Much of the epithelial barrier is formed by the rigid lipid bilayer of the enterocyte brush border. As in most cell membranes, this structure has appreciable solubility to lipid compounds but offers a strong barrier to water soluble constituents. The enterocyte balances its dual function as both an absorptive and barrier cell by embedding transport systems within this membrane for the water soluble compounds that it wishes to transport. However, at the junction between epithelial cells there is a potential route for solute traffic that is not regulated by brush border membrane transporters or channels. In order to regulate traffic through this paracellular pathway, mammalian epithelial cells form a series of intercellular junctions along their lateral margins. Closest to the luminal surface lies the tight junction and underneath is the adherens junction.
These structures are enormously complex in both their lipid and protein constituents. An ever expanding family of proteins are found in the vicinity of these junctions, forming fibrils that cross the plasma membrane to interact with proteins from the adjoining cell. These proteins also interact intracellularly with the actomyosin ring that encircles the enterocyte at the level of the tight junction through numerous smaller proteins. The fibrils between cells consist of at least two types of tetraspanning membrane proteins, occludin and members of the claudin family. The latter is comprised of at least 19 different but related proteins, their name coming from the Latin “to close”. It is of interest that defects in claudins have already been associated with human disease: a genetic mutation in claudin 16 appears to be involved in renal hypomagnesaemia, characterised by massive renal loss of magnesium.3
Furthermore, claudins 3 and 4 are targets for the bacterially produced toxin, Clostridia perfringens enterotoxin which dramatically increases tight junctional permeability in tissue culture systems.4
On the intracellular side of the membrane, the carboxy terminal end of these proteins interacts with the tight junction proteins ZO‐1, ZO‐2, and ZO‐3 (see fig 1). These proteins belong to the membrane associated guanylate kinase superfamily and possess an enzymatically inactive guanylate kinase‐like domain. Underneath the junctional complex lies a ring of actin microfilaments and contraction of this has been proposed to regulate paracellular permeability. Connecting this ring to the junctional complex (ZO family members) are a series of actin filaments, as schematically outlined in fig 1. In addition to these protein constituents, there are numerous other junctional proteins that have been described and a tremendous effort is underway to elucidate the physiological interactions of these proteins in terms of cellular and junctional function. Excellent reviews of tight junctional structure and the protein composition of these fascinating organelles are available.5,6,7,8
One emerging concept is that the relative abundance of the different claudin family members is important in determining the physiological properties of the junction. Along normal developmental axes (such as along the crypt‐villus axis) or during disease expression, the relative abundance of the claudins can change by up to 1000‐fold while other more structural protein components seem to change relatively little.9
Figure 1Tight junction structure. Homotypic association of claudin and occludin is illustrated. Numerous other proteins have been described and for a detailed description of their function and localisation, the reader is referred to the reviews (more ...)
It is also important to consider what factors alter permeability of the junction. We now recognise that the functional state of the tight junction, once considered a static parameter, is in reality incredibly dynamic. Epithelial tight junctions open and close all the time in response to a variety of stimuli. These include dietary state, humoral or neuronal signals, inflammatory mediators, mast cell products, and a variety of cellular pathways that can be usurped by microbial or viral pathogens.10,11,12,13,14,15,16,17,18,19
A complete discussion of all of these mechanisms is beyond the scope of this article but a few deserve closer attention as they are important in our understanding of disease pathogenesis.
The first are dietary factors. It is now generally appreciated that the permeability of the paracellular pathway can be modulated by transcellular absorptive processes. During activation of the sodium dependent glucose transporter (SGLT1), there is a physiological opening of tight junctions that allows for the movement of small molecules and peptides.20,21,22,23
This pathway will accommodate particulate sizes of the order of 2000 molecular weight (MW) but still exclude large particles such as horseradish peroxidase (MW ~40
Although this is a normal physiological event, the purpose it subserves remains unclear. However, as discussed in the next section, this observation is important in our understanding of how to understand permeability measurements.
A physiological pathway, relevant to disease, is the zonulin pathway. Many bacteria alter tight junction state, presumably to enhance their own growth requirements. Vibrio cholerae
secretes a variety of toxins and one of these, zonula occludens toxin, was recognised as increasing paracellular permeability. The mechanism by which this occurred was novel and involved binding to an apical membrane receptor on the enterocyte with subsequent activation of an intracellular pathway resulting in actomyosin contraction and increased paracellular permeability. The investigators speculated that it was unlikely that this pathway was present for the sole benefit of the bacteria and that a more likely scenario was that the pathway was a physiological one that bacteria had evolved to take advantage of. This proved to be true and in an elegant piece of work this bacterial toxin was used to identify the human homologue for this pathway now termed zonulin.25
It appears that in many scenarios where permeability is increased, a common pathophysiological event is upregulation of zonulin secretion from a lamina propria source into the lumen26,27,28
with inappropriate activation of this pathway. The end result is increased paracellular permeability. This is illustrated in fig 2.
Figure 2Zonulin pathway. Depicted is a schematic view of what is known about the regulation of zonulin. Multiple steps are illustrated. (1) A luminal trigger for zonulin release arises and interacts with receptors, presumably on the epithelial (more ...)
Measurement of permeability
Over the past several decades there has been a concerted effort to develop simple non‐invasive means to evaluate the permeability properties of the paracellular pathway. In order to rationally evaluate methods to quantitatively evaluate the paracellular pathway in vivo, it is important to keep in mind a few simple principles. Movement across this pathway occurs by a non‐carrier mediated process and as such depends on several features.
- The concentration gradient across the barrier.
- The surface area of the epithelium.
- The time available for permeation.
- The intrinsic permeability properties of the barrier.
