week).

Statistical Data Analysis
We used means (± standard deviation) and frequencies (%) to describe the general characteristics of the sample population. Associations between MetS and potential risk factors (e.g., age, sex, smoking history, socioeconomic status) were analyzed with Pearson's chi-squared test (χ2) or Student's t-test as appropriate. Associations between MetS and micronutrients and markers of oxidative stress and inflammation were analyzed by the Wilcoxon test for non-parametric values. All concentrations of micronutrients/phytochemicals and markers of oxidative stress and inflammation were tested for normal distribution using the Shapiro-Wilk test of normality. Concentrations were natural log-transformed to meet residual normality. We used the least absolute shrinkage and selection operator (LASSO) [29] to select (from a total of twenty variables) the variables included in the final model by using the GLMNET package on R. The objective was to obtain the L1 penalization (λ) parameter to find the simplest model (i.e., the smallest number of coefficients) and a reduction of the bias and variance. We did a cross-validation and applied the rule of one standard error. In order to avoid over-fitting, a nonparametric bootstrapping with 1000 iterations was performed, and the selected variables were those that appeared more than 80% in all bootstrap samples. Following the selection of the variables, we calculated the odds ratios (ORs) and their 95% confidence intervals (CIs) to study the association between MetS and micronutrients, markers of oxidative stress, and inflammation. All analyses were stratified by sex. Analyses included participants with complete information for all variables. We performed a sensitivity analysis in which we replaced censored values of CRP, insulin, and leptin with the values of the detection limit divided by two. All tests were two-tailed. We considered a p-value of 0.05 as statistically significant. All analyses were performed using SAS 9.4 (SAS Institute Inc., Cary, NC, USA) and R (version 3.5.3).

Participants' Characteristics
Men had higher values of weight, BMI, and waist circumference, and presented higher levels of cardiometabolic risk factors compared to women (e.g., higher systolic and diastolic blood pressure, concentration of triglycerides, total cholesterol and fasting glucose, lower concentrations of HDL-C). Nearly half of participants included in the present study had MetS (55.6% with MetS compared to 44.4% without MetS). In our study sample (N = 504), nearly 62% of men had MetS compared to 48% of women. Men also had more MetS components (e.g., hypertriglyceridemia, hyperglycemia, and low HDL-C) compared to women (Table 1). Younger participants with higher education, with a job, and who did aerobic physical activity ≥ 150 min per week were less likely to have MetS. The same results were observed when comparing men and women, with the exception of job status for women (Supplementary Table S1).   3 Total triglycerides ≥ 150 mg/dL or on related medication. 4 HDL cholesterol < 40 mg/dL for men and < 50 mg/dL for women or on related medication. 5 Systolic blood pressure ≥ 130 or diastolic blood pressure ≥ 85 mm Hg or on related medication. 6 Fasting plasma glucose ≥ 100 mg/dL or self-reported based on a physician diagnosis.

