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<reference>
ORIGINAL RESEARCH
published: 12 January 2022
doi: 10.3389/fnut.2021.799095

The Association Between Dietary
Antioxidant Micronutrients and
Cardiovascular Disease in Adults in
the United States: A Cross-Sectional
Study
Ting Yin 1† , Xu Zhu 1† , Dong Xu 2 , Huapeng Lin 3 , Xinyi Lu 1 , Yuan Tang 1 , Mengsha Shi 1 ,
Wenming Yao 1 , Yanli Zhou 1 , Haifeng Zhang 1,4*‡ and Xinli Li 1*‡
1

Edited by:
Monica Trif,
Centre for Innovative Process
Engineering, Germany
Reviewed by:
Eman Mehanna,
Suez Canal University, Egypt
Charles Apprey,
Kwame Nkrumah University of
Science and Technology, Ghana
*Correspondence:
Xinli Li
xinli3267@njmu.edu.cn
Haifeng Zhang
haifeng_zhang@163.com
† These authors have contributed

equally to this work and share first
authorship
‡ These authors have contributed
equally to this work
Specialty section:
This article was submitted to
Nutrition and Sustainable Diets,
a section of the journal
Frontiers in Nutrition
Received: 21 October 2021
Accepted: 15 December 2021
Published: 12 January 2022
Citation:
Yin T, Zhu X, Xu D, Lin H, Lu X, Tang Y,
Shi M, Yao W, Zhou Y, Zhang H and
Li X (2022) The Association Between
Dietary Antioxidant Micronutrients and
Cardiovascular Disease in Adults in
the United States: A Cross-Sectional
Study. Front. Nutr. 8:799095.
doi: 10.3389/fnut.2021.799095

Frontiers in Nutrition | www.frontiersin.org

Department of Cardiology, The First Affiliated Hospital of Nanjing Medical University, Jiangsu Province Hospital, Nanjing,
China, 2 Department of Vascular Surgery, Affiliated Hangzhou First People’s Hospital, Zhejiang University School of Medicine,
Zhejiang, China, 3 Department of Medicine and Therapeutics, The Chinese University of Hong Kong, Hong Kong, Hong Kong
SAR, China, 4 Department of Cardiology, The Affiliated Suzhou Hospital of Nanjing Medical University, Suzhou Municipal
Hospital, Gusu School, Nanjing Medical University, Suzhou, China

Background: Antioxidant micronutrients represent an important therapeutic option for
the treatment of oxidative stress-associated cardiovascular diseases (CVDs). However,
few studies have evaluated the relationship between the levels of multiple dietary
antioxidants and CVDs.
Objective: The study therefore aimed to evaluate associations between dietary
antioxidants and total and specific CVDs among a nationally representative sample of
adults in the US.
Design: In total, 39,757 adults (>20 years) were included in this cross-sectional study
from the 2005–2018 National Health and Nutrition Examination Survey. We analyzed
dietary recall of 11 antioxidant micronutrients in this population. Multivariate logistic and
weighted quantile sum (WQS) regression were both applied to examine the relationships
between these antioxidants, alone and in combination, with the prevalence of all CVDs
and specific CVDs. The linearity of these correlations was also explored using restricted
cubic spline (RCS) regression.
Results: Multivariate logistic models showed that, compared with the lowest quartile,
the levels of 11 antioxidants in the highest quartile were independently associated with
decreased total CVD (all P < 0.05). The WQS index showed that, when considered
together, the 11 micronutrients were negatively correlated with total CVD (P < 0.001)
and five specific CVDs (all P < 0.05), and selenium had the strongest association (weight
= 0.219) with total CVD. Moreover, the RCS model demonstrated that iron, zinc and
copper were all negatively and non-linearly correlated with total CVD, while the eight
other micronutrients had non-significant, linear, negative relationships with total CVD (P
for non-linearity >0.05). A piecewise binary logistic regression analysis showed that the
inflection points in the relationships between CVD and iron, zinc and copper were 7.71,
6.61, and 0.74 mg/day, respectively.

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Antioxidant Micronutrients and Cardiovascular Disease

Conclusions: Our findings suggested that high levels of combined dietary antioxidant
micronutrients are associated with decreased prevalence of CVDs, and that selenium
has the greatest contribution to this association.
Keywords: antioxidant micronutrients, dietary nutrient intake, cardiovascular disease, disease nutrition
interaction, weight quantile sum, restricted cubic spline, US adults

HIGHLIGHTS

(including retinol, carotene and cryptoxanthin) and E (αtocopherol) and the water-soluble vitamin C (ascorbic acid)
all have anti-inflammatory properties (10). These vitamins
interact with free radicals and reactive nitrogen species (RNS),
decreasing polyunsaturated fatty acid (PUFA) peroxidation
and thus protecting cell membrane phospholipids and plasma
lipoproteins, with beneficial effects on glutathione (GSH) status
and oxidation defense capabilities (11). Similarly, increasing
evidence indicates that essential metal micronutrients, such as
selenium, zinc, copper, and iron, play critical roles in a wide range
of physiological processes; they are integral to the enzymatic
system involved in the reduction of oxygen free radicals (12). It
is possible that inadequate intake of these nutrients may result in
increased ROS, which have an implied role in the mechanisms
and risk of CVD.
Observational studies and clinical trials have evaluated the
potential role of antioxidant micronutrients and their safe dose
for CVD prevention or treatment (13). Epidemiological studies
of individual micronutrients reported that dietary vitamin A,
carotenoids (14, 15), vitamin C (16, 17), vitamin E (18), selenium
(19, 20), zinc (21), iron (22), and copper (23) were associated
with lower CVD risk and reduced cardiovascular mortality.
These correlations were even more pronounced in the deficiency
state of subsequent complement micronutrients and the level
of dietary metal micronutrients are closely related to serum
concentrations. The metabolism and distribution of various trace
vitamins and minerals also alter cellular ion contents, which
in turn modulates the metabolic functions of macronutrients
(carbohydrates, lipids, and proteins) and levels of probiotic
bacteria; this affects intrinsic pathological mechanisms of CVD
and cumulative drug therapeutic effects in people with this
disease (22), and thus impacts long-term pharmacotherapy.
Notably, the effect of antioxidant micronutrients, alone or
in combination, in the prevention or reduction of CVD is
controversial. Analyses from 15 trials reporting data on 188,209
participants showed that supplementation of the antioxidant
vitamins E and C and β-carotene has no effect on the incidence of
major cardiovascular events, myocardial infarction, stroke, total
deaths, and cardiac-related deaths (24). Other studies have even
suggested that supplementation of antioxidants may increase
CVD risk owing to potential peroxidation (23). There are several
challenges to overcome in studies on the impact of dietary
antioxidants relating to dosage, duration of intervention, and
baseline micronutrient status of those receiving interventions,
in particular, the interactive effects between vitamins cannot
be ignored (25). There is a rapidly growing interest in the
health effects of dietary exposure to combinations of dietary
antioxidant micronutrients because mixed exposure better

