Vos, Miriam B.*; Colvin, Ryan†; Belt, Patricia‡; Molleston, Jean P.§; Murray, Karen F.||; Rosenthal, Philip¶; Schwimmer, Jeffrey B.#; Tonascia, James†; Unalp, Aynur†; Lavine, Joel E.**; On Behalf of the NASH CRN Research Group
Nonalcoholic fatty liver disease (NAFLD) is the most common chronic liver disease in children in the United States (1). The term NAFLD refers to a spectrum of disease, ranging from isolated steatosis to nonalcoholic steatohepatitis (NASH) to cirrhosis (2) and children can present at any stage along this continuum. Typically found in overweight individuals, NAFLD is associated with features of the metabolic syndrome including dyslipidemia, visceral adiposity, hypertension, and insulin resistance (3).
Similar to other constituents composing the metabolic syndrome, it is poorly understood why some obese children have normal livers whereas others develop steatosis alone or florid steatohepatitis. Although NAFLD is much more common in overweight children, a body mass index (BMI) above a certain threshold is a poor predictor of NAFLD severity (4).
Diet composition is an environmental factor that may influence NAFLD severity. Previous attempts have been made at correlating diet and NAFLD in children; however, precedent studies did not use liver biopsy or reliable imaging techniques for case ascertainment (5,6). The importance of specific micro- or macronutrients may lie more in their influence on cell injury, fibrosis, inflammation, or degree of steatosis rather than categorical change in liver fat or alterations in surrogate markers. These features can only be assessed with a biopsy-validated study design.
The National Institute of Diabetes and Digestive and Kidney Diseases organized and funded a Nonalcoholic Steatohepatitis Clinical Research Network (NASH CRN) beginning in 2002. Clinical, laboratory, anthropometric, demographic, and nutritional data collected as part of the NASH CRN provide a unique resource because liver histology underwent a systematic, masked central review by an expert panel of pathologists and clinical factors were assessed near the time of liver biopsy. The objective of the present study was to examine dietary data from a large cohort of NAFLD patients and correlate diet variables with specific histopathologic features. We hypothesized that children with decreased antioxidant vitamin consumption and increased sugar-sweetened beverage consumption would have worse pathologic features of NAFLD.
SUBJECTS AND METHODS
The NAFLD database is a prospective, observational registry of subjects with known or suspected NAFLD, which includes children 2 to 17 years. The NAFLD database studies have institutional review board approval at each of the clinical centers participating in the NASH CRN (Appendix). Written consent was obtained from a parent or guardian and written assent obtained from all children 8 years and older before participation. Participants were eligible for inclusion in this diet study if they had baseline clinical data collected within 6 months of liver biopsy (most were assessed within 2 months) and their liver biopsy specimens had undergone masked central review and scoring by the pathology committee of the NASH CRN by November 2007 (Table 1). Because liver biopsy was an inclusion criterion into the study, baseline clinical data including diet information was collected after the liver biopsy and diagnosis of NAFLD.
Demographic data were obtained via structured interview and questionnaires. Height, weight, waist, and hip measurements were taken in duplicate while standing and wearing lightweight clothing. Height and weight were measured without shoes to the nearest 0.1 cm and 0.1 kg, respectively. BMI was calculated as weight (kilogram) divided by height (meter) squared. BMI percentile was determined according to age and sex based on data from the Centers for Disease Control and Prevention and converted to a z score for comparison.
Diet information was collected using the complete Block Brief Questionnaire. This questionnaire includes 77 food items and was developed from the NHANES 1999–2002 dietary recall data. Individual portion size is asked, and pictures are provided improving quantification of nutrient intake and allowing detailed comparisons of diet and histopathology. Sugar-sweetened beverage consumption per week information was compiled by totaling the “glasses or cans” per year reported for fruit juices, Kool Aid, and similar drinks, fruit punches, and soda and dividing by 52 weeks. We chose to include 100% fruit juice because juices contain large amounts of fructose, a sugar known to induce fatty liver in animal models (7).
Fasting whole blood samples were obtained by venipuncture following overnight fast of more than 12 hours and processed for plasma and serum within 2 hours. Each clinical center performed reported laboratory assays on site.