As the last item is the characteristic of interest for measurement, it is important to try and avoid differences in the first three variables during the measurement process by carefully selecting the probes used and the method of their employment. Historically, a wide variety of probes have been utilised to determine paracellular permeability properties. Typically, they have several features in common; they are usually small, water soluble, non‐charged compounds that are not destroyed in the gut, are non‐toxic, not metabolised or sequestered once absorbed, and quantitatively cleared by the kidney into the urine. Finally, they should be easily detectable in urine and easily separated from similar endogenous or dietary compounds. This area has been extensively reviewed recently.29,30
Despite the proliferation of available probes, the majority of work in humans and experimental animals has employed a variety of small saccharide probes and/or Cr‐EDTA. This is simply because these probes satisfy the criteria above, are cheap, and easily detectable. However, it is important to recognise that several of these probes are destroyed by processes that take place in the lumen of the gut and this limits their exposure to the epithelium in a manner that can be advantageously used to evaluate permeability characteristics in a regional manner.
As an example, sucrose is a useful probe for determining permeability characteristics of the gastroduodenal region.31
Distal to the gastroduodenal region, sucrose is rapidly hydrolysed by sucrase‐isomaltase and therefore permeation of intact sucrose across the gastrointestinal mucosa must occur in the most proximal regions of the gut. In a similar manner, the traditional small intestinal permeability probes, lactulose, mannitol, rhamnose, or cellobiose, are degraded by the bacterial flora of the large intestine and yield no information regarding colonic permeability characteristics. Furthermore, under conditions of small intestinal bacterial overgrowth, loss of lactulose and mannitol is impossible to quantify and calls into question determination of permeability. In order to evaluate colonic permeability properties, probes must be selected that are stable in this environment. These include either Cr‐EDTA or sucralose.32
Although these probes are stable throughout the gastrointestinal tract, they preferentially provide information regarding colonic permeability because, under normal conditions of intestinal transit, they reside within the colon for the majority of their time in the gut. The principles and techniques of probe selection for regional permeability determinations are discussed more extensively elsewhere32
but the basic features of these probes are schematically depicted in fig 3.
Figure 3Region specific permeability measurements. By carefully choosing probes that have only a limited exposure to the gastrointestinal epithelium site, selective permeability determinations can be made. Sucrose is destroyed once it leaves (more ...)
Perhaps the most important issue to consider is what the permeation rate of the various probes actually means. The most concise hypothesis regarding the routes various probes take across the epithelium has been provided by Fihn and colleagues.33
These investigators provided data to suggest that there are a series of “aqueous pores” distributed along the crypt‐villus axis of the small intestine. At the tips of the villus are relatively abundant small channels (radius <6 A) while in the crypts there exist much larger channels (50–60 A) in low abundance. At the base of the villus are intermediate sized (10–15 A) channels. The channels at the tips of the villus are increased in number by addition of glucose22
and susceptible to solvent drag effects while those in the crypt are unaffected by these alterations. The intermediate sized channels seem to be unaffected by solvent drag effects, perhaps as under physiological conditions this part of the villus is not exposed to luminal contents. With these data it would appear that, under normal conditions, molecules the size of disaccharides (for example, lactulose) are restricted from moving across the villus tip whereas mannitol can do so with relative freedom. This hypothesis is illustrated schematically in fig 4 and provides one possible explanation for the marked difference in permeation rates for probes with small differences in physical size.
Figure 4Small intestinal permeation pathways. Depicted is a schematic representation of the crypt‐villus axis. The broken line is an estimated depth of “luminal exposure”—the limit to which solvent drag effects (more ...)
Traditionally, small intestinal permeability is expressed as the ratio of the fractional excretion of a larger molecule to that of a smaller one (for example, lactulose:mannitol). Using the paradigm from the preceding paragraph, this would represent the number of intermediate sized pathways as a proportion of the total number of aqueous channels. As the smaller channels are concentrated at the villus tips, permeation rates of compounds across this pathway become a rough assessment of mature small intestinal surface area. This is well established from a clinical perspective. In diseases where there is a marked reduction in mature small intestinal surface area, such as coeliac disease, there is a substantial reduction in the fractional excretion of small probes such as mannitol. Coupled with this there is an increase in the fractional excretion rates of larger probes such as lactulose. This would suggest either an increased number of intermediate sized channels per unit surface area or the appearance of an alternate pathway, accessible to lactulose, that is not evident under normal physiological circumstances. As intermediate sized channels appear to be present in the immature parts of the crypt‐villus axis, relative expansion of this crypt‐villus fraction (as observed in coeliac disease) is a possible explanation. However, there are also data demonstrating that the presence of either epithelial damage or increased rates of apoptosis provide alternate routes for the permeation of larger molecules such as lactulose.34,35,36
The net result of a reduction in the fractional excretion of mannitol and an increase in that of lactulose is a dramatic increase in the lactulose:mannitol ratio. The converse is seen during the healing process of the coeliac lesion. During treatment with a gluten free diet, the first sign of recovery is seen as an increase in the fractional excretion of mannitol, suggesting that recovery of mature surface epithelium precedes the reduction in apoptosis and damage.37
This interpretation fits well with the known pathophysiology of coeliac disease.
Therefore, a reasonable interpretation of permeability data, expressed as a lactulose:mannitol ratio, is the amount of epithelial damage as a proportion of mature small intestinal surface area. It is important to remember that this parameter provides no information about either colonic or gastric permeability. The former is due to the fact that both probes are destroyed in the colon and the latter since the surface area of the small intestine is so much greater than that of the stomach. In order to obtain this type of information, additional probes such as sucrose and sucralose are necessary and provide the necessary data without detracting from the measurement of small intestinal permeability. Additional probes to assess absorptive function can also be added to extend the usefulness of such testing.38,39