- We analyzed 11 dietary antioxidant micronutrients in
collaboration with total cardiovascular disease (CVD)
and specific CVDs in a nationally representative
United States population.
- Dietary intake of 11 antioxidant micronutrients were
independently associated with decreased odds of total CVD
and specific CVDs.
- Weighted quantile sum (WQS) analysis showed that, when
considered together, the 11 antioxidants were negatively
associated with the odds of total CVD and specific CVDs; of
all components, selenium was most strongly associated with
total CVD.
- Iron, zinc and copper were all non-linearly, negatively
correlated with total CVD; inflection points were at 7.71, 6.61,
and 0.74 mg/day, respectively.

INTRODUCTION
Cardiovascular disease (CVD) is the leading cause of death and
disability in both developed and developing countries, and has
become a serious public health problem (1, 2). Numerous studies
have been published on CVD risk factors in recent decades; these
have established that aging, smoking, obesity, cholesterol levels,
poor dietary habits, educational level, blood pressure, diabetes,
and genetics, all affect risk (3). At-risk populations would benefit
from the effective management of these risk factors. Strikingly,
43.9% of the US adult population is still projected to have some
form of CVD by 2030 (4). Epidemiological studies have also
shown that 75% of pre-mature CVDs are preventable through
early intervention (5). A more comprehensive understanding of
CVD etiology and its underlying mechanisms remain a priority.
Oxidative stress plays an important role in the progression
of various CVDs, including atherosclerosis, heart failure, cardiac
arrhythmia, and ischemia-reperfusion injury (6, 7). Increased
oxidative stress can modify DNA and proteins, induce subcellular
remodeling and Ca2+ -handling abnormalities, and lead to
functional hypoxia and disordered metabolism; this can result in
cellular inflammation and programmed cell death, necrosis, and
fibrosis, which are tightly regulated by reactive oxygen species
(ROS) production and intracellular defense mechanisms (8, 9).
There are a number of therapeutic options available for the
treatment of oxidative stress-associated CVDs. Among these,
micronutrients are critical for every stage of the antioxidant
response. Fundamental studies on the properties of antioxidant
micronutrients have shown that the fat-soluble vitamins A

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Antioxidant Micronutrients and Cardiovascular Disease

intakes for energy, nutrients, and other food components.
All NHANES participants were eligible for two 24-h dietary
recall interviews; the first was collected in person in a mobile
examination center and the second was collected by telephone, 3–
10 days after the first 24-h recall period (30). In the dietary recall
investigation in NHANES 2003–2018, two dietary interviews
were conducted with all sample members.
Data on intake for 11 antioxidant related micronutrients
[vitamin C (DRTVC), iron (DRTIRON); vitamin E as
α-tocopherol (DRTATOC); zinc (DRTZINC); β-carotene
(DRTBCAR); copper (DRTCOPP); α-carotene (DRTACAR),
vitamin A (DRTVARA); retinol (DRTRET); β-cryptoxanthin
(DRTCRYP); selenium (DRTSELE)] were included in our
analysis.
(https://wwwn.cdc.gov/Nchs/Nhanes/2017-2018/
DR1IFF_J.htm)
(https://wwwn.cdc.gov/Nchs/Nhanes/20172018/DR2IFF_J.htm#Appendix_2). The average of two 24-h
dietary recall values for each antioxidant in each 2-year cycle
from the 16 years of available data was analyzed. These amounts
reflected only nutrients obtained from foods, beverages, and
water, including tap and bottled water (32); they did not
include nutrients obtained from dietary supplements, antacids,
or medications.

represents the mixed diets that people eat in real-world situations.
Studies on micronutrient status and the effects of combined
supplementation are therefore worthy of further study, but
they pose unique challenges to inference (26, 27); few studies
have been conducted in this domain. Statistical techniques for
analyzing exposure to combined factors have been developed,
including weighted quantile sum (WQS), which permits the
assessment of the overall effect of combined treatments on health
outcomes, and crucially, the weighted contributions of each
component used to answer questions about projecting exposures
to a higher dimensional space (28).
The National Health and Nutrition Examination Survey
(NHANES), which collects detailed data on diet, nutritional
status, and chronic disease to inform nutrition and health
policy, is the cornerstone for national nutrition monitoring
in the US (29). The objectives of the current study were to
present an analysis using this large, cross-sectional, nationallyrepresentative database to assess the intake status of v antioxidant
micronutrients with a known role in CVD, and explore the effects
of combined exposure to these antioxidants on CVD.

METHODS
Study and Population

CVD Definition

This study used data from NHANES, a nationally-representative,
cross-sectional database on civilian, non-institutionalized
persons living in the United States, administered by the
National Center for Health Statistics (NCHS) at the Centers
for Disease Control and Prevention. NHANES surveys are
demographically based, with samples selected through a
complex, multistage survey design (30). A detailed sample
design is available at: https://wwwn.cdc.gov/nchs/nhanes/
tutorials/module2.aspx). Specifically, we used the NHANES
2003–2018 continuous survey. The data in this survey were
collected using a series of large, complex, stratified, multistage
probability samples with a 4-year design, with data released
in 2-year cycles. Our analysis used data on dietary recall of
vitamin intake, collected from the What We Eat in America
(WWEIA) component of the NHANES, which is conducted
by a partnership between the US Department of Agriculture
(USDA) and the US Department of Health and Human Services
(DHHS) (31).
Participants provided written informed consent and all study
procedures were approved by the National Center for Health
Statistics Research Ethics Review Board. None of the authors
of this study have ever been involved in the collection or
production of the NHANES database. Participants with fewer
than two valid 24-h dietary recalls, and those who were younger
than 20 years of age for whom data on CVD outcomes were
missing, were excluded from our analyses during the 2003–
2018 period.

Participants aged ≥20 years old were asked: “Has a doctor or
other health professional ever told you that you have X,” where
X was congestive heart failure (CHF), coronary heart disease
(CHD), angina, heart attack, or stroke. The CVD data in the
NHANES 2003–2018 survey was obtained from medical records,
laboratory data, and questionnaires. In addition, the data for
each CVD outcome were gathered for further analysis of their
association with dietary antioxidant micronutrients.

