Prevalence and metabolic determinants of abnormal alanine aminotransferase: A cross‐sectional study of Iranian adults, 2018–2022

Abstract Background Alanine aminotransferase (ALT) is an enzyme whose activity became the principal biomarker for liver disease. In the current study, we aimed to determine the prevalence of abnormal ALT, as a surrogate of nonalcoholic fatty liver disease (NAFLD) and its associated determinants using different criteria among Tehranian subjects between 2018 and 2022. Methods This is a cross‐sectional study on 5676 Tehranian individuals aged 20–70 years. The weighted prevalence of abnormal ALT was calculated using both the National Health and Nutrition Examination Survey in the United States (US‐NHANCE; ALT ≥30 U/L for females and ≥40 U/L for males) and the American College of Gastroenterology (ACG) guideline (ALT >25 U/L for females, and >33 U/L for males) thresholds. Moreover, uni/multivariable logistic regression analysis was performed to find the determinants of abnormal ALT. Results The weighted prevalence of abnormal ALT was 12.8% (7.6% females and 18% males) and 22.5% (17.7% females and 27.3% males) based on US‐NHANCE and ACG criteria, respectively. Our results showed every decade increase in age decreased the risk of abnormal ALT by 32%. We also found that generally male gender, being overweight/obese, central adiposity, TG ≥6.9 mmol/L, non‐HDL‐C ≥3.37 mmol/L, lipid‐lowering medications, pre‐diabetes/T2DM were associated with abnormal ALT using different cutoff points. Moreover, among men resting tachycardia (≥90 beats per min), hypertension, and females past‐smoker were also found as other determinants of abnormal ALT. Conclusion High prevalence of abnormal ALT among non‐elderly Iranian adults, especially among men, necessitates immediate multifaceted strategies by policymakers to prevent potential complications caused by NAFLD.


| INTRODUC TI ON
Alanine aminotransferase (ALT) is an enzyme that principally presents in the cytosol of the hepatocytes with more than three thousand activities compared to its serum activity. ALT also exists in other tissues including, the kidney, heart, pancreas, muscles, adipose tissues, intestines, colon, prostate, and brain with a much lower concentration than the liver. 1 Among different liver enzymes, serum ALT activity became the principal biomarker for liver disease 2 such as viral hepatitis, autoimmune hepatitis, and most importantly nonalcoholic fatty liver disease (NAFLD). 3,4 Based on the data from the National Health and Nutrition Examination Survey (NHANES), the prevalence of elevated ALT raised from 8.9% in 2002 (ALT >43 IU/L for both genders) to 11.4% in 2012 (>40 IU/L for males, >31 IU/L for females) in the U.S adults. 5,6 Also, there is a similar growing trend for the prevalence of elevated ALT in the Korean population; from 7.4% (>43 U/L) in 2009, to 7.8% (>40 U/L) in 2015. 7,8 In a cross-sectional study in the southeast of the Kerman province of Iran-2017, the prevalence of elevated ALT (> 40 U/L in males and >35 U/L in females) was 6.7% in females and 11.7% in males, and it was more prevalent among younger individuals. 9 In another study conducted in the north-east of Iran-2013, the prevalence of elevated ALT (> 40 U/L for both males and females) among individuals aged >50 years was 4% (4.9% in males and 3.5% in females) and more frequent among the younger population. 10 The difference in the reported prevalence might be related to the different definitions of abnormal ALT. 6,11,12 In addition to age and gender, abnormal ALT is associated with general and central adiposity and other cardiometabolic components including high blood pressure, 13,14 high blood glucose, abnormal values of triglycerides (TG), total cholesterol (TC), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C). 8,10,[15][16][17] Importantly, few studies found genderspecific differences in the association between cardiometabolic risk factors and abnormal ALT levels; however, this issue did not address in other studies. 18,19 Since the current first-line management of NAFLD rely on increasing physical activity levels, nutritional care, and lifestyle modification, early screening with detection of abnormal ALT could be useful for a variety of noncommunicable diseases such as metabolic syndrome, type-2 diabetes (T2DM), hypertension, and cardiovascular disease. [20][21][22][23][24] The level of abnormal ALT differs through populations due to variations in ethnicity, geographic region, and socioeconomic status. 10,15,25,26 In the current study, we aimed to determine the prevalence of abnormal ALT and its associated components using different criteria among Tehranian males and females in 2018-2022.

