Intestinal Permeability in Human Nonalcoholic Fatty Liver Disease

A Systematic Review and Meta-analysis

Toon J. I. De Munck; Pan Xu; Harm J. A. Verwijs; Ad A. M. Masclee; Daisy Jonkers; Jef Verbeek; Ger H. Koek

Disclosures

Liver International. 2020;40(12):2906-2916. 

In This Article

Discussion

In this systematic review, the association between in vivo IP and NAFLD and its severity was evaluated, based on eight studies in paediatric and six in adult NAFLD patients. In this study, we demonstrated that NAFLD patients have an increased IP compared to HC. Furthermore, we observed a positive correlation between IP and the degree of hepatic steatosis, while no clear association between IP and the presence of NASH, hepatic inflammation or fibrosis was demonstrated.

In the present systematic review, we included both studies in paediatric and in adults NALFD patients. Although paediatric NAFLD shows some different characteristic as compared to adults patients, such as histological features and progression rate to hepatic cirrhosis or HCC, they also show large overlap as both are associated with the metabolic syndrome, central obesity, dysregulated glucose metabolism, dyslipidemia, cardiovascular diseases and have similar genetic risk factors (eg PNPLA3 and GCKR).[25] In addition, gut microbiome dysbiosis, metabolic endotoxemia and systemic inflammation are considered to play a role in paediatric and adult NAFLD development.[26] To account for possible differences between adult and paediatric patients, subgroup analysis by age was performed and discussed below.

First, we observed that NAFLD patients had increased small intestinal permeability by means of L/M, L/R (seven studies) or serum zonulin levels (four studies) compared to HC. IP was also found to be increased in NAFLD compared to controls when only considering studies that matched for BMI or where BMI did not differ between groups. This indicates that the presence of NAFLD, independent of BMI, is associated with an increased IP. Furthermore, we observed a more prominent increase in small intestinal permeability by means of L/M in paediatric NAFLD patients compared to adult NAFLD patients. IP in NAFLD patients is believed to be influenced by, among others, microbiome perturbations, faecal short chain fatty (SCFA) acids levels and endogenous alcohol production.[27] Since microbiome composition is different in children compared to adults, altered production of SCFAs and alcohol by the intestinal microbiota and difference in IP are expected. In line, butyrate and propionate were observed to be enriched in faecal samples from adult NAFLD patients, while formate, acetate and valerate, were less abundant whereas butyrate and propionate were unaffected in faecal samples from paediatric NAFLD patients.[28,29] As IP was often investigated in a singular paediatric or adult study, subgroup analysis by age is not desirable. Therefore, future studies are needed to investigate differences in IP between adult and paediactric NAFLD patients.

Evidence is less convincing when comparing NAFL and NASH patients as investigated in six studies. In the pooled analysis, an increased small intestinal permeability was found by L/M (three studies) but not zonulin (three studies). It should be noted that results need to be interpreted with care because of substantial heterogeneity between studies for both parameters. Furthermore the number of study subjects in both adults or paediatric studies is very low. In previous studies, IP has also been associated with several metabolic abnormalities including obesity, dyslipidaemia and hyperglycaemia.[5] In NAFLD patients, increased IP is believed to induce hepatic steatosis, inflammation and fibrosis via translocation of bacterial products from the gut to the liver.[4]

Furthermore, we investigated the association between IP and NAFLD severity. We demonstrated that small intestinal permeability increases with the degree of hepatic steatosis while no association with hepatic inflammation, ballooning or fibrosis was observed in the included studies. Interestingly, it cannot be excluded that an increased IP is more important in the development of hepatic steatosis than of hepatic inflammation or fibrosis. Based on experimental and cross-sectional data, not only an association of increased IP with the degree of hepatic steatosis but also with hepatic inflammation and fibrosis was expected. The development of hepatic fibrosis and inflammation is believed to be triggered by bacterial translocation from the gut to the liver, however, a causal link has not been proven.[4,6,28] Mechanistically, translocation of bacterial products (eg LPS), leads to activation of toll-like receptor 4 in the liver and results in hepatic inflammation and fibrogenesis.[4,6,28] The amount of NAFLD subjects with significant hepatic fibrosis or inflammation in the included studies is relatively low what may explain why no association between IP and hepatic fibrosis or inflammation was found. Furthermore, most studies included in this review focus on small intestinal permeability, while colon permeability was not investigated. Microbiome perturbation in the colon have been associated with NAFLD presence and severity and are believed to harm the integrity of the gut barrier.[28,30] In mice, high fat diet feeding has been observed to induce metabolic abnormalities, systemic and liver inflammation which was accompanied by an increased colon permeability.[31] Furthermore, Pijls et al observed an increased colon permeability in patients with stable compensated cirrhosis compared to healthy controls while gastroduodenal and small IP were not altered.[32] Possibly colon permeability is linked to the degree of hepatic inflammation and fibrosis in NAFLD patients while small intestinal permeability is not. Therefore, future studies should also focus on the association between colon permeability and hepatic fibrosis and inflammation.

