Associations of exposure to multiple metals with blood pressure and hypertension: a cross- sectional study in Chinese preschool children concentrations and hypertension.

Background:Exposure to metals might be a risk factor for hypertension, which contributes largely to the global burden of disease and mortality. However, relevant epidemiological studies of associations between metals exposure with hypertension among preschoolers are limited. This study aimed to explore the associations of urine metals with blood pressure and hypertension among Chinese preschoolers. Methods: A total of 1220 eligible participants who had urine metals measurement, blood pressure measurements, and relevant covariates were included in this cross-sectional study. Urine concentrations of metals were measured by inductively coupled plasma mass spectrometer. The single and multiple metals regression models were used to investigate the associations of urine metal with blood pressure and the risk of hypertension after adjusting for potential confounders. Results: We observed urine concentrations of chromium, iron, and barium were negatively associated with levels of systolic blood pressure, diastolic blood pressure and the risk of hypertension in the single metal model (all P-FDR adjustment < 0.05). Signicant associations of urine chromium concentrations with systolic blood pressure, diastolic blood pressure and the risk of hypertension were found in the multi-metal model ((β or odds ratio (95% condence interval) was -3.07 (-5.12, -1.02) , -2.25 (-4.29, -0.22), and 0.51 (0.26, 0.97) for 3rd quartile, compared with 1st quartile, respectively). The same association was found for barium concentrations in the multi-metal model, while none of the associations among iron quartiles was signicant. In addition, urine chromium, iron and barium may have joint effects on levels of systolic blood pressure and diastolic blood pressure and risk of hypertension. Children’s age and body mass index could modify the associations of chromium, iron, and barium concentrations with blood pressure. Conclusions: Our ndings suggested that exposure to chromium, iron, and barium was inversely associated with blood pressure and hypertension among preschool children. These ndings need further validation in prospective studies. demonstrated an inverse association between urine metals concentrations (Cr, Fe and Ba) and and in preschool The association of Fe, and Ba concentrations with could be modied by children’s age and BMI. Further studies are warranted to our ndings in prospective cohorts and to elucidate the potential mechanisms underlying

increase in blood pressure during childhood would lead to adult hypertension and increase the risk of cardiovascular disease in adulthood [26][27][28][29]. If childhood hypertension is reversed before adulthood, the risk of cardiovascular disease in the future will be signi cantly reduced [30]. Therefore, prevention and control of blood pressure in childhood are of great signi cance [27,31]. In this study, we explored the potential association of 23 metal concentrations in urine with the levels of blood pressure as well as the risk of hypertension among preschool children in China. The multi-metal model was applied to investigate the simultaneous impacts of multiple metals on blood pressure and hypertension. Subgroup analysis was conducted to further explore the associations in various strati ed groups.

Study population
We used cluster sampling to investigate 7 kindergartens in Shiyan City, Hubei Province, China in 2019. A total of 1595 preschoolers aged 2 ~ 6 years old were included in the study. Brie y, an interviewer-administered questionnaire on demographic characteristics including basic information for children (e. g. sex, age, birth weight, physical activity, salt intake, etc.), and parental information (e. g. parents height, weight, education level, family income, and history of hypertension) was performed by trained interviewers. Parents/guardians of children were asked to complete the questionnaire and physical examinations with help of the researcher and to provide morning urine specimens (5 mL).
Physical examination including height, weight and blood pressure was performed by quali ed physicians. Body mass index (BMI) was calculated as body mass / height 2 (kg/m 2 ). Overweight and obesity was de ned by BMI using child growth standards established by the World Health Organization. After excluding those with unrecovered questionnaire (n = 50, a recovery rate of 96.87%), missing basic information (n = 110), missing physical examination (n = 206), insu cient urine samples (n = 9), a total of 1220 participants were eligible for further analysis. The study was approved by the Ethics Committee of Hubei University of Medicine (2019-TH-80), and consent from parents was obtained before completing the questionnaires.

