See “Nonalcoholic Fatty Liver Disease and Dyslipidemia: The Beleagured Hepatocyte” by Jackson and Book on page 287.
Nonalcoholic fatty liver disease (NAFLD) is the most common cause of chronic liver disease among children, affecting 10% of American youth (1,2). Nonalcoholic steatohepatitis (NASH), the progressive form of NAFLD, is well described in children and can result in cirrhosis (3). In addition, NAFLD confers an independent risk for cardiovascular disease (CVD) in adults (4–8). Children with NAFLD have an increased prevalence of risk factors for CVD including elevated low-density lipoprotein (LDL), increased total cholesterol, and decreased high-density lipoprotein (HDL), when compared with matched controls (9–11). The severity of NASH, as assessed by the NAFLD activity score (NAS), is associated with increased triglyceride (TG)/HDL, total cholesterol/HDL, and LDL/HDL ratios (12). Furthermore, children with NAFLD have greater carotid arterial intima-media thickness (CIMT), a risk factor for CVD development, as compared with obese children without NAFLD (13–15). Studies of dietary and pharmacologic interventions in children with NAFLD have shown conflicting effects on CVD risk factors (16–19). Thus, the impact of NAFLD and NASH regression on CVD risk factors in children remains uncertain.
The development of NASH is associated with dyslipidemia and alterations in hepatic lipid metabolism. The liver secretes TG-rich lipoproteins in the form of very-low-density lipoproteins (VLDL) and, less frequently, intermediate-density lipoproteins (IDL). Increased levels of VLDL and IDL have been described in NAFLD and are also associated with an increased CVD risk.
Non–high-density lipoprotein cholesterol (non-HDL-C) is a composite measure that encompasses VLDL, LDL, IDL, and lipoprotein(a) (20). Non-HDL-C is easily calculated from a commonly available lipid profile by subtracting HDL-C from the total cholesterol level (non-HDL-C = total cholesterol − HDL) and can be performed with no additional cost. We have shown that non-HDL-C is significantly higher in adults with NASH compared with in those with steatosis alone (21). Furthermore, non-HDL-C is a risk factor for CVD development, and is a superior predictor of CVD in adults when compared with the widely used LDL (22). Non-HDL-C is now considered an important secondary target of lipid-lowering therapy (23,24).
Non-HDL-C levels in childhood also predict CVD risk factors in adults. The Bogalusa Heart Study analyzed 1163 children ages 5 to 14 years during a 27-year period. Childhood non-HDL-C was a predictor of not only adult non-HDL-C but also adult dyslipidemia, hyperinsulinemia, and hyperglycemia, all of the important CVD risk factors (25). Furthermore, increased non-HDL-C levels correlate with the extent of coronary and aortic atherosclerotic disease in youth found on autopsy (26).
Similar to non-HDL-C, TG/HDL ratio correlates with small, dense LDL particles in children and adolescents and a ratio ≥3 predicts increased concentrations of small, dense LDL particles (27,28). TG/HDL is strongly associated with insulin resistance and independently predicts arterial stiffness in obese youth (29–31). TG/HDL ratio is also significantly associated with increased CIMT in youth with type 2 diabetes mellitus, although HDL-C was the only lipid to independently contribute to the prediction of CIMT (32). Both high TG and non-HDL-C that persist into adulthood are a strong predictive risk factor for CVD and development of type 2 diabetes mellitus in adulthood (33).
Although studied in adults with NAFLD, non-HDL-C as a CVD risk factor has not been assessed in children with NAFLD. Furthermore, the impact of histologic improvement in NASH and regression of NASH on lipid profiles including non-HDL-C and TG/HDL ratio has not been evaluated.
The Treatment of Nonalcoholic Fatty Liver Disease in Children (TONIC) trial was a randomized, double-blinded, placebo-controlled trial to evaluate the efficacy of vitamin E and metformin for 96 weeks in children with biopsy-proven NAFLD and elevated alanine aminotransferase (ALT) levels. Neither metformin nor vitamin E was found to be superior to placebo in improving ALT levels, the primary endpoint, although vitamin E was associated with an improvement in NAS and resolution of NASH. The TONIC trial, with its use of serial liver biopsies, allows for the evaluation of the impact of NASH resolution on lipid levels including non-HDL-C and TG/HDL.
The present study is based on post hoc analyses of children who participated in the TONIC trial and aims to examine the frequency of dyslipidemia in children and adolescents with NASH, evaluate the relation between baseline liver histology and lipid levels including non-HDL-C and TG/HDL in children with NAFLD, and assess the impact of the resolution of NASH and histologic improvement on lipids levels including non-HDL-C and TG/HDL.
