The association between serum copper concentration and prevalence of diabetes among US adults with hypertension (NHANES 2011–2016)

Abstract The objective of this study was to examine the association between the serum copper concentration and the prevalence of diabetes among US adults with hypertension using the data from the National Health and Nutrition Examination Survey (NHANES). The study population was selected from adults aged over 20 years old in the three survey cycles of NHANES from 2011 to 2016. Logistic regression model analyses were applied to determine the independent risky effect of copper to the prevalence of diabetes. Also, a restricted cubic spline (RCS) model was performed to explore the potential nonlinear association between serum copper concentration and the prevalence of diabetes. A total of 1786 subjects (742 cases and 1044 controls) were included, and 924 were men (51.7%), and 742 (41.5%) were diabetic. Compared with non‐diabetic individuals, the concentration of serum copper in diabetic patients with hypertension was higher. After adjusting for age, sex, race, education, marital status, body mass index (BMI), family poverty income ratio (PIR), smoking, alcohol drinking, physical activity, systolic blood pressure (SBP), diastolic blood pressure (DBP), and hyperlipidemia, the highest quartile of serum copper concentration significantly increased the risk of diabetes as compared with the lowest quartile (OR: 1.38, 95% CI: 1.01–1.92, p trend = 0.036). The results of RCS analysis showed significant non‐linear relationship between serum copper concentration and prevalence of diabetes (p‐non‐linear = 0.010). This study finds that serum copper concentration are significantly associated with risk of diabetes in hypertensive patients, which suggests copper as an important risk factor of diabetes development.

were applied to determine the independent risky effect of copper to the prevalence of diabetes.Also, a restricted cubic spline (RCS) model was performed to explore the potential nonlinear association between serum copper concentration and the prevalence of diabetes.A total of 1786 subjects (742 cases and 1044 controls) were included, and 924 were men (51.7%), and 742 (41.5%) were diabetic.Compared with non-diabetic individuals, the concentration of serum copper in diabetic patients with hypertension was higher.After adjusting for age, sex, race, education, marital status, body mass index (BMI), family poverty income ratio (PIR), smoking, alcohol drinking, physical activity, systolic blood pressure (SBP), diastolic blood pressure (DBP), and hyperlipidemia, the highest quartile of serum copper concentration significantly increased the risk of diabetes as compared with the lowest quartile (OR: 1.38, 95% CI: 1.01-1.92,p trend = 0.036).The results of RCS analysis showed significant non-linear relationship between serum copper concentration and prevalence of diabetes (p-nonlinear = 0.010).This study finds that serum copper concentration are significantly associated with risk of diabetes in hypertensive patients, which suggests copper as an important risk factor of diabetes development.

K E Y W O R D S
copper, cross-sectional study, diabetes, hypertension, NHANES Diabetes mellitus, as a chronic non-communicable disease, has a high epidemic intensity worldwide. 1The disease is characterized by hyperglycemia and is mainly caused by defective insulin secretion or impaired insulin bioactivity. 2The metabolic dysfunction may cause serious damage to many organs in the body, and is an important cause of premature death in the population. 3,4According to estimates, the global prevalence of diabetes among people aged 20-79 years will be 10.5% (536.6 million people) in 2021, and is expected to rise to 12.2% (783.2 million people) in 2045. 57][8][9] Absolute or relative insufficiency of insulin secretion and low insulin utilization efficiency were the main causes and mechanisms of T2DM. 10 Considering the complexity of the pathogenesis of diabetes, it is particularly important to find some of the factors that influence the onset of the disease.[13] Copper is an essential mineral element for the human body which can participate in physiological processes such as erythropoiesis, immune function and resistance to oxidative stress damage, energy production, glucose metabolism and neuropeptide synthesis. 14,157][18][19] Notably, the association between copper and diabetes has been widely recognized.A recent meta-analysis by Qiu et al. found that diabetic patients carried higher levels of copper than healthy individuals. 20However, in Samadi's study, they concluded that copper levels may not have a significant impact on the association between diabetes. 21Hypertension and diabetes frequently co-occur, posing a compounded risk for cardiovascular and metabolic complications.Considering that previous studies have reported that hypertensive patients have more than twice the risk of developing new-onset diabetes than healthy individuals, 22 and the controversy that exists in previous studies, 20,21 the objective of this study was to examine the association between the serum copper concentration and the prevalence of diabetes among US adults with hypertension using the data from the National Health and Nutrition Examination Survey (NHANES).By focusing on this specific demographic, the research seeks to uncover novel insights that could lead to more targeted and effective strategies for managing and preventing diabetes in hypertensive patients.

