Journal of Pediatric Gastroenterology & Nutrition:
Pediatric Nonalcoholic Fatty Liver Disease: A Critical Appraisal of Current Data and Implications for Future Research
Patton, Heather M.*; Sirlin, Claude†; Behling, Cynthia‡; Middleton, Michael†; Schwimmer, Jeffrey B.§; Lavine, Joel E.§
*Division of Gastroenterology, Department of Medicine, and †Department of Radiology, University of California, San Diego, CA; ‡Pacific Rim Pathology Group, Sharp Memorial Hospital, San Diego; §Division of Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, University of California, San Diego
Received June 15, 2006; accepted July 19, 2006.
Address correspondence and reprint requests to Joel E. Lavine, MD, PhD, Division of Gastroenterology, Hepatology, and Nutrition, Department of Pediatrics, University of California, San Diego, 200 West Arbor Drive, San Diego, CA 92103-8450. (e-mail: firstname.lastname@example.org).
Supported by grants T32DK07202, M01RR00827, R21DK71486, R01DK075128, and U01DK61734.
Although population prevalence is very difficult to establish, nonalcoholic fatty liver disease (NAFLD) is probably the most common cause of liver disease in the preadolescent and adolescent age groups. There seems to be an increase in the prevalence of NAFLD, likely related to the dramatic rise in the incidence of obesity during the past 3 decades. Despite an increase in public awareness, overweight/obesity and related conditions, such as NAFLD, remain underdiagnosed by health care providers. Accurate diagnosis and staging of nonalcoholic steatohepatitis (NASH) requires liver biopsy. The development of noninvasive surrogate markers and the advancements in imaging technology will aid in the screening of large populations at risk for NAFLD. Two distinct histological patterns of NASH have been identified in the pediatric population, and discrete clinical and demographic features are observed in children with these 2 patterns. The propensity for NASH to develop in obese, insulin-resistant pubertal boys of Hispanic ethnicity or a non-Hispanic white race may provide clues to the pathogenesis of NAFLD in children. The natural history of pediatric NASH has yet to be defined, but most biopsies in this age group demonstrate some degree of fibrosis. In addition, cirrhosis can be observed in children as young as 10 years. While the optimal treatment of pediatric NAFLD has yet to be determined, lifestyle modification through diet and exercise should be attempted in children diagnosed with NAFLD. A large, multicenter trial of vitamin E and metformin is underway as part of the NASH clinical research network.
The term nonalcoholic steatohepatitis (NASH) was introduced by Ludwig et al. in 1980 to describe a pattern of liver injury in adults resembling alcoholic hepatitis that occurred in the absence of significant alcohol consumption (1). NASH was first described in children 3 years later (2). As in adults, nonalcoholic fatty liver disease (NAFLD) is probably the most common cause of liver disease in the preadolescent and adolescent age groups. There seems to have been an increase in the prevalence of fatty liver disease along with the dramatic rise in obesity observed in the pediatric population during the past 3 decades, although this may partially represent increased recognition of this condition. Pediatric NASH is a global problem, with case series published from North America, Europe, Australia and Asia (3-12). The natural history of pediatric NAFLD remains largely unknown.
In reviewing pediatric NAFLD, it is essential to recognize that there are no data to support the extrapolation of adult studies to the pediatric population in this field. There are emerging data that significant differences exist between adult and pediatric NAFLD, thus extrapolation of adult data may lead to erroneous conclusions. The objective of this review is to summarize the literature regarding pediatric NAFLD while highlighting areas of need for further research effort.
The term NAFLD encompasses a spectrum of histological findings, ranging from bland steatosis (simple steatosis) to steatosis accompanied by inflammation and other evidence of cellular injury (NASH). Various degrees of fibrosis may be present in NASH, including cirrhosis. Significant differences exist in the particular histological patterns of injury observed in adults and children with NASH, and these are outlined in this article.
POPULATION TRENDS IN PEDIATRIC OBESITY
There has been a significant increase in both the prevalence and severity of obesity in the US population during the past 3 decades. Data from the National Health and Nutrition Examination Survey (NHANES) suggest a tripling of the prevalence of obesity among adolescents from 5% in 1960 to 15% in 2000, and this increase in prevalence has been particularly striking among non-Hispanic black and Mexican American adolescents (13). Data from NHANES 2003-2004 show that this trend continues, with 17.1% of US children and adolescents obese on this most recent survey (14). In addition, the average amount by which the obese population exceeds the obesity threshold has increased 247% during this same period (15).
These trends in obesity extend beyond the United States. Overweight and obesity among school-age children is a global problem (16). In conjunction with the alarming rise in obesity, population data indicate that 7% of US adolescents exhibit impaired glucose tolerance (17). The increased prevalence of obesity has also been implicated as a cause for increase in blood pressure among children and adolescents (18). Thus, the rise in the prevalence and severity of obesity is accompanied by an increase in obesity-related comorbidities, either during childhood or in adulthood because of early-onset, longer-duration obesity. Nonalcoholic fatty liver disease is an example of such obesity-associated conditions that may result in dramatic increases in liver-related morbidity and mortality as a downstream effect of increased prevalence of childhood obesity.
Despite alarming trends in overweight and obesity among children and adolescents, there are data suggesting low rates of diagnosis and management of overweight, obesity and related comorbidities by healthcare providers. Centers for Disease Control and Prevention analysis of data from NHANES 1999-2000 revealed that overall, only 36.7% of obese children and adolescents had been told by their physician or healthcare provider that they were obese (19). In another study, the frequency with which general pediatricians, pediatric endocrinologists and pediatric gastroenterologists at 2 academic centers diagnosed and managed obesity and obesity-related comorbidities was evaluated (20). Overall, children with a body mass index (BMI) percentile of 95 or greater were given a diagnosis of obesity at 48% of visits. Although pediatric gastroenterologists were more likely to screen for NAFLD in obese children compared with general pediatricians (prevalence, 23% vs 2%), screening only occurred in a small fraction of visits, suggesting that NAFLD is largely undiagnosed in practice.