Biopsies were evaluated for NAFLD features according to the validated histological scoring system for the NASH CRN (8): steatosis (grade 0 [<5% macrovesicular fat], grade 1 [5%–33%], grade 2 [34%–66%], grade 3 [>66%]), portal inflammation (grade 0–2), lobular inflammation (grade none, <2, 2–4, and >4), and ballooning degeneration (none, few, and many). A NAFLD activity score (NAS) was tabulated by summing scores for steatosis, lobular inflammation, and ballooning degeneration (score range 1–7). For analyses, cases with low NAS (range 1–3) were compared with those with high scores (range 4–8). A diagnostic categorization was also determined for each case: “not NASH,” “borderline zone 3,” “borderline zone 1,” or “definite NASH” (8). “definite NASH” unequivocally fulfills previously defined criteria for steatohepatitis, whereas the category of “not NASH” encompasses cases of NAFLD in which the changes are so mild or nonspecific that more specific classification cannot be made. The borderline zone 1 designation resembles a pattern of steatosis with predominant portal inflammation and portal fibrosis, without significant lobular inflammation or perisinusoidal fibrosis. This pattern is similar, but not identical, to that described previously in children as “type 2 NASH” (9).
Analyses included children 6 to 17 years of age at enrollment. Unadjusted comparisons of characteristics by steatohepatitis status, NAS, steatosis grade, lobular inflammation, and ballooning were conducted using either Wilcoxon rank sum tests and Kruskal-Wallis tests (categorical variables), or Fisher exact tests (measured variables). The medians of selected variables are presented with the first and third quartile to demonstrate distribution. Additional comparisons of steatohepatitis status limited to the “not NASH” and “definite NASH” groups were also conducted.
A total of 149 children were eligible and included for analyses. Of those subjects, 110 (73%) were boys and 79 (53%) were Hispanic, primarily Mexican American. The mean age was 13 ± 2.6 years and the mean BMI z score was 2.3 ± 0.4. The subjects were fairly evenly distributed over the 4 categories of NAS disease severity (Table 1). Demographic variables were similar between children when compared by NASH status, except age, which was less in both borderline zone 3 and borderline zone 1 groups. There were significant differences in baseline laboratory values as displayed in Table 1. Triglycerides, glucose, gamma-glutamyltransferase, and aspartate aminotransferase/alanine aminotransferase (ALT) tended to be higher with greater NASH severity, as reported previously (4). Uric acid was highest in the definite NASH group and had a significant variation across all 4 groups (P = 0.008), with the lowest level seen in the borderline Z1 group. Uric acid for “definite NASH” was higher compared with those with steatosis alone (not present), although this did not reach significance (P = 0.07).
In general, as shown in Table 2, total energy consumption was similar between groups and was lower than reported in nationally representative survey data for these age groups (10). For all children, most energy came from carbohydrates (median percentage of energy from carbohydrates was 50.4% (range 27%–74%)). Most consumed a moderate fat diet (median percentage of kilocalories from fat was 35% [range 18%–54%]). Fifty percent or fewer of the children in all 4 groups consumed >6 sugar-sweetened beverages per week and this was not different between groups. When the amount of specific daily nutrients was compared between the 4 groups, no significant differences were found (Table 2). All groups consumed greater than the recommended daily allowance (RDA) for vitamin C, vitamin A, and vitamin D (11). All groups consumed less than the RDA for folate (400 μg/day for boys and girls) and less than half of the RDA for vitamin E (22.5 IU/day) (11).
Comparison between those without NASH (not present) and those with definite NASH (definite) also showed no significant differences. We also compared the subjects by high and low NAS score and there was again no difference for total energy, percentage of kilocalories from fat, carbohydrates, or protein (data not shown). Reported consumption of saturated fat, unsaturated fat, and dietary cholesterol was similar when compared by both NASH status and NAS.
Diet variables were also compared for 3 histologic features: steatosis, lobular inflammation, and ballooning. Median consumption of vitamin E was lower in children with higher grade of steatosis (grade I: 8.4 [5.9–11.5], grade II: (6.1 [4.4–9.6], grade III: 6.9 [5.7–10.3], P = 0.05) (Table 3). Total energy, percentage of protein, fat, and carbohydrates, as well as vitamins C, A, and D were not significantly different by steatosis grade. When diet variables were examined by amount of lobular inflammation, only dietary fiber differed significantly and was increased in the 8 subjects with a score of 3 (highest level) compared with the 73 and 70 subjects with low and moderate inflammation (P = 0.03) (data not shown). Increased ballooning was seen in those with reduced vitamin C consumption: none: 106.9 (65.8–160.6), few: 139.9 (82.1–202.3), many: 72.6 (59.6–120.5) (P = 0.05) (Table 4). As shown, carbohydrate consumption in grams varied significantly for ballooning, although percentage of energy from carbohydrates was not significantly different between groups.
