Intestinal Permeability
Clinical Implications
The Intestinal Epithelial Barrier
The intestinal epithelium is the largest mucosal surface and provides an interface between the external environment and the human’s internal environment. Healthy, mature gut mucosa provide an essential epithelial barrier that permits the absorption of nutrients, electrolytes, and water but restricts the passage of larger, potentially toxic compounds from the lumen into systemic circulation. The intestinal epithelium mediates selective permeability through two major routes: transcellular and paracellular pathways. Transcellular permeability is predominantly regulated by selective transporters for amino acids, electrolytes, short-chain fatty acids, and sugars (Broer 2008, Ferraris 1997, Kunzelmann 2002). The paracellular route is the dominant pathway for passive solute flow across the intestinal epithelial barrier, and its functional state depends on the regulation of intercellular tight junctions (TJ) (Yu 2009). TJ are dynamic, multiprotein complexes that function as a selective/semipermeable paracellular barrier, which facilitates the passage of ions and solutes through the intercellular space while preventing the translocation of luminal antigens, microorganisms, and their toxins.
TJ serve as barriers and selectively regulate the passive diffusion of ions and small water-soluble solutes through the paracellular pathway. They are arranged in strands and are located in the apicolateral membrane of neighbouring epithelial cells, linking cells together (Laukoetter 2006). Several transmembrane proteins located in TJ strands include zona occludens-1 (ZO-1), occludin, tricellulin, junctional adhesion molecule, and the claudin family (Bazzoni 2003, Fanning 1998, Furuse 1993, Furuse 1998, Ikenouchi 2005, Morita 1999). Within the latter group, several members have been demonstrated in detail to specifically control paracellular barrier properties. Whereas some claudins (e.g., claudin-4 and -5) increase barrier function (Amasheh 2005, Van Itallie 2001), others such as claudin-16 induce paracellular channels (Kausalya 2006). TJ are supported by a dense perijunctional ring of actin and myosin that can regulate barrier function. In the presence of an intact epithelial cell layer, the paracellular pathway between cells remains sealed.
Intestinal Permeability
An impaired TJ system compromises the epithelial barrier and leads to a phenomenon called intestinal permeability (IP). An altered barrier function is accompanied by oxidative stress, inflammation, mucosal damage, and aberrant immune responses to antigens (Cereijido 2007, Fasano 2008). When the integrity of the TJ system is compromised, antigens can pass from the intestinal lumen to the gut submucosa through the paracellular pathway. This challenges the mucosal immune system, which in turn produces an immune response that is capable of targeting any organ or tissue (Fasano 2008, Groschwitz 2009).
Diseases Associated with Leaky Gut Syndrome
A fast-growing number of diseases are recognized to involve alterations in IP related to changes in TJ competency, including:
• type 1 diabetes (Mojibian 2009, Simpson 2009, Vaarala 2006);
• celiac disease (Dubois 2008, Duerksen 2005, Pearson 1982);
• inflammatory bowel disease (Heller 2005, Zeissig 2007);
• acute colitis (Boirivant 2008);
• Clostridium difficile-associated colitis (Nusrat 2001);
• irritable bowel syndrome (IBS) (Dunlop 2006, Zhou 2009);
• multiple sclerosis (Westall 2007);
• rheumatoid arthritis (Edwards 2008);
• asthma (Benard 1996, Hijazi 2004);
• liver disease (Cariello 2010, Farhadi 2008);
• atopic dermatitis (Rosenfeldt 2004);
• food allergy and sensitivity (Kalach 2001, Ventura 2006);
• chronic heart failure (Sandek 2007);
• autism (de Magistris 2010, D’Eufemia 1996);
• multiple organ dysfunction syndrome (Doig 1998); and
• post-trauma injury severity (Faries 1998).
TJ are also thought to be involved in cancer development and allergies (Cereijido 2007, Fasano 2001, Shen 2006).
Zonulin
The discovery of Zot, an enterotoxin elaborated by Vibrio cholerae that reversibly opens TJ (Fasano 1991), has increased understanding of the intricate mechanisms that regulate the intestinal epithelial paracellular pathway. Zonulin, a novel human protein analogue to Zot induces TJ disassembly and a subsequent increase in IP (Fasano 2000). Zonulin has been observed to be upregulated in several autoimmune diseases in which TJ dysfunction seems to be the primary defect (Clemente 2003, Drago 2006).