| Study population
The Tehran Lipids and Glucose Study (TLGS) is a communitybased longitudinal study performed on a Tehranian urban population aged ≥3 years to determine the prevalence and incidence of non-communicable diseases (NCD) and related risk factors.
Volunteer recruitment was done in two different phases, the first (1999-2001; n = 15,005)

| Clinical and laboratory measurements
Trained interviewers asked all participants about demographics, family history of diabetes (FH-DM), history of cardiovascular disease (CVD), medication history, education levels, and smoking habits. The subject's anthropometric parameters were measured with light clothing and without shoes. Weight was measured using a digital scale (Seca 707, Seca Corp; range 0.1-150 kg, sensitivity 0.1 kg).
Height was measured with a tape meter in a standing position and shoulders in normal alignment. WC at the umbilical was measured by an unscratched tape meter. After 15 min of rest, systolic and diastolic blood pressures (SBP and DBP, respectively) were measured twice on the right arm (with a time interval of 5 min) by a standardized mercury sphygmomanometer (calibrated by the Iranian Institute of Standards and Industrial Researches), and blood pressure was considered a mean of these measurements.
For all subjects after 12-14 h of overnight fasting, a venous blood sample was collected. Fasting plasma glucose (FPG) was measured using an enzymatic colorimetric method with glucose oxidase. TC was assayed using the enzymatic colorimetric method with cholesterol esterase and cholesterol oxidase. Measurement of HDL-C was done with a homogeneous method (HDL-C Immuno FS) in which non-HDL-C was removed using antibodies against human lipoproteins, and then HDL-C was measured by the enzymatic colorimetric cholesterol assay. TG was assayed using an enzymatic colorimetric method with glycerol phosphate oxidase. The intra-and inter-assay coefficients of variation (CVs) both were 2.2% for glucose. For both TC and HDL-Cholesterol, intra-and inter-assay CVs were 0.5% and 2%, respectively. Intra-and interassay CVs were 0.6% and 1.6% for TG, respectively. Serum creatinine (SCr) levels were assayed by the kinetic colorimetric Jaffe method. ALT was measured by an optimized UV-test according to the International Federation of Clinical Chemistry and Laboratory Medicine (IFCC) on a photometry system; their intra-and interassay CVs were 2.2% and 3.8% for ALT, respectively. Analysis was performed in the TLGS research laboratory on the same day as blood sampling using commercial kits (Pars Azmoon Inc., Tehran, Iran) and a Selectra 2 auto-analyzer (Vital Scientific, Spankeren, The Netherlands). To monitor the quality of measurements, lyophilized serum controls in two different normal and pathologically high concentrations (TruLab N and TruLab P; Pars Azmoon Inc.) were used. To assess the repeatability of all assays, in the first run, at least eight repeated measurements were performed and followed by repeated measurements on consequent days. Intraassay CV was calculated using data from the first run and interassay CV was calculated using data from all runs. Furthermore, the TLGS laboratory participates in an external quality assurance program.
Laboratory analyses of the study samples were performed when both internal and external quality control measures met the acceptance criteria.