PREVALENCE OF NAFLD/NASH
There are 2 inherent challenges in determining the population prevalence of NAFLD. The first challenge is in making a diagnosis of NAFLD. Diagnosis requires liver biopsy, which is not feasible in a population-based study. Therefore, most studies use serum aminotransferase elevations as a surrogate marker for fatty liver disease, ideally in conjunction with negative markers for other types of liver disease. The sensitivity and specificity of aminotransferases in the diagnosis of pediatric NAFLD is not established, and no other surrogate serum markers for NAFLD exist. Alternatively, radiographic modalities can be used, although the diagnostic accuracy of this approach has its limitations and this modality does not allow for distinction between simple steatosis and steatohepatitis. The second challenge in determining population prevalence is in selecting a representative study population with minimal bias.
There are few population-based prevalence studies of pediatric NAFLD. In the United States, data from NHANES III (N = 2450 children; age range, 12-18 years) found an elevated alanine aminotransferase (ALT) in 75 (3%) of adolescents (21). A similar prevalence of abnormal ALT (3.2%) was reported from the 1998 Korean National Health and Nutrition Examination Survey in which 1543 subjects ages 10 to 19 years were evaluated (22). A study of 810 northern Japanese children (age range, 4-12 years) using ultrasound found an overall prevalence of echogenic liver of 2.6% (5). Thus, although there are few population-based data available and there are limitations in the population-based approach as previously noted, the available data suggest an overall prevalence of at least 3% for suspected fatty liver disease among children and adolescents in the United States and Asia.
There are 2 population-based samples that have evaluated the overweight and obese subsets for prevalence of abnormal ALT. Data from NHANES III demonstrate that 6% of overweight adolescents and 10% of obese adolescents had elevated ALT (21). The Child and Adolescent Trial for Cardiovascular Health (CATCH) is a school-based trial that recruited third graders from public elementary schools in California, Louisiana, Minnesota and Texas (N = 2575) and followed them through the 12th grade. The prevalence of suspected fatty liver among obese adolescents (n = 127) from this cohort as determined by unexplained elevated ALT was 23% (23).
Most prevalence studies have been conducted in cohorts of children selected for overweight or obesity, many of whom were referred for medical evaluation of obesity. There are 2 such Japanese series that have evaluated aminotransferase levels. The first, published in 1984, reported abnormal aminotransferase levels in 12% (ALT level, ≥31 IU/L) (3); the second, published in 1997, reported a prevalence of 24% (ALT level, ≥35 IU/L) (7). Whether this difference is a reflection of differences in the populations studied, the sampling error, the values chosen for reference range of ALT or a temporal trend in the population prevalence of abnormal serum aminotransferases cannot be determined. In a more recent study of 84 obese Chinese children, fatty liver was suggested, as revealed by ultrasonography, in 77%, and 24% had both echogenic liver, as revealed by ultrasonography, and elevated ALT (24). There are 2 Italian studies using ultrasound to assess the prevalence of suspected fatty liver among obese children. The first (N = 72) found echogenic liver in 53%, 25% of whom also had elevated serum aminotransferases (6). The second study (N = 375) reported a prevalence of echogenic liver in 42% among prepubertal and pubertal children (8). There has only been 1 study to date assessing the prevalence of abnormal aminotransferase levels among children with type 2 diabetes (N = 48) (25). The prevalence of elevated ALT in this cohort was 48%.
Although liver biopsy cannot be used as a screening tool in population studies, autopsy series are another means of estimating prevalence based on a histological diagnosis within select populations. Wanless and Lentz (26) reviewed adult autopsy records from Toronto Western Hospital and found the overall prevalence of steatohepatitis to be 6.27%. Although this study was histology based, it shares the limitation of other prevalence studies in having been performed in a biased population (in this case, a hospital-based population selected for BMI). Given the significant challenges in determining the population prevalence of NASH, the most accurate approach taken regarding this problem is a study recently performed in San Diego County. Records from the county medical examiner's office were reviewed, along with liver histology from more than 800 children who died of unnatural death, to determine the prevalence of fatty liver and clinical correlates of histology findings. The results of this study confirm that fatty liver is the most common liver abnormality in children ages 2 to 19 years. The variables that were significantly associated with fatty liver in this study were increased BMI, older age, male sex, Hispanic ethnicity, and Asian race (27).
DIAGNOSIS OF NAFLD/NASH
In clinical practice, the diagnosis of NAFLD is usually suspected upon finding elevated aminotransferases and/or evidence of fatty liver on radiographic studies in the proper clinical setting. This requires exclusion of other causes of liver disease. Several studies have reported a fairly narrow mean age of presentation with NASH, ranging from 11.6 years to 13.5 years (4,9-11), although the diagnosis may be made in children as young as 2 years. Children may complain of abdominal pain (prevalence, 42%-59%), although they are often asymptomatic (9,11). Although hepatomegaly can often be detected on examination, this physical finding may be missed in clinical practice (28). Acanthosis nigricans, a velvety brown to black pigmentation found in skin folds and axillae and associated with hyperinsulinemia, has been reported in as many as 50% of children with NASH (10,11,29), although our clinical experience suggests that careful examination reveals some degree of this pigmentation in most children with NASH. A family history of NAFLD may also be relevant because familial clustering is often found (30).
