Association between Dietary Total Vitamin A, β-carotene, and Retinol Intake and Risk of cardiometabolic multimorbidity: Results from the China Health and Nutrition Survey, 1997–2015

Background The association between vitamin A and single cardiometabolic diseases has been extensively studied, but the relationship between dietary vitamin A intake and the risk of cardiometabolic multimorbidity (CMM) has not been studied. Therefore, the present study was conducted to explore the association with CMM risk by analyzing different sources of vitamin A. Methods This study utilized 13,603 subjects aged ≥ 18 years from 1997–2015 from the China Health and Nutrition Survey (CHNS). Dietary intake was calculated from 3 consecutive 24-h dietary recalls combined with a house hold food inventory. CMM is defined as the development of at least two cardiometabolic diseases. Results After a median follow-up of 9.1 years, there were 1050 new cases of CMM. The risk of CMM was significantly lower in those with higher vitamin A intake (Q1 vs Q5 HR 0.66, 95% CI 0.54–0.81). β-carotene (Q1 vs Q5 HR 0.82, 95% CI 0.66–1.02) and retinol (Q1 vs Q5 HR 0.59, 95%CI 0.48–0.73) intake had a similarly negative correlation. Using restricted cubic spline found an L-shaped relationship between retinol intake and CMM (p non-linear < 0.001). In subgroup analyses, protective effects were stronger for participants aged ≥ 44 years (HR 0.72, 95%CI 0.57–0.92) and for the female group (HR 0.62, 95%CI 0.45–0.84). Conclusion Dietary vitamin A was a protective factor for CMM, and this effect was stronger in age ≥ 44 years and in the female group. There was a ceiling effect on the protective effect of retinol intake on the risk of CMM.


Introduction
Multimorbidity can be de ned as the simultaneous occurrence of at least two diseases in the same person, and it can be a combination of several different chronic diseases 1 .The coexistence of multiple diseases complicates the management of this group of patients, with more comprehensive treatment needs leading to greater consumption of health resources, and prevention and improved management of multimorbidity are now key priorities in many countries 2 .Cardiometabolic multimorbidity (CMM), de ned as the coexistence of 2 or more cardiometabolic diseases, including hypertension, diabetes, stroke and cardiovascular disease, is one of the most common multimorbidity patterns 3 .The incidence of CMM has signi cantly increased over the past two decades with the rise in life expectancy and ongoing advancements in the management of cardiovascular diseases and diabetes 4 .It has been reported that CMM not only signi cantly increases the risk of adverse outcomes such as dementia, reduces the quality of life of patients, and results in shorter life expectancy, but also poses a signi cant challenge to the healthcare system [5][6][7][8][9] .In response to this challenge, research on CMM has intensi ed, and the role of nutrients at the level of modi able risk and preventive factors has gained widespread attention [10][11][12][13] .
Vitamin A refers to the compounds retinal, retinol and its esters, which is an indispensable micronutrient necessary for normal physiological function, plays an important role in cell differentiation, embryonic development, vision and immune function [14][15][16] .Carotenoids from plants (eg, fruits and vegetables) and retinol from animals (eg, liver, kidneys, dairy products) are the main dietary sources of vitamin A for most people 17,18 .In a prospective study focusing on hypertension, it was pointed out that the total dietary intake of vitamin A showed an L-shaped relationship with the incidence of new-onset hypertension 19 .Subsequently, in a study involving 17,111 participants with a median follow-up time of 11 years, the authors reported an inverse relationship between dietary intake of vitamin A and the risk of diabetes, particularly in men (HR 0.69, 95%CI 0.49-0.97) 20.A meta-analysis pooling the combined effects of 20 observational studies of risk indicators showed that vitamin A and its organic compounds were negatively associated with the risk of stroke (log OR -0.46, 95%CI -0.81 ; -0.12) 21 .Thus far, the majority of extensively reported associations have primarily centered on a single disease model, with insu cient credible evidence available for investigating the relationship between dietary intake of vitamin A and CMM.
Therefore, in order to ll the gaps in existing knowledge, we will use the data from the China Health and Nutrition Survey (CHNS), a longitudinal study spanning 18 years, to comprehensively investigate the prospective association between dietary total vitamin A, β-carotene, retinol intake, and CMM, providing important epidemiological evidence for the prevention of CMM at the level of knowledge.

