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Nonalcoholic fatty liver disease (NAFLD) is currently the most common form of chronic liver disease in both children and adults and threatens to become a serious public health problem (1,2). It is estimated that one in three adults and one in 10 children or adolescents in the United States have NAFLD (3,4), which is tightly associated with obesity and insulin resistance (IR). NAFLD encompasses a spectrum of disease ranging from simple steatosis in which triglycerides (TG) accumulate in hepatocytes in the absence of evidence for liver damage or inflammation to nonalcoholic steatohepatitis (NASH), defined by the association of lipid accumulation with evidence of cellular damage, inflammation, and different degrees of fibrosis (5).

At present, the most commonly used noninvasive tests to diagnose NAFLD include measurement of serum aminotransferases and liver ultrasound, alone or in combination (6,7). Although these tools are in general useful for the diagnostic of NAFLD, they lack specificity and sensitivity to distinguish NASH from simple steatosis and determine the presence and extent of liver damage and fibrosis (6,7). Thus, currently, a liver biopsy remains the only reliable way to distinguish NASH from simple steatosis.

A growing body of evidence from epidemiologic studies in both adult and children using either liver enzymes or ultrasound as surrogate markers of NAFLD has established NAFLD as an independent predictor for development of metabolic syndrome (MS), diabetes, and cardiovascular (CV) disease (8–11). CV disease remains the leading cause of death in the adult population, and it has been shown that the atherosclerotic process begins in childhood (12). In the Bogalusa heart study in children, investigators found that the extent to which the intimal surface was covered with atherosclerotic lesions was significantly associated with elevation of concentrations of total cholesterol (TC), LDL, TG, and lower concentration of HDL. Ratios of cholesterol ester-rich lipoprotein levels (TC/HDL and LDL/HDL) are well-established predictors of CV disease (13). More recently, the TG/HDL ratio has been shown to be a strong predictor of MS and CV disease (14,15). In addition, recent studies have suggested that apolipoprotein B (ApoB)/apolipoprotein A-1 (ApoA-1) ratio may be a good predictor of CV risk and the presence of MS and IR (16).

Recently, experimental studies have suggested that the presence and degree of liver damage and inflammation may play a crucial pathogenic role in the development of these conditions (17–19). However, there is currently a paucity of data regarding the possible association between liver histopathological changes and atherogenic risk in both adults and children. Thus, the aim of this study was to assess the relationship between severity of liver injury and atherogenic lipid profile in a large, well-characterized group of children with biopsy-proven NAFLD using different lipid ratios as predictors of CV risk.

PATIENTS AND METHODS

Patients.

A total of 118 consecutive patients diagnosed with NAFLD (60 male and 58 female) seen at Bambino Gesù Children's Hospital from January 2005 to April 2008 were included in the study. The study was approved by the Ethics Committee of the Bambino Gesù Children's Hospital and Research Institute, Rome, Italy. Informed consent was obtained from each patient or responsible guardian.

Inclusion criteria were persistently elevated serum aminotransferase levels, diffusely echogenic liver on imaging studies suggestive of fatty liver, and biopsy consistent with the diagnosis of NAFLD (20–22). Exclusion criteria were hepatic virus infections (Hepatitis A–E and G, cytomegalovirus, and Epstein-Barr virus), alcohol consumption, history of parenteral nutrition, and use of drugs that are known to induce steatosis (e.g. valproate, amiodarone, or prednisone) or to affect body weight and carbohydrate metabolism. Autoimmune liver disease, metabolic liver disease, Wilson's disease, and alpha-1-antitrypsin-associated liver disease were ruled out using standard clinical, laboratory, and histologic criteria.

The BMI and BMI Z score were calculated (23,24). MS was defined as the presence of ≥3 of the following five criteria (25): abdominal obesity (defined by waist circumference ≥90th percentile for age) (26); hypertriglyceridemia as TG >95th percentile for age, sex, and race (27); low-HDL cholesterol as concentrations <5th percentile for age and sex (27); elevated blood pressure as systolic or diastolic blood pressure >95th percentile for age and sex (28); and impaired fasting glucose or known type 2 diabetes mellitus (as defined in the next section). Physical maturation was assessed by two expert physicians (an endocrinologist M.M. and a pediatrician V.N.) according to Tanner (29). Subjects were divided into Tanner stage (T) as follows: prepubertal (T1), early pubertal (T2), midpubertal (T3–4), and late pubertal (T5).

Evaluation of lipid profiles.

