Several studies have provided evidence that gut-derived bacterial products are the necessary cofactors for development of ALD in a minority of alcoholics (6
). For example, alcoholics with ALD have high serum endotoxin levels, and serum endotoxin levels correlate with ALD severity (26
). Peripheral monocytes from ALD patients are primed for producing cytokines and oxidants (reactive oxygen and nitrogen species) and this priming seems to be due to endotoxin (27
). Still others showed that alcohol fed rats with liver damage have high endotoxin levels in the portal vein, and there is a strong correlation between endotoxin levels and the severity of liver damage in these rats (30
Indeed, it is well-established that alcohol exerts its damaging effect on the liver in synergy with endotoxin. For example, neither alcohol alone nor endotoxin alone (at least not low endotoxin levels) cause severe liver injury, but a combination of these two agents was sufficient to cause significant liver damage. Endotoxin can prime and activate macrophages (Kupffer cells) in rats chronically exposed to EtOH so that they overproduce cytokines such as TNF-α, IL-6, and IL-8 (8
). These cytokines not only injure hepatocytes directly, but also, they can initiate a hepatic necroinflammatory cascade, which includes migration of leukocytes, including neutrophils, into the liver (15
). These leukocytes produce injurious products, especially oxidants resulting from nitric oxide (16
) such as peroxynitrite, which can cause liver cell necrosis. The EtOH-endotoxin synergy, along with other direct metabolic effects of EtOH on the liver (e.g., hypoxia, perturbation of NO-dependent pathways), not only can initiate liver injury, but also, can create a vicious circle that sustains a chronic necroinflammatory process and hastens the onset of liver failure.
Nevertheless, these studies have not ruled out the possibility that endotoxemia is a consequence of and not a prerequisite for ALD. The finding that lowering serum endotoxin level by oral administration of non-absorbable antibiotics (7
) or lactobacillus (6
) attenuates alcohol-induced liver damage in alcohol fed rats support the idea that endotoxin is key for promoting severe liver injury but does not provide direct evidence for endotoxin as a trigger for ASH. Our time course study, for the first time, demonstrates that endotoxemia precedes ASH and thus supports the view that endotoxin is not the consequence of liver disease and could act as a trigger for development of ASH. Our finding that fatty liver occurs early and before significant endotoxemia supports the hypothesis that simple steatosis is primarily due to the direct effects of alcohol on hepatic lipid metabolism and is not dependent on endotoxin (32
). Unlike steatosis, steatohepatitis appears to be dependent on endotoxin. Although the mechanism of endotoxemia in alcoholics is not fully established, it is clear that the source of the endotoxin is the gut flora. Endotoxin, which is generated by bacteria in the gut lumen, permeates into the portal circulation and then travels to the liver where it is usually taken up and eliminated by Kupffer cells. Hence, abnormally high blood endotoxin levels (endotoxemia) could be due to: (i) increased production of endotoxin by small bowel bacterial overgrowth or abnormal gut flora (dysbiosis), (ii) increased permeation of endotoxin through the intestinal wall (gut leakiness), (iii) reduced liver clearance due to portal blood shunting that occurs in advanced liver disease and portal hypertension, and/or (iv) reduced endotoxin clearance due to defective liver Kupffer cell function. Gut leakiness may have a particularly large effect because it can potentiate the other three mechanisms. This could happen because integrity of the intestinal barrier is a gate keeper. Changes in the integrity of the intestinal barrier can blunt or accentuate the effect of the other possible mechanisms of endotoxemia.