Despite substantial scientific interest in dietary influences on NAFLD, little information is available about diet in children with NAFLD compared with the histologic features of the disease. In this registry-based study, we were able to examine self-reported diet and compare this to rigorously measured histology. We found that diet (after the diagnosis of NAFLD) did not differentiate simple steatosis and NASH. We also found several interesting associations that suggest areas for further investigation, including low consumption of vitamin E, weak associations between vitamins E and C and increased histologic severity, and finally, strong associations between uric acid (a surrogate marker for fructose consumption) and histologic severity.
We did not find any significant differences in diet when we compared children with “NASH” to “not NASH.” Each group reported similar consumption of fat, sugar-sweetened beverages, antioxidants, and other micronutrients. Diet comparisons by NAS score, representing a composite of these findings, also did not differ significantly. These findings suggest that diet is not the primary cause of whether a child with NAFLD has NASH or not.
In our cohort of children with NAFLD, the median reported consumption of vitamin E in all groups was less than half of the US RDA of vitamin E (for adolescents 22.5 IU/day) (11). Because of the registry design, our study did not include obese children without NAFLD, so we do not have a disease-free control group for comparisons. In general, obese children have not been identified as having lower reported intake of vitamins E and C. Data from the National Health and Nutrition Examination Survey (1988–1994) demonstrated that children who were obese had similar reported intake of these vitamins as well as fruits and vegetables compared with nonobese children (12).
In our study, we found weak associations with vitamins E and C consumption compared with steatosis and ballooning, respectively. Both vitamin C and vitamin E function as scavengers of hydoxyl, peroxyl, and superoxide radicals and protect against plasma lipid and low-density lipoprotein peroxidation (13) and other oxidative stress, and thus could be important in preventing the progression of NAFLD. Vitamin E has been tested as a treatment for NAFLD (14–17) in part because antioxidant deficiency may lead to increased lipid peroxidation and cell death due to mitochondrial compromise (evident as ballooning on liver biopsy). In the recently published Pioglitazone or Vitamin E for Nonalcoholic Steatohepatitis randomized controlled trial comparing pioglitazone, vitamin E, and placebo in adults with NASH, vitamin E therapy was associated with improved ALT, aspartate aminotransferase, steatosis, lobular inflammation, and ballooning (18), suggesting a protective role in hepatocytes. In the Treatment of Nonalcoholic Fatty Liver Disease in Children treatment trial of adolescents with NAFLD, 58% of those treated with vitamin E had resolution of NASH or borderline NASH, which was significantly better than those taking placebo (28%), and those taking vitamin E demonstrated significant improvement in NAFLD Activity scores (19).
There are several previous nonpathology-based published studies that also examine the diet of children with and without presumptive NAFLD based on surrogate markers. Quiros-Tejeira et al found a small increase in consumption of dietary cholesterol in the suspected NAFLD children compared with normal ALT subjects (20). De Piano et al (6) studied 43 adolescents, including 13 with NAFLD (based on ultrasound evaluation), and found no significant differences in total energy, percentage of protein, percentage of carbohydrates, percentage of fat, or cholesterol consumption compared with obese adolescents without echogenic livers. Papandreou et al (21) compared adolescents with and without NAFLD (using ultrasound for fat assessment) and found that total energy, percentage of protein, and percentage of fat were similar; however, they found an increase in carbohydrates and sugar intake in children with NAFLD.