Diagnosis of NAFLD requires exclusion of other forms of chronic liver disease, including hepatitis B and C, Wilson disease, α1-anti-trypsin deficiency, autoimmune hepatitis, drug-induced liver injury (valproate, methotrexate, tetracycline, amiodarone, prednisone) and total parenteral nutrition. Although an elevated ALT and/or an enlarged, echogenic liver, as revealed by ultrasonography, in the setting of overweight or obesity and/or evidence of insulin resistance are highly suggestive of NAFLD, histological evaluation remains the only means of accurately assessing the degree of steatosis, necroinflammatory lesions and fibrosis found in NASH and in distinguishing NASH from simple steatosis. Furthermore, alternative or additional diagnoses may be made on liver biopsy; thus, this is the only definitive means to evaluate children with suspected NAFLD.
SCREENING FOR NAFLD/NASH IN THE PEDIATRIC POPULATION
Although clinical series suggest that most patients with NAFLD have elevated transaminases, there are reports of obese children with radiographic evidence of fatty liver who have normal aminotransferases (31,32). There are also children who have normal aminotransferases in the setting of biopsy-proven NAFLD (9,10,33). Whether the reference range of serum aminotransferase levels is appropriate has also been called into question because reference ranges are determined by population sampling that includes many individuals with undiagnosed liver diseases such as NAFLD (34). Thus, the sensitivity, specificity and the predictive values of serum aminotransferases and various imaging techniques remain undetermined. Cost analysis is not possible without information regarding performance characteristics of various screening modalities. Therefore, data to determine the optimal screening strategy for NAFLD/NASH in various populations are unavailable. Development of noninvasive surrogate markers for NAFLD/NASH with improved sensitivity and specificity compared with serum aminotransferases will make screening at-risk populations feasible and more reliable.
HISTOPATHOLOGY OF PEDIATRIC NAFLD/NASH
In adults, the histological features of NASH are well described (35). A typical adult pattern of NASH includes macrovesicular steatosis, lobular inflammation and ballooning degeneration, often with poorly formed Mallory hyaline and, if present, a perisinusoidal deposition of collagen with a zone 3 (perivenular) distribution. Several case series have suggested that distinct histological features may be observed in the pediatric population with NASH (4,9,11). Recent comprehensive evaluation of biopsy-proven NAFLD in children ages 2 to 18 years was undertaken to evaluate specific and unique patterns of injury observed in the pediatric population and to assess clinical correlates of these patterns (33). In contrast with typical findings in adult NASH, portal inflammation was present in most (70%) biopsy specimens, and portal fibrosis was present in 60% of biopsies. A distinct injury pattern consistent with the diagnosis of steatohepatitis was defined from this cohort. In contrast with the pattern typically described in adults (steatosis with ballooning degeneration and/or perisinusoidal fibrosis in the absence of portal features), the second pattern was composed of steatosis with portal inflammation and/or fibrosis, without perisinusoidal fibrosis or evidence of ballooning degeneration (Fig. 1). This pattern, termed type 2 NASH, was present in 51% of the cohort, whereas typical adult (type 1) NASH was present in 17%. There are distinct clinical and demographic differences between children with type 1 and type 2 histology (Fig. 2). This suggests that there may be important pathophysiological differences between adult- and pediatric-type NASH. Children with type 2 NASH were younger and had greater severity of obesity than children with type 1 NASH. Boys were significantly more likely to have type 2 NASH than do girls. Type 2 NASH was predominant among children of Asian or Native American race and those of Hispanic ethnicity. These data demonstrate that it is imperative for research efforts to address the pathogenesis, natural history and treatment of pediatric NASH as an entity distinct from adult NASH, and to distinguish among its subtypes.
IMAGING EVALUATION OF PEDIATRIC NAFLD/NASH
Although liver biopsy is considered the diagnostic criterion standard for NAFLD/NASH, it does have significant limitations, including cost and risk. Although the severity of steatosis on biopsy was shown to be reasonably reproducible, the variability in staging additional features of NASH has been shown in adult subjects (36). Thus, there is a need to develop noninvasive imaging methods for screening, diagnosis and longitudinal assessment of patients. Such methods, ideally, would be safe and inexpensive, and would evaluate the entire liver for fat, fibrosis and inflammation. The inexact but primary imaging modalities for the assessment of pediatric NAFLD are ultrasound and magnetic resonance (MR) imaging (31,37-43). Computed tomography may have a role in adults (44,45) but is not appropriate as a longitudinal diagnostic tool in children because of ionizing radiation.
The basis of sonographic evaluation of fatty liver is that fat within the liver simultaneously scatters and attenuates the ultrasound beam. The scattering causes the liver to appear hyperechoic (or bright), and the attenuation causes progressively greater signal loss with depth from the skin surface. Thus, the presence of fat in the liver can be inferred if the liver is both hyperechoic and associated with depth-dependent signal reduction (46,47). In clinical studies in adults, ultrasound has been shown to have 60% to 94% sensitivity and 73% to 93% specificity for the diagnosis of liver fat (40-43,48,49). The sensitivity falls when evaluating patients with less than 30% steatosis on biopsy (43% in the study by Hepburn et al.) (45,48,49). Although the accuracy of ultrasound for liver fat detection in children has not been established, the underlying physical principles are the same; thus, it is anticipated that ultrasound will have similar diagnostic performance in the pediatric age group.
Ultrasound has several important limitations. Ultrasound is operator and machine dependent, and the results are not necessarily reproducible. The assessment of hyperechogenicity and signal attenuation is inexact and influenced by confounding variables, including body habitus (50). For example, in overweight patients, fat outside a healthy liver may attenuate the ultrasound beam within the liver and lead to a false-positive diagnosis of fat deposition. Moreover, coexisting liver disease, such as fibrosis and inflammation, affects liver echogenicity (41,43,46,48-51). Thus, ultrasound is not as accurate as MR on grading the severity of liver fat (37,38,52,53) and is unsuitable for monitoring disease progression.