Serum TG (intra-assay coefficient of variation 1.5% and interassay coefficient of variation 1.8%), TC (intra-assay coefficient of variation 0.8% and interassay coefficient of variation 1.7%), and HDL cholesterol (intra-assay coefficient of variation 0.95% and interassay coefficient of variation 1.3%) were measured by routine colorimetric assays (Roche/Hitachi Modular P/D, Can 433, Milan, Italy). LDL cholesterol was calculated using the Friedewald formula (LDL = TC − TG/5 − HDL). Serum ApoA-1 and ApoB levels were measured using ADVIA-1650 Chemistry System (Bayer Corporation, Tarrytown, NY). None of our patients was on lipid-lowering medications at the time of lipid profiles measurements.

Glucose metabolism and insulin sensitivity.

A 2-h oral glucose tolerance test (OGTT) was performed with the standard 1.75 g of glucose per kg of body weight or a maximum of 75 g. Glucose tolerance status was determined according to the classification of the American Diabetes Association in which fasting plasma glucose levels up to 99 mg/dL are considered normal; impaired fasting glucose is defined by a fasting plasma glucose of 100–125 mg/dL; impaired glucose tolerance is defined by a 2-h plasma glucose of 140–199 mg/dL; and diabetes mellitus is defined by a fasting plasma glucose ≥126 mg/dL or a 2-h plasma glucose ≥200 mg/dL (30,31). Plasma glucose was measured in triplicate by the glucose oxidase technique on a Beckman glucose analyzer (Beckman, Fullerton, CA); and plasma insulin was measured by a specific radioimmunoassay (MYRIA Technogenetics, Milan, Italy).

The degrees of IR and sensitivity were determined, respectively, by the homeostatic model assessment insulin resistance using the formula: insulin resistance = (insulin × glucose)/22.5 and by the insulin sensitivity index derived from OGTT using the formula: insulin sensitivity index = [10,000/square root of (fasting glucose × fasting insulin) × (mean glucose × mean insulin during OGTT)] (32,33).

Liver histology.

The clinical indication for biopsy was either to assess the presence of NASH and degree of fibrosis or other likely independent or competing liver diseases. Liver biopsy was performed in all children, after an overnight fast, using an automatic core biopsy 18-gauge needle (Biopince, Amedic, Sweden) under general anesthesia and ultrasound guidance. A Sonoline Omnia Ultrasound machine (Siemens, Germany) with a 5-MHz probe (5.0 C 50, Siemens) was used. Two biopsy passes within different liver segments were performed for each subject. The length of liver specimen (in millimetres) was recorded. Only samples with a length ≥15 mm and including at least 10–11 complete portal tracts were considered adequate for the purpose of the study (34). Biopsies were evaluated by a single liver pathologist. Biopsies were routinely processed (i.e. formalin fixed and paraffin embedded). Sections of liver tissue, 5-μm thick, were stained with hematoxylin and eosin, Van Gieson, Periodic acid-Schiff, digested Stain, and Prussian blue stain. Immunohistochemical staining with antibodies to alpha-1-antitrypsin was used to exclude alpha-1-antitrypsin deficiency-associated liver disease. Liver biopsy features were graded according to the NAFLD activity scoring (NAS) system proposed by Kleiner et al. (35). Briefly, grade of steatosis was scored as 0 = <5%, 1 = 5–33%, 2 = >33–66%, and 3 = >66%; grade of lobular inflammation was scored as 0 = no foci, 1 = <2 foci/200×field, 2 = 2–4 foci/200×field, and 3 = >4 foci/200×field; and grade of ballooning was scored as 0 = none, 1 = few ballooning cells, and 2 = many cells/prominent ballooning. The grade of steatosis (0–3), lobular inflammation (0–3), and ballooning (0–2) were then combined to determine the NAFLD activity score (0–8) as proposed. Fibrosis was scored as 0 = none; 1 = periportal or perisinusoidal fibrosis; 2 = perisinusoidal and portal/periportal fibrosis; 3 = bridging fibrosis; and 4 = cirrhosis (35). The liver biopsy samples were then classified as definitive NASH (unequivocally fulfils previously described criteria for steatohepatitis), borderline diagnosis (some but not all histologic features of steatohepatitis), and simple steatosis (isolated fat deposition in hepatocytes).

Statistical analysis.