Intestinal Permeability Urine Test
Lactulose to mannitol ratio measurement is one of the methods most widely used to diagnose IP defects (Dastych 2008). It is based on an oral challenge with lactulose and mannitol, two non-metabolized sugar molecules. Both lactulose and mannitol are absorbed as whole molecules by the human small intestine. With a radius of 0.52nm, lactulose is too large for intracellular diffusion and therefore relies on paracellular diffusion for intestinal absorption. The intestinal capacity for absorption of lactulose is a direct measure of the tightness of junctional complexes. Mannitol is much smaller than lactulose with a molecular radius of 0.4nm. It is absorbed readily through mucosal epithelial cell membranes by passive diffusion (transcellular uptake) so its absorption is less dependent on intestinal integrity. Ingestion of lactulose and mannitol simultaneously controls for fluctuations in gastric emptying, intestinal fluid volume, and intestinal transit time, allowing direct measurement of the paracellular absorptive capacity of the gut. Attaining these molecules for measurement is easily performed with a six-hour collection of urine and the ratio between them are used as indicators of IP and mucosal barrier function.
Clinical Therapeutics for the Treatment of Intestinal Permeability
Food Restriction
The relationship between food allergies and IP is unclear. Patients with atopic food allergies have baseline permeability measurements that are higher than control levels according to lactulose to mannitol ratio measurement (Andre 1987). This finding suggests that IP may be a result of food allergy or may play a role in the pathogenesis of food allergy. Therefore, patients experiencing IP may benefit from the identification of hidden food allergies and their subsequent removal from the diet. A clearer relationship has been found between IP and the consumption of gliadin, a glycoprotein within gluten that is found in wheat and some other grains, such as oats, rye, barley, and millet. Gliadin has been shown to increase IP by releasing preformed zonulin (Clemente 2003, Drago 2006). Intestinal cell lines exposed to gliadin released zonulin in the cell medium with subsequent zonulin binding to the cell surface, rearrangement of the cell cytoskeleton, loss of occludin-zonula occludens-1 (ZO1) protein–protein interaction, and increased monolayer permeability (Drago 2006). Therefore, a gliadin-free diet could be an important dietary component of an IP treatment protocol.
Glutamine
Glutamine is the primary source of amino acids for the intestinal mucosa (Windmueller 1982). It is an important energy source for cells of the intestinal mucosa and has been shown to be conditionally essential for normal mucosal structure and function (Klein 1990). Deprivation of glutamine has been shown to decrease claudin-1, occludin, and ZO-1 expression in cell studies (Li 2004) while supplementation has been shown to improve intestinal barrier function in animal models of endotoxin-induced permeability (Dugan 1995). Numerous human clinical trials support these positive findings. In a prospective, double-blind, multiple-center study conducted on 120 patients submitted to major elective abdominal surgery, the addition of alanine-glutamine dipeptide (equivalent to 0.34 grams glutamine/kg/day) for 6 days minimized the intensity of the increase in IP during the postoperative period (Jiang 1999). Similar results were obtained in 20 patients exposed to severe burns. The administration of glutamine dipeptide (equivalent to 0.34 grams glutamine/kg/day) reduced the lactulose/ mannitol ratio by the third day and normalized it by the sixth day. Normal levels were maintained through the twelfth day of treatment (Zhou 2003). Peng (2004) confirmed these results by demonstrating that the accentuated increase in IP of 25 patients exposed to severe burns was reduced by the oral administration of 0.5 grams glutamine/kg/day. Finally, the administration of glycyl-L-glutamine (equivalent to 0.23 grams glutamine/kg/ day) prevented exacerbation of the increase in IP in patients with chronic intestinal inflammatory disease or with intestinal neoplasias. All patients who received total parenteral nutrition without the addition of glutamine experienced an increase in IP (van der Hulst 1993).
Polyunsaturated Fatty Acids
The integrity of intestinal barrier function is substantially affected by the fatty acid profile of membrane phospholipids (Zhao 2008). The proportion of saturated to unsaturated fatty acids and the ratio between omega-6 and omega-3 polyunsaturated fatty acids (PUFA) exert a considerable effect on membrane fluidity, eicosanoid synthesis, mucosal health, and epithelial barrier function (Calder 2008). The effects of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), and gamma-linolenic acid (GLA) on IP have been reported in a number of studies. EPA has been found to be effective in supporting barrier integrity (Willemsen 2008), while improving TJ permeability through an increased expression of occludin (Jiang 1998, Yamagata 2003). DHA has also been found to be effective in supporting barrier integrity (Willemsen 2008), while affecting TJ permeability in a concentration-dependent manner (Usami 2003), increasing intestinal absorption (Kajita 2000, Usami 2003), and protecting against increased IP caused by methotrexate administration (Horie 1998). GLA has been found to improve TJ permeability associated with an upregulation of occludin (Jiang 1998, Yamagata 2003) while also affecting TJ permeability in a concentrationdependent manner (Usami 2003). When Usami (2003) combined their results with previous results, the effects of three identical dosages of PUFA on paracellular permeability was noted to be in the order of GLA ≥ EPA ≥ DHA (Usami 2001).