The most commonly used MR technique in assessment of liver fat is phase-shift imaging (39,52). In this method, MR images of the liver are acquired at time points in which protons in fat and protons in water are in phase (signals from fat and water add up) and at time points in which they are out of phase (signals from fat and water cancel). By comparing the signal intensity of liver tissue between in-phase and out-of-phase images, the presence of liver fat can be ascertained and the quantity estimated (Fig. 3) (54,55). This method is rapid (data can be acquired in a single breath hold), reproducible, operator independent and widely available on routine clinical scanners (31,56-58). The major limitation is that this method incorrectly assumes that the signals from fat and water are directly proportional to the amounts of fat and water (59). Magnetic resonance imaging methods that can calculate more accurate fat fractions are in development (59).
Magnetic resonance spectroscopy (MRS) can measure the quantity of water and fat in the liver more precisely than do MR imaging methods. The basis of MRS measurement of fat fraction is that protons in water and fat resonate at slightly different frequencies. At the magnetic field strength of most clinical MR scanners, water protons resonate at a frequency about 220 Hz higher than do fat protons. This results in a typical fat-water spectrum (Fig. 3) with 1 main peak from water protons, separated from a series of peaks from fat protons. The fat-water signal ratio is then just the ratio of the area under the fat peaks to the area under the water peak. To obtain the fat-water weight ratio, correction is made for the different decay rates (the so-called T1 and T2 relaxation rates) of signal from fat and water protons during the time the spectrum is being acquired. Liver MRS has been shown to be reproducible in a population of 2349 adults as part of the Dallas Heath Study, and the resulting fat-water ratios correlated well with pathologic findings (60). Magnetic resonance spectroscopy offers a potentially more precise assessment of the fat-water ratio than do the imaging methods, allowing better quantitative estimates of fat fraction, especially in livers with less than 10% fat. However, MRS demonstrates some limitations in that it evaluates only selected sections of liver. Thus, MRS and MR imaging techniques may be complementary regarding quantitative accuracy and sampling homogeneity, respectively.
Developing technology attempts to visualize liver fibrosis by MR imaging with contrast agents. In small clinical studies of patients with various causes of liver disease, 2 different MR contrast agents have been evaluated for detection of liver fibrosis (61). Low molecular weight chelates of gadolinium accumulate preferentially within areas of liver fibrosis and cause signal enhancement (62). Superparamagnetic iron oxides preferentially accumulate within Kupffer cells in liver parenchyma and cause signal loss (63). Individually, each agent is of limited efficacy for depiction of liver fibrosis; however, in combination, the 2 agents are complementary. Liver fibrosis appears as a meshwork of high-signal reticulations superimposed on low-signal parenchyma (64). In preliminary clinical studies, the reticulations have been assessed qualitatively and quantitatively, and high correlation with histological fibrosis stage was achieved in a subset of 46 adults with NAFLD/NASH (65). Validation of this technology will be required in children with NASH.
DISEASE ASSOCIATIONS: RISK FACTORS AND INDICATORS OF PATHOGENESIS
A number of variables have been associated with fatty liver disease in the pediatric population, and these offer potential clues to the pathogenesis of NASH. Many of these are similar to risk factors that have been identified in the adult population, including obesity, visceral adiposity, insulin resistance, race/ethnicity and the presence of other features of the metabolic syndrome. Other variables, such as sex distribution and the progression of pubertal development, are unique to pediatric NASH and thus may provide insight in understanding the underlying pathogenesis of NASH in this age group.
The most widely accepted paradigm of the pathogenesis of NASH is that of the "2-hit" theory in which NASH results from fatty infiltration of the liver due to obesity and insulin resistance, followed by inflammatory insults, potentially due to oxidative stress (66). Overweight and obesity are consistently identified as significant risk factors for NAFLD/NASH in studies from North America, Europe and Asia (4,5,8-12,24,67). Data from NHANES III (N = 2450; age range, 12-18 years) found that 6% of overweight adolescents had elevated ALT (OR, 3.5; 95% CI, 3.4-12.8) and 10% of obese adolescents had elevated ALT (OR, 6.7; 95% CI, 3.5-12.8), suggesting a dose-response effect (21). Using data from obese adolescents enrolled in the CATCH trial, a multivariate model significantly predicted the serum ALT level using the combination of sex, race/ethnicity and BMI, and accounted for 36% of the individual variance (23). Only 1 study has included data on the duration of obesity. A series of 11 obese Japanese children with 2- to 7-year obesity duration (mean, 5 years) found simple steatosis in 5 children, steatosis with inflammation in 3 and fibrosis in 3; none of the children had cirrhosis (68). The significance of the age of onset and/or the duration of obesity in children as a factor in development and progression of NASH is not established.
As in adults, fat distribution may be as important or more important than total fat mass in determining the susceptibility to NAFLD, likely due to the association of visceral fat with insulin resistance (5,8,24,69,70). In a recent study, MR imaging was used to estimate adipose tissue distribution. Hepatic fat fraction on MR imaging had a weak positive correlation with visceral adipose tissue (r = 0.37; P < 0.05), but not with BMI or subcutaneous adipose tissue (69). Subcutaneous fat thickness measured with ultrasound, waist-hip ratio and MR imaging have been used to assess visceral adiposity in children. However, in children and adolescents, the correlation between anthropometric measurements, such as waist-hip ratio and waist circumference, and intraabdominal adipose tissue, as measured by imaging techniques, is not strong (71). Future investigations of fat distribution in pediatric NASH may best be conducted using imaging techniques.