Covariates
Potential confounding variables for the CVD outcome measures
were collected. Sociodemographic characteristics included
age in years (modeled continuously), sex (male or female),
race/ethnicity (“non-Hispanic white,” “non-Hispanic black,”
“Mexican American,” “other Hispanic,” and “other,” including
multi-racial), educational level (above high school, high school,
and below high school), working status, and poverty status.
Health-related covariates included body mass index (BMI;
kg/m2 ); smoking status, as defined by responses to two questions
(“Have you smoked at least 100 cigarettes during your entire
life?”) (https://wwwn.cdc.gov/Nchs/Nhanes/2017-2018/SMQ_
J.htm); and habitual drinking status, in which someone was
defined as drinking if they answered “yes” to “In your entire life,
have you had at least 12 drinks of any kind of alcoholic beverage?”
and “In the past 12 months did you have at least 12 drinks of
any kind of alcoholic beverage?” (https://wwwn.cdc.gov/Nchs/
Nhanes/2017-2018/ALQ_J.htm). We also collected data on
comorbidities, including diabetes mellitus and hypertension, as
determined through a questionnaire administered by NHANES
personnel (Subcommittee of Professional and Public Education
of the American Heart Association Council) (https://wwwn.cdc.
gov/Nchs/Nhanes/2017-2018/DIQ_J.htm); in addition, dietary

Measurement of Two 24-h Diet Recalls
Dietary intake data for all participants were used to estimate the
types and amounts of foods and beverages (including all types
of lipids volume) consumed during the 24-h period prior to a
dietary recall interview (midnight to midnight), and to estimate

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Antioxidant Micronutrients and Cardiovascular Disease

RESULTS

supplement intake was recorded in another questionnaire
(https://wwwn.cdc.gov/nchs/nhanes/Search/default.aspx.) (33).

Study Population Characteristics
According to Total CVD Status

Statistical Analyses

In total, 80,312 people were initially enrolled. Ten thousand
seven hundred and thirteen participants with fewer than two
valid 24-h dietary recalls and 29,842 people younger than
20 years of age with missing data on CVD outcomes were
excluded, leaving a total of 39,757 participants who were included
in our analyses (Figure 1), comprising 19,279 (48.5%) male
and 20,460 (47.5%) female participants, with a mean age of
49.6 ± 18.0 years old. For the purposes of our analysis,
we separated these into those without CVD (n = 35,280,
47.5%) and those with CVD (n = 4,477, 56.6%). Table 1
shows the demographic characteristics of these two groups.
Participants with CVD were generally older (66.7 ± 13.0
vs. 47.5 ± 17.4), comprising a greater proportion of men
(56.6 vs. 47.5%), more high-school educated individuals (25.3
vs. 23.0%), more non-Hispanic participants (55.5 vs. 42.5%),
higher poverty states (23.1 vs. 21.6%), a greater proportion of
smokers (61.2 vs. 43.3%), higher BMI (7.0 vs. 6.8%), greater
dietary supplement use (59.1 vs. 49.9%), and greater rates of
diabetes (33.0 vs. 10.1%) and hypertension (73.4 vs. 31.1%).These
differences between the two groups were statistically significant
(all P < 0.05).

Eleven antioxidant micronutrients were analyzed for their
association with CVD overall and with CHD, CHF, heart attack,
stroke and angina separately. Normality of continuous variables
was assessed using Kolmogorov–Smirnov-tests. Continuous
variables were expressed as mean [standard deviation (SD)]
and were compared using unpaired t-tests. Categorical or
dichotomous variables were expressed as absolute value
(percentage) and were compared using χ 2 -tests. The correlation
coefficients for all antioxidant micronutrient dietary intakes
were calculated using the Pearson correlation method. The
metabolites of antioxidant micronutrients were divided into
quartiles, and the lowest quartile was used as a reference
category. Concentrations of each of the 11 micronutrients
were log-transformed to normalize their distributions for
further analysis.
Multivariate logistic regression models were used to calculate
odds ratios (ORs) and 95% confidence intervals (CIs) to assess
the aggregate specific CVDs prevalence associated with each of
the 11 micronutrients. Three models were used, with increasing
levels of adjustment for confounding variables: in model 1, data
were adjusted for age and sex; model 2 was based on model 1, with
additional adjustments for race, education levels and poverty;
and model 3 was based on model 2, with additional adjustments
for smoking, drinking, BMI, total cholesterol, dietary supplement
use, diabetes, and hypertension.
Because of high correlations between dietary intake values
for various vitamins, we performed WQS regression using the
gWQS in R v.3.6.1 (34). These analyses were used to assess
associations between the level of all antioxidant micronutrients
in combination and CVD, and to evaluate these as predictors
of CVD in logistic regression models. Each micronutrient was
assigned a weight within the index that indicates its contribution
to the overall association (35). In the gWQS function, we used
deciles for exposure weighting, 1,000 bootstrap repetitions, a
random seed set to 2018, and a binomial distribution for the
general linear model. A constraint of this approach is that, in each
case, weights are estimated by pooling effects only in the positive
or the negative direction. Individual vitamin weights of ≥0.1
were considered significant contribution rate. The value of 0.1
was chosen for ease of comparison across models that included
different numbers of antioxidants.
The shape of the relationship between dietary micronutrient
intake and risk of CVD were explored using the restricted cubic
spline (RCS) regression model with three knots (10th, 50th,
and 90th) percentile of antioxidants and analysis of variance
(ANOVA) was used to test for non-linearity (36). If nonlinearity was detected, segmented regression was used to fit the
piecewise-linear relationship between micronutrients and total
CVD or specific CVDs, and to calculate the threshold inflection
point using a recursive algorithm, as described previously (37).
The significance of each interaction (P interaction) was tested
using the likelihood ratio test. Significance was set to P <
0.05 (two-sided).

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Distribution of and Correlation Between
Antioxidant Micronutrient Levels
Overall, Supplementary Table S1 shows the detected
concentrations and distribution of the 11 antioxidant
micronutrients, with the highest levels observed for vitamin C,
followed by iron, vitamin E, zinc, β-carotene, copper, α-carotene,
vitamin A, retinol, β-cryptoxanthin, and lowest levels observed
for selenium.
Correlation analyses showed that most of the antioxidant
micronutrients were moderately correlated with the other 10
vitamins (Spearman’s rank r ≥ 0.3). An r value of ≥0.7 was
found in the correlations between zinc and iron, α-carotene and
β-carotene, selenium and zinc, and zinc and copper (all P <
0.001). An r-value of ≥0.5 was found between iron and copper,
β-carotene and α-carotene, selenium and iron, vitamin A and βcarotene, selenium and copper, retinol and vitamin A, vitamin
c and β-carotene, vitamin E and copper, copper and vitamin A,
and vitamin c and β-cryptoxanthin (all P < 0.001), as shown in
Figure 2.