Study population
The CHNS is an ongoing multipurpose longitudinal study that began in 1989-2015 and includes 288 communities in 12 provinces/autonomous municipalities in China, with follow-up visits scheduled every two to four years.According to the 2010 census, the provinces included in the CHNS sample accounted for 47% of China's population by 2011.The CHNS was established as a joint project of the University of North Carolina at Chapel and the Chinese Academy of Preventive Medicine (now the China Center for Disease Control and Prevention [CCDC]).Each round of surveys collected data on sociodemographic factors, diet, physical activity, health and behavioral changes at the household and individual level in relation to urbanization and social and economic changes at the community level 22 .A detailed description of the methodology of the survey and design is presented in other literature 23-25 .This study used data from 1997 to 2015 and included a total of 22,545 subjects who participated in at least 2 surveys.

Assessment of nutrient intake
In each survey, dietary assessment is conducted face-to-face by trained nutritionists.Individual dietary data are collected using standardized questionnaires, with a 24-hour dietary recall conducted on each of three consecutive days randomly assigned each week.Meanwhile, a combination of weighing and measurement techniques is used to measure household food consumption based on stock changes from the beginning to the end of each day, with dietary intake expressed as per capita daily consumption.The consumption of cooking oil and seasonings by each individual in the household is estimated through weighted household food intake.Previous detailed descriptions of dietary measurements have been published, and the accuracy of dietary assessment methods has been validated 26,27 .
The compositional database for assessing nutrient intake was obtained from the China Food Composition Table (CFCT).In view of the fact that it is often necessary to test for different forms of vitamin A in food and that each form has its own different biological activity, the biological activity of vitamin A is generally expressed in terms of retinol equivalents(RE) 28,29 .The intake of each nutrient is represented by calculating the cumulative average value from the baseline to the last available round before the nal round.

Covariates
In our current study, the covariates selected were factors known or suspected to be associated with the risk of CMM or variables that differed signi cantly between different sources of vitamin A intake, which were obtained in a structured questionnaire, including age, sex (male/female), district (rural/urban), region (north/south), education (Illiteracy/Primary school/Middle school /High school or above), smoking status (Current smoker/Former/Never), alcohol drinking (no/yes), individual income level (Low/Medium/High/Very high), body mass index (<18.5/18.5-24/24-28/≤28),physical activity (light/moderate/heavy/unknown), and dietary intakes of total energy, protein, carbohydrate, dietary ber, sodium to potassium ratio, calcium, zinc (all in quintiles).

Assessment of CMM
The main outcome was CMM, de ned as progression to at least two of the following cardiometabolic diseases: hypertension, diabetes, stroke and myocardial infarction.The participant's disease history was synthesized by responses to the following questions at each follow-up visit: "What was the doctor's diagnosis of your illness or injury?" "Has a doctor ever told you that you suffer from high blood pressure/diabetes/myocardial infarction/stroke?" "How old were you when the rst event occurred?""Has the event happened in the past year?""Did you use any of these treatment methods?" "Are you currently taking anti-hypertension drugs?"Among the diagnosis of hypertension are two additional criteria: 1) an average of 3 systolic BP (SBP) measurements≥140 mmHg; 2) an average of 3 diastolic BP (DBP) measurements≥90 mmHg.