| Definitions
Age groups were defined as; 20 to <30 years (as reference), 30 to <40 years, 40 to <50 years, 50 to <60 years, and ≥60 years in both genders. BMI was calculated as weight (kg) divided into the square of height (m 2 ). BMI was categorized into three groups: 1. <25 kg/m 2 (as reference); 2. 25-30 kg/m 2 , and ≥30 kg/m 2 . Abdominal obesity was defined as WC ≥95 cm as recommended by The Iranian National Committee of Obesity and based on multiple cross-sectional and prospective studies. 28 Marital status is categorized as single (as reference), married, or widowed/divorced. Education was categorized into three groups: 1. <6 years (reference); 2. 6-12 years; and 3.
≥12 years (as reference). Smoking status was defined in three groups; 1. Current smokers as participants who smoked cigarettes daily or occasionally as well as those who used water pipe or pipe; 2. Past smokers as participants who smoked in the past; and 3. Those who never smoked (as reference). A Modifiable Activity Questionnaire (MAQ) was used to collect data on physical activity. 29,30 We used the average metabolic equivalent of a task (MET) score to define physical activity values and it was categorized as ≥1500, 600-1500, and <600 MET mins/wk (as reference). Positive family history of diabetes was defined as having at least one parent or sibling with diabetes. History of CVD was defined as previous ischemic heart disease and/or cerebrovascular accidents. Diabetes status was defined as follows: normoglycemia (FPG <5.55 mmol/L as reference), prediabetes (FPG: 5.55 to <7 mmol/L), and type 2 diabetes (FPG ≥7 mmol/L or taking diabetes-lowering medications). Hypertension status was classified as follows: normal (SBP < 120 and DBP < 80 as reference), prehypertension (SBP: 120-140 and DBP: 80-90), and hypertension (SBP ≥140 or DBP ≥90 or taking antihypertensive medications).
Heart rate was measured through palpation. High triglycerides were defined as TG ≥6.9 mmol/L. Low HDL-C was defined as HDL-C <1.29 mmol/L for females and <1.04 mmol/L for males. Non-HDL-C was calculated as TC minus HDL-C, and it was categorized non-HDL-C as <2.59 mmol/L (as reference); 2.59 to <3.37 mmol/L, and ≥3.37 mmol/L. 31 Pulse rate was classified as, < 60 beats per minute, 60-90 beats per minute (as reference), and ≥90 beats per minute. 32 Chronic kidney disease (CKD) is defined as either kidney damage or estimated glomerular filtration rate (eGFR) <60 mL/min/1.73 m 2 for >3 months. 33 eGFR was estimated from serum creatinine (SCr) values using CKD-EPI equations 34 : First, SCr values were multiplied by 0.95 before eGFR calculation to standardize SCr 35,36 : where κ is 0.7 for females and 0.9 for males and α is −0.329 for females and −0.411 for males.

| Statistical analysis
Baseline characteristics of the study population were shown as mean (standard deviation: SD) and number (%) for categorical variables.
For covariates with a skewed distribution, the median (interquartile range: IQR) was reported. A comparison of baseline characteristics between normal and abnormal ALT according to two definitions was done by student's t-test for continuous variables, chi-square test for categorical variables, and Mann-Whitney test for skewed variables.
The weighted prevalence of abnormal ALT was calculated using both US-NHANCE and ACG thresholds. Data were weighted directly to the 2016 urban population of Tehran, 40 based on the 2016 national Iranian census, to match the age (10-year strata) and gender strata.
The association of different categorical variables with abnormal ALT (for both US-NHANCE and ACG definition) was assessed by calculating multivariable-adjusted odds ratios (ORs) with a 95% confidence interval (CI) using binary logistic regression analysis. A univariable analysis was done for each potential covariate including gender (female as reference), age categories, marital status, education levels, smoking status, physical activity groups, BMI categories, abdominal obesity, glucose tolerance status, blood pressure categories, CKD, high TG, low HDL-C, non-HDL-C categories, lipid-lowering medications, a steroid medication, FH-DM, and history CVD. Then, those covariates with a p-value less than 0.2 in the univariable analysis were selected to enter the multivariable model. We evaluated the eGFR = 141 × the minimum of standardized SCr (mg∕dL)∕κ or 1 × the maximum of standardized SCr (mg∕dL)∕ or 1 − 1.209 effect modification of gender and each variable in a multivariable model. All analyses were performed in each gender separately in addition to the total population. All analyses were conducted using STATA version 17 SE (StataCorp, TX, USA), and a two-tailed p < 0.05 was considered significant.

| RE SULTS
The study population consists of 5318 participants (male = 2434) with a mean age (SD) of 45.2 years (12.8). The baseline characteristics of participants by ALT levels for both defined thresholds are shown in Table 1. There were significant differences in baseline characteristics between normal and abnormal ALT, except for marital status, education levels, physical activity status, CKD, steroid medication, history of CVD, and FH-DM. Compared to normal ALT participants in the abnormal group were younger and had higher values for BMI, WC, SBP, DBP, FPG, TG, and non-HDL-C; moreover, they also had higher frequencies of smokers and using lipid-lowering medication.
In the total population, the weighted mean (95% CI) value of ALT level for individuals aged 20-30 years was significantly higher com-  (Table S1). As shown in Figure 1, the weighted prevalence of abnormal ALT according to ACG guideline was significantly higher compared to US-NHANCE suggested threshold. Considering US-NHANCE suggested threshold the weighted prevalence (%) (95% CI) of abnormal ALT was 18.0 (16.6-19.8), 7.6 (6.7-8.7), and 12.8 (11.9-13.8) in males, females, and total population, respectively. Also, individuals with abnormal ALT were more likely to be younger in males, females, and total population, respectively (Table S1). As the same as the US-NHANCE suggested threshold, the prevalence of abnormal ALT is higher among younger individuals. Since BMI is known to be a significant source of risk for abnormal ALT, as a sensitivity analysis, BMI-adjusted prevalence was also calculated and the results remained essentially the same (data not shown).
Results of the uni-and multivariable logistic regression analysis for abnormal ALT using different definitions are illustrated in Tables 2 and 3