We identified only two human studies evaluating whole gut permeability in NAFLD patients.[12,21] In the study of Miele et al whole gut permeability was increased in adult NAFLD patients compared to HC and was associated with more advance steatosis but not with hepatic inflammation, ballooning or fibrosis.[12] In the study of Farhadi et al no association between whole gut permeability and NAFLD presence or severity was observed.[21] More research is needed to elucidate the role of whole gut permeability in NAFLD patients.

Finally, we wanted to elucidate clinical factors that correlated with IP in NAFLD. However, data on this topic are scarce. Associations between small intestinal permeability and serum liver function tests, pro-inflammatory cytokines, and metabolic factors are underinvestigated in the NAFLD population. In the general population, elevated levels of pro-inflammatory markers, dyslipidaemia, hyperglycaemia, insulin resistance, anthropometric measurements resembling obesity and the consumption of a Western-style diet have been identified as confounding factors for IP.[5] These factors may vary between different chronic diseases and because of the limited data confounding factors for increased IP in NAFLD patients remain to be identified.

When comparing study results, IP test characteristics must be taken in mind. Urinary recovery of enteral administrated non-digestible markers (different sugars or 51Cr-EDTA) are widely used to assess IP at different sites of the gastrointestinal tract (depending on type of marker and collection time). Duals sugar tests (eg L/M and L/R) have greater clinical value than the administration of one marker alone (sucralose or 51Cr-EDTA) as they are less influenced by differences in renal function, intestinal transit time or gastric emptying time between study subjects. Recently serum zonulin has emerged as a marker to assess the small intestinal epithelial integrity. However, Ohlsson et al suggest that serum zonulin might rather be a biomarker for low-grade inflammation than for IP, because zonulin is identical to prehaptoglobin-2, not enterocyte specific and associated with overweight, obesity and hyperlipidemia.[10,11,33,34] Furthermore, in the study of Linsalata et al serum zonulin did not correlate with the L/M but did correlate with serum IL-6 and serum IL-8 concentrations in 91 subjects (39 irritable bowel syndrome, 32 coeliac disease and 20 HC). Finally, to date zonulin is the only known regulator of intestinal tight junction but it is likely that other zonulin unrelated pathways are also important in this process. Caution must be taken when using serum zonulin as a biomarker for small IP. Therefore, studies using zonulin as marker for small intestinal IP were analysed separately.[11]

This systematic review has some limitations. Firstly, because of the observational nature of all included studies in this systematic review only associations and not causalities were investigated. Secondly, substantial inter-study heterogeneity was noted in most analyses. In this review, only studies investigating in vivo IP by means of urinary excretion of orally administered substances or serum zonulin levels were included. Studies using circulating LPS levels, the major component of the outer membrane of Gram-negative bacteria, as marker for IP were not included as circulating LPS measurements are not site-specific and have a high false-positive rate.[35] Thirdly, because of the small number of studies included in the meta-analysis the presence of publication bias cannot be ruled out and subgroup analysis is not desirable. Finally, only 14 studies were included, which were small in terms of sample size, focused on both paediatric and adult NAFLD and most of them had poor quality.

In conclusion, small intestinal permeability appears to be increased in NAFLD patients compared to healthy controls and appears to be positively associated with the degree of hepatic steatosis. However, included studies where small in sample size, had poor quality and showed high heterogeneity. To date, no clear evidence is available that small intestinal or whole gut permeability increases with NAFLD severity (presence of NASH, hepatic inflammation or fibrosis). Future studies should also focus on colonic permeability in NAFLD patients.

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