Statistical analysis
The follow-up time is calculated from the start of participants' involvement in the survey to the diagnosis by CMM, death, or the end of follow-up in these three states, whichever comes rst.In all subsequent analyses, participants were divided into ve groups according to the quintiles (Q1-Q5) of total vitamin A intake.Continuous variables in baseline characteristics were summarized as mean and standard deviation (SD) or median (IQR), categorical variables were expressed as frequency and percentage, and differences between different quintiles of vitamin A intake were compared using ANOVA tests or Chi-square tests.
To assess the relationship between total intake of vitamin A, β-carotene, and retinol in the diet and the risk of cardiovascular disease, three multivariate Cox proportional hazards regression models were established.Model 1 was unadjusted.Model 2 was adjusted for age, sex, regions, district, education, and Model 3 was adjusted for age, sex, regions, district, education, smoking, alcohol, income, BMI classi cation, physical activity, total energy, total protein, total carbohydrates, total dietary ber, sodium to potassium intake ratio, calcium, zinc intake.Missing covariates were imputed using the MICE package in R Studio 4.2.3 software 30 , employing the Predictive Mean Matching (PMM) imputation method with ve iterations to generate datasets for education (0.9%), individual income level (4.8%), body mass index (0.3%), and physical activity (0.3%).The linear trends were tested by assigning a median value to each quintile of the vitamin A intake.Moreover, the dose-response associations of dietary total vitamin A, β-carotene, retinol intake and the risk of CMM were examined using restricted cubic spline regression with ve knots (Three knots were used in retinol intake and the risk of CMM), adjusted for the confounding variables mentioned above.
To investigate whether the associations between dietary total vitamin A, β-carotene, retinol intake and the risk of CMM by these strati cation variables, the potential effect modi cation was examined using the interaction models.In addition, we excluded participants who developed CMM during the rst 2 years of follow-up to perform an assessment of the robustness of the results.All analyses were performed using R (version 4.3.2).All statistical tests were twotailed and considered signi cant at p < 0.05.

Characteristics of study participants
This study included a total of 13,603 participants, consisting of 6,534 males (48.0%) and 7,069 females (52.0%).The mean baseline age of the participants was 43.9 (SD 14.8).The median dietary intake of total vitamin A, β-carotene, and retinol is 530.5 (301.0,998.2), 2602.0 (1418.8,4730.4), and 110.4 (44.1, 288.7) μg RE/d.Table 1 shows the main baseline characteristics of participants in the vitamin A intake quintile group.Compared to the rst group, participants in the highest intake group were more likely to reside in the northern region, have a higher proportion of urban residents, possess higher levels of education and income, and have a higher likelihood of alcohol consumption.In terms of dietary nutrient intake, the intake of all nutrients increased in the quintile groups, while the ratio of sodium to potassium intake showed a decreasing trend.

Associations between vitamin A intake and cardiometabolic multimorbidity
During a median follow-up period of 9.0 years, we ascertained 1050 incident cases of CMM.Table 2 shows the association between vitamin A intake and CMM risk.After adjusting for all covariates, the multivariate-adjusted HRs and 95% CIs from lowest to highest vitamin A intake were 1.00 (ref), 0.59 (0.48-0.73), 0.82 (0.67-1.00), 0.66 (0.54-0.81), and 0.80 (0.64-0.99), revealing a negative association, but the p-trend value (0.733) showed a non-signi cant linear trend.Therefore, we further analyzed the nonlinear relationship based on Model 3 by setting intake as a continuous variable and using restricted cubic spline.In Figure 2A the association between vitamin A intake and CMM risk appeared to be U-shaped, but not signi cantly (p non-linear 0.191).

Associations between retinol intake and cardiometabolic multimorbidity
In the analysis of retinol intake, the risk of CMM was consistently lower in Q2-Q5 than in Q1 in all models.fully adjusted HRs (95% CI) were 0.70 (0.57-0.85), 0.66 (0.54-0.80), 0.66 (0.54-0.81), 0.59 (0.48-0.73), respectively, p for trend 0.001 (Table 2).Spline models with fully adjusted covariates were constructed to pro le a more direct relationship between retinol intake and cardiometabolic multimorbidity.As depicted in Figure 2C the results of multivariate Cox regression with restricted cubic spline (3 knots) found an L-shaped relationship (p non-linear<0.001).With an increase in retinol intake, the risk of CMM shows a decreasing trend until it stabilizes at around 500 μg/d.