Association between MetS and Markers of Inflammation, Oxidative Stress, and Micronutrients
We observed differences in blood concentrations of micronutrients/phytochemicals and inflammatory/oxidative stress markers between participants with and without MetS ( Table 2). Concentrations of vitamin E and A, leptin, CRP, insulin, and 8-iso-prostaglandin F2α were higher in participants with MetS. Concentrations of adiponectin and Ox-LDL were lower in participants with MetS. When stratified by sex (Figure 2, Supplementary Table S2), concentrations of vitamin E, leptin, CRP, and insulin were higher in men with MetS, whereas adiponectin and Ox-LDL concentrations were lower. Regarding women, concentrations of vitamins E and A, leptin, and CRP were higher for those with MetS, while concentrations of adiponectin and beta-carotene were lower.
In both men and women, we observed a positive correlation between CRP and leptin and insulin and vitamin E, and a negative correlation between CRP and adiponectin and vitamin D and beta-carotene ( Figure 3). Leptin was positively correlated with insulin, CRP, and vitamin E, and negatively correlated with adiponectin and beta-carotene. We also observed a positive correlation between adiponectin and vitamin D and beta-carotene and cortisol, and a negative correlation between adiponectin and leptin and insulin and CRP in women.
In both men and women, we observed a positive correlation between CRP and leptin and insulin and vitamin E, and a negative correlation between CRP and adiponectin and vitamin D and beta-carotene ( Figure 3). Leptin was positively correlated with insulin, CRP, and vitamin E, and negatively correlated with adiponectin and beta-carotene. We also observed a positive correlation between adiponectin and vitamin D and beta-carotene and cortisol, and a negative correlation between adiponectin and leptin and insulin and CRP in women. Leptin, adiponectin, CRP, vitamin A, age, sex, and physical activity were selected by the LASSO logistic regression as the best predictors for MetS (Table 3). For both men and women combined, a one-unit increase in log CRP was associated with a 51% increase in the odds of having MetS (OR 1.51 (95% CI: 1.16, 1.98)), and a one-unit increase in log adiponectin was associated with a 76% decrease in the odds of having MetS (0.24 (0.13, 0.46)). Moreover, adults with a one-unit increase in log leptin were 3.10 times more likely to have MetS (3.10 (2.10, 4.72)), and a one-unit increase in vitamin A was associated with a 2% increase in the odds of having MetS (1.02 (1.01, 1.03)). We observed that, in men, a one-unit increase in log CRP was associated with a 71% increase in the odds of having MetS (1.71 (1.19, 2.51)), and men with a one-unit increase in log leptin were 3.34 times more likely to have MetS (3.34 (2.15, 5.42)). For women, a one-unit increase in log CRP was associated with a 38% increase in the odds of having MetS (1.38 (0.94, 2.06)), and a one-unit increase in vitamin A was associated with a 3% increase in the odds of having MetS (1.03 (1.01, 1.05)). Moreover, women with a one-unit increase in log leptin were 3.31 times more likely to have MetS (3.31 (1.63, 7.20)), and, in women, a one-unit increase in log adiponectin was associated with an 82% decrease in the odds of having MetS (0.18 (0.07, 0.46)).  Leptin, adiponectin, CRP, vitamin A, age, sex, and physical activity were selected by the LASSO logistic regression as the best predictors for MetS (Table 3). For both men and women combined, a one-unit increase in log CRP was associated with a 51% increase in the odds of having MetS (OR 1.51 (95% CI: 1.16, 1.98)), and a one-unit increase in log adiponectin was associated with a 76% decrease in the odds of having MetS (0.24 (0.13, 0.46)). Moreover, adults with a one-unit increase in log leptin were 3.10 times more likely to have MetS (3.10 (2.10, 4.72)), and a one-unit increase in vitamin A was associated with a 2% increase in the odds of having MetS (1.02 (1.01, 1.03)). We observed that, in men, a oneunit increase in log CRP was associated with a 71% increase in the odds of having MetS (1.71 (1.19, 2.51)), and men with a one-unit increase in log leptin were 3.34 times more likely to have MetS (3.34 (2.15, 5.42)). For women, a one-unit increase in log CRP was associated with a 38% increase in the odds of having MetS (1.38 (0.94, 2.06)), and a oneunit increase in vitamin A was associated with a 3% increase in the odds of having MetS (1.03 (1.01, 1.05)). Moreover, women with a one-unit increase in log leptin were 3.31 times more likely to have MetS (3.31 (1.63, 7.20)), and, in women, a one-unit increase in log adiponectin was associated with an 82% decrease in the odds of having MetS (0.18 (0.07, 0.46)).

