The association between serum copper concentrations and elevated blood pressure in US youths: National Health and Nutrition Examination Survey 2011-2016

Li Li (  650500@hospital.cqmu.edu.cn ) The Third A liated Hospital of Chongqing Medical University https://orcid.org/0000-0003-2453-558X Changsong Liu Department of Cardiology,The Third A liated Hospital of Chongqing Medical University Yanfen Liao Department of Stomatology,The Seventh A liated Hospital Sun Yat-sen University Zongyuan Zhu Department of Huiqiao Building,Nanfang Hospital,Southern Medical University Lili Yang Department of Cardiology,The Third A liated Hospital of Chongqing Medical University Qin Zhang Department of Cardiology,The Third A liated Hospital of Chongqing Medical University


Results
After multiple adjustments, dose-response analyses revealed that EBP was associated with progressively higher serum copper concentrations in a nonlinear trend. In comparison with the lowest quartile of serum copper concentrations, the adjusted odds of EBP for the highest quartile was 5.26 (95% con dence interval [CI], 2.76-10.03).

Conclusion
Our results suggested that high serum copper concentrations were signi cantly associated with EBP in US youths.
Background Elevated Blood Pressure (EBP) among youths has become an important public health challenge in the United States [1]. Recent epidemiological studies have suggested that accumulation of essential trace metals may play a critical role in the development of hypertension [2][3][4].
Copper is an essential trace metal with antioxidant properties mediated through many redox enzymes [5].
Overload of this metal leads to Fenton-type redox reactions, resulting in oxidative injury [6]. Results of pathological accumulation of copper are observed in Wilson's disease, a recessively inherited disorder of copper metabolism [7]. Additionally, evidence for the relationship between high copper concentrations and heart failure, Parkinson's disease, and ischemic stroke were found in epidemiological studies [8][9][10].
However, evidence regarding the association between serum copper and blood pressure (BP) have been inconsistent or con icting and most research has been conducted in adult populations [11][12][13][14]. A recent study found serum copper to be associated with several cardiovascular disease risk factors in US youths, including increased total cholesterol, glycohemoglobin and fasting insulin. Nevertheless, no association was found with BP levels [15]. However, this study used BP as a continuous variable and ignored the impact of age, sex and height on the de nition of blood pressure level in youths.
In the current study, using the data from the National Health and Nutrition Examination Survey (NHANES) 2011-2016, and the new 2017 American Academy of Pediatrics guidelines (AAP) [1], we aimed to evaluate the association and dose-response relationship between serum copper and EBP in US youths.

Study population
NHANES is a nationally representative, multistage survey of the noninstitutionalized US civilian population. All participants provided written informed consent (parental consent was obtained for those < 18 years) and NHANES was approved by the National Center for Health Statistics' Ethics Review Board.
Detailed information on NHANES data collection and survey procedures used in this analysis are publicly available and can be found elsewhere [16].
Participants who had completed blood pressure reading and serum copper measure were included in our analyses. Individuals missing important covariates were excluded. The process of data inclusion is presented in Figure 1.

Study exposure
Serum specimens were processed, stored at appropriate temperatures (-70 °C), and shipped to the Division of Laboratory Sciences, National Center for Environmental Health, Centers for Disease Control and Prevention, Atlanta, GA for analysis. Inductively coupled plasma dynamic reaction cell mass spectrometry (ICP-DRC-MS) was used for trace level elemental analysis. The isotopes measured included zinc (m/z 64), copper (m/z 65), and selenium (m/z 78) and the internal standard gallium (m/z 71). Serum samples were diluted 1+1+28 with water and diluent containing gallium (Ga) for multi-internal standardization. If you want, you can reference the technical stuff: https://www.cdc.gov/nchs/data/nhanes/2013-2014/labmethods/CUSEZN_H_met.pdf.
De nition of EBP BP measurements were obtained by a certi ed examiner using an appropriate cuff size and a mercury manometer. Three BP readings were obtained after the participant had rested for 5 minutes in a seated position with feet at on the oor. Mean systolic and diastolic BP for each participant were calculated from the recorded readings. Using the 2017 American Academy of Pediatrics guidelines, we classi ed participants as having EBP or normal BP consistent with previous reports [17]. For children aged 1-13 years, EBP was de ned as the mean systolic and/or diastolic BP percentile ≥ 90th percentile for sex, age, and height. For adolescents aged 13-17 years, EBP was de ned as SBP at least 120 mmHg and/or DBP at least 80 mmHg.

