This is the first study to investigate the effects of probiotic treatment on obesity-related liver abnormalities in children. The study was designed as a short-term pilot trial to circumvent the frequently reported unpredictable effects of lifestyle changes that usually confound the results of a long-term study in this population. In fact, even minimal weight and lifestyle changes may affect the biochemical parameters of NAFLD (4–6). Here we show that a short course of probiotic treatment significantly improved ALT values, which in most cases became normal, and that this effect was independent of weight changes.
Small intestinal bacterial overgrowth has been implicated in the pathogenesis of NAFLD (11–13). The results of the glucose H2BT, which is a surrogate test for SIBO evaluation, in our children did not confirm the increased prevalence of SIBO reported in morbidly obese adults (12). However, the level of PG-PS IgA antibodies, another surrogate for SIBO, was significantly higher than that of a group of 20 age-, sex-, and weight-matched obese nonhepatopathic controls (Table 1), and moreover they significantly decreased only in the children treated with probiotics and not in the placebo-treated group. Because PG-PS polymers are integral cell wall components of most bacterial species, including intestinal Bacteroides, which are the major determinants of SIBO (24,25), the decrease of PG-PS IgA antibodies after treatment may imply abnormalities in the patients' small intestinal microflora. Wigg et al (11) found that levels of endotoxin (ie, another SIBO marker) did not differ between patients with NAFLD and healthy controls. In a more recent work, endotoxin levels in the peripheral circulation were increased in patients with NAFLD, but they were unrelated to disease severity (26). Given the differences between peripheral and portal blood values and potential methodological difficulties caused by the short half-life of endotoxins, data obtained from the measurement of serum endotoxin levels should be interpreted with caution (11,26). For this reason, we did not measure endotoxin levels in the present study.
TNF-α is believed to be one of the protagonists of the inflammatory response in NAFLD after stimulation via Toll-like receptors by endotoxins, which reach the liver via portal flow through a more permeable intestinal barrier. However, similar to a previous study (11), the TNF-α values in our hepatopathic obese patients overlapped those of the 20 age- and weight-paired obese individuals. They tended toward a mild decrease in both treatment arms without, however, reaching statistical significance. Lactobacillus GG–induced modulation on studied parameters involved in NAFLD pathogenesis will probably require long-term studies. The effects of probiotics on NAFLD have been evaluated in animal models and, although to a lesser extent, in human adults. Probiotic VSL#3 treatment in NAFLD ob/ob mice lowered the transaminase level and affected histological liver inflammation without modifying steatosis (14). The same treatment in mice with diet-induced NAFLD attenuated fibrosis without affecting steatohepatitis (15,27). The unchanged US hepatorenal ratios in our patients are in line with these data. Another study showed that probiotics had a beneficial effect on several types of human liver disease including NAFLD, probably acting on the steatosis component (16). Contrary to what may be expected from the results of animal studies, a recent preliminary report on human NAFLD found a reversible increase in magnetic resonance imaging–calculated fat liver content during probiotic treatment (17). However, details of possible simultaneous weight changes were not reported. Given the above, individuals enrolled in such studies should undergo accurate anthropometric monitoring to ensure that other factors do not confound the effect of treatment on the outcome.
Our results do not shed light on the mechanism(s) underlying the improvement of our patients' ALT values. Recent evidence indicates that there are quantitative (ie, SIBO) and qualitative differences in gut microbiota and microbiome between lean and obese individuals (28). The fact that our patients were completely asymptomatic from a gastrointestinal viewpoint may argue against the existence of a true SIBO. Because positive H2BTs in the exceedingly large percentage of Wiggs' patient series has been questioned by several investigators (29), it is feasible that in our study, Lactobacillus GG exerted a beneficial effect on obesity-related liver disease by modulating an altered bacterial composition rather than reducing its quantity (30). However, methodological aspects may have affected the results of the glucose H2BT in detecting SIBO; namely, the dose of the sugar or the duration of the test may not have been sufficient to exclude a bacterial contamination restricted to the distal small bowel. (21) Future large-scale intervention trials investigating the potential benefit of probiotics should include a combination of gastrointestinal dysfunction tests, including lactulose H2BT and intestinal permeability studies.
In view of the excellent tolerance and the remarkable reduction of transaminase after probiotics in our double-blind, placebo-controlled, short-term pilot study, Lactobacillus rhamnosus strain GG could be considered a potential therapeutic tool for pediatric obesity-related liver disease in children who are unable to lose weight. Our promising data provide a rationale for starting a larger study on possibly biopsy-proven pediatric NAFLD.
We are grateful to Patrizia Colicchio, MD, and Brunella Aceto, dietician, for assistance in the clinical management of patients; Maria Passaretti, PhD, and Luciano Rapacciuolo, PhD, for laboratory assistance; and Francesco Manguso, MD, for critical evaluation of the manuscript. We are indebted to Jean Ann Gilder (Scientific Communication srl) for editing the text. Lactobacillus rhamnosus strain GG (ATCC 53103) with verified composition and indistinguishable placebo were supplied by Dicofarm SpA, Rome, Italy.
1. Yanovski SZ, Yanovski JA. Obesity prevalence in the United States—up, down, or sideways? N Engl J Med 2011; 364:987–989.
2. Younossi ZM, Stepanova M, Afendy M, et al. Changes in the prevalence of the most common causes of chronic liver diseases in the United States from 1988 to 2008. Clin Gastroenterol Hepatol
. 2011 Apr 11 [Epub ahead of print].
3. Schwimmer JB, Deutsch R, Kahen T, et al
. Prevalence of fatty liver in children and adolescents. Pediatrics 2006; 118:1388–1393.