| Study design and population
For this cross-sectional research, the publicly available data from the 2011 to 2016 National Health and Nutritional Examination Surveys (NHANES) was obtained.In brief, the NHANES is a series of representative cross-sectional survey conducted in the United States by the Centers for Disease Control and Prevention (CDC) in order to provide information on nutrition and health. 23,24

| Measurements of serum copper concentration
Serum copper was tested using inductively coupled plasma dynamic reaction cell mass spectrometry at the Environmental Health Sciences Laboratory of the National Center for Environmental Health of the Centers for Disease Control and Prevention, in accordance with extensive quality control procedures.In the binary logistic regression models, serum copper was categorized into four groups, according to the tertiles of the detected values.

| Assessment of diabetes and hypertension
Diagnosis of diabetes mellitus is based on (1) symptoms of diabetes mellitus combined with a random blood glucose ≥11.1 mmol/L or (2)   fasting blood glucose ≥7.0 mmol/L or (3) a positive oral glucose tolerance test. 25The criteria for diagnosing hypertension are as follows

| Other variables
Covariates included sociodemographic, behavioural and health characteristics that were a priori considered potential confounders.
Sociodemographic variables included age groups (20-39 years, 40-59 years, and ≥ 60 years), sex (male and female), race (Mexican American, non-Hispanic Black, non-Hispanic White and other race), education level (lower than high school, high school or equivalent and college or above), marital status (married/cohabiting, widowed/ divorced/separated and never married), family poverty income (<1.3,1.3-3.5 and >3.5).Behavioural characteristics consisted of smoking status (no or yes), alcohol consumption (no or yes), physical activity (no, moderate or vigorous).Health factors consisted of BMI (normal, overweight or obese) and hyperlipidemia (no or yes).

| Statistical analysis
The relationship between serum copper and the risk of diabetes was determined using the using R4. 1. 3 software.Due to the skewed distribution of serum copper concentrations, the data were logarithmically (Ln) transformed.The demographic characteristics of the diabetic and non-diabetic groups were described by the composition ratio (%), and the chi-squared test was used to compare the differences between the two groups.Continuous variables were compared between groups using the Student ttest or Mann-Whitney U-test based on the normality of the distribution.
In this study, we utilized logistic regression analysis due to its effectiveness in handling binary outcome variables.Logistic regression is particularly advantageous for estimating the odds ratios (ORs), allowing us to understand the strength and direction of the association between serum copper levels and diabetes risk, while adjusting for potential confounders.So, serum copper levels were categorized into four groups (Q1 ~ Q4) according to quartiles, and logistic regression was performed with group Q1 as the reference group.Model 1 was a crude model, model 2 was adjusted for gender and age, and model 3 was complementally adjusted for race, education, marital status, PIR, BMI, smoking, alcohol drinking, physical activity, SBP, DBP, and hyperlipidemia.
Additionally, we employed restricted cubic splines (RCS) to explore potential non-linear relationships between serum copper levels and diabetes risk.RCS is an advanced statistical technique that enables the modelling of complex, non-linear relationships without assuming a specific functional form.By using RCS, we can better capture and illustrate any potential thresholds or curve patterns in the relationship between serum copper levels and diabetes risk, which might be overlooked in linear analyses.The dose-response association was finally calculated by RCS in fully adjusted model 3.

| Characteristics of study participants
A total of 1786 hypertensive patients (Figure 1), including 924 men and 862 women, were enrolled in this study.742 of the participants were diagnosed with diabetes, accounting for 41.5% of the total.Compared with the non-diabetic population, the diabetic population had lower education and income levels, higher BMI (p < 0.05), and higher proportions of older adults, alcohol drinkers and hyperlipidemia (p < 0.05).
But there were no significant differences between the diabetic and non-diabetic populations in gender and the proportion of smokers (p > 0.05).More detailed information was shown in Table 1.