Data for the present study were obtained from participants in the TONIC trial (34). TONIC was a pediatric treatment trial of NAFLD conducted by the NASH Clinical Research Network. The TONIC trial study design has been previously described, and the ClinicalTrials.gov identifier is NCT00063635 (35). Briefly, children ages 8 to 17 years with biopsy-confirmed NAFLD within 6 months of randomization and persistently elevated ALT levels were eligible for study inclusion. NAFLD was defined as liver histology with >5% of hepatocytes exhibiting macrovesicular steatosis. Children with diabetes mellitus, monogenic inborn errors of metabolism, viral hepatitis, alcohol use, pregnancy, cirrhosis, or other causes of chronic liver disease were excluded. Children with biopsy-proven NAFLD were randomized at a 1:1:1 ratio to vitamin E 400 U twice daily, metformin 500 mg twice daily, or double-dummy placebo for 96 weeks. The primary endpoint was significant and sustained reduction in ALT level compared with placebo. Secondary endpoints included the resolution of NASH in children with NASH on baseline biopsy and histologic improvement.
Participants underwent biopsies within 6 months before study randomization and 96 weeks later. Biopsies were centrally evaluated by a panel of NASH Clinical Research Network pathologists. Liver biopsies were assessed using the NAS. The NAS is a composite score ranging from 0 to 8 points composed of steatosis (0–3), hepatocyte ballooning (0–2), and lobular inflammation scores (0–3) (36). Fibrosis stage was scored on a scale of 0 to 4. The presence of NASH was assessed independently of the NAS score based on the pattern of injury and was categorized as definite, borderline, or no NASH.
Fasting serum total cholesterol, HDL, TG, and LDL levels were measured locally at baseline and week 96. From these measurements, non-HDL-C was calculated (non-HDL-C = total cholesterol − HDL). Definitions for high, borderline-high, acceptable, and low lipid levels were derived from the National Heart, Lung and Blood Institute Expert Panel Guidelines for Cardiovascular Health and Risk Reduction in Children and Adolescents (37).
Histologic improvement was defined by a decrease in the NAS of ≥2 with no worsening of fibrosis. Resolution of NASH was defined as a diagnosis of no steatohepatitis at 96 weeks among children with borderline or definite NASH at baseline. An example of the histology of NASH and subsequent NASH resolution is presented in Figure 1.
Mean lipid levels and lipid level elevations were compared between children with NASH and children with borderline or no NASH at baseline using t tests and Fisher exact test. Linear regression was used to assess the association between histologic improvement and resolution of NASH and mean baseline and 96-week lipid levels. Treatment group (metformin or vitamin E) versus placebo, baseline body mass index (BMI), and ethnicity were included in the multivariable model. For the comparison of the mean change in lipid levels between groups, P values were derived in the same manner with the addition of the baseline value of the lipid measure to the multivariable model. All of the analyses were carried out using SAS 9.3 (SAS Institute, Cary, NC) and Stata 12 (StataCorp, College Station, TX). Nominal, 2-sided P values were used and were considered statistically significant if P < 0.05.
All of the children included in the present study have been described in a previous publication (34). Baseline demographics, anthropometrics, laboratory data, and liver histologic characteristics were evenly distributed across treatment groups. In the TONIC trial, no significant difference in sustained reduction of ALT was seen among the vitamin E, metformin, and placebo groups. Significant differences were found in the vitamin E–treated group compared with placebo in significant histologic measures such as resolution of NASH and decrease in NAS score. Altogether, 56 of 146 patients (38%) who underwent repeat liver biopsy had histologic improvement in NASH (defined as a decrease of ≥2 points on NAS and without worsening fibrosis). A total of 52 of 121 patients (43%) with NASH at baseline experienced resolution of NASH.
Baseline Lipid Levels
Dyslipidemia was frequent in this cohort at baseline (Table 1). Definitions for high and low lipid levels were derived from the National Heart, Lung, and Blood Institute Expert Panel Guidelines for Cardiovascular Health and Risk Reduction in Children and Adolescents (37). Hypertriglyceridemia and low HDL levels were the most common abnormalities. High TG levels (TG ≥130 mg/dL) were present in 50.3% of patients. The mean TG level was 153 mg/dL (acceptable <90 mg/dL). Low HDL levels (<40 mg/dL) were found in 61.8% of patients with a mean HDL of 38 mg/dL. Elevated total cholesterol (total cholesterol ≥200 mg/dL) was present in 23.7% of patients, and mean total cholesterol was 176 mg/dL (acceptable <170 mg/dL). Elevated LDL (LDL ≥130 mg/dL) was present in 21.5% of patients, although the mean LDL was within the acceptable range at 109 mg/dL (acceptable <110 mg/dL). Elevated non-HDL-C (non-HDL ≥145 mg/dL) was found in 35.2% of patients. Mean non-HDL-C was 138.5 mg/dL (acceptable <120 mg/dL). TG/HDL >3.0 was present in 57.2% of patients. Baseline lipid levels did not differ among three treatment groups at baseline or at 96 weeks. A total of 144 patients (83%) had some form of lipid abnormality. During the course of the mean total cholesterol, LDL and HDL decreased significantly, whereas TG levels increased significantly.
