Results from this cohort of children and adolescents with obesity in Sweden, derived from 11,776 patients, presented a prevalence of mildly increased ALT of 37.9% and markedly increased ALT of 10.6%. Moreover, the results demonstrated evidence of sex differences in the associations of age with both mildly and markedly increased ALT. We also found dose-response associations of degree of obesity with both mildly and markedly ALT. Regarding metabolic parameters, high triglycerides, high LDL, high total cholesterol, and low HDL were associated with both mildly and markedly increased ALT. However, impaired fasting glucose was only associated with markedly increased ALT, but not with mildly increased ALT.
The prevalence of mildly and markedly increased ALT in this study was similar to a population of children and adolescents with overweight and obesity in European German-speaking countries , where the prevalence of mildly and markedly increased ALT were 35.7 and 13.5%, respectively. When considering mildly and markedly increased ALT as an entity, a lower prevalence of increased ALT (16.8%) was reported in an epidemiological study of children and adolescents with obesity in Israel . Compared to our finding, this lower prevalence of increased ALT may be due to hereditary and ethnic differences, as well as different dietary patterns, as they are known to influence NAFLD risk [13, 34, 35].
Although ALT is commonly used as a proxy for NAFLD, liver biopsy remains the gold standard in the diagnosis of NAFLD [14, 24]. Compared with the prevalence of markedly increased ALT of 10.6% in our study, an autopsy-based study in the US reported a higher prevalence of NAFLD based on liver biopsy, which was 38% in children and adolescents with obesity . However, since the sample in the US study was deceased individuals whose causes of death were mostly due to accidents, homicide, and suicide, the finding in the US study might not represent general pediatric population with obesity. Although ALT is an important indicator for childhood NAFLD, it is not enough as a single marker. Thus, a combination of clinical, biochemical, biomarkers and non-invasive imaging tests may replace liver biopsy in the future. On the other hand, liver function tests and non-invasive measurements, such as transient elastography together with controlled attenuation parameter, may improve the accuracy of non-invasive diagnosis of those children with obesity and NAFLD-fibrosis who should undergo a liver biopsy. The indication of liver biopsy is not only to diagnose NAFLD per se, but to rule out steatohepatitis which is more likely to progress to advanced fibrosis and cirrhosis .
Although several studies have investigated the association of elevated ALT with age and sex among children and adolescents with obesity [15, 16, 33], previous studies did not take sex-age interaction into account in the analyses. The present study confirms that both sex and age are independently associated with elevated ALT. In addition, the present study provides evidence of sex difference in the associations of age with both mildly and markedly increased ALT. The odds of markedly increased ALT in adolescent males with obesity was increased dramatically over the years, while the corresponding pattern in adolescent females with obesity was minuscule. Considering increased ALT as a marker for NAFLD, the sex-age interaction in NAFLD has been previously shown in adult population [17, 18, 36]; NAFLD is more common in males during young and middle adulthood, yet after the ages of 50 the NAFLD prevalence is higher in females. Although the mechanism of the age-sex interaction in NAFLD is still uncertain, a possible explanation is that estrogen may protect the liver from injuries and steatosis [17, 36]. Moreover, mice models showed that, in high-fat environment, the innate immune cells from males are activated and thereby promote liver inflammation, while macrophage in females produce higher anti-inflammatory lipid mediators. The different inflammatory responses between male and female mice appeared to be due to sex hormone differences as the anti-inflammatory response in female mice was lost with ovariectomy . Furthermore, female is suggested to be more responsive to browning of white adipose tissue which would increase free fatty acid metabolism and thus may reduce the risk for hepatotoxicity . Another possible explanation for the sex-age interaction in NAFLD among children and adolescent with obesity is that insulin resistance, which play important role in NAFLD pathogenesis, is more prevalent in male adolescents with obesity compared to female adolescents with obesity . Understanding the sex-age interaction in pediatric NAFLD would support future pediatric NAFLD screening guidelines based on age and sex.
In accordance with other studies [15, 16], the present study found a dose-response association between the degree of obesity and increased ALT among children and adolescents with obesity. Moreover, we have been able to show that the dose-response association is stronger in markedly increased ALT compared to mildly increased ALT. Compared to individuals with class I obesity, those with class III obesity had 2.2-fold higher odds of having mildly increased ALT and 3.7-fold higher odds of having markedly increased ALT. There are some probable mechanisms of the positive association between the degree of obesity and increased ALT. Firstly, a higher degree of obesity contributes to higher ectopic fat accumulation in the liver leading to liver injury . Secondly, a higher degree of obesity is associated with more elevated circulating inflammatory cytokines . Furthermore, the degree of obesity is correlated with insulin resistance , and insulin resistance is linked with the inflammatory response .
