The ω-3 fatty acid density of the diet was largely reflective of ALA intake, because ALA constituted 96% (IQR 94%–98%) of ω-3 fatty acid intake (data not shown). According to the dietary reference intakes, the acceptable macronutrient distribution range for ALA in children and adolescents is 0.6% to 1.2% of energy, which equates to 0.67 to 1.33 g/1000 kcal of ω-3 fatty acids when applying an Atwater factor of 9 kcal/g of ALA (25). The ω-3 fatty acid density of the diets observed here (0.72 g/1000 kcal [IQR 0.59–0.93 g/1000 kcal]) was concentrated around the lower end of the acceptable macronutrient distribution range (Fig. 2, Table 1). The ω-3 fatty acid intake was also low in relation to ω-6 fatty acids (median ω-6 to ω-3 ratio 9.0 (IQR 7.4–10.2) (Table 1). Although a desirable ω-6 to ω-3 fatty acid ratio has not been determined for pediatric NAFLD, the observed ratio of 9:1 greatly exceeds most references for disease prevention, which range from approximately 3:1 to 6:1 (19).
Despite greater reported energy intake in boys (1772 kcal [IQR 1369–2406 kcal]) compared with girls (1220 kcal [IQR 949–1860 kcal]) (P < 0.001), there were no significant differences in total, fried, and nonfried fish consumption by sex (supplemental Table 1 [http://links.lww.com/MPG/A244]). Girls appeared to consume a more ω-3 fatty acid–rich diet than boys in relation to energy intake (0.77 g/1000 kcal [0.61–0.98 g/1000 kcal] vs 0.71 g/1000 kcal [0.58–0.88 g/1000 kcal], P = 0.05]), and ω-6 fatty acid intake (ω-6 to ω-3 ratio 8.6 [7.1–10.0)] vs 9.1 [7.6–10.3], P = 0.05) (supplemental Table 1 [http://links.lww.com/MPG/A244]). Larger disparities in fish and ω-3 fatty acid intake were observed between ethnic groups than between sexes (supplemental Table 2 [http://links.lww.com/MPG/A245]). Subjects from the Other ethnicity group consumed greater amounts of fish (12.8 oz/month [3.7–31.3 oz/month]) than Hispanics (4.4 oz/month [0.0–13.0 oz/month], P = 0.03) and non-Hispanic whites (3.3 oz/month [0.7–13.6 oz/month], P = 0.02). Additionally, non-Hispanic whites reported diets that were lower in ω-3 fatty acid content compared with subjects in the Hispanic and Other ethnicity groups (supplemental Table 2 [http://links.lww.com/MPG/A245]). Age was not associated with any of the fish or ω-3 fatty acid parameters measured (data not shown).
Higher fish and ω-3 fatty acid intakes were generally associated with lower ALT values, although none of the correlations were strong or statistically significant (P > 0.05); however, when additional factors including demographic, anthropometric, and dietary variables were accounted for in a linear regression model, the relations between serum ALT and long-chain ω-3 fatty acid intake tended toward significance (P = 0.08, data not shown).
The dietary fish and long-chain ω-3 fatty acid intake and ω-6 to ω-3 fatty acid intake ratio were also examined in relation to histological features of NAFLD. The main significant findings of these analyses were related to inflammation (Table 2). There appeared to be a protective effect of fish intake on hepatic inflammation that was significant for portal inflammation (P = 0.02) and tended toward significance for lobular inflammation (P = 0.08) (Table 2). Fish and long-chain ω-3 fatty acid consumption and ω-6 to ω-3 fatty acid ratio were not associated with the other histological parameters or a diagnosis of definite NASH (data not shown). Adjustment for demographic, anthropometric, and dietary variables did not influence the findings for fish intake and hepatic inflammation, but resulted in a stronger association between long-chain ω-3 fatty acid intake and lobular inflammation (P = 0.004, Table 2).
There is emerging evidence that long-chain ω-3 fatty acids may be important mediators of NAFLD pathogenesis (7). The findings from this registry-based study offer insight into the dietary intake of fish and ω-3 fatty acids in children with documented NAFLD in the United States. This provides valuable contextual information and a useful perspective from which to examine the diet–disease relation.
Most of the subjects in this sample were not consuming the recommended amounts of fish and ω-3 fatty acids (Table 1) (24,25). The observed low intake of ω-3 fatty acids are consistent with the results of 3-day food records collected from 35 pediatric patients with NAFLD in Toronto, which reported average intakes of ALA that were less than two-thirds the adequate intake level (12). Together, these findings indicate that diet may be contributing to the low EPA and DHA content of cell membranes that has been observed in patients with NAFLD (9). Although this study did not have a control group for comparison, the frequency of fish consumption was less than that reported from a sample of >1000 adolescents from 5 public schools in Rhode Island, which had 36% of subjects who indicated eating fish at least once per week (vs 26%) and 17% who reported never eating fish (vs 23%) (28). Of interest, the Other ethnicity group reported approximately 3 times greater fish consumption than the Hispanic and non-Hispanic white subjects; however, this was a diverse group of non-Hispanic Indian, Asian, Pacific Islander, black, and Mixed ethnicities that contained only 16 subjects, which precluded meaningful subgroup analysis (supplemental Table 2 [http://links.lww.com/MPG/A245]).