| DISCUSS ION
In a population-based study of middle-aged Tehranian residents, the weighted prevalence of abnormal ALT ranged from 12.8% to 22.5% applying US-NHANCE and ACG criteria, respectively. We also found that generally younger age, male gender, being overweight/obese, central adiposity, TG ≥6.9 mmol/L, non-HDL-C ≥3.37 mmol/L, lipidlowering medications, and pre-diabetes/T2DM were associated with abnormal ALT using different definitions. Moreover, among men resting tachycardia, and hypertension, and for females, pastsmoker were also found as another determinant of abnormal ALT.
Our reported prevalence of abnormal ALT in the current study was generally higher than those reported in developed countries, estimated at 11.2% in the Australian population 42  the prevalence of elevated ALT increased to 38%. 10 In another casecontrol study conducted in the north of Iran, the prevalence of ALT >40 U/L was about 7.8%. 15 Several cross-sectional studies have investigated the association of abnormal ALT with gender and the majority of them showed that male gender is an independent risk factor for ALT elevation.
Our results showed that the male gender had higher odds of having TA B L E 1 Baseline characteristics of study population according to different categories of ALT in total population: Tehran lipids and glucose study.  Considering lipid profiles, TG ≥6.9 mmol/L, non-HDL-C ≥3.37 mmol/L, and lipid-lowering medications were independently associated with abnormal ALT in the total population. A genderspecific analysis shows that high triglyceridemia was associated with abnormal ALT only among males, while the last two variables were associated only with females. In previous Iranian studies, hypertriglyceridemia was associated with elevated ALT in both genders. 10,15 The influence of hypertension on abnormal ALT was observed only among men using the US-NHANCE threshold. This positive association among men was reported by the CHIEF Study 52 in the Our study showed positive associations between abnormal serum ALT levels and heart rate ≥90 beats per minute in the total population using US-NHANCE threshold and among males in ACG guideline definition even after multiple adjustments with metabolic risk factors as well as physical activity levels that did not remain as an independent factor. This significant association was reported by Laine et.al 53 among overweight/obese Finish adults.
In another study, Straznicky et al. 54 reported that ALT was positively associated with resting HR in obese subjects with metabolic syndrome. Moreover, Kim et al. 55 reported a positive association between resting HR and NAFLD among post-menopausal women.
In our analysis, this association remained a significant event after adjustment with central and general obesity, hypertension, diabetes, and lipid profiles, which may be a representative marker Abbreviations: ACG, the American College of Gastroenterology; ALT, alanine aminotransferase; BMI, body mass index; CI, confidence interval; CKD, chronic kidney disease; CVD, cardiovascular disease; FH-DM, family history diabetes; HDL-C, high-density lipoprotein cholesterol; MET, metabolic equivalent of a task; OR, odds ratio; T2DM, type 2 diabetes.

| CON CLUS IONS
In conclusion, we found that out of every 100-person, about 18-27 males and 8-18 females have abnormal ALT. We also observed male sex and younger age showed significant risk factors for abnormal ALT among the Tehranian population. Obesity, prediabetes/T2DM, hypertriglyceridemia, high non-HDL-C in both genders, as well as resting tachycardia and hypertension for men and past-smoker for women had the most consistent association with abnormal ALT.
Hence, the high prevalence of abnormal ALT among non-elderly Iranian adults, especially among men, necessitates immediate multifaceted strategies by policymakers to prevent potential complications caused by NAFLD.

CO N FLI C T O F I NTE R E S T S TATE M E NT
None declared.

DATA AVA I L A B I L I T Y S TAT E M E N T
The datasets used and/or analyzed during the current study are available from the corresponding author upon reasonable request.