As in adult NAFLD/NASH, insulin resistance and hyperinsulinemia are thought to be critical factors in the pathogenesis of pediatric fatty liver disease. A Japanese study of obese children identified hyperinsulinemia as the variable most strongly associated with an elevated ALT (7). In a retrospective evaluation of children with biopsy-proven NAFLD from San Diego, fasting hyperinsulinemia was present in 75% of subjects. Ninety-five percent of subjects met the criteria for insulin resistance by homeostasis model assessment of insulin resistance or by quantitative insulin sensitivity check; insulin resistance was predictive of steatosis, inflammation and fibrosis (11). A study of obese Chinese children confirmed a relationship between insulin resistance, also using homeostasis model assessment of insulin resistance and quantitative insulin sensitivity check, and suspected fatty liver as revealed by ultrasonography (24).
Hypertriglyceridemia/Other Features of the Metabolic Syndrome
Nonalcoholic steatohepatitis is considered the hepatic manifestation of the metabolic syndrome in adults. Although criteria for the metabolic syndrome in children and adolescents have not been formally defined, components of the metabolic syndrome in adults (obesity, hypertension, insulin resistance, hypertriglyceridemia and low level of high-density lipoprotein [HDL] cholesterol) have been assessed in children (72). Elevated triglycerides have been found to be associated with hepatic steatosis in several series of children (9,12,24,73), and 1 study reported significantly lower HDL cholesterol level in obese adolescents with suspected fatty liver (intrahepatic fat content, >5% by MRS) compared with obese controls (70). The Korean National Health and Nutrition Examination Survey found abnormal ALT levels in 3.2% of 1543 children ages 10 to 19 years, and participants with 3 or more risk factors for the metabolic syndrome had an odds ratio of 6.2 (95% CI, 2.3-16.8; P < 0.001) for an elevated ALT level (22).
CHILDHOOD OBSTRUCTIVE SLEEP APNEA SYNDROME
There is a growing body of literature from human and animal studies implicating sleep apnea in the pathogenesis of impaired glucose metabolism (74). In adults, the severity of sleep apnea has been associated with insulin resistance, independent of BMI and waist circumference (75). One study in obese children likewise found that the severity of obstructive sleep apnea (OSA) correlated with fasting insulin levels, independent of BMI (76). Leptin-deficient (ob/ob) mice (an animal model of obesity and insulin resistance) exposed to intermittent hypoxia develop increased insulin levels and worsened glucose tolerance that is eliminated by the administration of leptin (77). Studies in ob/ob mice have also suggested that OSA may alter hepatic lipid homeostasis. Long-term exposure to hypoxia-reoxygenation, modeling the oxygenation patterns of severe OSA, resulted in increased fatty infiltration of the liver; this may occur because of increased expression of lipogenesis genes (78). Because of its association with obesity, insulin resistance and impaired lipid homeostasis, OSA can be hypothesized to play a role in the pathogenesis of hepatic steatosis. OSA may also contribute to the second hit of NASH by generation of reactive oxygen species, activation of NF-κB and release of inflammatory cytokines such as interleukin (IL)-6 and tumor necrosis factor-α (74,79,80).
Evaluation of the relationship between OSA and NAFLD has been minimal, and there are no investigations of the potential role of OSA in children with NAFLD. In a study of 60 adult Chinese subjects with NASH, the prevalence of OSA was reported to be 18% (81). An evaluation of adults with suspected OSA found BMI and severe OSA independently associated with elevated serum aminotransferases, and most of the patients (13/18) who underwent liver biopsy in this study had steatosis (82). Another study in obese adults with OSA reported that treatment with nasal continuous positive airway pressure was associated with a decrease in serum aminotransferase levels (83). Although controversy exists regarding the criteria to be used to diagnose sleep-disordered breathing in childhood (84), this is an area that should be investigated because of its potential for furthering the understanding of the pathogenesis of NASH and providing a novel approach to the treatment of fatty liver disease.
Data from our center collected from obese adolescents enrolled in the CATCH trial found that the highest rate of elevated ALT occurred in Hispanic adolescents (36%), followed by non-Hispanic whites (22%) and blacks (14%; P < 0.01) (23). A retrospective review of children with biopsy-proven NAFLD evaluated at the Children's Hospital in San Diego found a significant predominance of Hispanic (53%) and non-Hispanic white (25%) children in comparison with the reference population of children ages 5 to 19 years in San Diego (32.8% and 49%, respectively) (11). In contrast, NAFLD seemed underrepresented among non-Hispanic black children (5% of study population compared with 7.4% in San Diego). Another recent study found abnormal ALT level in obese children to be 4 times more common among non-Hispanic white (20.6%) versus non-Hispanic black children (5.4%) (29). Thus, race and ethnicity seem significant determinants of susceptibility to childhood NAFLD/NASH. Furthermore, Hispanic ethnicity has been identified as a risk factor for more advanced fibrosis (33).
The basis for the racial/ethnic disparity in NASH prevalence is not determined but may be related to differences in body composition, insulin sensitivity, adipocytokine profile or other unidentified genetic and environmental factors. From NHANES III to NHANES 1999-2000, a marked difference in the prevalence of overweight according to race/ethnicity among adolescent boys has emerged, from 10.7%, 11.6% and 14.1%, to 20.7%, 12.8% and 27.5% among non-Hispanic black, non-Hispanic white and Mexican American adolescents, respectively (13). The prevalence of impaired fasting glucose in NHANES III was significantly higher among Mexican American adolescents compared with non-Hispanic black adolescents (13% vs 4.2%), even after adjusting for age, sex and BMI (17). The lipid profiles of obese children have also been shown to vary according to race, although the significance of this in the risk for fatty liver disease is unknown. Moderately and severely obese black children have been shown to have lower triglyceride and higher HDL cholesterol levels compared with white and Hispanic children (72).