Measurement of blood pressure and hypertension
The blood pressure measurements were performed by a trained operator as described in 2018 Chinese guidelines for the management of hypertension. Blood pressure determinations were taken in a sitting position after 5 min rest using an electronic sphygmomanometer. Hypertension was de ned as systolic blood pressure (SBP) and/or diastolic blood pressure (DBP) ≥ P 99 + 5 mmHg of blood pressure in children of the same sex, age, and height according to the 2018 Chinese guidelines for the management of hypertension.
All the collected samples were sent to the laboratory within 4 h, and then stored at − 80°C before further analysis.
Before analysis, frozen urine samples were thawed at room temperature before centrifugation. Total 200 µL of the supernatant was transferred to 5 mL polypropylene tubes and acidi ed with 40 µL nitric acid (Thermo Fisher, USA) at 4°C overnight. The urine samples were allowed to stand at room temperature for 30 min the next day, brought to room temperature and diluted to 4 mL with butanol, and then centrifuged (3000 r/min, r = 20 cm, 10 min). The accuracy of ICP-MS was checked by analyzing multi-element reserve solution and internal standard liquid (United States), urinary sample quality controls (Sero, Billingstad, Norway)) and standard reference materials 1640a (Trace Elements in Natural Water, National Institute of Standards and Technology, Gaithersburg, MD) in every 50 samples. We further utilized a spiked pooled urine sample (100 samples randomly pooled together) as an inter-laboratory comparison to ensure the precise and accurate detection of titanium, iron, rubidium, strontium, molybdenum, barium, tungsten, uranium (no available certi ed reference agents). The standard recovery rate was in the range of 73.51 126.44% and the regression coe cients (r 2 ) of the calibration standard solutions were greater than 0.999. The limits of detection (LOD) of each element were in the range 0.10 134.57 ng/L, and values below the LOD were given the value LOD/2.

Statistical analysis
Baseline characteristics were compared using t-tests or Mann Whitney U tests for continuous variables and chi-square tests for categorical variables. We used Spearman's rank correlation analysis to explore correlations between urine metal concentrations, after natural log-transformation to account for their right-skewed distributions. Multivariate linear regression models were used to test linear regression coe cient (β) and 95% con dence intervals (CIs) for SBP and DBP among the participants and individual urine metals categorized into quartiles according to distributions. The lowest quartiles were assigned to be the reference groups. We also used multivariate logistic regression models to estimate odds ratios and 95% CIs for hypertension and urine metals. Linear trend P-values were derived by modeling the median value of each metal quartile as a continuous variable in the regression model. Model 1 was adjusted for sex (boys, girls) and age (continuous), and model 2 was further adjusted for children's BMI (continuous), birthweight (continuous), salt intake (little, normal and salty), physical activity (yes, no), paternal BMI (continuous), maternal BMI (continuous), paternal education (middle school or below, high school, and university and above), maternal education (middle school or below, high school, and university and above), family income (RMB) (< 4999, 5000-7999 and > 8000), family history of hypertension (yes, no) and urine creatinine (continuous).
False discovery rate (FDR) corrections of 23 hypothesis tests were conducted based on an available spreadsheet software [33]. Restricted cubic spline (RCS) analysis was used to explore non-linear associations. For metals with signi cant trends both for SBP, DBP and hypertension after FDR corrections in the multivariate regression model, we further evaluated the simultaneous effects of the co-exposure to multiple metals on the levels of SBP and DBP and risk of hypertension by constructing multi-metal regression models considering potential confounders. In addition, a sensitivity analysis was performed among preschool children with no family history of hypertension. Strati ed analyses according to sex (boys, girls), age (2-3 years old, 4 years old, 5years old, 6years old), and children's BMI (overweight and obesity or not) were conducted. We also evaluated the interaction between two metals that were signi cant in the multi-metal models. There was statistical signi cance for P value ≤ 0.05. All data were analyzed using R (3.6.2). Table 1 shows the characteristics of preschool children. Among the 1220 preschool children, 1125 (92.2%) individuals had normal blood pressure, while 95 (7.8%) individuals had hypertension. There were no statistical differences between the non-hypertension group and the hypertension group in sex, age, children's BMI, birthweight, salt intake, paternal BMI, maternal BMI, paternal education, maternal education, family income, family history of hypertension, and urine creatinine. Compared with the non-hypertension group, children with hypertension were more likely to have a higher percentage of physical activity, and higher levels of SBP and DBP (all P < 0.05).