Subgroup analysis
To further explore the potential in uencing factors of the relationship between vitamin A intake and CMM, we rst conducted a strati ed analysis by age and gender.As can be seen in Table 3, age may have altered the relationship between vitamin A intake and CMM risk due to interaction effects, which can be more cautiously interpreted to mean that the negative association between vitamin A intake and CMM risk may be stronger in older subjects compared with younger subjects (<44 vs ≥44 years, P-interaction=0.049), and the same was seen in β-carotene and Retinol intake showed the same.Secondly, for male patients, the protective effects of vitamin A and β-carotene are retained, but Retinol was found to still play a role in reducing CMM risk in certain groups.In the terms of female patients, whether it is β-carotene, Retinol, or total vitamin A intake, the higher the intake compared to the low intake group, the lower the risk of CMM.However, the interaction was not signi cant.
Supplemental Figure 1 shows the different groups of CMM.Among all participants, the top 5 groups were hypertension and diabetes (4.04% of the total population), hypertension and cardiovascular disease (1.24% of the total population), hypertension and stroke (1.13% of the total population), hypertension, diabetes, and cardiovascular disease (0.54% of the total population), and hypertension, diabetes, stroke (0.35% of the total population).There still exists an association between vitamin A and different combinations of CMM, although not signi cant in some categories (Supplemental Table 1).In order to minimize reverse causality as much as possible, after excluding patients who developed CMM during the rst two years of follow-up, the analysis results were consistent with the main analysis results (Supplemental Table 2).

Discussion
This study is grounded in a national population-based prospective cohort, utilizing information from 1997-2015, involving 13,603 subjects.According to our knowledge, this study is the rst to report the association between different dietary sources of vitamin A and CMM, as well as different CMM clusters.We observed that both total dietary vitamin A, β-carotene and retinol intake, were signi cantly negatively associated with the risk of CMM.
In strati ed analyses, we observed that intake of more vitamin A may have a greater effect on reducing the risk of CMM in patients aged ≥ 44 years.The same is true for gender, where the bene cial effect of dietary vitamin A intake on CMM prevention may be more pronounced in female patients compared with male patients.Unfortunately, the protective effect of the CMM was preserved when analyzing different clusters of the CMM, especially the groups that included cardiovascular disease, which may be in uenced by the small number of cardiovascular disease cases, and a larger population should be included in future studies for analysis.Furthermore, we observed a ceiling effect between retinol intake and the incidence of CMM, where the risk of developing CMM decreased as retinol intake increased, but eventually plateaued.
Research on the cardiometabolic effects of diets with high antioxidant capacity has been in the spotlight to date, and previous studies have reported extensively on the effects of vitamin A on cardiometabolic health at the level of serum concentrations, supplements, and dietary intake, respectively, but very few studies have examined the effects of dietary intake of vitamin A on the risk of CMM.According to previous reports, many studies have yielded results that increased intake of vitamin A is strongly associated with a reduced risk of hypertension [31][32][33] , diabetes 34 , cardiovascular disease 35,36 , and stroke 21 , and that vitamin A has bene cial cardiovascular effects by attenuating lipid peroxidation and free radical-induced damage 37 .Chi-Ho Lee and colleagues used data from the Hong Kong Cardiovascular Risk Factor Prevalence Study (CRISPS) to reported that intake of vitamins A through diet rather than supplementation not only did not increase cardiovascular events but also resulted in a lower risk of adverse cardiovascular outcomes after a median follow-up of 22 years (HR 0.68, 95%CI 0.53-0.88).A prospective cohort that systematically evaluated the relationship between 29 nutrients and CVD reported that dietary vitamin A intake was signi cantly negatively associated with CVD (HR 0.70, 95%CI 0.54-0.91).We added to the above evidence by directly examining the association between dietary vitamin A intake and CMM.
While vitamin A has received widespread attention, carotenoids, as the main components of vitamin A, not only possess the highest vitamin A activity 38 , but blood concentrations of carotenoids are also considered biomarkers of fruit and vegetable intake 39,40 , making them a focal point in research.Despite numerous studies exploring the association between carotenoids and cardiac metabolism, consensus has not yet been reached on the results.A meta-analysis of 69 prospective studies showed a non-linear relationship between total dietary carotenoid intake and cardiovascular disease (p non-linearity = 0.002), with most risk reductions ranging from 4,000 to 6,000 µg/d 41 .In a meta-analysis that included 10 trials and 16 reports, it was shown that slightly increased cardiovascular morbidity was observed when β-carotene supplementation was given alone (RR 1.04, 95% CI: 1.00-1.08) 42.When people obtain natural β-carotene through the intake of plants and fruits, the bioavailability may be reduced due to changes in cell wall structure during food processing, as well as interactions with other dietary components and phytochemicals in the gastrointestinal tract 38,43 .Direct provision of supplemental intake through interventions may improve utilization, but it is also important to consider that the tolerable upper intake level may be in uenced by the nutritional status and cardiovascular risk of the baseline population 42 .Excessive intake of β-carotene can lead to excessive depletion of free radicals, which can adversely affect cardiovascular health 44 .This may partly explain the inconsistent results between clinical trials and observational studies.In addition, Yi-Wen Jiang found an association between high dietary βcarotene intake and low risk of type 2 diabetes mellitus in 77,643 subjects from the West by integrating the results of six cohort studies (RR 0.78, 95%CI 0.70-0.87) 45.A meta-analysis of stroke also reported that increased levels of βcarotene were more effective in reducing stroke risk at higher ages (log OR -0.61, 95%CI -1.09 ; -0.12) 21 .This corroborates with the observation of a negative association between dietary intake of β-carotene and CMM in our current study.