Studies comparing the body composition of European American, African American and Mexican American boys and girls, ages 3 to 18 years, by dual-energy x-ray absorptiometry found that lean body mass was higher in black than in white boys and girls. Hispanic children had higher fat mass and fat percentage than did non-Hispanic white children, even when adjusting for body size (85,86). Another study comparing black obese adolescents with white obese adolescents found that despite similar BMI, total body fat and body fat percentage, black obese adolescents had approximately 30% less visceral adiposity than did white obese adolescents (87). High visceral adiposity was associated with a significant and equal decrease in insulin sensitivity (about 38%) in both races. However, the compensatory insulin response to this varied by race with a more robust insulin response in white obese adolescents compared with black obese adolescents. These data indicate a higher diabetogenic risk in black obese adolescents, but may also indicate an increased risk for complications of hyperinsulinemia in white obese adolescents, such as fatty liver disease.
In a large cohort of Hispanic children, fasting serum adiponectin was shown to be highly heritable, with genes accounting for 93% of the variance in its circulating levels (88). This is markedly different from what was observed in a cohort of northern European adults in which genes were estimated to account for 46% of variance (89). African American youth were recently reported to have lower adiponectin levels compared with whites, even after controlling for Tanner stage, sex, abdominal and visceral adipose tissue and leptin (90). This difference in adiponectin levels in African American youth is contrary to what would be predicted on the basis of the relative frequency of NAFLD reported in African American versus white children. Thus, the impact of race on prevalence of NASH may be mediated in part through inherent differences in adiponectin levels, but this needs to be assessed in a large multiracial study population while controlling for other factors known to have an impact on adiponectin.
Most published case series have found boys more commonly diagnosed with NAFLD than girls (3-5,8-11,24,67,68). Male predominance has also been demonstrated in a national school-based study using ALT as a surrogate marker (44% of boys affected vs 7% of girls) (23) and in an autopsy study in San Diego assessing liver biopsy (10.5% of boys affected vs 7.4% of girls) (91). As previously noted, boys are also more likely to have type 2 NASH and girls to have type 1 NASH, suggesting that sex hormones may play a significant role in the predisposition to and/or expression of fatty liver disease (33). In this study, girls with type 2 NASH were several years younger (mean age, 10.5 years) than girls with type 1 NASH (mean age, 13.3 years). Although the Tanner stage was not assessed, girls with type 2 NASH were more likely prepubertal and thus have a hormonal profile more similar to boys, who predominantly had type 2 NASH. In contrast, girls with type 1 NASH were more likely pubertal and thus have higher estrogen levels.
Sex hormones are attractive candidates for mediators of the development of and/or protection from steatohepatitis, with data suggesting a permissive role for testosterone and a protective role for estrogen. Nonalcoholic fatty liver disease is twice as common in postmenopausal compared with premenopausal women, and estrogen replacement therapy decreases the risk of NAFLD in postmenopausal women (92). In women with polycystic ovarian syndrome, hyperandrogenism, as determined by hirsutism, was shown to have a strong relationship to fatty liver, independent of insulin sensitivity and BMI (93). Further evidence for the potential importance of sex hormones in NASH comes from aromatase deficiency. Men with aromatase deficiency caused by a homozygous mutation of the CYP19 gene have congenital estrogen deficiency (aromatase catalyzes formation of aromatic C18 estrogens from C19 androgens). In a case report of a man with aromatase deficiency, progressive insulin resistance with eventual type 2 diabetes, acanthosis nigricans, premature atherosclerotic disease and biopsy-proven NASH developed after treatment with supraphysiological doses of testosterone for more than 2 years. After 1-year estrogen therapy, all of these conditions, including the appearance of the liver on repeat biopsy, improved (94). Hepatic steatosis has also been observed in aromatase knockout (ArKO) male mice. Only male ArKO mice develop hepatic steatosis, and estrogen replacement results in decreased hepatic triglyceride levels and steatosis (95). Aromatase knockout mice treated with estrogen from birth do not develop fatty liver at all (96). Thus, the estrogen-testosterone ratio seems a potentially important mediator in the development of insulin resistance and hepatic steatosis. Short of aromatase deficiency, an aberration in aromatase activity or an abnormality in estrogen receptor function could result in unfavorable regulation of sex-steroid hormone-dependent genes, which have an impact on insulin resistance and hepatic steatosis.
Several studies have reported a fairly narrow mean age of presentation with NASH, ranging from 11.6 to 13.5 years, suggesting that age or, more specifically, developmental stage is a significant variable in the onset of fatty liver disease (4,9-11). The role of sex hormones and insulin resistance at puberty may account for the significance of developmental stage in onset of fatty liver. Pubertal stage has only been evaluated in 1 study from Italy in which the prevalence of suspected fatty liver, as assessed by ultrasound, was highest in Tanner stage IV (47%), intermediate in stages II to III (36%) and lowest in stage I (33%) (8). Puberty is associated with modest insulin resistance (decrease in insulin sensitivity, 25%-30%) that is compensated for by an increase in insulin secretion. This decrease in sensitivity occurs early in puberty, between Tanner stages I and II, with a nadir at Tanner stage III and recovery by stage V (97,98). A longitudinal study comparing adolescents who progressed from Tanner stage I to stage III/IV (n = 31) to those who remained at stage I (n = 29) during a mean follow-up period of 2.0 ± 0.6 years was undertaken to investigate the relative contributions of growth and pubertal development to insulin resistance (99). Insulin sensitivity decreased by 32% in children who progressed to Tanner stage III/IV compared with only 15% in those who remained at stage I. The effect of pubertal development on insulin sensitivity remained significant after controlling for age, fat-free mass and body fat content (99). In girls, BMI and growth hormone have been shown to be predictive of insulin sensitivity, whereas in boys, BMI and testosterone levels have been shown to be predictive (97). The relative contributions of pubertal stage and obesity to the development of insulin resistance need to be carefully assessed in future studies designed to evaluate the onset of NAFLD in children.