Covariates
Demographic information included age, gender, race (Mexican American, other Hispanic non-Hispanic White, non-Hispanic Black, and other race) and family monthly poverty level index category. Other covariates included body mass index (BMI), serum cotinine, physical activity, energy intake and other trace metals (serum selenium, serum zinc, urinary manganese, urinary lead, urinary strontium, urinary arsenic and urinary mercury). BMI was calculated as measured weight in kilograms divided by measured height in meters squared, and BMI percentiles were calculated based on the CDC's BMI-for-age sexspeci c growth charts. BMI categories were de ned as follows: "normal," BMI < 85th percentile; "overweight" and "obese," BMI ≥ 85th percentile. Physical activity was assessed by the amount of television, video game and computer usage daily and was classi ed as high (≤ 2) and low (> 2) in concordance with previous report [18]. Energy intake data were obtained from two 24-hour dietary recall interviews and calculated as an average of 2-day energy intake. Serum cotinine, the primary proximal metabolite of nicotine, is generally regarded as the marker of exposure to environmental tobacco smoke.

Statistical analysis
Differences between groups were tested by the Chi-square test for categorical data and the independent Student t-test, Mann-Whitney-U test, analysis of variance, or the Kruskal-Wallis test for continuous data, as appropriate. Binary logistic regression models were performed to estimate the association between serum copper and EBP. In multivariate logistic regressions, model 1 adjusted for age and sex, model 2 further adjusted for race, family monthly poverty level category, physical activity, BMI, total energy intake, serum cotinine, and other trace metals. We assessed for collinearity between adjustment variables by calculating variance in ation factors. The dose-response relationship was conducted by restricted cubic spline with three knots. We performed tests for linear trend by entering the median value of each category of copper as a continuous variable in the models. All analyses were performed using Stata 15.1 and R 3.3.0 software. All reported probabilities (p-values) were two-sided with p < 0.05 considered as signi cant.

Results
Overall, the study cohort included a total of 1242 US youths aged 8-17 years at baseline. Table 1 presents the characteristics of the study participants. The mean serum copper concentrations was 114.17 µg/dL. The overall prevalence of EBP was 14.0%. EBP was more likely to occur in males (132 versus 42, P < 0.01) and in Non-Hispanic Black participants (P < 0.01). Participants with EBP tend to be older (12.9 versus 12.0 years, P = 0.01), overweight and obese (P < 0.01), consumed more calories (2152 versus 1878 kcal, P < 0.01) and had lower amount of physical activity (P = 0.01) compared to those without EBP. There was no difference in serum copper concentrations between genders (male versus female, 114.3 versus 114.1 µg/dL, P = 0.89). Non-Hispanic blacks had higher mean serum copper concentrations compared to non-Hispanic whites, Mexican-Americans and other Hispanic (122.4, 112.1, 110.9 and 109.5 µg/dL, respectively, P < 0.01). Mean serum copper concentrations were higher in participants with EBP compared to those without EBP (125.5 versus 112.3 µg/dL, P < 0.01). Abbreviations: BMI, body mass index. Table 2 presents the odds ratios of EBP based on quartiles of serum copper concentrations. In crude logistic regression analyses, high levels of serum copper were signi cantly associated with increased risk of EBP. After adjustment for age and sex (model 1), serum copper concentrations had a signi cantly positive association with EBP. After further adjustment for race, family monthly poverty level category, physical activity, BMI, total energy intake, serum cotinine, and other trace metals (model 2), the results remained stable and statistically signi cant. Dose-response relationship between serum copper and EBP were shown in Fig. 2. In restricted cubic spline model, excessive serum copper increased the risk of EBP in a linear manner (P = 0.029).