Associations Between 11 Antioxidant
Micronutrients and Total CVD
The 11 antioxidant micronutrients were divided into quartiles,
and the reference category was considered to be the lowest
quartile. The results from the multivariable logistic regression
models adjusted for the covariates to assess the prevalence rates
of total CVD associated with antioxidant micronutrients are
shown in Table 2. Using model 1, 11 antioxidant micronutrients
were found to have negative association with total CVD when
comparing the second, third, and fourth quartiles with the
reference quartile, respectively (all P for trend <0.05). Further

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FIGURE 1 | Study flow diagram.

TABLE 1 | Sociodemographic characteristics of the study population.
Variable

Total
(n = 39,757)

Non-CVD
(n = 35,280)

CVD
(n = 4,477)

P-value

Age, years, Mean ± SD

49.61 ± 18.04

47.46 ± 17.43

66.73 ± 12.9

<0.001

Male, N (%)

19,279 (48.49%)

16,746 (47.47%)

2,533 (56.58%)

<0.001

10,033 (25.24%)

8,524 (24.16%)

1,509 (33.71%)

Education level, N (%)
Below high school

<0.001

High school

9,264 (23.30%)

8,131 (23.05%)

1,133 (25.31%)

Above high school

20,460 (51.46%)

18,625 (52.79%)

1,835 (40.98%)

6,413 (16.13%)

5,948 (16.89%)

465 (10.39%)

Race/ethnicity, N (%)
Mexican American

<0.001

Other Hispanic

3,482 (9.66%)

3,193 (9.05%)

289 (6.45%)

Non-Hispanic White

17,481 (43.97 %)

14,996 (42.51%)

2,485 (55.51%)

Non-Hispanic Black

8,511 (21.41%)

7,546 (21.39%)

965 (21.55%)

Other race

3,870 (9.73%)

3,597 (10.20%)

273 (6.10%)

Poverty, N (%)

8,315 (20.91%)

7,280 (20.63%)

1,035 (23.12%)

0.007

Smoker, N (%)

18,021 (45.32%)

15,281 (43.31%)

2,740 (61.20%)

<0.001

Drinking, N (%)

27,962 (70.33%)

24,947 (70.71%)

3,015 (67.34%)

Body mass index, kg/m2 , Mean ± SD

29.15 ± 6.87

29.04 ± 6.77

30.33 ± 7.29

Total cholesterol, mmol/L, Mean ± SD

4.86 ± 1.13

5.14 ± 1.13

4.72 ± 1.07

<0.001

20,241 (50.91%)

17,596 (49.88%)

2,645 (59.08%)

<0.001

Dietary supplement use, N (%)

<0.001

Diabetes, N (%)

5,039 (12.67%)

3,562 (10.10%)

1,477 (32.99%)

<0.001

Hypertension, N (%)

14,242 (35.82%)

10,956 (31.05 %)

3,286 (73.40%)

<0.001

CVD, cardiovascular disease.
Data are presented as mean (SD) or median (interquartile range), or n (%).

In model 3, when comparing the fourth quartiles of each
antioxidant micronutrients with the reference quartile, vitamin
E (OR, 0.74; 95% CI, 0.67–0.82); retinol (OR,0.85; 95% CI,
0.77–0.94); vitamin A (OR, 0.75; 95% CI, 0.68–0.83), α-carotene
(OR, 0.74; 95% CI, 0.67–0.82), β-carotene (OR, 0.75; 95% CI,
0.68–0.83), and β-cryptoxanthin (OR, 0.90; 95% CI, 0.81–0.99),

analysis known risk factors were used as covariates to reduce
false positives induced by multiple corrections both in model 2
and 3. The second, third, and fourth quartiles of 11 antioxidant
micronutrients were independently associated with the decreased
prevalence of total CVD compared with the lowest reference
quartile (all P for trend < 0.05).

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FIGURE 2 | Pairwise Pearson correlation coefficients between dietary intake of 11 antioxidant micronutrients in adults from the United States, collected from the
National Health and Nutritional Examination Survey (NHANES) database 2003–2018.

Selenium was found to have the greatest contribution to the
combined effect of the micronutrients (weight = 21.60%) in the
total CVD model. Selenium, copper, β-carotene, vitamin E, and
iron had weights of >0.1 in the total CVD analysis; additionally,
weights of >0.1 were also observed for selenium in CHF, angina
and stroke; copper in angina, heart attack, and stroke; vitamin E
in CHF, CHD, and heart attack; and iron in CHF and stroke. βcryptoxanthin had the greatest contribution of all antioxidative
micronutrients in stroke (weight = 23.80%); the weight of
vitamin C did not exceed 0.1 in the total CVD analysis or those of
specific CVDs (as shown in Table 4; Supplementary Figure S1).

vitamin C (OR, 0.81; 95% CI, 0.73–0.90), iron (OR, 0.74; 95% CI,
0.66–0.82), zinc (OR, 0.76; 95% CI, 0.68–0.85), selenium (OR,
0.67; 95% CI, 0.60–0.75) and copper (OR, 0.67; 95% CI, 0.60–
0.75) had a lower odds ratio, respectively. The results showed
significant protective correlation existed between 11 antioxidant
micronutrients and total CVD.

WQS Regression Analysis of Negative
Relationships Between the 11 Antioxidant
Micronutrients in Combination and Total
and Specific CVDs
The negative relationships between the combined antioxidant
micronutrients and prevalence rates of total and specific CVDs
were analyzed using WQS regression analysis. The combined
index for the 11 antioxidant micronutrients was independently
correlated with total CVD (adjusted OR, 0.79; 95% CI, 0.74–
0.84; P < 0.001), CHF (adjusted OR, 0.82; 95% CI, 0.73–0.91;
P < 0.001), CHD (adjusted OR, 0.87; 95% CI, 0.79–0.96; P
= 0.005), angina (adjusted OR, 0.89; 95% CI, 0.79–0.99, P =
0.037), heart attack (adjusted OR, 0.86; 95% CI, 0.79–0.94, P =
0.001), and stroke (adjusted OR, 0.73; 95% CI, 0.66–0.80, P <
0.001), as shown in Table 3. WQS constrains exposure–outcome
associations to a negative direction.