Descriptive statistics were computed for all factors. These included medians and percentiles for continuous factors and frequencies for categorical variables. Spearman correlation coefficients were estimated to evaluate correlations between lipid ratios (including TG/HDL, cholesterol/HDL, LDL/HDL, non-HDL cholesterol/HDL, and ApoB/A ratios) and several continuous and ordinal factors. In addition, Wilcoxon rank-sum tests were used to assess associations between lipid ratios and several categorical variables such as MS. ANOVA was performed to study the association between NAFLD and lipid ratios; the natural logarithm transformation of each lipid ratio was modeled as the outcome, and post hoc analyses were performed using Bonferroni's adjustment for multiple comparisons (36). In addition, analysis of covariance was performed to adjust for potential confounders such as age, sex, puberty development, and MS components. A p <0.05 was considered statistically significant. SAS version 9.2 software (The SAS Institute, Cary, NC) and R version 2.4.1 software (The R Foundation for Statistical Computing, Vienna, Austria) was used for all analyses.

RESULTS

Patient characteristics.

The main anthropometric, clinical, and laboratory data of our patient population are summarized in Table 1. The median age was 11 years (interquartile range, 9–13 years), there was an equal proportion of boys and girls, and two-thirds were obese. The features of the MS were commonly present with more than two-thirds having a waist circumference ≥90th percentile, 29 patients (24.6%) presented with hypertriglyceridemia, 28 (23.7%) with low-HDL cholesterol, 30 (25.4%) had glucose intolerance, and 27 patients (22.9%) suffered from hypertension. One-hundred twelve children (95%) presented with at least one feature of the MS, whereas overt MS (i.e. ≥3 features) was present in 26 children (22%) (Table 2).

Table 1 Demographic, clinical, and laboratory features in children with NAFLD
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Table 2 Metabolic syndrome and histological characteristics in children with NAFLD
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Liver histology.

The main liver biopsy features are summarized in Table 2. Steatosis was present in all cases, and it was of moderate-to-severe grade in most patients. Lobular inflammation was of mild severity in most children. Fifty-five percent of cases revealed ballooning of the hepatocytes that was in most cases of grade 1 (few balloon cells). The median NAS was 4 (interquartile range, 3–5). Seventeen patients (14.4%) were classified as simple steatosis, 58 patients (49.1%) as borderline disease, and 43 patients (36.4%) as definitive NASH. Some degree of fibrosis was present in 64% of cases including mild fibrosis (stage 1) in 58 patients (49%) and moderate fibrosis (stage 2) in 11 patients (9%), whereas 6 patients (5%) showed advanced fibrosis (stage 3). No patient had established cirrhosis. Interface hepatitis or other features suggestive of autoimmune hepatitis were not present in any of the cases.

Association of severity of liver disease with atherogenic lipid profile in children with NAFLD.

To study the potential association of severity of liver disease with atherogenic risk in children with NAFLD, we first assessed the correlation of the individual histologic features (steatosis, inflammation, ballooning of hepatocytes, and fibrosis) and the NAS with established predictors of atherogenic risk profile. The NAS and its individual components showed a significant positive correlation with TG/HDL, TC/HDL, and LDL/HDL ratios (p <0.05 for all) but not with ApoB/ApoA-1 ratio (p = 0.58; Table 3). Similarly, a positive correlation was found between these scores and stage of fibrosis (Table 3 and Fig. 1). After adjusting for potential confounders including BMI, homeostatic model assessment index, impaired glucose tolerance, and presence of MS, both NAS and stage of fibrosis remained independent predictors of an atherogenic lipid profile (Table 4).

Table 3 Correlations between clinical characteristics, liver histological features, MS, and lipid markers of CV risk
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Figure 1
figure 1

Association between lipid markers of CV risk and liver histology. Vertical axis represents the lipid ratio, and horizontal axis represents patient groups according the NAS. The box represents the interquartile range (the 25th and 75th percentiles) from the median (horizontal line) and the bars represent the 95% confidence interval. All lipid ratios were significantly increased in patients with definitive NASH compared with patients with simple steatosis or borderline diagnosis.

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Table 4 NAFLD, fibrosis, and lipid ratios: ANCOVA
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All lipid ratios, except for ApoB/ApoA-1, were found to be markedly higher in children with established NASH compared with those patients with simple steatosis or borderline disease (p <0.05 for all; Fig. 2). There was no evidence to suggest a significant difference in lipid ratios between patients with simple steatosis and those with borderline disease. Thus, these data strongly suggest that in children with NAFLD, NASH, and fibrosis are at particular risk for CV disease associated with an atherogenic lipid profile.

Figure 2
figure 2

Association between lipid ratios and the stage of fibrosis. Vertical axis is the lipid ratio, and horizontal axis is the fibrosis stage. The box represents the interquartile range (the 25th and 75th percentiles) from the median (horizontal line) and the bars represent the 95% confidence interval. Lipid ratios were significantly higher in patients with moderate-to-severe fibrosis (stage 2–3) compared with those patients with no or mild fibrosis (stage 0–1).