Lipid Levels in Relation to Liver Histology
There was no difference in the proportion of children with definite NASH and those with borderline or no NASH with elevated LDL (LDL ≥110 mg/dL or ≥130 mg/dL), total cholesterol (total cholesterol ≥170 mg/dL), or non-HDL-C (non-HDL-C ≥120 mg/dL or ≥145 mg/dL) at baseline (Table 2). There was a nonsignificant increase in the proportion of children with NASH who had TG levels ≥130 mg/dL compared with those with borderline or no NASH (58.9% vs 44.0%, P = 0.06). There was a significant increase in the proportion of children with NASH with HDL ≤40 mg/dL compared with those with borderline or no NASH (75.3% vs 52.0%, P = 0.003). In addition, TG/HDL >3 was more frequent in children with NASH than in those without NASH (65.8% vs 51.0%, P = 0.05).
Lipid Levels and Resolution of NASH
Although baseline lipid levels did not differ significantly by histology, patients whose NASH resolved experienced an improvement in multiple lipid indicators compared with those without resolution of NASH (Table 3; Fig. 2). Patients whose NASH resolved had a significant decrease in total cholesterol levels from baseline compared with those who did not experience a resolution of NASH (mean change [95% confidence interval, CI] −10.0 mg/dL [−18.0 to −2.0] vs. −0.9 mg/dL [−8.0 to 6.3], P = 0.02). Furthermore, patients whose NASH resolved had a significant decrease in non-HDL-C levels compared with patients without resolution of NASH who had no change in non-HDL-C from baseline (mean [95% CI] change −7.3 mg/dL [−14.9 to 0.2] vs 1.1 [−5.7 to 7.9], P = 0.01). These results remained significant when controlled for baseline lipid level, BMI, ethnicity, and treatment group. Patients whose NASH resolved had a nonsignificant decrease in LDL when compared with patients whose NASH did not resolve (mean change [95% CI] −11.2 mg/dL [−19.1 to −3.3] vs −2.1 mg/dL [−7.6 to 3.4], P = 0.06). TG levels increased in patients with and without resolution of NASH, but the increase did not differ between the 2 groups (mean change [95% CI] 21.7 mg/dL [−2.2 to 45.6] vs 18.9 mg/dL [−1.7 to 39.5], P = 0.28). There was no significant change in HDL or TG/HDL during the study period or difference in HDL or TG/HDL change by histologic response (P = 0.42 and 0.35, respectively).
Lipid Levels and Histologic Improvement
Histologic improvement was also associated with an improvement in multiple lipid indicators (Table 4; Fig. 3). Children who experienced histologic improvement had a significant decrease in total cholesterol levels from baseline compared with those who did not experience histologic improvement (mean change [95% CI] −11.4 mg/dL [−18.9 to −3.9] vs −1.9 mg/dL [−7.7 to 3.9], P = 0.04). In addition, LDL significantly decreased in those with histologic improvement compared with in those without histologic improvement (mean change [95% CI] −11.2 mg/dL [−19.1 to −3.3] vs −2.1 mg/dL [−7.6 to 3.4], P = 0.04). Non-HDL-C levels also decreased significantly in children with histologic improvement in NASH when compared with those in children without histologic improvement (mean [95% CI] change −8.8 mg/dL [−15.5 to −2.0] vs 0.5 [−5.1 to 6.2], P = 0.03). There was no significant change in TG, HDL, or TG/HDL levels by histologic improvement.
Change in BMI and Change in Lipid Levels
Change in BMI during the study duration was positively correlated with changes in non-HDL-C (P = 0.02). There was no correlation between change in BMI and change in LDL and total cholesterol levels (P = 0.13 and 0.09, respectively). Thus, although change in non-HDL-C may be driven by change in BMI, change in total cholesterol and LDL was independent of change in BMI. No difference was seen in change in BMI by treatment group.
The results of the present study demonstrate that dyslipidemia, characterized by hypertriglyceridemia, hypercholesterolemia, elevated non-HDL-C, and low HDL levels, is frequent in children and adolescents with NAFLD. Furthermore, the present study demonstrates that non-HDL-C, a powerful cardiovascular risk marker, is elevated in children and adolescents with NAFLD. Finally, our study establishes that both resolution of NASH and histologic improvement in NASH are associated with improvements in non-HDL-C, LDL, and total cholesterol. Interestingly, although multiple lipid indicators improved in the setting of histologic improvement and NASH resolution, no change was seen in HDL, TG, or TG/HDL. Thus, although histologic improvement and NASH resolution improve some measures of CVD risk, other important risk factors persist.
