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.
Definition of EBP
BP measurements were obtained by a certified 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 flat on the floor. Mean systolic and diastolic BP for each participant were calculated from the recorded readings. Using the 2017 American Academy of Pediatrics guidelines, we classified participants as having EBP or normal BP consistent with previous reports [17]. For children aged 1- 13 years, EBP was defined as the mean systolic and/or diastolic BP percentile ≥ 90th percentile for sex, age, and height. For adolescents aged 13-17 years, EBP was defined 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 sex-specific growth charts. BMI categories were defined 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 classified 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 inflation 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 significant.