Discussion
In this study, using a nationally representative large-scale database and updated classi cation system for pediatric BP, we found that serum copper was associated with increased risk of EBP in the US population aged between 8 to 17 years old. The association was statistically signi cant despite adjustments for the impact of co-exposure to multiple metals [19,20].
In animal experiments, plasma copper concentrations were signi cantly higher in hypertensive rats [21]. Other animal studies have demonstrated implanting a copper cuff results in neointimal thickening in response to vascular injury [22], while copper chelators inhibit the development of vascular in ammation and new intima formation in response to vascular injury [23,24]. In humans, evidence linking serum copper and blood pressure has been inconsistent. Most research conducted thus far has been in adults. Taneja, S.K et al [25]. conducted a study with 500 adult participants which noted higher levels of copper in urine in hypertensive subjects (2.70 ± 0.10 versus 5.14 ± 0.15 mg/dL). Conversely, Li Y et al [26]. found no difference. Additionally, Kim MH et al [27]. found that copper intake was inversely associated with SBP and DBP in 258 adult subjects.. There is a dearth of studies investigating the association between copper and blood pressure in youths. In a cross-sectional study conducted among 1427 US youth, no association was found between serum copper concentrations and blood pressure [15]. Another study among European children suggested a positive association between serum copper and diastolic blood pressure [28]. However, a primary de cit of these studies was that they did not use blood pressure percentiles to de ne hypertension, which is the current standard for delineating normotension from hypertension in youths. Using the 2017 hypertension diagnostic criteria, our results indicate that high serum copper concentrations increase the risk of EBP in youths.
The mechanisms behind the association between serum copper and BP remain unclear. It has been reported that Reactive oxygen species (ROS) disrupts the balance of oxidation and antioxidant systems. Excessive copper can affect the activity of ROS [29]. The resultant increase in superoxide may lead to the impairment and dysfunction of endothelial structure, and consequently result in the development of EBP [30]. Furthermore, high copper concentrations can reduce myosin-ATPase activity, resulting in calcium overload [31]. Increased Ca 2+ in smooth muscular layer of vessels may cause an increase in the arterial wall tension nally resulting in EBP [32].
As suggested by previous literature, serum copper appears to re ect the status of copper nutrition in both depleted and replete populations. Pediatric reference intervals for serum copper concentrations are not well established [33]. Only a few studies have measured serum copper concentrations in children and adolescents [33]. The reference values of serum copper concentrations in US were higher than the counterparts in Mexico and Egypt and was the same as in China. In our study, the average serum copper concentrations in participants with EBP was 125.5 ± 32.8 µg/dL and in those with normal BP was 112.3 ± 24.2 µg/dL, which were within the US reference range. We provide the rst evidence of a correlation between serum copper and EBP in US youths.
Our study has several strengths. This is the rst study to explore the relationship between serum copper and EBP since the standards for pediatric blood pressure were updated in 2017. In addition, we investigated the dose-response relationship between serum copper and EBP. Furthermore, we considered the simultaneous impacts of various metals in our analyses to capture the complex nature of environmental exposure as a whole.
However, this study also has some limitations. First, this is a cross-sectional study, and it is di cult for us to determine causality. Second, although a number of potential confounders were controlled, we cannot rule out the possibility of unmeasured confounding factors. Third, the complicated interactions of many trace metals with copper warrants further investigation.

Conclusions
In conclusion, our study has found a positive and non-linear association between serum copper and elevated blood pressure in US children and adolescents. We hope that this study can help policy makers develop safer reference ranges in US youths. Further prospective studies are needed to con rm our results and elucidate the mechanisms involved.

Declarations
Ethics approval and consent to participate All participants provided written informed consent (parental consent was obtained for those < 18 years) and NHANES was approved by the National Center for Health Statistics' Ethics Review Board.

Consent for publication
Not applicable.

Availability of data and materials
Anonymized data are available from the National Center for Health Statistics (https://www.cdc.gov/nchs/nhanes/about_nhanes.htm) or from the corresponding author on reasonable request.

Competing interests
The authors declare that they have no competing interests.
Author's Contribution Changsong Liu collected the data and drafted the manuscript; Yanfen Liao and Zongyuan Zhu revised the manuscript critically for important intellectual content; Lili Yang and Qin Zhang analyzed and interpreted the data; Li Li were the designer of the manuscript and approved to submit the manuscript nally.