Study design and participants
The study population was selected from the 2017–2018 cycle of NHANES, a complex cross-sectional survey program conducted in the US by the National Center for Health Statistics of the Centers for Disease Control and Prevention. Answers to the structured household interviews, such as demographic data, medical history, and dietary and PA information, were self-reported. Physical examinations, including anthropometric measurements and blood samples, were performed at a mobile examination center. Publicly available data collection descriptions, analytical guidelines, and full datasets for NHANES 2017–2018 are available[24,25].
Diagnostic criteria and definition of groups
MAFLD and non-MAFLD
MAFLD is diagnosed based on radiologically diagnosed hepatic steatosis and the presence of overweight/obesity, diabetes mellitus, or metabolic dysregulation[26]. Metabolic dysregulation was defined as the presence of two or more of the following: 1) waist circumference ≥102 cm in men and 88 cm in women; 2) blood pressure ≥130/85 mmHg or antihypertensive treatment; 3) triglyceride level ≥1.70 mmol/L or specific drug treatment; 4) high-density lipoprotein cholesterol (HDL-C) level <1.0 mmol/L for men and <1.3 mmol/L for women; 5) prediabetes (fasting blood glucose level between 5.6 and 6.9 mmol/L or 2-hour post-load glucose level between 7.8 and 11.0 mmol/L or HbA1c between 5.7% and 6.4%); 6) homeostasis model assessment-insulin resistance (HOMA-IR) score ≥2.5; and 7) C-reactive protein (CRP) level >2 mg/L. Patients who did not meet the criteria comprised the non-MAFLD population.
Smoking and alcohol consumption
Smoking status was categorized as current, former, and never-smoker. Drinking status was categorized as never (<12 drinks in lifetime), former (had ≥12 drinks in any year and did not drink last year, or did not drink last year but drank ≥12 drinks in lifetime), current light/moderate (average of ≤1 drink per day for women and ≤2 drinks per day for men over the past year), and current heavy (average of >1 drink per day for women or >2 drinks per day for men over the past year) drinker[27].
Demographic variables
The following demographic variables were self-reported per the NHANES design: age (18–34, 35–49, 50–64, ≥65 years), sex, race/ethnicity (White, Black, Mexican, Asian, other), educational attainment (EA) (<high school, high school or GED, ≥college), poverty income ratio (PIR; <1.30, 1.30–3.49, ≥3.50), insurance (yes, no), and comorbidity data, including metabolic and cardiovascular disease, hypertension, and cancer (all types). We defined obese participants as those with a BMI ≥30 kg/m2 (for non-Asians) or ≥27.5 kg/m2 (for Asians based on the World Health Organization cut-off values for Asians)[28]. Non-obese participants were defined as those who had a BMI of <30 kg/m2 for non-Asians and <27.5 kg/m2 for Asians. Normal-weight or lean participants were defined as those who had a BMI of <25 kg/m2 for non-Asians and <23.0 kg/m2 for Asians. The waist circumference cut-off criteria for metabolic syndrome were also lower for Asians (90 cm for men and 80 cm for women) than for Caucasians (94 cm for men and 80 cm for women)[29]. Blood pressure was measured using standardized equipment in the sitting position. Hypertension was defined as a systolic blood pressure ≥140 mmHg and diastolic blood pressure ≥90 mmHg or the use of antihypertensive medications. Blood samples were collected from each participant, and levels of blood urea nitrogen, creatinine, white blood cell count, hemoglobin, platelet count, hypersensitive CRP, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transferase, total cholesterol, HDL-C, low-density lipoprotein cholesterol, triglycerides, plasma fasting glucose, fasting insulin, HbA1c, and HOMA-IR were measured. The participants were examined using whole-body dual-energy X-ray absorptiometry and analyzed using Hologic Discovery Software version 12.1 to derive the fat mass.