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Multiple Logistic Regression Analysis for
Selenium, Copper, β-Carotene, Vitamin E,
and Iron and Specific CVDs
The weight of selenium, copper, β-carotene, vitamin E, and iron
were exceeded 0.1, which were significant contribution in CVDs,
and the relationship between the 5 antioxidants and specific
CVDs was further assessed using multiple logistic regression.
After adjustment using model 3, compare with the lowest
quartile, as the maximum weight in total CVD model the highest
selenium level remained significantly negatively associated with
CHF (adjusted OR, 0.70; 95% CI, 0.58–0.84; P = 0.001), angina

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TABLE 2 | Adjusted regression coefficients with 95% confidence intervals (95% CIs) in a multiple regression analysis for total CVD model and 11 dietary antioxidant
micronutrients in adults from the United States, collected from the National Health, and Nutritional Examination Survey (NHANES) database, 2003–2018.
Micronutrients

Q1

Q2

Q3

Q4

P for
trend

OR

OR (95% CI)

OR (95% CI)

OR (95% CI)

Model 1

1

0.83 (0.76–0.91)

0.72 (0.65–0.79)

0.70 (0.64–0.77)

<0.001

Model 2

1

0.86 (0.79–0.94)

0.76 (0.69–0.83)

0.75 (0.68–0.82)

<0.001

Model 3

1

0.85 (0.77–0.93)

0.75 (0.68–0.83)

0.74 (0.67–0.82)

<0.001

Model 1

1

0.89 (0.81–0.98)

0.87 (0.80–0.96)

0.81 (0.74–0.90)

<0.001

Model 2

1

0.91 (0.83–1.00)

0.89 (0.81–0.98)

0.82 (0.74–0.90)

0.001

Model 3

1

0.93 (0.85–1.03)

0.89 (0.81–0.99)

0.85 (0.77–0.94)

0.015

Model 1

1

0.87 (0.79–0.95)

0.80 (0.72–0.88)

0.68 (0.62–0.75)

<0.001

Model 2

1

0.89 (0.81–0.98)

0.83 (0.75–0.91)

0.71 (0.64–0.78)

<0.001

Model 3

1

0.89 (0.81–0.99)

0.85 (0.76–0.94)

0.75 (0.68–0.83)

<0.001

Model 1

1

0.77 (0.69–0.84)

0.76 (0.69–0.83)

0.65 (0.59–0.72)

<0.001

Model 2

1

0.81 (0.73–0.89)

0.82 (0.75–0.91)

0.72 (0.65–0.80)

<0.001

Model 3

1

0.81 (0.73–0.89)

0.81 (0.73–0.89)

0.74 (0.67–0.82)

<0.001

Model 1

1

0.83 (0.75–0.91)

0.72 (0.65–0.79)

0.65 (0.59–0.71)

<0.001

Model 2

1

0.88 (0.80–0.97)

0.78 (0.71–0.86)

0.71 (0.64–0.78)

<0.001

Model 3

1

0.86 (0.78–0.95)

0.77 (0.70–0.85)

0.75 (0.68–0.83)

<0.001

Model 1

1

0.88 (0.80–0.96)

0.79 (0.72–0.87)

0.77 (0.70–0.85)

<0.001

Model 2

1

0.91 (0.83–1.01)

0.84 (0.76–0.92)

0.83 (0.76–0.92)

<0.001

Model 3

1

0.92 (0.83–1.01)

0.85 (0.77–0.94)

0.90 (0.81–0.99)

0.012

Model 1

1

0.79 (0.72–0.87)

0.70 (0.63–0.76)

0.66 (0.60–0.73)

<0.001

Model 2

1

0.84 (0.76–0.92)

0.75 (0.68–0.82)

0.72 (0.66–0.80)

<0.001

Model 3

1

0.84 (0.76–0.93)

0.78 (0.71–0.86)

0.81 (0.73–0.90)

<0.001

Model 1

1

0.82 (0.75–0.90)

0.75 (0.68–0.82)

0.69 (0.63–0.77)

<0.001

Model 2

1

0.85 (0.77–0.93)

0.78 (0.71–0.86)

0.73 (0.66–0.80)

<0.001

Model 3

1

0.86 (0.78–0.95)

0.79 (0.72–0.87)

0.74 (0.66–0.82)

<0.001

Model 1

1

0.83 (0.75–0.90)

0.79 (0.72–0.87)

0.73 (0.66–0.81)

<0.001

Model 2

1

0.86 (0.78–0.94)

0.83 (0.75–0.91)

0.76 (0.69–0.85)

<0.001

Model 3

1

0.85 (0.77–0.93)

0.85 (0.77–0.93)

0.76 (0.68–0.85)

<0.001

Model 1

1

0.84 (0.77–0.91)

0.77 (0.70–0.84)

0.65 (0.59–0.72)

<0.001

Model 2

1

0.86 (0.79–0.94)

0.80 (0.73–0.88)

0.70 (0.63–0.78)

<0.001

Model 3

1

0.84 (0.76–0.92)

0.77 (0.70–0.85)

0.67 (0.60–0.75)

<0.001

Model 1

1

0.77 (0.71–0.85)

0.72 (0.65–0.79)

0.57 (0.52–0.63)

<0.001

Model 2

1

0.81 (0.74–0.89)

0.78 (0.71–0.85)

0.63 (0.57–0.70)

<0.001

Model 3

1

0.82 (0.74–0.90)

0.79 (0.72–0.87)

0.67 (0.60–0.75)

<0.001

Vitamin E (mg)

Retinol (µg)

Vitamin A (µg)

α-carotene (µg)

β-carotene (µg)

β-cryptoxanthin (µg)

Vitamin C (mg)

Iron (mg)

Zinc (mg)

Selenium (µg)

Copper (mg)

CVD, cardiovascular disease; OR, Odd ratio; CI, confidence interval; O, quartile.
Multivariable logistic regression was conducted, and ORs were calculated while comparing the second, third, and fourth quartiles of each chemical with reference to the first
exposure quartile.
Model 1 was adjusted as age and sex.
Model 2 was adjusted as model 1 plus race, education levels and poverty.
Model 3 was adjusted as model 2 plus smoking, drinking, BMI, total cholesterol, dietary supplement use, diabetes and hypertension.

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Supplementary Figures S2–S6. Among these, the association of
β-cryptoxanthin with CHF, and iron, zinc, and copper with heart
attack (non-linearity, P < 0.05) were all negative and non-linear.
Linear, negative associations were found between each of the 11
microelements and stroke (all P for non-linearity >0.05).