In line with other studies in children and adolescents with obesity [20, 21], the present study demonstrated the association of markedly increased ALT with impaired fasting glucose, independent of the degree of obesity. This confirms that insulin resistance plays an important role in the pathogenesis of pediatric NAFLD, as explained by the “multiple-hit model”; considered as the first hit, insulin resistance and obesity are the main factors inducing the first lipid accumulation in the liver . Furthermore, systemic and hepatic insulin resistance also contributed to oxidative damage, leading to hepatocellular death and progression of liver steatosis to non-alcoholic steatohepatitis, fibrosis and cirrhosis .
While pediatric NAFLD has been positively associated with atherogenic lipid components in the general population [44,45,46,47], study on this association in children and adolescents with obesity demonstrated diverging results [15, 21, 23, 48]. A hospital-based study indicated association between NAFLD and dyslipidemia , while a large population-based study did not detect such association . Other hospital-based studies found association of NAFLD with triglycerides [21, 23] and total cholesterol , but not with other lipid components. These diverging results may be due to various cut-offs values for ALT and dyslipidemia components used in those studies. The present study has been able to divide ALT and each lipid components into varying degrees. Our results indicated stepwise patterns of the association between each atherogenic lipid component with increased ALT - the higher the total cholesterol, LDL, and triglyceride levels, the higher the odds of having mildly increased ALT. When the outcome was markedly increased ALT, its association with those lipid components were stronger. Our results are consistent with recent findings from a cohort of children and adolescents with obesity in Israel . However, the associations in the Israeli study were not adjusted, while our results were adjusted for the degree of obesity, age, and sex.
Compared with other lipid components, the present study showed that triglyceride levels had the strongest association with both mildly increased ALT and markedly increased ALT. While the present study used ALT as a surrogate marker for NAFLD, other studies using ultrasound [21, 23] and liver biopsy  to diagnose NAFLD also indicated that high level of triglycerides was associated with pediatric NAFLD in obese population. Further, a study in a cohort of pediatric patients with biopsy-proven NAFLD suggested that a high level of triglycerides was a risk factor for developing non-alcoholic steatohepatitis .
Limitations and strengths of the study
The gold standard of NAFLD diagnosis is liver biopsy. Yet, it is not possible to perform liver biopsy in a large epidemiological study due to its invasiveness. We have used ALT as a marker of suspected NAFLD, and hence few considerations should be considered in interpreting the results. Firstly, other conditions than liver disease, e.g., viral infections, muscle disease, and drug-induced liver injury , may affect ALT levels in children and adolescents. Especially in children younger than 5 years of age, increased transaminases are likely to have other causes . In addition, among children aged 3–10 years, other liver diseases (e.g., Wilson’s disease, celiac disease, viral hepatitis) are usually more probable than NAFLD . Secondly, although ALT have been known to have high sensitivity in identifying NAFLD in children with overweight and obesity , 17–22% cases of pediatric liver steatosis had normal ALT level [28, 50]. Moreover, degree of ALT elevation does not correlate with steatosis severity . Individuals in the advanced stage of NAFLD, i.e., fibrosis and cirrhosis, may have normal values of ALT. Misclassification bias therefore might occur in the present study. Thirdly, as ALT was measured in a single time point in the present study, we might miss the within-individual biological variation. Yet, the National Health and Nutrition Examination Survey (NHANES) involving adolescents with obesity in US found that diurnal variation did not affect the prevalence of elevated ALT . Another study found that children and adolescents with ALT > 2 times URL tend to have persistently elevated ALT . In the present study, we did not use AST as concomitant marker to ALT, as it has not been evaluated in pediatric obesity-related NAFLD.
We chose the first recorded ALT obtained in obesity treatment. Nevertheless, obesity duration before the initiation of treatment remains unknown since the BORIS register only provides data during treatment and not historical measures. In addition, we had no information on ethnicity, whereas some studies indicated association of pediatric NAFLD with ethnicity [13, 34]. Hence, the generalizability of the results in the present study to other populations may be limited. Nevertheless, approximately one-third of individuals in the BORIS register have a non-Nordic background .
A primary strength of the study is the data taken from a national clinical register. This strengthens the external validity of the study. Moreover, the large sample size made it possible to categorize the individuals into varying degrees of increased ALT, degree of obesity severity, and dyslipidemia to show the patterns of association between increased ALT and degree of obesity as well as metabolic parameters.