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Exploring the association between theobromine intake and hepatic steatosis in young people

Statement of ethics

All participants gave their informed agreement, and the project was authorized by the National Center for Health Statistics Research Ethics Review Board.

Study population

In order to achieve nationwide representation, NHANES, a thorough and ongoing cross-sectional national survey in the US, uses a stratified, multistage, clustered random sampling to collect diet and health data from the whole population16. Out of 15,560 participants in the 2017–2020 NHANES cycle, 9698 had available CAP data. We excluded 2451 participants testing positive for hepatitis B antigen, hepatitis C antibody, or hepatitis C RNA, 799 with significant alcohol consumption (4, 5, or more drinks daily), 2728 lacking theobromine intake data, and 1924 participants age > 45 years old. Ultimately, 1796 participants (ranging in age from 12 to 45 years) were included in the study. Figure 1 illustrates the sample selection flowchart.

Figure 1
figure 1

Flowchart of participant selection. NHANES National Health and Nutrition Examination Survey, CAP controlled attenuation parameter.


The investigation focused on dietary theobromine intake as the exposure factor. Theobromine intake assessment involved two 24-h food recall interviews. Three to ten days after the first interview, which took place at a mobile exam facility, there was a telephone interview. The 24-h dietary questionnaire collected the type and quantity of all beverages and foods (including foods such as chocolate) in the 24 h prior to the interview. The United States Department of Agriculture’s Food and Nutrient Database for Dietary Studies was the source of information on nutrient intakes, including theobromine intake17. More details are available at Theobromine intake per participant was averaged from two days of dietary recall data when available, or based on a single day’s data otherwise (among all, 1654(92%) participants completed both the 24-h recalls).

The study’s outcome variable, CAP, was measured using the FibroScan® 502 V2 Touch equipped with liver ultrasonography transient elastography. This device records CAP by measuring ultrasonic attenuation, which reflects hepatic steatosis and indicates liver fatness. According to a recent key study, there is 90% sensitivity in detecting different degrees of hepatic steatosis when CAP values ≥ 274 dB/m, which indicate NAFLD status, are present18. This study, which is based on three earlier investigations, classifies CAP ≥ 302 dB/m as a sign of severe steatosis in instances of NAFLD19,20,21.

Our study incorporated categorical covariates such as gender, race/ethnicity, education level, smoking status, hypertension, diabetes, and cholesterol levels. Continuous covariates in our analysis included age, body mass index (BMI), alanine aminotransferase (ALT), aspartate aminotransferase (AST), γ-glutamyl transpeptidase (GGT), serum creatinine, serum albumin, and uric acid. Detailed data on dietary theobromine intake, CAP, and other variables are publicly accessible at

Statistical analysis

We took high volatility in our data set into account by using a weighted variance estimation technique. A weighted multivariate logistic regression model was employed to examine the correlation between theobromine intake and CAP (Model 1: no covariates were adjusted; Model 2: age, gender, and race/ethnicity were adjusted; Model 3: age, gender, race/ethnicity, education level, body mass index, smoking status, and the existence of diabetes, hypertension, and high cholesterol level were adjusted; Model 4: age, gender, race/ethnicity, education level, body mass index, smoking status, and the existence of diabetes, hypertension, and high cholesterol level, aspartate aminotransferase, alanine aminotransferase, γ- glutamyl transpeptidase, serum albumin, serum creatinine and uric acid were adjusted). The weighted χ2 test was utilized for categorical data in order to assess group differences, and the weighted linear regression model was applied for continuous variables. Subgroup analysis was performed using stratified multivariate regression analysis. Subgroups were divided according to age, sex, and race/ethnicity. The continuous variable age was divided into two groups (< 18 years; 18–45 years). Using generalized additive models and smooth curve fits, the nonlinear relationship between theobromine consumption and CAP was investigated. After finding nonlinearity, we used a recursive technique to calculate the inflection point in the theobromine intake and CAP connection. We next used a two-piecewise linear regression model to both sides of this point. All analyses were conducted using R ( and EmpowerStats (, with a P value < 0.05 considered statistically significant.

Ethics approval and consent to participate

The studies involving human participants were reviewed and approved by CDC’s National Center for Health Statistics Institutional Research Ethics Review Board. The patients/participants provided their written informed agreement to participate in this study. All our methods followed the guidelines of the Helsinki Declaration. And secondary analysis does not require additional institutional review committee approval.

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