TABLE 3 | WQS regression model to assess the protective association of the
mixture of 11 antioxidant micronutrients with individual CVDs and total CVD risk in
adults from (NHANES) database, 2003–2018.
Subgroup

OR

95% CI

P

CVD

0.79

(0.74–0.84)

<0.001

Congestive heart failure

0.82

(0.73–0.91)

<0.001

Coronary heart disease

0.87

(0.79–0.96)

0.005

Angina

0.89

(0.79–0.99)

0.037

Heart attack

0.86

(0.79–0.94)

0.001

Stroke

0.73

(0.66–0.80)

<0.001

DISCUSSION
Our analysis of 11 dietary antioxidant micronutrients in
39,757 US adults from a prospective, nationally representative
U.S. cohort demonstrated that, after adjustment for common
confounding variables, all individual 11 micronutrients were
independently negatively associated with total CVD. In addition,
the WQS index model showed a negative correlation between
all the antioxidative micronutrients in combination and total
CVD, with the greatest influence on this relationship from
selenium. Negative associations were also observed between
selenium and several specific CVDs (CHF, CHD, angina, heart
attack, stroke). Non-linearity regression indicated a U-shaped
correlation between iron, zinc and copper and total CVD, with
inflection points at 7.71, 6.61, and 0.74 mg/day, respectively.
Oxidative stress exceeds the buffering capacity of
the antioxidant defense systems ultimately resulting in
cardiovascular dysfunction (22, 38). Laboratory data shown
exogenous antioxidants, as a part of a diet are able to protect
tissues from ROS and reactive RNS-induced injury (3, 39), and
thus protect cells and organ systems against free radical damage.
For instance, Vitamins A, C, and E and carotenoids direct the
neutralization of free radicals; break the chain reaction of lipid
peroxidation (40), lowering total cholesterol and low-density
lipoprotein and CVD risks (41); copper, zinc, iron and selenium
are required for the activity of superoxide dismutase (SOD),
catalase and glutathione peroxidase (GPx) diminish excessive
oxidative stress (42, 43). Current our partial outcomes were
consistent with previous population large meta-analyses and
a few randomized controlled clinical trials, which have shown
that single magnesium, copper and zinc (42, 44), selenium,
carotenoids (17), vitamin C (44), and vitamin E (45, 46),
are all associated with reduced prevalence of CVDs, and
CVD-related death.
Despite the theoretical that these antioxidants must exert
beneficial effects against oxidative stress, other prospective
population trials have yielded inconsistent results on their ability
to prevent CVD (47). Dosage was one of the main reasons
proposed for this inconsistency. Intake of β -carotene at 15–
50 mg/day had no beneficial effects on CVD outcomes in
adults in a meta-analysis consisting of 15 clinical trials (40,
48). It’s also notable that immoderate removal of ROS or RNS
and their derived products by antioxidant supplementation
may upset cell signaling pathways and might be increase the
risk of chronic disease (49). Research on the effect of excess
antioxidant supplementation on CVDs reported that selenium
in the diet at >400 µg/day induced selenosis and heart hazards
(50), and 4 years of supplementation with 20–30 mg/day βcarotene was associated with increased risk of CVD (51).
Moreover, an upper limit for vitamin A (retinol) at 3,000

CVD, cardiovascular disease; WQS, weighted quantile sum; OR, odds ratio; CI,
credibility interval.
WQS regression model was adjusted as age, sex, race, education levels, poverty,
smoking, drinking, BMI, total cholesterol, dietary supplement use, diabetes
and hypertension.

(adjusted OR, 0.70; 95% CI, 0.57–0.85; P = 0.002), heart attack
(adjusted OR, 0.71; 95% CI, 0.61–0.84, P < 0.001), and stroke
(adjusted OR, 0.66; 95% CI, 0.55–0.78; P < 0.001), respectively
(Supplementary Table S2). In addition, copper in the highest
quartile remained significant protectively associated with CHF
(adjusted OR, 0.73; 95% CI, 0.61–0.87; P = 0.002), angina
(adjusted OR, 0.74; 95% CI, 0.61–0.89; P = 0.017), heart attack
(adjusted OR, 0.70; 95% CI, 0.58–0.84; P = 0.001) and stroke
(adjusted OR, 0.70; 95% CI, 0.58–0.84; P = 0.001), respectively
(Supplementary Table S3). In the adjusted model, a significant
association between fourth quartile of β-carotene and vitamin E
and decreased 5 specific CVDs prevalence, respectively (all P for
trend <0.05) (Supplementary Tables S4, S5); Besides, iron in the
second, third, and fourth quartiles decreased risk of CHF, heart
attack, and stroke compared to those in the lowest (all P for trend
<0.05) (Supplementary Table S6).

Dose Response Relationship Between 11
Antioxidant Micronutrients and the
Prevalence Rates of Total CVD and
Specific CVDs
The median intake level of dietary iron, zinc and copper were
13.2, 9.9, and 1.1 mg, respectively (Supplementary Table S1).
RCS and multivariate logistic regression analyses were used to
flexibly model and visualize the U-shaped relationships of iron,
zinc and copper with the prevalence of CVD. Iron (P for nonlinearity = 0.006), zinc (P for non-linearity = 0.024) and copper
(P for non-linearity = 0.013) all had a non-linear and negative
correlation with total CVD, and total CVD events increased
rapidly for iron, zinc and copper below those levels (Figure 3).
Models predicted that the concurrent decrease in risk of CVD
with decreased concentrations of iron, zinc and copper flattened
out at levels of 7.71, 6.61, and 0.74 mg, respectively (Table 5). All
eight other micronutrients had linear, negative associations (all P
for non-linearity >0.05) (Figure 3).
The relationships between each of the 11 microelements and
specific CVDs (CHD, CHF, heart attack, angina, and stroke)
had negative non-linear and linear outcomes as shown in

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TABLE 4 | WQS regression analysis of 11 antioxidant micronutrients weights of total CVD and specific CVDs.
CVD (%)

Congestive heart failure (%)

Coronary heart disease (%)

Angina (%)

Heart attack (%)

Stroke (%)

Selenium

21.60

24.80

2.80

Copper

17.10

9.07

2.48

33.00

4.64

14.10

11.40

27.70

β-carotene

16.30

19.10

25.00

7.86

8.14

5.10

Vitamin E

11.90

9.47

11.20

33.00

6.04

10.90

Rion

3.82

10.60

22.50

8.09

1.860

3.01

12.20

Vitamin C

8.62

0.14

5.50

0.29

2.53

4.25

Vitamin A

4.74

4.43

11.90

17.50

18.20

0.54

α-carotene

4.49

6.60

13.60

12.80

2.61

6.78

Retinol

2.24

0.61

2.87

6.33

18.80

0.04

Zinc

1.46

0.96

10.40

15.70

5.83

0.03

β-cryptoxanthin

0.94

0.51

1.49

00.11

0.71

23.80

CVD, cardiovascular disease; WQS, weighted quantile sum.

