Nonalcoholic steatohepatitis (NASH) is a serious liver disease associated with obesity. Characterized by metabolic syndrome, hepatic steatosis, and liver inflammation, NASH is believed to be under the influence of the gut microflora. Here, the composition of gut bacterial communities of NASH, obese, and healthy children was determined by 16S ribosomal RNA pyrosequencing. In addition, peripheral blood ethanol was analyzed to monitor endogenous ethanol production of patients and healthy controls. UniFrac-based principle coordinates analysis indicated that most of the microbiome samples clustered by disease status. Each group was associated with a unique pattern of enterotypes. Differences were abundant at phylum, family, and genus levels between healthy subjects and obese patients (with or without NASH), and relatively fewer differences were observed between obese and the NASH microbiomes. Among those taxa with greater than 1% representation in any of the disease groups, Proteobacteria, Enterobacteriaceae, and Escherichia were the only phylum, family and genus types exhibiting significant difference between obese and NASH microbiomes. Similar blood-ethanol concentrations were observed between healthy subjects and obese non-NASH patients, but NASH patients exhibited significantly elevated blood ethanol levels. Conclusions: The increased abundance of alcohol-producing bacteria in NASH microbiomes, elevated blood-ethanol concentration in NASH patients, and the well-established role of alcohol metabolism in oxidative stress and, consequently, liver inflammation suggest a role for alcohol-producing microbiota in the pathogenesis of NASH. We postulate that the distinct composition of the gut microbiome among NASH, obese, and healthy controls could offer a target for intervention or a marker for disease. (HEPATOLOGY 2013;57:601-609) N onalcoholic fatty liver disease (NAFLD), the hepatic manifestation of metabolic syndrome, is the most common cause of elevated liver enzymes in the United States.1 NAFLD with inflammation and fibrosis is known as nonalcoholic steatohepatitis (NASH) because it resembles alcoholic liver disease (ALD) without a history of alcohol ingestion. The incidence of NASH has been increasing over the past 20 years. 3 In the United States, the current prevalence of NAFLD and NASH could be as high as 46% and 12%, respectively. 4 Without an effective available treatment, the prognosis of NASH is not optimistic. NASH is responsible for approximately 10% of liver transplants in the United States and is projected to become the most common indication for liver transplantation in the near future.

Previous studies using culture-based methods suggested an association between constipation and altered abundance of certain taxa of the colonic microbiome. We aim to examine the global changes in gut microbial composition of constipated patients. A cross-sectional pilot study using 16S rRNA gene pyrosequencing was performed to compare stool microbial composition of eight constipated patients and 14 nonconstipated controls. Only obese children were enrolled so that the microbiome features associated with constipation would not be obscured by those associated with obesity. The sequencing reads were processed by QIIME for quantitative analysis of the microbial composition at genus and above levels. Dietary intake for all the individuals was assessed by dietary recalls and a food frequency questionnaire. The ecological diversities of fecal microbiome of the constipated patients differed from those of the controls. Significantly decreased abundance in Prevotella and increased representation in several genera of Firmicutes were observed in constipated patients compared with controls. The conventional probiotic genera Lactobacillus and Bifidobacteria were not decreased in the microbiomes of the constipated patients. These alterations in the fecal microbiome of constipated patients suggested that a novel probiotic treatment including certain Prevotella strains may be more effective than conventional probiotic products incorporating Lactobacillus or Bifidobacterium species. While it is possible that the observed changes in the microbiome in constipated subjects are a consequence of a low-fiber diet, these changes also predict a different pattern of bacterial fermentation end-products, such as increased butyrate production, which may contribute to pathogenesis of constipation.

We conclude that vitamin B12 and folate deficiencies are rare in children with newly diagnosed IBD in the United States and we question whether routine monitoring is warranted. Vitamin A and zinc deficiency are common in patients with newly diagnosed IBD and levels should be assessed at the time of diagnosis so that enteral repletion can commence. Vitamin D deficiency is common in all of the children in the Buffalo, NY, area, and routine screening for this deficiency is warranted.

The exact pathophysiology of non-alcoholic steatohepatitis (NASH) is not known. Previous studies suggest that dietary advanced glycation end products (AGEs) can cause oxidative stress in liver. We aim to study the effects of dietary AGEs on liver health and their possible role in the pathogenesis of NASH. METHODS: Two groups of mice were fed the same diet except the AGE content varied. One group was fed a high AGE diet and the second group was fed a regular AGE diet. Liver histology, alanine aminotransferase, aspartate aminotransferase, fasting glucose, fasting insulin, insulin resistance and glucose tolerance were assessed. RESULTS: Histology revealed that neutrophil infiltration occurred in the livers of the high AGE group at week 26; steatosis did not accompany liver inflammation. At week 39 livers from both groups exhibited macro- or micro-steatosis, yet no inflammation was detected. Higher insulin levels were detected in the regular AGE group at week 26 (P = 0.034), compared to the high AGE group. At week 39, the regular AGE group showed higher levels of alanine aminotransferase (P<0.01) and aspartate aminotransferase (P = 0.02) than those of the high AGE group. CONCLUSIONS: We demonstrate that a high AGE diet can cause liver inflammation in the absence of steatosis. Our results show that dietary AGEs could play a role in initiating liver inflammation contributing to the disease progression of NASH. Our observation that the inflammation caused by high AGE alone did not persist suggests interesting future directions to investigate how AGEs contribute to pro-oxidative and anti-oxidative pathways in the liver.

Our data suggest that the gut microbiome composition does not contribute to the incidence of endotoxemia in NASH, and endotoxemia is not required in the pathogenesis of NASH. Our observations highlight the current concept that multiple factors contribute to the development of NASH.