Association between serum heavy metal levels and diabetic retinopathy in NHANES 2011–2020 | Scientific Reports – Nature Journal

Sample and overall

The National Center for Health Statistics (NCHS) conducted a population-based cross-sectional NHANES to collect data on the nutritional and health conditions of children and adults in the United States of America via interviews, physical examinations, and laboratory tests. NHANES employs a categorized, multistage probability sampling design to select a sample of an unstructured population to evaluate health and nutritional status. NHANES procedures and protocols were approved by the NCHS Research Ethics Review Committee and written consent was obtained.

Inclusion and exclusion criteria

The study population was mainly over 30 years of age. This cross-sectional analysis used data from five NHANES survey cycles (each lasting two years) between 2011 and 2020. Of the 45,462 participants in this study, participants aged < 30 years (n = 23,484), those with unknown diabetes-related conditions, and those lacking information on the glycated hemoglobin (HbA1c) state or the HbA1c level was less than 6.5% (n = 18,955) were eliminated. Diabetic patients aged < 30 years (n = 910) were eliminated to lower the likelihood of having type 1 diabetes. Besides, participants lacking information on serum levels of heavy metals (n = 517) and DR (n = 13) were disqualified. We filled in the remaining missing values in using the average of the entire sequence in the spss software. Finally, 1583 participants were included in the analysis. The study design flow is depicted in Fig. 1.

Diagnosis of DR

The following criteria were used to identify diabetes: (1) a prior diagnosis from a medical expert, (2) fasting blood glucose (FBG) levels of > 7.0 mmol/L, (3) HbA1c levels of > 6.5%, or (4) receiving diabetes medication¹³. DR was self-reported using a dichotomous classification, whereby the respondent had been informed by their physician that diabetes was affecting their eyes.

Determination of heavy metals

Whole blood specimens were collected by physicians at the NHANES Mobile Examination Center (MEC) via venipuncture in EDTA-coated tubes, centrifuged on-site, and stored frozen at − 30 °C for transportation to the Centers for Disease Control and Prevention (CDC) laboratory in California, where they were stored in a frozen state until further analysis. First, in the sample dilution step, a small amount of whole blood is taken from a larger sample and mixed to distribute the cellular components evenly. This mixing is critical because certain metals (e.g., lead) are primarily associated with red blood cells. Samples with clots or microclots are identified and excluded from analysis due to concerns about sample inhomogeneity. Diluted blood samples are prepared by mixing 1 part of the sample, 1 part of water, and 48 parts of diluent. The diluent contains chemicals that release metals from red blood cells, reduce ionization inhibition, prevent clogging, and enable the use of internal standards. The diluted sample is then passed through an inductively coupled plasma (ICP) ion source into the mass spectrometer. The liquid blood sample is converted into aerosol droplets that are vaporized, atomized, and ionized in the plasma region. The resulting ions enter the mass spectrometer with argon gas for analysis. The Dynamic Reaction Cell (DRC) plays a vital role in the selective reaction by removing interferences or enhancing the ion signal of specific elements. The ions passing through the DRC are electrically selected and directed to the analytical quadrupole. The electrical signal generated by the ion impact detector is processed into digital information to determine the elemental concentration. For the values under LODs, an imputed fill value (LOD/√2) was adopted to fill up the vacancy in data preprocessing. Quality assessment and quality control (QA/QC) processes for data collection from blood samples complied with the Therapeutic Laboratory Development Act of 1988¹⁴. Detail information of the NHANES laboratory procedure is available at https://www.cdc.gov/nchs/nhanes/index.htm.

Covariates

Data on demographics (age, gender, and race/ethnicity), academic achievement, poverty-to-income proportion, smoking, body mass index (BMI), waist circumference, serum ALB, FBG, HbA1c, triglycerides, and total cholesterol were collected using standardized questionnaires. Participants were divided into Caucasian, Black, Hispanic, Mexican American, and other race categories. Height (H, m) and weight (W, kg) were measured according to norms, and BMI was calculated as W/H² (kg/m²). Smokers were defined as individuals who smoked ≥ 100 cigarettes¹⁵. Smoking status was divided into three categories: never smoked (< 100 cigarettes in their lifetime), gave up smoking (previously smoked > 100 cigarettes but no longer smoke), and still smoking (previously smoked > 100 cigarettes and still smoking). Hypertension was defined as systolic blood pressure > 140 mmHg or diastolic blood pressure > 90 mmHg, use of blood pressure drugs, or self-reported high blood pressure¹⁶.

Statistical analysis

Categorical variables were expressed as proportions and frequencies and were statistically analyzed using Chi-square analyses. Continuous variables were expressed as mean and standard errors and were analyzed using linear regression models. Multifactorial logistic regression analysis was used to examine the relationship between heavy metal exposure and DR. Additionally, subgroup analysis of heavy metal concentration and DR was performed to test whether the effect of serum levels of heavy metal on DR could be altered by age, sex, and BMI. All data analyses were performed using R (version 4.0.2) and SPSS software (version 24.0). p < 0.05 was considered statistically significant.

Institutional review board statement

Our study used five cycles of open NHANES database (2011–2020), National Center for Health Statistics granted the study procedures of Ethics Review Board. These data could be accessible at the following URL: https://www.cdc.gov/nchs/nhanes/irba98.htm#print.

Ethics statement

All survey participants signed a declaration of consent form after being made aware of the nature of the poll. The informed consent was approved after evaluation by the Committee of the National Center for Statistics on Health Ethics Assessment Board. To make the best use of these resources, all of the data is made publicly accessible after formal anonymization. These data could be accessible at the following URL: https://www.cdc.gov/nchs/nhanes/irba98.htm#print.