| The association between serum copper concentration and prevalence of diabetes among adults with hypertension
We investigated the relationship between serum copper levels and risk of diabetes using multiple logistic regression models.In model 1, without adjusting for any variables, it was found that higher level of serum copper was not associated with higher risk of diabetes, but the trend test was statistically significant (p for were presented in Table 2.
Besides, we used RCS curves to further elucidate the relationship between serum copper concentration and diabetes after adjusting for all covariates.As shown in Figure 2, the potential non-linear association between serum copper concentration and the prevalence of diabetes was investigated.

| Subgroup analyses
To explore whether there are differences in the association between serum copper concentration and the prevalence of diabetes in different subgroups.We performed logistic regression analyses stratified by characteristics such as age, gender, and so forth.
The results of the multivariate logistic regression model showed that there was a significant positive correlation between serum levels of copper and diabetes only in women, the trend test was statistically significant (p for trend<0.05).However, no statistical correlation was found between in males.Moreover, the results of Diabetes is a common endocrine system disease.It is a metabolic syndrome characterized by prolonged hyperglycemia due to defects in insulin secretion and action resulting in metabolic disorders of carbohydrates, fats and proteins. 27The incidence of diabetes mellitus has been on the rise in recent years, and its chronic complications have a serious impact on the quality of patients. 28The pathogenesis of diabetes mellitus mainly includes genetic, environmental and immunologic factors. 29Studies have showed that oxidative stress causes pancreatic islet cell dysfunction and insulin resistance, which resulted in tissue cell damage. 30 the meantime, more and more studies are focusing on the role of trace elements in the development of diabetes.Although trace elements are found at extremely low levels in the body, they are involved in the composition of molecular structures such as hormones, nucleic acids and catalytic enzymes. 31Trace elements are closely related to the biological functions of molecules, and the abnormal levels can affect the metabolism and functions of tissues and cells. 32,33For example, in a meta-analysis of 22 cohort studies, Zhao et al. identified   an inverse association between magnesium intake and diabetes. 34so, Safarzad et al. found that lower serum zinc level is associated with higher fasting insulin in T2DM. 11Not only that, but the role of copper is coming into focus.High plasma copper concentrations have been reported to mediate the formation of damaging ROS, which are associated with insulin resistance. 35,36A population-based study reported an association between copper levels and hypertension, and Darroudi et al. found that serum copper levels within a certain range may be associated with elevated blood pressure. 37Given that the cooccurrence of hypertension and diabetes was prevalent among the adults, 38 this study aimed to explore the association between serum copper concentration and the prevalence of diabetes among hypertensive adults using the NHANES database.
This study offers a novel examination in the US hypertensive adults of the role of serum copper concentration regarding its association with diabetes and considering the confounding role of various factors.It is worth noting that non-Hispanic Black, participants received education of college or above, obesity and drinkers were more likely to show such association.Before this study, there was a previous published article assessing the association between plasma copper concentration and diabetes in hypertensive Chinese adults. 39wever, given the racial differences as well as environmental differences, it remains unclear whether the above conclusions are analogous among Americans.Therefore, our study is likely to have an important role to evaluate the influence of copper in adults of different regions.
A large population-based prospective cohort study concluded that dietary intakes of copper was associated with a higher risk of diabetes in Japanese population. 40Considering the potential recall bias of dietary intake surveys and individual differences in copper intake, absorption and metabolism, more previous studies have measured copper levels in biological samples to reflect copper exposure and accumulation in human body.In Ekin's study, they found that copper levels were higher, in diabetic patients, than in the controls. 41sides, a multicentre study by Raudenska demonstrated that copper was significantly increased in the diabetics. 42The conclusions of the above articles are consistent with the findings of this study.
However, a case control study in Norway generated different conclusion.This might be related to the smaller sample size and the difference in the region. 43r study's subgroup analysis reveals significant demographic Haber-Weiss reaction, 44 both of which may generate large amounts of hydroxyl radicals and further lead to elevated levels of ROS in the body.This may directly cause macromolecular damage or indirectly lead to oxidative stress, 45 resulting in abnormal insulin secretion and decreased β cell function decline. 46 the process involved in the aggregation of human pancreatic islet amyloid polypeptides into amyloidogenic fibrils, 47 which is critical for the progressive degeneration of pancreatic islet cells in T2DM.
The advantages of this research included the first report of serum copper concentration being positively associated with the risk of diabetes, the evaluation of a dose-response association between serum copper concentration and diabetes in hypertensive adults in US, the presentation of stratified analyses.This study specifically examines hypertensive individuals, a group with a heightened risk of developing diabetes.By analysing the relationship between serum copper levels and diabetes risk within this particular demographic, the research provides crucial insights into the interplay between hypertension and metabolic disorders, which is essential for understanding the link between these common health issues.Moreover, the findings from this study could reveal serum copper as a potential marker for diabetes risk in hypertensive patients.This opens avenues for early intervention strategies that target copper levels, potentially aiding in the prevention or management of diabetes in this high-risk group.
However, this cross-sectional study also has other drawbacks because of its observational nature.First, the cause-and-effect relationship between serum copper concentration and the occurrence of diabetes cannot be well established from this study.Thus, further epidemiological cohort studies or controlled trials would be required to evaluate the relationship between copper and diabetes in hypertensive adults.Second, while this study offers valuable insights into the relationship between serum copper levels and diabetes risk in hypertensive individuals, it is important to acknowledge certain methodological considerations.Notably, the analysis was conducted without the application of NHANES' complex survey weighting.
However, this approach allowed for a more straightforward analysis and interpretation of the data.Future studies might incorporate these weights to provide a more representative analysis of the national trends and associations.Third, recall bias is a concern in any study relying on self-reported data, such as NHANES.