Although available data indicate that most children with NASH present during adolescence, there is also a subgroup of children who present with fatty liver disease at a younger age. Nonalcoholic fatty liver disease has been identified in children as young as 2 years (11,100), and a study of nonobese Japanese infants (age, 3-11 months), in which echogenic liver was diagnosed by ultrasound in 2.2% to 5% of subjects between 1999 and 2003, was recently published (101). Whether early-onset NAFLD is the same disease entity developing in children with the same risk factors at a younger age or whether there is something fundamentally different about this subgroup is not known. Relatively low birth weight followed by rapid early postnatal weight gain may place children at risk for later obesity, central fat deposition and insulin resistance (102). Adiponectin levels are significantly reduced in small for gestational age children (age, 8-10 years) compared with short, healthy or obese children, and this difference is particularly pronounced in children with postnatal catch-up growth (103). The role, if any, of birth weight, early weight gain and feeding practices in subsequent development of fatty liver disease has not been investigated.
The identification of factors that determine the susceptibility to fatty liver among obese, insulin-resistant children is not clear at this time. An Italian study evaluated obese children ages 7 to 14 years, 20 of whom had elevated transaminases (ALT level, ≥1.5 × upper limit of normal) and a bright liver, as revealed by ultrasonography, and compared them with 30 children with normal ALT level and ultrasound result. Markers of inflammation (CRP and ferritin) were elevated in the group with evidence of fatty liver. No statistically significant difference in inflammatory cytokines (tumor necrosis factor-α, IL-6) or markers of oxidative stress (glutathione peroxidase) was detected in this study (104). In another study comparing obese insulin-resistant children with insulin-sensitive children pair-matched for BMI, sex, lean body mass, body fat percentage and pubertal status, fasting insulin and triglycerides levels were significantly higher, whereas adiponectin level was significantly lower in the insulin-resistant children (105). An Italian study of 54 severely obese adolescents, aged 11 to 18 years, assessed whole-body energy homeostasis using MR spectroscopy to measure steatosis. Indirect calorimetry demonstrated impaired whole-body fat oxidation in obese adolescents with NAFLD compared with obese adolescents without NAFLD. Reliance on fat oxidation in the fasting state was lower in adolescents with NAFLD, whereas their ability to suppress fat oxidation after glucose administration was impaired in comparison with obese adolescents without NAFLD (70). Whether the differences observed in markers of inflammation, fat oxidation, insulin and adipocytokines are mediated by genetic and/or environmental variables is unknown, but the propensity for certain obese children to develop complications such as NASH is an important question for future research.
Adiponectin exerts effects on glucose and lipid metabolism, has anti-inflammatory properties and is thought to play a role in the pathogenesis of adult NASH (106). Adiponectin levels are lower in obese versus age-matched lean adolescents and inversely related to inflammatory factors (CRP, IL-6), fat mass, insulinemia and insulin resistance (107). Adiponectin levels are lower in obese children with evidence of fatty liver when compared with obese children without evidence for fatty liver (29,108); however, because these studies are cross-sectional in nature, they are not able to assess directionality with respect to cause and effect. In a cohort of lean children, a significant negative correlation was observed between adiponectin and pubertal stage in boys. This decline in serum adiponectin levels in boys led to a significantly lower adiponectin level in boys compared with girls at the completion of puberty, to differences similar to what is observed in adults. A strong negative correlation of adiponectin level with testosterone was observed in boys, whereas estradiol levels in girls were not associated with adiponectin (109). Thus, changes in adiponectin levels during pubertal development could account for a greater vulnerability to the development of NAFLD in adolescent boys compared with that in girls, although this will require evaluation in longitudinal studies.
In summary, during the normal course of puberty, adiponectin levels decrease, insulin sensitivity level decreases, sex hormones increase and body fat distribution changes, all of which may predispose the subject to the development of hepatic steatosis. In children who enter puberty with excess fat and/or an underlying genetic predisposition to insulin resistance, these hormonal changes that occur as a normal part of growth and development may "tip the scales" toward the onset of fatty liver disease.
NATURAL HISTORY OF NASH IN THE PEDIATRIC POPULATION
Clinical series have reported fibrosis in 53% to 100% of liver biopsies from children with NAFLD, including several reports of children with cirrhosis (4,9-11). In fact, cirrhosis secondary to NASH has been reported in children as young as 10 years (10). A recent case report described a young man dying of complications of liver failure secondary to NASH cirrhosis at the age of 34 years (110). The incidence rate of cirrhosis secondary to pediatric NASH is unknown at this time. Predictors of advanced histology include severity of obesity and insulin resistance (11). There are no published longitudinal studies of NAFLD/NASH in children. In our own clinical experience, however, we have observed children with liver biopsies demonstrating significant histological progression during the course of a few years (Fig. 4).
TREATMENT OF NASH IN THE PEDIATRIC POPULATION
No drug therapies have been developed specifically for the treatment of fatty liver disease in either children or adults. The strategies that have been used in the treatment of NASH thus far are based on the current best understanding of the pathophysiology of this disease, the so-called "2-hit theory" (66). Thus, weight loss/decreased visceral fat, improved insulin sensitivity and antioxidant therapy have been the approaches explored in therapy for both adult and pediatric NASH.