Definition of liver steatosis and significant fibrosis
In this study, attenuation parameter (CAP) and liver stiffness measurement (LSM) cutoffs were used to identify individuals with hepatic steatosis or clinically significant fibrosis (CSF; fibrosis stage ≥2)[30]. A CAP cutoff ≥285 dB/m (based on 80% sensitivity and 77% specificity) was defined as hepatic steatosis, whereas LSM ≥8.6 kPa (based on 66% sensitivity and 80% specificity) was defined as CSF.
Furthermore, two composite secondary ordinal outcomes (LSM cutoffs with a sensitivity of 90% [≥5.6 kPa] and a specificity of 90% [≥6.5 kPa]) were assessed and analyzed in conjunction with the primary outcome.
Dietary assessment and calculation
The dietary intake data used in the analyses were obtained using two 24-h dietary recalls[31]. Using the US Department of Agriculture Food Patterns Equivalents Database, we calculated the HEI-2015 score, which is closer to the current Dietary Guidelines for Americans (DGA) 2015–2020[32]. The database comprises 13 components, nine of which assess the adequacy of food consumption recommended as part of a healthy diet (including total fruits, whole fruits, total vegetables, greens and beans, whole grains, dairy, total protein foods, and fatty acids) and four of which assess moderation (refined grains, sodium, added sugar, and saturated fat)[32]. For adequacy, higher scores were associated with higher intake, whereas for moderation, higher scores were linked to lower consumption. Seven components were each scored on a 0–10 scale, and the other six components were scored on a 0–5 scale. The HEI scores are summed to obtain the total score with a maximum of 100 points; higher scores indicate better DQ and reflect closer alignment with the DGA regarding adequacy.
PA assessment
A global PA questionnaire was administered as part of the NHANES survey, which measures the time spent sitting and that spent participating in typical PA during the past week. In terms of surveillance, the questionnaire has also been validated[33]. The total time spent on each PA was calculated based on the frequency of participation and the time spent per episode. The PA quantities were then indexed using metabolic equivalents (metabolic equivalent of task [MET]-minutes). MET-minutes were calculated by multiplying the MET value by the duration of the activity. A list of MET values assigned by the NHANES for specific activities can be found on the survey website. We created the following categories of total PA to investigate dose-response effects on MAFLD risk based on previously published data: <600, 600–7,999, and ≥8,000 MET-minutes/week[34]. PA was further classified into active (≥600 MET-minutes/week) and inactive (<600 MET-minutes/week). A PA level ≥600 MET-minutes/week reportedly results in significant health benefits[35].
Statistical analysis
The NHANES uses a complex, multistage, probability sampling design to select participants representative of the civilian non-institutionalized US population. Certain population subgroups were oversampled to increase the reliability and precision of the health status indicator estimates for these groups. Therefore, weights were created in the NHANES to account for the complex survey design (including oversampling), survey non-response, and post-stratification. We integrated weights in our statistical analysis so that our estimates are representative of the US civilian non-institutionalized population.
Chi-square tests were performed to compare the distribution of categorical variables, such as age group, sex, race/ethnicity, education level, and comorbidities, among patients with MAFLD who had and did not have obesity and in the integration of PA and DQ. Student’s t-tests were performed to compare the distribution of continuous variables such as blood biochemical and physical measurements.
Univariate and multivariate logistic regression analyses were conducted to calculate odds ratios (ORs) and 95% CIs of either the HEI, PA, age, sex, ethnicity, EA, or PIR with risks across the obese and non-obese MAFLD population. Furthermore, we explored the combined effects of PA (active vs. inactive) and the highest tertile of the HEI score (<58.72 vs. ≥58.72) on the prevalence and risk estimates of obese and non-obese MAFLD using multiple logistic regression models. All multiple logistic regression models were adjusted for age, race/ethnicity, education, ratio of total family energy intake, HEI, PA, smoking status, EA, PIR, and alcohol intake status.
The p-values for all statistical analyses were two-tailed with a significance level of 0.05. All analyses were performed using R Studio software (version 4.2.0).