Probiotics
Probiotic bacteria can directly alter epithelial barrier function by influencing the structure of TJ. A cell study found that S. thermophilus and L. acidophilus independently increased transepithelial resistance (a sensitive measure of mucosal barrier), decreased permeability, and induced activation of occludin and ZO-1 (Resta-Lenert 2003). Similarly, conditioned medium from several bacteria strains were found to independently increase transepithelial resistance. B. infantis exerted the biggest effect, decreasing claudin-2 protein expression and increasing ZO-1 and occludin total protein expression (Ewaschuk 2008). An animal in vivo study demonstrated increased ZO-1 expression upon colonization with E. coli Nissle 1917 (Ukena 2007), a probiotic sold in Europe to prevent infectious diarrhea and treat functional bowel disorders (Zyrek 2007). Pretreatment with the E. coli strain significantly reduced dextran sulfate sodium-mediated dye uptake into the colonic mucosa, which indicates reduced IP. Increased permeability of the epithelial barrier can also be caused by apoptosis (Chin 2002) and probiotics have been found to modulate apoptosis initiation by harmful stimuli. S. boulardii pretreatment prevented enterohaemorrhagic E. coli-induced apoptosis in a cell study (Dalmasso 2006) and a cell study found that two proteins (p40 and p75) secreted from L. rhamnosus inhibited cytokineinduced apoptosis (Yan 2007). Additionally, apical or basolateral pretreatment with either p40 or p75 protected several cell lines from H2O2-induced disruption of barrier function, as measured by transepithelial resistance and paracellular permeability (Seth 2008).
The ability of probiotics to improve IP has been investigated in a number of human clinical trials. A randomized singleblind, placebo-controlled study in 30 IBS patients found that a probiotic- fermented milk containing L. acidophilus, B. longum, and other lactic acid bacteria decreased small bowel permeability (Zeng 2008). In addition, a double-blind, placebo-controlled, crossover study found that six weeks of treatment with a probiotic supplement containing L. rhamnosus 19070-2 and L. reuteri DSM 12246 decreased the lactulose to mannitol ratio in 41 children with atopic dermatitis, increased IP, and gastrointestinal symptoms (Rosenfeldt 2004).
Zinc
Zinc is a trace element that is essential for the survival and function of cells as an important component of DNA polymerase and other enzymes involved in cell replication and differentiation. Dietary zinc appears to play a critical role in the maintenance of normal IP and control of inflammation. Zinc deficiency has been shown to cause ulcerations of the small intestine (Mengheri 1999) and disrupt mucosal barrier function by inducing a decrease in transepithelial resistance and alterations of TJ, specifically with delocalization of ZO-1, occludin and the transmembrane proteins, beta-catenin, and E-cadherin (Finamore 2008). There are also preliminary human data showing the benefit of zinc supplementation in helping to repair intestinal integrity. In a randomized crossover trial, 10 patients were given 37.5 mg of prophylactic zinc carnosine to determine if this would prevent the loss of intestinal integrity when administered indomethacin (Mahmood 2007). No loss of intestinal integrity was reported in those receiving zinc, while controls experienced a three to four–fold increase in IP. The zinc-treated group also had 75% reduction in gastric and small bowel injury and 50% less villous shortening.
Potential Therapeutic Agents
Quercetin (Suzuki 2009), a naturally occurring flavonoid, and phosphatidylcholine (Mitzscherling 2009), an abundant phospholipid in the plasma, have been shown to enhance intestinal barrier functions in human cells. In addition, N-acetyl- L-cysteine (NAC), an antioxidant, detoxifier, and precursor for glutathione synthesis, has been shown to prevent increased IP following intestinal ischemia and reperfusion in animals (Sun 2002).
Given the evidence, reinforcing the intestinal barrier, and more particularly the paracellular pathway, may be an excellent therapeutic strategy to treat or prevent diseases driven by luminal antigens.
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