Distributions of the urinary metals
Concentrations (µg/L) of urine 23 urine metals are presented in Table S1. Undetection rates (N% < LOD) of all the metals were < 1.3%. Compared with non-hypertension, children with hypertension had lower concentrations of Cr, Fe, and Ba (all P < 0.05, see Table S2). In the Spearman's rank correlation analysis, we found positive and signi cant associations among most metals, with r s ranging from 0.05 to 0.92 (P < 0.05) (Fig. S1).

Associations between metal exposure and blood pressure
The associations of metals in urine with the levels of SBP and DBP were investigated in the multivariable linear regression model ( Fig. 1 and  (Fig. 2). Meanwhile, we observed a decreasing non-linear trend of SBP levels with Cr concentrations (P for non-linearity = 0.016). In addition, there were signi cant and negative associations of urine Cd with SBP levels, and urine Al and As with DBP levels, while positive associations of U quartiles with increasing SBP levels. After FDR-adjustment, the associations remained signi cant (all FDR-adjusted P < 0.05) (Fig. 1).
For metals (Cr, Fe, and Ba) that were statistically signi cantly associated with levels of SBP and DBP and risk of hypertension among preschool children, we included them together in the multi-metals model. After adjusting for potential confounders, we found signi cant inverse associations of metals quartiles with SBP and DBP levels in the multi-metal models, except for Fe (all P trend < 0.05) ( The above observed associations were robust and did not drastically change in the sensitivity analyses (Table S4).

Associations between metal exposure and risk of hypertension
The association of urinary metals with the risk of hypertension was analyzed as both continuous and categorical variables with adjustments for potential covariates ( Fig. 1 and Table S3). In fully adjusted single-metal regression models, there were signi cant associations of hypertension risk with urine V, Cr, Fe, Co, Cr, Sn and Ba concentrations (all P < 0.05). Similar associations of urine metals and hypertension were observed in the linear model. After FDR adjustments at 5% alpha level, we found consistent results of Cr, Fe, and Ba quartiles with those from the models of single metal (all FDR adjusted P < 0.05). Compared with the lowest quartile, children in the highest quartile of Cr, Fe and Ba had a 0.48-fold (95% CI: 0.25 to 0.88), 0.48-fold (95% CI: 0.24 to 0.90) and 0.28-fold (95% CI: 0.13 to 0.57) decreased odds of hypertension, respectively. We further evaluated these signi cant associations in single-metal models using RCS analysis. A negative dose-response relation was observed for urine Cr, Fe and Ba with hypertension risk (P for overall association = 0.0004, 0.029, and 0.007, respectively) (Fig. 2).
We explored the associations between urinary metals and the risk of hypertension in the multi-metal model, and found the metals were not signi cantly related to hypertension, except for Ba ( Table 2). Quartiles of Ba in urine were suggested to be inversely related to the risk of hypertension, with the OR to be 0.31 (95% CI: 0.14 to 0.63) for the highest quartile. The association between urinary Ba and hypertension was similar in the sensitivity analysis excluding those who had a family history of hypertension (Table S4).

Subgroup analyses and interaction analyses
As shown in Table 3 respectively). However, no signi cant interaction was found between urine Cr, Fe, and Ba after being adjusted for confounders (all P interaction > 0.05).   (Table S7).

Discussion
In this study, we explored the relationships of urinary 23 metals with blood pressure as well as hypertension in Chinese preschool children. Overall, we observed the negative associations of Cr, Fe and Ba quartiles with levels of SBP and DBP and risk of hypertension. These linear dose-response associations were con rmed in the RCS models. Joint effect to Cr, Fe and Ba could greatly decrease the levels of SBP and DBP, and the risk of hypertension. Besides, modi cation effects of children's age and BMI on the associations of Cr, Fe, and Ba concentrations with blood pressure have also been suggested in the subgroup analysis.
Essential metals are suggested to be critical factors to normalize blood pressure levels and possess protective effects on hypertension [10,11,34]. The de ciencies of these trace metals have been suggested to decrease oxidative defense and exacerbate the adverse effects of toxicants [35]. suggested that low Cr was signi cantly associated with high blood pressure [37]. Our data provided a supporting result on the negative association of urine Cr with blood pressure as well as hypertension, which are in accordance with the previous epidemiological studies. In addition, signi cant interactions were found between the urine Cr concentration and age in our study. Urine high Cr quartiles were more strongly associated with lower DBP levels in children aged 2-4 years old.

