Independent associations of alanine aminotransferase (ALT) levels with cardiovascular risk factor clustering in Chinese adolescents

Results The prevalence of elevated ALT levels was 3.2% and 5.9% if abnormal ALT levels were defined as >40 IU/L and >30 IU/L respectively. Using the <25th, 25–75th and >75th percentile values of ALT, all subjects were divided into 3 ALT strata. Using the lowest ALT stratum as referent, the top ALT stratum was associated with obesity and clustering of cardiometabolic–inflammatory risk markers in both genders. After adjusting for age and body mass index (BMI), the highest ALT stratum remained independently associated with diastolic blood pressure and insulin resistance (fasting insulin and Homeostasis Model Assessment, HOMA-IR, HOMA-beta) in boys ( p < 0.05); and serum triglyceride and HOMA-beta ( p = 0.008 and 0.014, respectively) in girls. Repeat analysis after excluding subjects with ALT > 30 ( n = 123) or >40 IU/L ( n = 67) yielded comparable results. Conclusions In adolescents, top ALT stratum, albeit within normal range, is associated with clustering of cardiovascular risk factors, independent of obesity. Abbreviations ALT alanine aminotransferase BMI body mass index HOMA homeostasis model assessment IR insulin resistance NAFLD non-alcoholic fatty liver disease Keywords Alanine aminotransferase Cardiovascular risk factors Chinese adolescents 1 Introduction The prevalence of obesity in children is increasing worldwide [1–4] . Obesity is associated with clustering of cardiovascular risk factors, increased risk of morbidity and premature mortality in adulthood [5–8] . In the United States (US), numbers of overweight children and adolescents aged 6–19 years have tripled. In 2003, 17.1% of US children and adolescents were overweight [2,9,10] . In 2000, the prevalence of overweight (BMI ⩾ 24 kg/m 2 ) and that of obesity (BMI ⩾ 28 kg/m 2 ) in Chinese youth (7–18 years) were 17.0% and 10.0% for boys and 9.5% and 6.5% for girls, respectively [11] . Non-alcoholic fatty liver disease (NAFLD) is commonly associated with obesity and is emerging as the commonest chronic liver abnormality in US children. NAFLD is not a benign condition and is associated with increased cardiovascular and non-cardiovascular morbidity and mortality [12,13] . In a retrospective review of 742 autopsy cases of child deaths, aged 2–19 years, 13% had histologically proven NAFLD with the highest rate in the frankly obese group (38%) [14] . In US adolescents aged 12–19 years, the prevalence of elevated ALT (defined as >30 IU/L) was 8.0% (7.4%, 11.5% and 6.0% among white, Mexican American and black adolescents, respectively) [15] . Apart from ethnicity [15] , elevated ALT was also associated with obesity and fasting insulin [15,16] . In the Bogalusa Heart Study, persistent elevation of ALT within the reference range (0–55 IU/L) was related to an adverse cardiovascular risk profile in young adults [8] . To date, despite the epidemic of diabetes and obesity in China, there are limited data on ALT and its associations with obesity and cardiometabolic risk factors in this population. Besides, although there are close associations between markers of hepatic inflammation, obesity and cardiometabolic risk [17] , it remains uncertain whether ALT is independently associated with cardiometabolic risk or mediated via obesity. The aims of this study were: (1) to establish the prevalence of elevated ALT in a community-recruited cohort of Hong Kong Chinese adolescents and (2) to explore the nature of associations amongst ALT, obesity and cardiometabolic risk factors. 2 Patients and methods 2.1 Study population and design The study was conducted in 2003. Details of the methodology have been described previously [18,19] . In brief, 53 schools were randomly selected from among 477 secondary schools in Hong Kong using a computer-generated coding system. Of these 53 schools, 14 participated in the survey with consent from school principals. Six classes, 1 from each year grading (Form 1 to Form 6), were randomly selected in each school. All participants were healthy volunteers of Chinese ethnicity and were not taking any medications. The study was approved by the Clinical Research Ethics Committee of the Chinese University of Hong Kong. Informed consent was obtained from all participants and their parents. 