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DISCUSSION

The principal findings of this study relate to the link between severity of liver disease and atherogenic risk in children with NAFLD. The results show that the degree of liver injury and stage of fibrosis positively correlates with established markers of atherogenic risk. The association between liver damage and an atherogenic profile is independent of obesity, IR, and the presence of MS in children with NAFLD. The atherogenic profile is particularly prominent in patients with established NASH diagnosis as opposed to those with simple steatosis. Taken together, these results suggest a significant effect of the severity of NAFLD on CV risk in children.

With the growing prevalence of childhood obesity and overweight, pediatric NAFLD has become the most frequent chronic liver disease in children and adolescents in industrialized countries. A recent large autopsy study found that 9.6% of Americans aged 2 to 19 y have NAFLD, and this figure increased to 38% among those who were obese (4). Similar high figures have been reported among children from other countries in Europe and Asia (37,38). Currently, a liver biopsy remains the gold standard for NAFLD diagnosis (39). A liver biopsy provides key information regarding the degree of liver damage and remains the only reliable way of distinguishing NASH from simple steatosis.

There is a growing body of evidence suggesting an association between NAFLD and CV disease (8–11,40,41). Recent longitudinal long-term studies from both the United States and Europe have demonstrated that CV mortality is increased in adult patients with NAFLD (42–44). Moreover, these studies have suggested that the increased in CV mortality occurs in patients with NASH and fibrosis but not in those with simple steatosis.

Several cross-sectional studies have shown that patients with NAFLD have increased carotid artery atherosclerosis with enlarged intima-media thickness and increased prevalence of carotid plaques (45–47). Although others have demonstrated a link between NAFLD and the presence of endothelial dysfunction as assessed by brachial artery flow-mediated vasodilation (48), two recent studies in children attempted to evaluate the relationship between early atherosclerosis and NAFLD. Similar to adults, children with NAFLD were found to have increased carotid intima-media thickness compared with matched controls (49,50). A central limitation of these studies was the fact that the diagnosis of NAFLD was based on evaluation of liver enzymes or liver ultrasound; thus, the potential impact of the spectrum of NAFLD such as the presence of NASH versus simple steatosis and the relationship between the extend of liver damage and CV risk could not be assessed.

Recent experimental studies in mice have suggested that hepatic inflammation and hepatic IR may contribute to the development of dyslipidemia and increased susceptibility to atherosclerosis (17–19). We were able to show that this is also true in children with NAFLD. Our results identified a potential link between severity of liver damage and atherogenic lipid profile in children with biopsy-proven NAFLD. Indeed, we showed that the presence of NASH and fibrosis on liver biopsy in these patients correlates with lipid markers of atherogenic and CV risk. This association was independent of obesity, IR, and the presence of MS. This result is not surprising, because not all our patients are obese, suggesting the possible involvement of other risk factors associated with NAFLD, including genetic predisposition and dietary habits. Moreover, the TG/HDL ratio was the best lipid ratio to predict the presence of advanced liver disease (higher NAS and fibrosis stage). This is a novel finding and if validated in other studies, the TG/HDL ratio could help in selecting NAFLD patients with higher risk for disease progression who might benefit from liver biopsy and more aggressive follow-up. The ApoB/ApoA-1 ratio did not show the same correlation with liver histology as the other lipid ratios that may be explained by the fact that the liver is the major site for producing these lipoproteins.

The main strengths of our study are the inclusion of a large group of consecutively recruited children with liver biopsy-proven NAFLD with the full spectrum of disease along with an extensive characterization of their metabolic profile. However, our study has some limitations including the fact that patients were seen at a large referral tertiary care medical center. Although the results may be extrapolated to other similar medical centers, the results may not apply to children with NAFLD from the community. Second, most of our children were white, making it difficult to determine whether the association between NAFLD severity and atherogenic risk is different among other ethnic groups. Finally, whether chronic liver inflammation and injury is a primary mechanism promoting a proatherogenic dyslipidemia cannot be elucidated by this study and warrants further investigations in animal models. An increase in TG secretion form the liver in the form of LDL is likely responsible for the increase in serum TG concentrations commonly noted in patients with NAFLD (51).

In summary, our results reveal for the first time that the severity of liver injury in children with NAFLD is strongly associated with increased atherogenic risk. Future long-term longitudinal studies are needed to evaluate development of CV disease in children with NAFLD and to determine the most appropriate therapeutic strategies for these patients. At this point, we recommend that atherogenic risk factors should be carefully evaluated and aggressively treated in patients with NAFLD, especially when the disease progresses to its more severe forms (NASH and fibrosis) as they may be associated with higher risk for early atherosclerosis and consequent CV disease.