We were also interested in examining sugar consumption in our subjects because added sugars are known to be associated with dyslipidemia (22) and fructose can be used to induce fatty liver in animal models (23). Adolescents are the highest consumers of both added sugars and fructose, making them a high-risk group for potential effects (24). Because the Block Brief Questionnaire lacks a detailed breakdown of sugar, we used sugar-sweetened beverages [the largest source (24)] as a surrogate marker of fructose intake. Interestingly, we did not find any difference in reported sugar-sweetened beverage consumption between groups. Previous studies of adult NAFLD patients, including a registry study using a design similar to ours (25), have found increased reported intake of sugar-sweetened beverages, elevated uric acid levels (26–29), and associations between uric acid and fibrosis severity (25). Uric acid levels increase with fructose intake (30), and intake of fructose correlates with uric acid levels in the general population (31). Because of this it has been used as a surrogate marker of fructose intake. Despite the lack of difference in reported sugar-sweetened beverage consumption, our groups differed significantly in uric acid level, with the highest levels found in the definite NASH group (P = 0.008). When definite NASH was compared with steatosis alone (not NASH), there was a trend toward a higher uric acid level in those with definite NASH (P = 0.07). Because sugar-sweetened beverages only account for an average of ∼40% fructose in the diet (24), our subjects may have substantial fructose intake from other sources (eg, processed foods) that we were unable to measure given the limitations of our dietary instrument. In addition, it is possible that uric acid has an independent effect in NAFLD, unrelated to fructose intake. Further studies with both histology and more detailed diet information will be needed to understand the relation of fructose to NAFLD and more specifically to NASH in children.
A possible limitation of our study may be a reporting or recall bias because most participants reported a relatively healthy diet. For example, median sugar-sweetened beverage intake was reported as 1 glass or can or less per week. In the United States, average intake of sugar-sweetened beverages for children age 12 to 19 years represented 356 cal/day (32), which would translate to 2.5 cans of a typical 12-oz can of soda or more than three 8-oz glasses of fruit-like drink per day. Thus, our subjects report a much lower than average consumption of sugar-sweetened beverages. In addition, less than one-fourth of our subjects reported >40% fat intake, the definition of a high-fat diet. There are several factors that may account for this finding. All of our participants had already undergone a substantial medical procedure (liver biopsy) and had been given a diagnosis of NAFLD. This event may have been an effective trigger for lifestyle improvements, including alterations in diet. At the time of diagnosis, many patients are instructed to decrease intake of sugar-sweetened beverages, increase intake of fruits and vegetables, and reduce fat intake as part of standard therapy. Most of our subjects were evaluated at 1 to 2 months after the liver biopsy, possibly a peak time for implementing an improved diet. The Block Brief Questionnaire asks participants to reflect on their diet during the last year; however, the recall may be more influenced by their present diets or they may wish to appear in conformance with recently provided nutritional advice for a healthy diet. Overall, these effects may be less important because it would likely affect all participants, regardless of histologic variation.
Several of our findings had P values of 0.05. Use of multiple comparisons could lead to P values identified as significant that are actually random; however, there is a pathophysiological basis for these findings, and the results support work by Strauss that obese children are low in vitamin E (12) and the Pioglitazone or Vitamin E for Nonalcoholic Steatohepatitis trial. In our study, the trends for uric acid, vitamin E, and vitamin C were not consistent across the 3 groups. This is likely as a result of the difficulties of assigning scores and cutoffs to continuously variable pathology findings. An alternative hypothesis is that vitamins may have threshold levels and are associated with worsened pathology only when they fall below certain levels.
The Block Brief Questionnaire has inherent limitations. Cullen et al (33) compared a similar Block survey (Block Kids) in children with two 24-hour dietary recalls and found that the Block overestimated the percent energy from carbohydrates and found significant differences in the means for most food groups and nutrients between the 2 methods. They found that the Block was more accurate for children older than 12 years of age and for nutrients (compared with food groups), both bolstering the findings in the present study because our mean age was older than 13.0 years and we examined specific nutrients, not food group servings. Mexican American diets are not well represented in the Block Brief Questionnaire, and some of our subjects are Mexican Americans.
In summary, macronutrients did not differentiate between simple steatosis and NASH in the children in our study. We found that children with NAFLD consumed less than the recommended amounts of vitamin E, and that there was a weak association between lower consumption of both vitamins E and C and pathologic severity of NAFLD. Uric acid, a surrogate marker of dietary fructose, was significantly increased in those children with definite NASH compared with milder forms of NAFLD. Prospective studies are needed to evaluate diet in potential subjects before diagnosis and nutritional counseling (and ideally before NAFLD onset) to better confirm dietary contributors to this disease.