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The present study observed inverse associations of urine Fe concentrations with blood pressures and hypertension. A  [39]. Although there are numerous studies to explore the association between Fe levels and hypertension risk, the association was still controversial. For example, a prospective cohort study included 1303 participants reported no association between urine Fe concentrations with blood pressure in adults [40]. Furthermore, a case-control study of 502 hypertension patients and 502 healthy participants indicated urine Fe was associated with increased odds of hypertension [23]. Similar associations were found for another cross-sectional study [24]. Therefore, whether Fe concentrations were associated with hypertension risk needs to be investigated in further studies.
Additionally, there are substantial differences in study design, study populations (e.g., location and age groups), metals sample (i.e., blood or urine), and blood pressure assessment protocols, which may account for the variability observed in the existing literature. with SBP or DBP levels [42]. In this study, an inverse association between urinary Ba concentrations and blood pressure levels and hypertension risk was found. Previous literature in drinking water suggested high-Ba communities had lower cardiovascular disease and total mortality [43,44], which was similar to our results. Additionally, this study showed a signi cant interaction effect on blood pressure between urine Ba and overweight and obesity among children, which was consistent with Swayze et al. study [45]. The study included 12256 participants from the continuous National Health and Nutrition Examination Survey 1999-2016 and suggested a greater effect of barium on hypertension in those with obesity. Padilla et al [46] using data from National Health and Nutrition Examination Survey 1999-2002 highlighted toxic metal of Ba was positively related to BMI. These associations suggested the possibility that environmental exposure to metals may contribute to variations in human weight gain. The potential mechanistic pathways for these associations may be related to oxidative stress. Reactive oxygen species generated by metals can inhibit the normal mitochondrial metabolic function, and prevent the mitochondria from producing energy, which would cause the liver to divert metabolites to lipogenesis [47].
This study evaluated the potential associations of urine 23 metal concentrations with blood pressure levels and hypertension risk among Chinese children aged 2-7 years old. However, several limitations should be noticed. First, the participants of our study were selected from an urban area, which might lead to selection bias. Second, spot urine samples were used to measure the internal exposure levels of metals, which might lead to exposure misclassi cation. Third, measurements of 23 metals in the same urine sample might lead to measurement errors and increase the false positive rates, although FDR-method had been applied. Finally, the current ndings were based on a cross-sectional study, thus we could not ensure the causal relationship of metal exposure with blood pressure and hypertension.
Further studies with prospective design are required to con rm our ndings.

Conclusion
Our study demonstrated an inverse association between urine metals concentrations (Cr, Fe and Ba) and blood pressure and hypertension in Chinese preschool children. The association of Cr, Fe, and Ba concentrations with blood pressure could be modi ed by children's age and BMI. Further studies are warranted to con rm our ndings in prospective cohorts and to elucidate the potential mechanisms underlying the relationship between metals and hypertension. Availability of data and materials

Abbreviations
The datasets generated and/or analysed during the current study are not publicly available due [REASON WHY DATA ARE NOT PUBLIC] but are available from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests. Associations of metal concentration with blood pressure and risk of hypertension.
Page 18/19 The quartiles of metals were classi ed according to the natural log-transformed metal concentrations in urine (μg/L), with the rst quartiles to be the reference groups. The single metal model were adjusted with sex, age, children's BMI, birthweight, salt intake, physical activity, paternal BMI, maternal BMI, paternal education, maternal education, family income, family history of hypertension, and urine creatinine. Squares = coe cients or ORs; horizon lines = 95% CIs; vertical gray solid lines = 0 or 1; p = p-values for metals; p-FDR = FDR-adjusted p-values for metals; * p-FDR < 0.05.

Figure 2
Adjusted restricted cubic spline for associations of metal concentrations (log-transformed) with blood pressure and hypertension.
The lines represent coe cients (95%CI) based on restricted cubic splines for the log-transformed urine Cr, Fe, and Ba in the single metals regression model, after adjusting for sex, age, children's BMI, birthweight, salt intake, physical activity,