4. Franzese A, Vajro P, Argenziano A, et al
. Liver involvement in obese children. Ultrasonography and liver enzyme levels at diagnosis and during follow-up in an Italian population. Dig Dis Sci 1997; 42:1428–1432.
5. St George A, Bauman A, Johnston A, et al
. Effect of a lifestyle intervention in patients with abnormal liver enzymes and metabolic risk factors. J Gastroenterol Hepatol 2009; 24:399–407.
6. Nobili V, Alisi A, Raponi M. Pediatric non-alcoholic fatty liver disease: preventive and therapeutic value of lifestyle intervention. World J Gastroenterol 2009; 15:6017–6022.
7. Bellentani S, Scaglioni F, Marino M, et al
. Epidemiology of non-alcoholic fatty liver disease. Dig Dis 2010; 28:155–161.
8. Caldwell S, Argo C. The natural history of non-alcoholic fatty liver disease. Dig Dis 2010; 28:162–168.
9. Musso G, Gambino R, Cassader M, et al
. A meta-analysis of randomized trials for the treatment of nonalcoholic fatty liver disease. Hepatology 2010; 52:79–104.
10. Socha P, Horvath A, Vajro P, et al
. Pharmacological interventions for nonalcoholic fatty liver disease in adults and in children: a systematic review. J Pediatr Gastroenterol Nutr 2009; 48:587–596.
11. Wigg AJ, Roberts-Thomson IC, Dymock RB, et al
. The role of small intestinal overgrowth, intestinal permeability, endotoxaemia, and tumor necrosis factor α in the pathogenesis of non alcoholic steatohepatitis. Gut 2001; 48:206–211.
12. Sabaté JM, Jouët P, Harnois F, et al
. High prevalence of small intestinal bacterial overgrowth in patients with morbid obesity: a contributor to severe hepatic steatosis. Obes Surg 2008; 18:371–377.
13. Miele L, Valenza V, La Torre G, et al
. Increased intestinal permeability and tight junction alterations in nonalcoholic fatty liver disease. Hepatology 2009; 49:1877–1887.
14. Li Z, Yang S, Lin H, et al
. Probiotics and antibodies to TNF inhibit inflammatory activity and improve nonalcoholic fatty liver disease. Hepatology 2003; 37:343–350.
15. Esposito E, Iacono A, Bianco G, et al
. Probiotics reduce the inflammatory response induced by a high-fat diet in the liver of young rats. J Nutr 2009; 139:905–911.
16. Loguercio C, Federico A, Tuccillo C, et al
. Beneficial effects of a probiotic VSL#3 on parameters of liver dysfunction in chronic liver diseases. J Clin Gastroenterol 2005; 39:540–543.
17. Solga SF, Buckley G, Clark JM, et al
. The effect of a probiotic on hepatic steatosis. J Clin Gastroenterol 2008; 42:1117–1119.
18. Sabir N, Sermez Y, Kazil S, et al
. Correlation of abdominal fat accumulation and liver steatosis: importance of ultrasonographic and anthropometric measurements. Eur J Ultrasound 2001; 14:121–128.
19. Vajro P, Fontanella A, Perna C, et al
. Persistent hyperaminotransferasemia resolving after weight reduction in obese children. J Pediatr 1994; 125:239–241.
20. Mancini M, Prinster A, Annuzzi G, et al
. Sonographic hepatic-renal ratio as indicator of hepatic steatosis: comparison with (1)H magnetic resonance spectroscopy. Metabolism 2009; 58:1724–1730.
21. Gasbarrini A, Corazza GR, Gasbarrini G, et al
. Methodology and indications of H2-breath testing in gastrointestinal diseases: the Rome Consensus Conference. Aliment Pharmacol Ther 2009; 29(suppl 1):1–49.
22. Hazenberg MP, de Visser H, Bras MJ, et al
. Serum antibodies to peptidoglycan-polysaccharide complexes from the anaerobic intestinal flora in patients with Crohn's disease. Digestion 1990; 47:172–180.
23. Schrijver IA, De Man YA, Melief MJ, et al
. Reduced systemic IgG levels against peptidoglycan in rheumatoid arthritis (RA) patients. Clin Exp Immunol 2001; 123:140–146.
24. Lichtman SN, Keku J, Schwab JH, et al
. Evidence for peptidoglycan absorption in rats with experimental small bowel bacterial overgrowth. Infect Immun 1991; 59:555–562.
25. Lumsden AB, Henderson JM, Kutner MH. Endotoxin levels measured by a chromogenic assay in portal, hepatic and peripheral venous blood in patients with cirrhosis. Hepatology 1988; 8:232–236.
26. Harte AL, da Silva NF, Creely SJ, et al
. Elevated endotoxin levels in non-alcoholic fatty liver disease. J Inflamm (Lond) 2010; 7:15.
27. Velayudham A, Dolganiuc A, Ellis M, et al
. VSL#3 probiotic treatment attenuates fibrosis without changes in steatohepatitis in a diet-induced nonalcoholic steatohepatitis model in mice. Hepatology 2009; 49:989–997.
28. Turnbaugh PJ, Gordon JI. The core gut microbiome, energy balance and obesity. J Physiol 2009; 587:4153–4158.
29. Riordan SM, Duncombe VM, Thomas MC, et al
. Small intestinal bacterial overgrowth, intestinal permeability, and non-alcoholic steatohepatitis. Gut 2002; 50:136–138.
30. Abu-Shanab A, Quigley EM. The role of the gut microbiota in nonalcoholic fatty liver disease. Nat Rev Gastroenterol Hepatol 2010; 7:691–701.