2.2 Anthropometric and biochemical assessment Anthropometric indexes were measured and blood samples were obtained from each participant during the field study by a team of trained research nurses and assistants. Waist circumference (WC), body weight (measured to the nearest 0.1 kg by Tanita physician digital scale, Tanita Corp., Tokyo, Japan) and height (measured to the nearest 0.1 cm using a portable standiometer) were measured. Body mass index (BMI) (kg/m 2 ) was calculated from body weight (kg) divided by the square of body height (m 2 ). Blood pressure (BP) was measured after at least 5 min of rest using an electronic device validated against standard auscultatory mercury sphygomomanometry [20] (Omron, Omron Healthcare Inc., Tokyo, Japan). After an overnight fast of at least 8 h, blood samples were collected for measurement of fasting plasma glucose (FPG), total cholesterol (TC), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), insulin, high sensitivity C-reactive protein (hs-CRP), complete blood count, and renal and liver functions. 2.3 Laboratory assays All blood samples were kept in ice at 0 °C before being transported back to the laboratory. Assays for lipids, FPG, complete blood picture, and renal and liver functions were performed within 6 h of collection of samples. Aliquots of serum for other assays were stored at −70 °C. Plasma glucose (hexokinase method), TC (enzymatic method), TG (enzymatic method without glycerol blanking), HDL-C (direct method using PEG-modified enzymes and dextran sulfate), renal and liver function tests including plasma ALT levels were measured on a Roche Modular Analytics system (Roche Diagnostics GmbH, Mannheim, Germany) using standard reagent kits supplied by the manufacturer of the analyzer. The precision was within the manufacturer’s specifications. Low-density lipoprotein cholesterol (LDL-C) level was calculated using the Friedewald’s formula for TG < 4.5 mmol/L [21] . Serum concentrations of hs-CRP and insulin were measured by chemiluminescence immunoassays using IMMULITE ® 2000 Analyser (Diagnostic Products Corporation, Los Angeles, CA, USA). The sensitivities of the hs-CRP assay and insulin were 0.1 mg/L and 2 mIU/L, respectively. Insulin resistance (IR) was calculated using the Homeostasis Model Assessment (HOMA-IR) = FPG (mmol/L) × fasting insulin (mIU/ml) ÷ 22.5; HOMA-beta = [20 × fasting insulin (mIU/ml)] ÷ [FPG (mmol/L) − 3.5] [22] . 2.4 Statistical analysis Data are presented as means (standard deviation, SD), median (interquartile range, IQR) or frequency (%) as appropriate. Fasting serum insulin, HOMA-IR, HOMA-beta and TG were naturally log-transformed to correct skewness before analysis. Since the highly skewed hs-CRP could not be rendered to normal-like distribution using common transformations, nonparametric methods were used for hs-CRP. All subjects were divided by gender-specific ALT percentiles into 3 strata: (i) <25th percentile, (ii) 25th–75th percentile and (iii) >75th percentile. Trends in anthropometric indexes and cardiometabolic risk factors across the three ALT strata were assessed using the ANOVA linear trend test and Jonckheere–Terpstra trend test was used for the highly skewed hs-CRP. Jonckheere–Terpstra test is a nonparametric test for evaluating ordered differences among 3 or more groups. The Student’s t -test (for variables with normal distribution or skewed variables after being log-transformed) and Mann–Whitney test (for hs-CRP) were used for comparisons between the lowest and highest ALT strata, followed by adjustment for age and/or BMI using the multiple regression model. Ordinal regression was used to compare hs-CRP (that divided into quintiles) between the lowest and highest ALT strata adjusting for age and/or BMI. Overweight and obesity were defined by BMI greater than Hong Kong local age and gender-specific 85th (or 25 kg/m 2 , whenever smaller) and 95th (or 30 kg/m 2 , whenever smaller) percentile, respectively. The local percentile standards were based on the latest local population survey [23] . Clustering of cardiovascular risk factor was defined by either (A) the set of criteria our group adopted [19] which was based on modified definitions used in National Health and Nutrition Education Survey (NHANES) [24] (⩾3 of the following risk factors: (1) HDL-C ⩽ 1.03 mmol/L, (2) LDL-C ⩾ 2.6 mmol/L, (3) TG ⩾1.24 mmol/L, (4) FPG ⩾ 6.1 mmol/L and (5) age-, sex- and height-adjusted systolic or diastolic BP ⩾ 90th percentile) or (B) the International Diabetes Federation (IDF) criteria to define clustering of cardiovascular risk factors [25] (WC ⩾ 90th percentile or adult cutoff if