Although NAFLD risk could not be determined from this analysis, it was possible to explore the relation of fish and ω-3 fatty acid consumption to serological and histological indicators of disease. The Toronto study noted a strong inverse relation between EPA and DHA intake and ALT (12). A similar, although nonsignificant, inverse association between fish and long-chain ω-3 fatty acid intake and ALT was observed in this sample. More important, lack of fish and long-chain ω-3 fatty acid consumption was associated with greater portal and lobular inflammation (Table 2). Although fish and long-chain ω-3 fatty acid intake were not associated with a diagnosis of definite NASH, inflammation is known to predispose to fibrosis and progressive liver disease (29). The failure to detect a relation between fish intake and definite NASH may be related to the fact that histologic parameters for steatohepatitis in pediatric biopsies are not as well understood as they are in adults (30).
There are several anti-inflammatory and proresolution mechanisms of EPA and DHA in fish that would support the observation of protective effects on both portal and lobular inflammation. Historically, the effects of long-chain ω-3 fatty acids on inflammation have been largely attributed to the shift from ω-6 to ω-3 fatty acid–derived eicosanoids (19). In recent years, additional EPA and DHA-derived lipid mediators, including protectins and resolvins, have been identified, and are thought to be important anti-inflammatory mediators (31). A series of experiments by Oh et al (32) also demonstrated that DHA suppresses activation of the nuclear factor-κB inflammatory pathway in macrophages through a G-protein–coupled receptor. CRP, an indicator of systemic inflammation, was measured in a subset of subjects in this study (n = 151). Although CRP was not associated with fish consumption, there was a weak inverse correlation with long-chain ω-3 fatty acid intake (Spearman ρ = −0.155, P = 0.058, data not shown).
Subjects consuming greater amounts of fish and long-chain ω-3 fatty acids did not have lower levels of hepatic steatosis (Table 2). The profound effects of EPA and DHA on lipid metabolism that have been reported in experimental animal models of obesity have been given as a major rationale for their importance in NAFLD (6). Moreover, this finding conflicts with the results of a double-blind, randomized trial of pediatric patients with NAFLD in Italy, which noted dramatic reductions in ultrasound liver steatosis grade among subjects that were receiving DHA compared with germ oil (11). One possible explanation for this discrepancy may be the dose of long-chain ω-3 fatty acids required to induce an effect. The DHA supplements in the Italian study were 5 times greater than the median intake of long-chain ω-3 fatty acids that was reported by our subjects (11). Alternatively, the effect of ω-3 fatty acids on hepatic steatosis may have been confounded by genetic factors. Recently, a study of obese adolescents noted that hepatic fat fraction detected by magnetic resonance imaging was associated with the ω-6 to ω-3 fatty acid ratio of the diet, but only among participants who were homozygous for the G allele of rs738409 in the PNPLA3 gene, which codes for adiponutrin (33).
In interpreting the results of this study, there are limitations that should be considered. The diet was assessed using an FFQ that was administered after subjects were diagnosed as having NAFLD. There was differential inclusion of subjects by sex and by level of steatosis and hepatocyte ballooning, which may have overestimated the association of including a more diseased population. The relatively low intake of sugar-sweetened beverages observed in the analysis of children in the NASH CRN database by Vos et al (17) suggests that respondents may have already modified their diets before filling out the FFQ, and/or were misreporting their intake to appear healthier. Although the NASH CRN Standards of Care for Pediatric Patients with Fatty Liver Disorders makes no reference to fish consumption, there are recommendations to limit fried food, which may have prompted subjects to reduce the amount of fried fish they were eating or reporting. At the same time, most adolescents consider fish to be healthy, so overall consumption of fish may have actually increased (28). The validity of the Block Brief FFQ has not been evaluated in adolescents, but a moderate amount of error can be inferred based on the subject's low energy intakes (Table 1); however, for the size and purpose of this study, an FFQ may be superior to short-term dietary assessment instruments such as 24-hour dietary recalls or food diaries for episodically consumed foods such as fish. Although data were collected on both fried and nonfried fish intake, the long-chain ω-3 fatty acid content varies considerably by species (34). Furthermore, no data were available on the use of supplements containing ω-3 fatty acids. Finally, care must be taken when attempting to extrapolate these findings to other pediatric NAFLD populations, because there appeared to be some selection bias.