Appendix: Members of the Nonalcoholic Steatohepatitis Clinical Research Network
Baylor College of Medicine, Houston, TX: Stephanie Abrams, MD; Diana Arceo, MD, MS; Denise Espinosa; Leanel Angeli Fairly, RN
Case Western Reserve University Clinical Centers: MetroHealth Medical Center, Cleveland, OH: Carol Hawkins, RN; Yao-Chang Liu, MD; Margaret Stager, MD. Cleveland Clinic Foundation, Cleveland, OH: Arthur McCullough, MD; Srinivasan Dasarathy, MD; Ruth Sargent, LPN
Seattle Children's Hospital & Research Institute, Seattle, WA: Melissa Coffey; Karen Murray, MD; Melissa Young
Children's National Medical Center, Washington DC: Parvathi Mohan, MD; Kavita Nair
Duke University Medical Center, Durham, NC: Manal Abdelmalek, MD; Anna Mae Diehl, MD; Marcia Gottfried, MD (2004–2008); Cynthia Guy, MD; Paul Killenberg, MD (2004–2008); Samantha Kwan; Yi-Ping Pan; Dawn Piercy, FNP; Melissa Smith
Indiana University School of Medicine, Indianapolis, IN: Prajakta Bhimalli; Naga Chalasani, MD; Oscar W. Cummings, MD; Lydia Lee, Linda Ragozzino, Raj Vuppalanchi, MD
Riley Hospital for Children, Indianapolis, IN: Elizabeth Byam; Ann Klipsch, RN; Jean Molleston, MD; Girish Subbarao, MD
Johns Hopkins Hospital, Baltimore, MD: Kimberly Pfeifer; Ann Scheimann, MD; Michael Torbenson, MD
St Louis University, St Louis, MO: Sarah Barlow, MD (2002–2007); Jose Derdoy, MD (2007-); Joyce Hoffmann; Debra King, RN; Andrea Morris; Joan Siegner, RN; Susan Stewart, RN; Brent A. Tetri, MD; Judy Thompson, RN
University of California, San Diego: Cynthia Behling, MD, PhD; Janis Durelle; Joel E. Lavine, MD, PhD; Susana Mendoza; Jeffrey B. Schwimmer, MD; Claude Sirlin, MD; Tanya Stein, MD; Zobeida Palomares
University of California, San Francisco: Bradley Aouizerat, PhD; Kiran Bambha, MD; Nathan M. Bass, MD, PhD; Linda D. Ferrell, MD; Danuta Filipowski, MD; Raphael Merriman, MD (2002–2007); Mark Pabst; Monique Rosenthal; Philip Rosenthal, MD; Tessa Steel (2006–2008).
University of Washington Medical Center, Seattle: Matthew Yeh, MD, PhD
Virginia Commonwealth University, Richmond, VA: Sherry Boyett, RN; Melissa J. Contos, MD; Michael Fuchs, MD; Amy Jones; Velimir AC Luketic, MD; Bimalijit Sandhu, MD; Arun J. Sanyal, MD; Carol Sargeant, RN, MPH; Kimberly Selph; Melanie White, RN
Virginia Mason Medical Center, Seattle, WA: Kris V. Kowdley, MD; Jody Mooney, MS; James Nelson, PhD; Sarah Ackermann; Cheryl Saunders, MPH; Vy Trinh; Chia Wang, MD
Washington University, St Louis, MO: Elizabeth M. Brunt, MD
National Cancer Institute, Bethesda, MD: David Kleiner, MD, PhD
National Institute of Child Health and Human Development, Bethesda, MD: Gilman D. Grave, MD; Terry TK Huang, PhD, MPH
National Institute of Diabetes, Digestive and Kidney Diseases, Bethesda, MD: Edward Doo, MD; James Everhart, MD, MPH; Jay Hoofnagle, MD; Patricia R. Robuck, PhD, MPH (Project Scientist); Leonard Seeff, MD
Johns Hopkins University, Bloomberg School of Public Health (Data Coordinating Center), Baltimore, MD: Patricia Belt, BS; Frederick L. Brancati, MD, MHS; Jeanne M. Clark, MD, MPH; Ryan Colvin, MPH; Michele Donithan, MHS; Mika Green, MA; Rosemary Hollick (2003–2005); Milana Isaacson; Wana Kim; Alison Lydecker, MPH (2006–2008), Pamela Mann, MPH; Laura Miriel; Alice Sternberg, ScM; James Tonascia, PhD; Aynur Ünalp-Arida, MD, PhD; Mark Van Natta, MHS; Laura Wilson, ScM; Katherine Yates, ScM.
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