FIGURE 3 | Restricted cubic spline (RCS) analysis with a multivariate -adjusted association associations between dietary 11 antioxidant micronutrients and the
prevalence of total CVD. Eleven specific antioxidant micronutrients index are modeled as restricted cubic splines with knots at the 10th, 50th, and 90th percentiles
shown the non-linear association. Non-linearly related inflection points of iron, zinc and copper were annotated. The solid line is the adjusted HR; 10th percentile is
used as the reference (HR = 1). The shaded area is the 95% CI of the HR. Iron, zinc and copper shown non-linearity association with total CVD model (P for
non-linearity < 0.05).

(45). Previous studies highlighted those whole grains, nuts,
seeds, fruits and vegetables are rich in essential antioxidative
micronutrients, which as role in the primary prevention of
patients with CVD (9), and those who are at risk (11). Higher
vegetable and nut intakes have been associated with a lower

µg/day in adults is extrapolated from a small number of case
reports (15, 52).
It is difficult to obtain such high levels of trace elements
from conventional foods; thus, micronutrients obtained in the
diet rather than through supplements could be considered safer

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TABLE 5 | Threshold effect analysis of iron, zinc and copper on the prevalence of total CVD risk using piecewise binary logistic regression models.
Inflection point
Iron (mg)
Zinc (mg)
Copper (mg)

7.71
6.61
0.74

group

Mean difference (95% CI)

P-value

P for log likelihood ratio test
0.221

≤7.71

0.83 (0.71–0.97)

0.018

>7.71

0.90 (0.84–0.97)

0.007

≤6.61

0.75 (0.66–0.86)

<0.001

>6.61

0.90 (0.83–0.98)

0.012

≤0.74

0.71 (0.61–0.83)

<0.001

>0.74

0.86 (0.80–0.94)

<0.001

0.019
0.025

OR, Odd ratio; CI, confidence interval.
Iron, zinc and copper were log 2 transformed for fitting the piecewise binary logistic regression model.
Analyses was adjusted for age, sex, race, education levels, poverty, smoking, drinking, BMI, total cholesterol, dietary supplement use, diabetes and hypertension.

that the combination of 11 antioxidant micronutrients were
negatively associated with CVD. The current study focuses on
overall and accurate dietary antioxidant micronutrients data
collection, preliminary exploring correlations between individual
or combined micronutrients and CVDs. Also found that
selenium has the greatest influence on the association between
all 11 micronutrients in combination and total CVD.
Selenium is considered a cornerstone of the body’s antioxidant
defense mechanism; this is because it is incorporated in various
enzymes with antioxidant and anti-inflammatory functions.
Thirteen prospective cohort (59) and observational studies (60)
found a moderate inverse relationship between plasma/serum
selenium and CHD (61). A U-shaped relationship between
serum selenium levels and cardiovascular mortality may account
for conflicting observational reports from NHANES study
(62). RCS regression in our study showed linear, negative
associations of selenium with both total CVD and specific
CVDs (stroke, CHD, CHF, heart attack and angina). Our study
investigated the 95th percentiles of dietary selenium intake,
which was 207.8 µg/day; large-scale, randomized controlled trials
have investigated supplementation with a selenium antioxidant
cocktail with a daily dose between 75 and 200 µg or selenium
supplementation at 100 µg/day over 12 consecutive months
did not show any major benefits of selenium cardiovascular
endpoints or left ventricular systolic dysfunction, likely due to
underdosage (20).
Our study has some strengths. We selected WQS over
alternative approaches in our study because it accounts
for exposure-outcome correlations, as well as correlations
between exposures, highlighting the contribution of individual
components of the combination (63). WQS regression also
conserves statistical power and prevents unstable regression
coefficients, which might otherwise occur if the highly-correlated
antioxidant micronutrients were included simultaneously
in traditional regression models. A diet-based, rather than
supplement-based, approach to nutritional interventions in
CVD had proven to be an effective strategy resulting in strong
and tangible results. Micronutrients in the diet have synergistic
effects, and incorporating this synergy in the development of
dietary recommendations is therefore likely to provide the
maximum obtainable benefit obtainable from nutrition.
Our study also has limitations to consider. First, WQS
regression requires a directional homogeneity assumption, which

risk of stroke: an increase of 1 serving per day of green leafy
vegetables intake yielded a relative risk (RR) of 0.79 (95%
CI, 0.62–0.99) (42, 53); similar effects were demonstrated for
myocardial infarction (0.5 servings per week vs. once a week, RR
= 0.49) (39, 54). To date, antioxidants with safer dietary profile
[vitamin A at 3,000 µg/day or 7,500 µg/week; α-carotenoids at up
to 20 mg/day for lutein and 75 mg/day for lycopene; β-carotene
at 2–4 mg/day (14, 15, 48); selenium at 55 µg/day (19) or 200
µg/day for 12 weeks (20)]; vitamin C at 500–700 mg/day (13, 14)
have been confirmed, and reductions in CVDs and markers of
cardiometabolic risk in adults have been observed with their use.
In our study, the antioxidant micronutrients came from food
rather than supplementation. Supplementary Table S1 shown
the daily 95th percentile of antioxidants (vitamin E, 21.8 mg;
retinol, 1,201.0 µg; vitamin A, 1,414.0 µg; α-carotene, 1,785.1
µg; β-carotene, 7,816.1 µg; β-cryptoxanthin, 397.5 µg; vitamin
C, 233.9 mg; iron, 28.7 mg; zinc, 22 mg; selenium, 207.8 µg; and
copper, 2.3 mg) within safe limits had a cardiovascular protective
effect after adjustment for confounding factors in our multiple
logistic regression. Our non-linear regression indicated that
intakes of iron, zinc and copper of 7.71, 6.61, and 0.74 mg/day
may decrease total CVD risk, and also provides reference dose
levels for future prospective studies.
Another important factor to consider is the antioxidants
ability to prevent CVD relies on the internal antioxidant
network mechanism of multiple micronutrients. For example,
the fat-soluble vitamins A and E have been evaluated for
their synergistic effects on GSH homeostasis and antioxidant
properties (55). Vitamin C is known to act with vitamin E
to regenerate α-tocopherol in membranes and lipoproteins,
playing an important role in protein thiol group protection
against oxidation (56). The pro-oxidant vs. antioxidant activity
of beta-carotene and lycopene has also been found to depend
on their interaction with other co-antioxidant molecules such
as vitamin C or E in biological membranes (57). In a mineral
antioxidant study, vitamins also showing a protective association
between zinc metabolic level influence cardiometabolic risk
factor (58). Thus, the single-nutrient approach to nutritional
epidemiology is far from sufficient to explain the biological effects
of antioxidant micronutrients. The field of nutritional health is
shifting toward studying the effects of exposure to combinations
of nutrients, and the contribution of its individual components
on health outcomes. In this study, WQS regression index showed