The results of this study show that pediatric patients with NAFLD consume less than the recommended amount of fish and ω-3 fatty acids (24,25). Promoting the intake of fish may help to reduce both portal and lobular inflammation, but further research is needed to test this hypothesis and to determine the necessary amount and best sources. Based on the present fish consumption, dietary supplements may be a good option for increasing long-chain ω-3 fatty acid intake to recommended levels (25). Advances in food biotechnology may offer opportunities for alternative sources of ω-3 fatty acids in the future, but this remains to be seen (35). Until additional clinical trials evaluating the effectiveness of long-chain ω-3 fatty acid supplements on pediatric NAFLD are conducted, patients should be encouraged to increase fish intake to meet general health recommendations.
MEMBERS OF THE NONALCOHOLIC STEATOHEPATITIS CLINICAL RESEARCH NETWORK
Pediatric Clinical Centers
Baylor College of Medicine, Houston, TX: Stephanie H. Abrams, MD, MS; Ryan Himes, MD; Rajesh Krisnamurthy, MD; Leanel Maldonado, RN (2007–2012); Beverly Morris
Cincinnati Children's Hospital Medical Center, Cincinnati, OH: Kimberlee Bernstein, BS, CCRP; Kim Cecil, PhD; Stephanie DeVore, MSPH (2009–2011); Rohit Kohli, MD; Kathleen Lake, MSW (2009–2012); Daniel Podberesky, MD; Crystal Slaughter, BA, CCRP; Stavra Xanthakos, MD
Columbia University, New York, NY: Gerald Behr, MD; Joel E. Lavine, MD, PhD; Ali Mencin, MD; Nadia Ovchinsky, MD; Elena Reynoso, MD
Emory University, Atlanta, GA: Adina Alazraki, MD; Rebecca Cleeton, MPH; Saul Karpen, MD, PhD; Nicholas Raviele; Miriam Vos, MD, MSPH
Indiana University School of Medicine, Indianapolis, IN: Elizabeth Byam, RN; Oscar W. Cummings, MD; Cynthia Fleming, RN, MSN; Ann Klipsch, RN; Jean P. Molleston, MD; Kumar Sandrasegaran, MD; Girish Subbarao, MD
Johns Hopkins Hospital, Baltimore, MD: Kimberly Pfeifer, RN; Ann Scheimann, MD; Michael Torbenson, MD
Johns Hopkins University, Bloomberg School of Public Health (Data Coordinating Center), Baltimore, MD: Patricia Belt, BS; Jeanne M. Clark, MD, MPH; Erin Corless, MHS; Michele Donithan, MHS; Milana Isaacson, BS; Kevin P. May, MS; Laura Miriel, BS; Alice Sternberg, ScM; James Tonascia, PhD; Aynur Ünalp-Arida, MD, PhD; Mark Van Natta, MHS; Ivana Vaughn, MPH; Laura Wilson, ScM; Katherine Yates, ScM
Mount Sinai Kravis Children's Hospital, New York, NY: Ronen Arnon, MD; Mariel Boyd, CCRP
Northwestern University Feinberg School of Medicine/Ann & Robert H. Lurie Children's Hospital of Chicago: Katie Amsden, Mark H. Fishbein, MD; Elizabeth Kirwan, RN; Saeed Mohammad, MD; Ann Quinn, RD (2010–2012); Cynthia Rigsby, MD; Peter F. Whitington, MD
Saint Louis University, St Louis, MO: Sarah Barlow, MD (2002–2007); Jose Derdoy, MD (2007–2012); Ajay Jain MD; Debra King, RN; Pat Osmack; Joan Siegner, RN; Susan Stewart, RN; Dana Romo
University of California, San Diego: Sandra Arroyo; Cynthia Behling, MD, PhD; Jennifer Collins; Janis Durelle; Joel E. Lavine, MD PhD (2002–2010); Michael Middleton, MD, PhD; Kimberly Newton, MD; Melissa Paiz; Jeffrey B. Schwimmer, MD; Claude Sirlin, MD; Patricia Ugalde-Nicalo
University of California, San Francisco: Bradley Aouizerat, PhD; Linda D. Ferrell, MD; Shannon Fleck; Ryan Gill, MD, PhD; Camille Langlois; Emily Rothbaum Perito, MD; Philip Rosenthal, MD; Patrika Tsai, MD
University of Washington Medical Center and Seattle Children's Hospital, Seattle, WA: Kara Cooper; Simon Horslen, MB ChB; Evelyn Hsu, MD; Karen Murray, MD; Randolph Otto, MD; Deana Rich; Matthew Yeh, MD, PhD; Melissa Young
Washington University, St Louis, MO: Elizabeth M. Brunt, MD; Kathryn Fowler, MD
National Cancer Institute, Bethesda, MD: David E. Kleiner, MD, PhD
National Institute of Child Health and Human Development, Bethesda, MD: Gilman D. Grave, MD
National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, MD: Edward C. Doo, MD; Jay H. Hoofnagle, MD; Patricia R. Robuck, PhD, MPH (2002–2011); Averell Sherker, MD
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adolescents; fatty acid; fish; nonalcoholic fatty liver disease; ω-3
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