| CON CLUS ION
In summary, our results revealed that serum copper concentration may be associated with the risk of diabetes among US adults with hypertension.In the future, larger prospective cohort studies are warranted to explore such finding and to investigate causal inference.Longitudinal studies could provide deeper insights into how serum copper levels influence the progression from hypertension to diabetes.

F I G U R E 2 | 7 of 9 WU
variations in the association between serum copper levels and diabetes risk.This aspect of our research provides a deeper understanding of how this relationship may differ across various population segments.For instance, we found a notable positive correlation between high serum copper levels and diabetes risk among non-Hispanic Black participants, those with a higher education level, and individuals classified as obese.These findings align with previous studies indicating the heightened vulnerability of specific demographic groups to metabolic disorders.Previous research exploring the mechanism of influence of serum copper concentrations on diabetes provided multiple possible explanations for the study's findings.First, due to its redox properties, copper is able to catalyse the Fenton reaction and the The restricted cubic spline (RCS) plot.et al.
Notably, stratified multistage sampling design was used to get a representa-

1
Flow chart.The characteristics of the study participants.WU et al.race, education, marital status, PIR, BMI, smoking, alcohol drinking, physical activity, SBP, DBP and hyperlipidemia).It was found that the positive association between higher copper level and diabetes remained stable, and the trend test had statistical significance (p for trend<0.05).When taking the lowest quartile (Q1) as a reference, the level of serum copper concentration in the highest trend<0.05).In model 2, after adjusting for gender, age, it was found that higher serum copper concentration was positively associated with diabetes, and the trend test was statistically significant (p for trend<0.05).Compared with Q1, the OR of the highest serum copper levels group (Q4) was 1.49 (95% CI: 1.11-2.01).After adjusting all the covariate factors (including age, sex,TA B L E 1Note: The bold values means statistical significance.| 5 9 ity, SBP, DBP, and hyperlipidemia) suggested that ln-transformed serum copper concentration was significantly associated with the increased occurrence of diabetes.The OR of the model and its 95% confidence interval (CI) were 2.09(1.22,3.62).The effect value of the model can be interpreted as a corresponding 109% increase in the probability of developing diabetes with increasing 1 unit of ln-transformed serum copper concentration.More detailed results The association between serum copper levels and prevalence of diabetes in 1786 US adults with hypertension by logistic regression analyses.
(95% CI: 1.01-3.34).Notably, Table3demonstrated that the positive association between the highest serum copper concentration category and occurrence of diabetes appeared stronger among non-Hispanic Black, participants received education of college or above, obesity and drinkers.The ORs of these groups both had an obvious decreasing trend (All p for trend <0.05).TA B L E 2 Second, Cu 2 + interacts with pancreatic islet amyloid polypeptides to produce H 2 O 2 and mediates TA B L E 3 The results of subgroup analyses.
Note:The results were adjusted for age, sex, ethnicity, education levels, marital status, body mass index (BMI), family poverty income ratio (PIR), smoking status, alcohol consumption, physical activity, SBP, DBP, and hyperlipidemia.The bold values means statistical significance.