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AUTHOR CONTRIBUTIONS

assumes that either all exposures have adjusted associations
with the outcome that are all in a positive or a negative
direction (or can be coded a priori to meet this assumption), or
associations are null. However, the antioxidant micronutrients in
the study were all shown to be individually negatively associated
with CVD, satisfying this assumption. Second, WQS regression
also assumes the individual micronutrients have linear and
additive effects. Little is known about the benefits of these
assumptions and whether they have adverse impacts on studies of
epidemiologic data, in which such assumptions can never be met
precisely (27). However, this method provides a parsimonious,
parametric inference of the effect of combinations of factors,
and was therefore a highly appropriate method to use in this
study. Third, although the NHANES use of 24-h dietary recall
was the preferred choice of reference method. Reliance on
memory with twice recall is a well-documented limitation might
induce bias for micronutrients calculation. Comprehensive and
additional dietary data in NHANES have much more detailed
information would be conducive to minimize possibility of selfreporting bias (28). Fourth, although the potential role of the 11
antioxidant micronutrients in the development and progression
of CVDs has been investigated, the accuracy of self-reported
outcomes in NHANES is not well-characterized. The chemical
forms of antioxidant micronutrients (as organic and inorganic
compounds) and sustained and dynamic intakes should also
be considered. Building on our study, which has demonstrated
important synergistic effects of antioxidant micronutrients,
randomized trials with standardized antioxidants and protocols
would help to improve our understanding of the effects of
different food combinations on cardiovascular outcomes.

TY and XZ designed research, drafted the manuscript, and
performed statistical analysis. DX and HL extracted the data and
conducted analyses. XLu, YT, and MS took charge of software
operation. WY and YZ reviewed the manuscript. XLi and HZ
conceptualized the study. All authors reviewed, edited, and
finalized the final version of the manuscript.

FUNDING
This work was supported by Key Disciplines of The First
Affiliated Hospital of Nanjing Medical University.

ACKNOWLEDGMENTS
We thank the National Center for Health Statistics (NCHS) of
the Center for Disease Control (CDC) and Prevention, and all
participants who enrolled in the NHANES.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found
online at: https://www.frontiersin.org/articles/10.3389/fnut.2021.
799095/full#supplementary-material
Supplementary Figure S1 | Weights from the weighted quantile sum (WQS)
regression index for dietary intake of 11 antioxidant micronutrients and the
prevalence of total cardiovascular disease (CVD). A protective
(negatively-associated) model is shown, adjusted for age, sex, race, education
level, smoker status, poverty, body mass index (BMI), and diabetes mellitus status.
Supplementary Figures S2–S6 | Restricted cubic spline (RCS) analysis with a
multivariate -adjusted association associations between dietary eleven antioxidant
micronutrients and the prevalence of specific CVDs (CHD, CHF, heart attack,
angina and stroke). Eleven antioxidant micronutrients index are modeled as
restricted cubic splines with knots at the 10th, 50th, and 90th percentiles shown
the non-linear association. The solid line is the adjusted HR; 10th percentile is
used as the reference (HR = 1). The shaded area is the 95% CI of the HR. The
β–cryptoxanthin shown non-linearity association with CHF model (P for
non-linearity <0.05), and other 10 micronutrients shown linearity correlation with
CHF (P for non-linearity > 0.05). The iron, zinc and copper shown non-linearity
association with heart attack (P for non-linearity <0.05), others shown linearity
correlation relationship with heart attack (P for non-linearity >0.05). All 11 specific
antioxidant micronutrients shown linearity association with CHD, angina, and
stroke (P for non-linearity > 0.05).

CONCLUSION
Our findings suggested that higher levels of antioxidative
micronutrients in combination are associated with a decreased
total CVD risk, and that selenium has the greatest contribution to
this effect. Significant negative linear and non-linear correlations
exist between the 11 antioxidative micronutrients and total CVD
or specific CVDs. Future research is needed to better understand
the interactions and complexities of multiple micronutrients
and their combined effects on cardiovascular health, and to
assess and identify optimal intake levels to reduce CVD in
at-risk populations.

Supplementary Table 1 | Concentrations and distribution of the eleven
antioxidant micronutrients.

DATA AVAILABILITY STATEMENT

Supplementary Table 2 | Multiple logistic regression model to assess the
association between selenium and specific CVD risk.

Publicly available datasets were analyzed in this study. This data
can be found here: https://www.cdc.gov/nchs/nhanes/index.htm.

Supplementary Table 3 | Logistic regression model to assess the association
between copper and specific CVD risk.

ETHICS STATEMENT

Supplementary Table 4 | Logistic regression model to assess the association
between β-carotene and specific CVD risk.

Ethical review and approval was not required for the study on
human participants in accordance with the local legislation and
institutional requirements. The patients/participants provided
their written informed consent to participate in this study.

Supplementary Table 5 | Logistic regression model to assess the association
between vitamin E and specific CVD risk.

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Supplementary Table 6 | Logistic regression model to assess the association
between iron and specific CVD risk.

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</reference>

<statements>
1. Cross-sectional analyses of dietary antioxidant micronutrients in US adults show that higher combined intakes of micronutrients such as vitamin C, iron, zinc, selenium, and copper are associated with decreased prevalence of total CVD and several specific CVDs. Selenium appears to have the strongest inverse association with CVD prevalence, and dietary iron, zinc, and copper display non-linear (U-shaped) relationships, suggesting an optimal intake range and potential harm at both low and high intakes.
2. Observational studies of dietary zinc intake show a non-linear inverse association with CVD, with an apparent optimal intake around 6–7 mg/day and possible increased risk at both lower and higher intakes.
3. Observational data indicate that higher dietary and plasma selenium are associated with lower CVD prevalence and mortality, though high selenium levels have also been linked to dyslipidemia and diabetes risk in some populations, underscoring a narrow optimal range.
4. Zinc and selenium supplementation can cause toxicity at high doses or with prolonged use; high selenium status has been associated with dyslipidemia and diabetes risk, and excessive zinc can interfere with copper metabolism.
5. Zinc and selenium are promising in specific contexts, particularly where deficiency is present, and may reduce oxidative stress or improve certain risk factors, but evidence for event reduction is limited and potential risks at high exposures require caution.
</statements>

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