lower, plus ⩾2 of the following risk factors: (1) TG ⩾ 1.7 mmol/L, (2) HDL-C < 1.03 mmol/L, (3) BP ⩾ 130/85 mmHg, and (4) FPG ⩾ 5.6 mmol/L). Association between the 3 ALT strata, categories of normal weight, overweight and obesity, and the presence of cardiovascular risk factor clustering was examined using the Chi-square test or Fisher’s exact test, as appropriate. Association between median ALT level and obesity status, and the presence of cardiovascular risk factor clustering was assessed by Kruskal–Wallis or Mann–Whitney test. Statistical analyses were performed using SPSS 14.0 (SPSS Inc., Chicago, IL). All statistical tests were two-sided and a p -value <0.05 was considered statistically significant. Repeat analysis was performed by excluding subjects with elevated ALT level, defined as >30 IU/L ( n = 123) or >40 IU/L ( n = 67). 3 Results Of the 2102 adolescents (958 boys and 1144 girls) recruited, 123 (89 boys and 34 girls) had ALT > 30 IU/L and 67 (43 boys and 24 girls) had ALT > 40 IU/L. The prevalence of elevated ALT levels was 3.2% (4.5% boys and 2.1% girls) and 5.9% (9.3% boys and 3.0% girls) if normal ALT levels were defined as ⩽40 IU/L and ⩽30 IU/L, respectively. Table 1 shows the demographic and clinical characteristics of the entire cohort with a median age of 16 years (IQR: 14–17 years). Boys in the top ALT stratum were older, more obese (body weight, BMI, WC and waist–hip ratio), had higher systolic and diastolic BP, worse dyslipidemia (high LDL-C and TG) and were more likely to be insulin resistant (higher fasting insulin, HOMA-IR and HOMA-beta) and had higher inflammatory markers (higher hs-CRP and white cell counts) than those in the lowest ALT stratum ( Table 2a ). In girls, ALT top stratum was also associated with increased age, obesity indexes, systolic and diastolic BP, hs-CRP, dyslipidemia and insulin resistance ( Table 2b ). Multiple regression analysis, after adjustment for age and BMI showed that the top ALT stratum remained significantly associated with increased diastolic BP ( p = 0.006), fasting insulin ( p = 0.002), HOMA-IR ( p = 0.005) and HOMA-beta ( p = 0.001) in boys ( Table 2a ). In girls, the top ALT stratum was independently associated with increased TG ( p = 0.008) and HOMA-beta ( p = 0.014) ( Table 2b ). Both continuous and categorized ALT levels were associated with obesity and clustering of cardiovascular risk factors (IDF or modified NHANES criteria) in both boys and girls ( Tables 3a and 3b ). To allow for comparison with published data, we repeated analysis after excluding subjects with ALT > 30 IU/L ( n = 123) or ALT 40 IU/L ( n = 67) which yielded comparable results ( Table 4 ). 4 Discussion In this community-based cohort of school children, our results showed that the top ALT stratum, albeit within normal range, was associated with obesity and clustering of cardiovascular risk factors. In boys, the top ALT stratum was associated with diastolic blood pressure and insulin resistance (fasting plasma insulin, HOMA-IR and HOMA-beta) independent of obesity and age. In girls, fasting TG and HOMA-beta were also independently associated with ALT. There are relatively few community-based surveys to examine the prevalence of elevated ALT levels, as a surrogate marker of NAFLD, in adolescents. When we used a cut-off value of ALT > 40 IU/L, the prevalence of elevated ALT levels in Korean boys and girls were 3.6% and 2.8%, respectively [26] as compared to 4.5% and 2.1% in our cohort. Using a cut-off value of ALT > 30 IU/L, we found out that the respective figures in US adolescents were 12.4% in boys and 3.5% in girls [15] . While there is ongoing debate on the ‘normality’ of ALT level, the lower normal cut-off of 30 IU/L proposed by Prati [27] was criticized as impractical which may overwhelm the health care system [28] . In most published literatures since the 1980s, ALT level ⩽40 IU/L has been used to define normal ALT level. In our study, we analyzed the data in the entire cohort as well as after exclusion of subjects with high ALT level and drew similar conclusions. In agreement with findings from the US and Korean adolescents, we observed a male preponderance of elevated ALT levels which were associated with age, obesity indexes and cardiometabolic risk factors. Taken together, our findings support the importance of risk assessment as a continuum in youth as in the adult population. The liver is a critical organ in maintaining glucose and lipid homeostasis. ALT is a simple biochemical