Several case series and uncontrolled trials have demonstrated the efficacy of weight loss secondary to hypocaloric diet and exercise in improving or normalizing transaminases and the appearance of the liver, as revealed by ultrasonography (6,9,67,73). There are no published trials of diet and exercise demonstrating improved liver histology with weight loss in children. The strongest evidence for this approach comes from a trial of intensive nutritional counseling with the goal of improved insulin resistance and gradual weight loss (40%-45% of daily calories from carbohydrates with emphasis on complex carbohydrates with fiber, 35%-40% of daily calories from fat with emphasis on monounsaturated and polyunsaturated fats, and 15%-20% of daily calories from protein) in adults with biopsy-proven NASH (111). Of the patients who underwent repeat liver biopsy (15/16), 9 had histological response (decrease in total NASH score by at least 2 points), 6 had stable scores and none demonstrated worsening. Patients with improved scores had significantly greater weight reduction (mean change in BMI, −2.25 kg/m2 vs 0.58 kg/m2) and decrease in waist circumference (mean reduction, 6.94 cm vs −0.52 cm) compared with patients with stable histology. Unfortunately, the measures that were taken in this study are difficult to implement in clinical practice (patients were seen by a dietitian weekly for 8 weeks, biweekly for 3 months and then monthly for 6 months); despite this, 40% of patients merely stabilized rather than made an improvement in their condition. Despite the scarcity of data in children, optimization of a healthy diet in conjunction with exercise should be attempted in all children diagnosed with NAFLD. The particular type of dietary modification that may be beneficial in treating NAFLD should be explored.
As with weight reduction, insulin-sensitizing agents may be successful in treating NASH by decreasing hepatic steatosis. Metformin has been evaluated in an open-label pilot study of 10 children with biopsy-proven NASH and elevated ALT level (112). After 6 months of therapy (dosage, 500 mg twice per day), significant improvement was observed in serum ALT and hepatic steatosis, as assessed with MR spectroscopy. No adverse effects were observed in this study, and the National Institute of Diabetes and Digestive and Kidney Diseases-sponsored NASH Clinical Research Network (CRN) is now investigating metformin as monotherapy in a randomized controlled trial. Although treatment of adults with thiazoladinediones seems promising, with large studies underway as a part of the NASH CRN, the lack of safety data on this class of drugs in children with liver disease warrants caution in trying these drugs in children currently with NASH.
Antioxidant therapy has also been studied in children with NASH as a means of addressing the second hit thought to be caused by increased oxidative stress. Interestingly, the serum levels of the antioxidants β-carotene and α-tocopherol were found significantly lower in obese compared with those in children with normal weight participating in NHANES III (113). An open-label pediatric trial of oral vitamin E in dosages ranging from 400 to 1200 units per day for 2 to 4 months resulted in the normalization of ALT in all 11 of the obese children studied (114). Studies of vitamin E in adults with NASH have also demonstrated efficacy in improving transaminases and liver histology (115,116). Vitamin E is being studied as a monotherapy in children and adults as part of the NASH CRN (117).
Ursodeoxycholic acid (UDCA) is a cytoprotective agent that has been studied as a potential therapy in both adult and pediatric NAFLD. An Italian study evaluated the efficacy of UDCA in 31 obese children (mean age, 8.7 years; range, 4-14 years) with abnormal serum aminotransferase levels. The study included 4 children with abnormal aminotransferase levels and normal ultrasound result who were not evaluated with liver biopsy to confirm a diagnosis of NAFLD. The children were assigned to treatment groups based on their anticipated success with lifestyle modification. Children were treated with UDCA (dosage, 10-12.5 mg/kg/day) with or without weight-reduction diet in comparison with diet alone or no intervention. At 6 months, the addition of UDCA to diet was no more effective than diet alone in reducing serum aminotransferases or the appearance of steatosis, as revealed by ultrasonography. No difference was observed between children treated with UDCA and those receiving no intervention; however, the children assigned to these treatment arms were those who were judged unlikely to comply with a diet and exercise program (118). Because of selection bias, potential inclusion of children with diagnoses other than NAFLD and the use of ultrasound to assess the severity of steatosis in this study, the efficacy of UDCA in pediatric NAFLD remains to be determined.
Future treatment trials in pediatric NASH should be designed as controlled, randomized studies in patients with biopsy-proven disease. The optimal endpoints for such trials should include clinically significant parameters such as liver histology. Identification of biomarkers of disease activity and accurate noninvasive imaging techniques may facilitate the assessment of therapeutic efficacy without the need for liver biopsy.
Since its initial description in the 1980s, pediatric NAFLD has become the most common form of liver disease in the preadolescent and adolescent age groups. The dramatic rise in obesity observed in recent decades has been accompanied by an increase in the prevalence of obesity-related comorbidities, including NAFLD. Age, sex and race/ethnicity are significant determinants of risk for fatty liver disease in children, and future investigations of the pathogenesis of pediatric NAFLD should take into account the role of sex hormones, insulin sensitivity and adipocytokines. Liver biopsy remains the criterion standard for the diagnosis and staging of NAFLD, and there seems to be a histological dichotomy between pediatric- and adult-type histopathology in NAFLD that deserves further study. Development of noninvasive, surrogate markers of NASH and imaging techniques will facilitate improved screening practices and will aid in assessing natural history and response to treatment. Optimal lifestyle interventions in pediatric NAFLD are yet to be defined, and large, multicenter studies of insulin-sensitizing and antioxidant therapy are under way. The goals for future research in pediatric NAFLD are summarized in Table 1.
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