Discussion
In the present study, we have provided insights into the factors that may influence the development of MetS. In particular, our results reveal the necessity to target a variety of factors, such as dietary micronutrients, inflammation, and oxidative stress. Elevated circulating concentrations of CRP, leptin, insulin, and vitamin A were all positively associated with MetS and MetS components, whereas adiponectin was inversely associated. To our knowledge, this is the first study that uses the LASSO technique to identify specifically which circulating micronutrients/phytochemicals and inflammatory/oxidative stress markers are associated with MetS.
Regarding dietary micronutrients, in our study, we included serum vitamin E, polyphenols, and carotenoids based on their frequent presence in plant-based diets [30], and vitamin A and D for their occurrence in animal-based diets [31]. Contrary to observations made in other studies [9,10], our results showed higher concentrations of vitamins E and A in individuals with MetS. In the final model, only vitamin A remained statistically significant, with a positive association observed in women only. It should be noted that, compared to other studies where participants had vitamin deficiencies, in our sample, almost all participants showed concentrations of vitamins A/E within the normal values (>20 µg/dL and >500 µg/dL, respectively). The evidence regarding the association between vitamin A and MetS, however, remains inconclusive. In a recent meta-analysis, there was no association observed between vitamin A and MetS [19]. Ford et al. [10] and Beydoun et al. [32] observed similar results to the present study, although the positive association between vitamins A/E and MetS disappeared or changed after adjusting for cholesterol and triglycerides, a result that we did not observe. In this particular case, adjusting for cholesterol and triglycerides could be justified, since vitamin E is transported through LDL-C and HDL-C. This allows us to exclude variations caused by these transporters as the underlying reason. In Western societies, individuals generally obtain vitamin A from two main dietary sources: preformed vitamin A, i.e., retinol (ca. 75% of total vitamin A intake) or provitamin A in the form of carotenoids (e.g., beta-carotene, ca. 25%) [31]. From our study, based on mainly healthy participants from the general population, vitamin A (mainly coming from animal sources, e.g., meat, dairy products, fish, and associated oils) could be an indicator of their type of diet. This would explain why participants with MetS showed higher concentrations of vitamin A, since their diet might be rich in animal sources and low in fruit-and vegetable-derived carotenoids. Further on this hypothesis and the results observed in other studies, we also observed lower serum levels of beta-carotene in individuals with MetS [19]. Studies have shown that a diet rich in carotenoids can have a protective function in the development of chronic diseases [10,15,19]. This effect could be mediated by carotenoids acting as antioxidants, modulating transcription factors while also regulating inflammation [18]. However, in our study, beta-carotene was not selected in our final model. As for carotenoids, studies have observed that higher intakes of polyphenols are associated with reduced cardiometabolic risk factors [33]. In our study, we did not observe this association, although it is possible that i) our analytical test was not specific enough for dietary polyphenols, since it also detected other reduced compounds, and ii) the low bioavailability of many polyphenols impeded clear findings [33].
Our results emphasize the inflammatory response related to MetS, since biomarkers of inflammation, such as CRP, adiponectin, and leptin, were selected in our final model. Levels of CRP are increased in response to inflammation and are considered an indicator of coronary risk and metabolic problems [34]. On the other hand, levels of adiponectin (secreted by adipose tissue) are reduced in response to chronic inflammation [35]. The role of adiponectin was mainly observed in women. Sex-based differences in the strength of the association between adiponectin and MetS could be explained by the fact that women had higher concentrations of circulating adiponectin compared to men. These sex differences seemed to be related to body composition and fat distribution, since women usually have less visceral adipose tissue and more subcutaneous adipose tissue than men [36]. Both CRP and adiponectin are considered as key players in the development of MetS, characterized by an imbalance of pro-and anti-inflammatory response in favor of the first. Furthermore, we observed that leptin had the strongest association with MetS, although the main association was observed with abdominal obesity as expected, since concentrations of leptin are correlated with total body fat [37]. This association was observed in both men and women regardless of concentrations being higher in women than men. Leptin was also associated with high blood pressure in men and hyperglycemia in both men and women. Cross-sectional and prospective studies have observed an association between leptin concentrations and MetS independent of obesity [38,39]. Leptin has different biological roles, including energy balance and endocrine function [40]. Studies have observed that a dysfunctional adipose tissue produces adipokines (e.g., adiponectin and leptin) in abnormal concentrations and is associated with cardiometabolic problems [37]. Leptin resistance has also been identified as a risk factor associated with MetS, similar to insulin resistance [41]. Insulin concentrations were also higher in adults with MetS, though in the final model the association was not statistically significant. Although insulin resistance is a characteristic of individuals with MetS, MetS may appear without the presence of elevated insulin concentrations [42]. This could explain why insulin was selected in our model, but its association was not statistically significant.
While inflammation was related to MetS, no marker of oxidative stress (including OxLDL, MDA, or 8-iso-prostaglandin F2α) was associated with MetS or its components. This contrasts with previous findings that have associated these markers with MetS [43,44]. It is possible that the limited differences in dietary status and the overall relatively healthy population prevented an imbalance in oxidative stress homeostasis.
Limitations of the present study include the cross-sectional design of our study, which does not allow drawing conclusions about causality. Moreover, we only analyzed data from participants with complete measurements, and therefore lost information by excluding participants with incomplete data. Strengths include the individual and objective measurement of a large number of risk factors. In addition, the LASSO selection technique allowed us to select the variables that best explained the association.

Conclusions
The present study contributes to a better understanding of the key determinants (selected from a large set of possible factors of MetS). Oxidative stress, inflammation, and nutritional status are among several factors that may influence the onset of MetS, and this study emphasizes that chronic inflammation appears to best characterize individuals with MetS.