Conclusions
In conclusion, our study suggests that dietary intake of vitamin A, β-carotene, and retinol is negatively associated with the risk of CMM, and that this negative association is stronger in age ≥ 44 years and in the female population.Among these, there was a ceiling effect on the protective effect of retinol intake on CMM.  2 P-trend was detected by assigning a median value to each quintile of vitamin A intake.

Financial
disclosure: This work was supported by the National Natural Science Foundation of Hubei (No. 2023AFB955 and 2022CFD163).Declarations of interest: The authors have no relevant nancial or non-nancial interests to disclose.Author Contribution YDT and YX participated in writing the manuscript and analyzing the data.FY and XLG participated in the design and conceptualization of the study.XHZ and GYQ approved the nal version of the manuscript.All authors were involved in revising the manuscript and read and approved the submitted version.This research uses data from China Health and Nutrition Survey (CHNS).We are grateful to research grant funding from the National Institute for Health (NIH), the Eunice Kennedy Shriver National Institute of Child Health and Human Development (NICHD) for R01 HD30880 and R01 HD38700, National Institute on Aging (NIA) for R01 AG065357, National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) for R01 DK104371 and P30 DK056350, National Heart, Lung, and Blood Institute (NHLBI) for R01 HL108427, the NIH Fogarty grant D43 TW009077, the Carolina Population Center for P2C HD050924 and P30 AG066615 since 1989, and the China-Japan Friendship Hospital, Ministry of Health for support for CHNS 2009, Chinese National Human Genome Center at Shanghai since 2009, and Beijing Municipal Center for Disease Prevention and Control since 2011.We thank the National Institute for Nutrition and Health, China Center for Disease Control and Prevention, Beijing Municipal Center for Disease Control and Prevention, and the Chinese National Human Genome Center at Shanghai.

Table 1
Baseline characteristics of the study population according to Vitamin A intake quintiles and cumulative mean nutrient intake1

Table 2
The relationship between cumulative mean intake of vitamin A, β-carotene and retinol and cardiometabolic multimorbidity risk1 1Values are HRs (95% CIs) based on mixed-effects Cox proportional hazards models.