Both small bowel and colonic bacteria can be the source of endotoxemia. Thus, it is important to assess both small bowel and colonic permeability to determine whether gut leakiness is the source of endotoxemia. Unfortunately, there is no universally accepted method to assess in vivo
intestinal permeability. But, use of poorly absorbed sugars to assess small and large bowel permeability has now been validated in several studies in both healthy and disease states (33
). Mannitol (M) and lactulose (L) are poorly absorbed in the small bowel and their concentration in the urine (or L/M ratio) is a valid marker of small bowel leakiness. Since both lactulose and mannitol are extensively metabolized by colonic bacteria, they are not appropriate substrates to assess colonic permeability. In contrast, a poorly absorbed sugar, sucralose, is not metabolized by bacteria and thus is an appropriate marker for assessing total gut (small bowel and colon) permeability (35
We used the sugar test to address the question whether gut leakiness is involved in alcohol-induced endotoxemia and if so, where is the site of leakiness in the gut. We now report that gut leakiness indeed occurs after daily alcohol feeding and the time course of gut leakiness and endotoxemia suggests that gut leakiness is, at least in part, responsible for endotoxemia in alcohol fed rats. Our current finding confirms our prior observation in alcoholic subjects (10
) and alcohol-fed rats (11
). Gut leakiness as the source of endotoxemia in alcoholics was also suggested by others (15
). Indeed, other reports show that alcohol consumption substantially disturbs intestinal mucosal structure and function in both man and animals (18
). Our time course study provides yet another and more direct evidence for the importance of gut leakiness in alcohol-induced endotoxemia. We also report, for the first time, that alcohol disrupts barrier function throughout the gut. The deleterious effect of alcohol on small bowel barrier function (increased urinary lactulose) appears to be more rapid and occurs after only 2 weeks of exposure. In contrast, longer exposure to daily alcohol is required for disruption of colonic barrier (increased urinary sucralose). It is interesting that the magnitude of endotoxemia in the first 4 weeks of alcohol feeding, when the small bowel was leaky, was modest; while endotoxemia was more severe in the second month (4–8 weeks) of alcohol exposure when total gut leakiness was present. This finding is compatible with permeation of the high endotoxin content of the colon. But, the contribution of defective clearance of endotoxin by an injured liver should also be considered. Further studies are needed to determine the mechanism for the differences of time courses between disruption of small bowel and colonic barrier function.
How alcohol causes gut leakiness is not yet established. The integrity of intestinal barrier function depends on both healthy epithelial cells and an intact paracellular pathway, which appears to be the main route for permeation of macromolecules such as endotoxin (33
). Oxidative injury to key proteins regulating this paracellular pathway is a plausible mechanism because oxidative stress is responsible for alcohol-induced tissue injury and organ failure (40
). More specifically, alcohol increases oxidative stress and we (16
) and others (45
) have shown that oxidative stress disrupts the monolayer barrier. Our data now support the key contribution of tissue oxidative injury in alcohol-induced gut leakiness. Our data also suggest that alcohol–induced oxidative injury to the gut is a result of upregulation of iNOS. Upregulation of iNOS causes overproduction of NO and then peroxynitrite and superoxide. NO can generate superoxide via a process called NOS uncoupling that refers to uncoupling of NADPH oxidation as well as by disruption of cytochrome C function (47
). Eventually peroxynitrite and superoxide anion cause nitration and carbonylation of key proteins responsible for integrity of barrier resulting in gut leakiness. Our in vitro
) suggest the following cascade of events: alcohol activates NF-kB resulting in upregulation of iNOS. Increased iNOS increases NO production and high levels of peroxynitrite and superoxide resulting in nitration and carbonylation of tight junctional and cytoskeletal proteins. This oxidative damage to these key proteins disrupts integrity of intestinal barrier. Our rat model can not differentiate between luminal versus bloodstream effects of alcohol on the gut. But, since both apical and basolaterally applied alcohol promote monolayer hyperpermeability, one can conclude that both local and the systemic effects of alcohol could be involved in gut leakiness.
In summary, our findings provide the first evidence that alcohol-mediated gut leakiness and endotoxemia precede the development of alcoholic liver disease and thus might be the necessary trigger for development of ASH. Further studies are needed to determine whether inhibition of iNOS upregulation in alcohol fed rats prevents gut leakiness, endotoxemia and steatohepatitis. Controlled clinical trials in alcoholics to see if interventions that prevent gut leakiness and endotoxemia prevent ALD must wait until an effective intervention to prevent gut leakiness is developed. We acknowledge that the best approach to prevent ALD is abstinence but unfortunately the long term success of maintaining sobriety is very poor. Thus, alternative approaches such as preventing gut leakiness would still have a positive impact in patients’ quality of life of sober alcoholics at risk for or suffering from ALD.