marker of liver function and liver disease which can be readily measured. In a multi-ethnic cohort including 392 white, African-American and Hispanic obese adolescents, elevated ALT level was associated with reduced whole body insulin sensitivity index derived from oral glucose tolerance test parameters [29] . In adults, NAFLD is now considered the hepatic manifestation of the metabolic syndrome [30] closely associated with insulin resistance [31–33] . While obesity and insulin resistance are often proposed to be the linking factors for the clustering of cardiometabolic and inflammatory markers [34] , there is emerging evidence pointing to the causal role of hepatic resistance in this complex syndrome. The underlying mechanisms include increased lipoprotein and glucose production as well as hepatic fatty infiltration and micro-inflammation [17,35,36] . In support of this notion, we observed graded increases in inflammatory marker (hs-CRP), insulin resistance (HOMA-IR), compensatory hyperinsulinemia (fasting insulin and HOMA-beta), obesity indexes and blood pressure across the ALT strata in boys. In girls, similar risk association was observed for inflammatory markers, obesity and lipid profile. In the Korean adolescent cohort, elevated ALT level (>40 IU/L) was also associated with cardiovascular risk factor clustering although the authors did not examine insulin resistance and inflammatory markers. In our cohort, top ALT stratum remained independently associated with diastolic blood pressure and insulin resistance in boys, as well as triglyceride and compensatory pancreatic hyperfunctioning in girls. These findings suggest a possible causal relationship between these risk factors and liver inflammation, although studies are required to elucidate the exact mechanisms. From a clinical standpoint, both obesity and NAFLD are associated with latent complications including liver cirrhosis and hepatocellular carcinoma [37–41] . While the natural history of childhood NAFLD remains to be clarified, there have been sporadic reported cases of childhood NAFLD progressing to liver cirrhosis [38] and hepatocellular carcinoma [41] . Taking into consideration the adult epidemiological data, our results have added new dimension to our fight against obesity to prevent young onset chronic liver disease in addition to other co-morbidities including diabetes and cardio-renal complications [42] . Several limitations in this study need to be acknowledged. The onset of puberty is associated with insulin resistance. In Hong Kong, the mean age of onset of puberty is 11.4 years in boys and 9.78 years in girls [43,44] . Given the mean age of 15–16 years of our cohort, the majority of adolescents were well into their puberty. Due to the invasive nature of the procedure and for ethical reasons, liver biopsy was not performed. Despite its endemic nature in Asia, we did not measure markers of chronic viral hepatitis B infection since a territory-wide vaccination program had been implemented in Hong Kong since 1985. Thus, babies born to women who were hepatitis B carriers had been vaccinated since birth. We also did not screen for chronic hepatitis C virus infection which affects 0.035–0.099% of the general population except in children with transfusion-dependent haematological diseases [45] . In this regard, all our study subjects had no significant past medical history. Although occult alcohol consumption not detected by history taking remains a possibility, local studies have reported a very low rate of habitual alcohol drinking in Hong Kong students [46] . Lastly, the cross-sectional nature of these data precludes the exploration of the causal relationship among ALT levels, obesity and cardiometabolic risk factor clustering. In conclusion, there is a clustering of cardiometabolic and inflammatory risk factors in Chinese adolescents with high normal ALT, especially amongst boys. This association was largely attributed to obesity although there were independent associations amongst ALT, BP and insulin resistant state. Prospective studies and interventional trials are required to delineate the independent and additive effects of obesity and liver inflammation on disease manifestation. Acknowledgements We thank all school personnel, parents and participants for making this study possible. Special thanks are extended to Ms. Delanda Wong, Ms. Yee Mui Lee and Mr. Stanley Wong for conducting the survey. 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