Limited data have suggested that NAFLD in children is associated with dyslipidemia. Schwimmer et al evaluated 150 adolescents with biopsy-proven NAFLD and 150 overweight control individuals for cardiovascular risk factors (9). Children with NAFLD had higher total cholesterol, LDL, and TG levels compared with BMI-matched controls. Nobili et al evaluated 118 children with biopsy-proven NAFLD. This group found that NAS and fibrosis stage were positively correlated with TG/HDL, total cholesterol/HDL, and LDL/HDL ratio and were predictors of an atherogenic lipid profile. Our study confirms these findings, demonstrating a high prevalence of dyslipidemia in children with biopsy-proven NAFLD and NASH. Our study also provides longitudinal data on children with NAFLD and NASH that adds to these cross-sectional studies and demonstrates the impact of NASH resolution and histologic improvement on CVD risk factors. Although several studies have assessed the impact of weight loss interventions in children with NAFLD, none have been performed with serial liver biopsies and, thus, could not assess the relation between histologic changes and lipid indicators (16,17). Our study addresses these limitations by assessing biopsy-proven NASH serially, allowing for assessment of the relation between histologic changes in NASH and improvement in lipid indicators. We were able to show that resolution of NASH and histologic improvement in NASH are associated with parallel improvements in LDL and total cholesterol with persistence of low HDL levels and hypertriglyceridemia.
We also demonstrate that elevated non-HDL-C is frequent in NAFLD and declines with histologic improvement. Non-HDL-C is an accepted risk factor for the development of CVD in adults and a target of lipid-lowering therapy (23,24). Childhood non-HDL-C predicts dyslipidemia, hyperglycemia, and hyperinsulinemia in adulthood and correlates with atherosclerosis on autopsy (26). Thus, an improvement in liver histology in children with NASH is associated not only with an improvement in traditional lipid indicators but also with an improvement in non-HDL-C, a powerful predictor of future CVD.
Interestingly, the TG levels increased in all of the groups during the study duration and no improvement was seen in TG/HDL. The methods used for TG measurement vary, and the final TG value can exclude or include free glycerol level, altering the final value (38,39). Following NAFLD and NASH resolution, there may be a decrease in TG formation because of alterations in available free fatty acids. This would result in increased levels of free glycerol and, using a 1-step method, falsely elevate TG measurements. Further evaluation of TG levels using both methods is needed to clarify this issue.
The present study has several important limitations. Although dyslipidemia and elevated non-HDL-C are validated markers of CVD risk, they remain surrogate markers of CVD. Long-term follow-up is needed in children with NAFLD and NASH to determine whether resolution of NAFLD and/or NASH decreases CVD events and CVD-related mortality in adulthood. Furthermore, our study demonstrates only an association between liver histology and lipid levels and cannot demonstrate causality. Improvements in dyslipidemia may be the result of improvements in insulin resistance and the metabolic syndrome in addition to alterations in hepatic lipid metabolism. In addition, the change in dyslipidemia may be affected by vitamin E use. Although data exist to suggest that vitamin E supplementation may make LDL particles more resistant to oxidative stress, clinical trials of vitamin E supplementation in adults at risk for or with CVD have shown no impact on circulating lipid levels (31–33). Furthermore, evaluation of the impact of vitamin E on lipid levels in children is needed. Finally, although the present study demonstrated an improvement in non-HDL-C, LDL, and total cholesterol, TG and HDL levels did not improve. Adolescents with NAFLD tend to have delayed clearance of plasma TG, likely from intestinal chylomicrons (40). In the groups with improvement and/or resolution of NASH, one would expect the kinetics of chylomicron TG to have been normalized. This, however, was not the finding in the present study. The fact that TG remain high in the responders would suggest that the kinetics of intestinal TG may not have been normalized (or affected) by the intervention or that production of TG from the liver remains elevated despite the improvement in the liver disease.
In summary, we have demonstrated that dyslipidemia, including elevated non-HDL-C levels and increased TG/HDL, is frequent in children with NAFLD and NASH. We have also shown that resolution of NASH and histologic improvement are independently associated with an improvement in non-HDL-C, total cholesterol, and LDL levels, suggesting that an improvement in NASH and NASH resolution may improve CVD risk in children with NAFLD. Although an improvement was shown in non-HDL-C, a similar improvement in TG and TG/HDL was not seen and the data remain inconclusive for long-term atherosclerotic risk. Further studies using direct measurement of lipoprotein particle size and characteristics will be needed. Although the improvement in non-HDL-C is promising, the lack of improvement in TG/HDL demonstrates complexity in the relation between peripheral lipids and NAFLD. Larger sample sizes would allow improved comparison between improvements in steatosis versus inflammation in the liver and the relation with peripheral lipids.
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