Intestinal Permeability Tests

An Introduction to the Intestinal Barrier

The small intestine has the task of protecting our internal milieu against molecules from the outside world in addition to being the primary site of nutrient digestion and absorption. It does this not only on a chemical level, but also via a single-celled mucosal layer that exists to form a functional barrier.¹

Luminal contents pass through this single layer of cells by either the transcellular or paracellular pathways. Transcellular passage is carrier-mediated or accomplished through passive diffusion, active transport, or engulfment by the cell membrane.² Paracellular transport is regulated by protein complexes known as tight junctions that form a seal between cells.³ The permeability of this seal is regulated by zonula-occludens, intracellular proteins that connect the tight junctions to the cytoskeleton of the adjacent cells.⁴ The cytoskeleton is also made up of proteins, which comprise a network of thin, overlapping fibers known as the actin-myosin network.⁴ This partnership between the actin-myosin network and the zonula-occludens proteins controls the permeability of the tight junctions, and thus the intestinal barrier.

Barrier Changes

Various forms of injury can occur to the intestinal barrier, including changes in the microbiome, epithelial cells, and/ or tight junctions themselves. Whenever any of these structural changes occur, the composition and function of the mucosal barrier are modified and there is a risk of increased permeability and an associated risk of gastrointestinal and extra-intestinal sequelae.³ As the awareness of this association expands, more and more non-invasive laboratory tests to assess intestinal permeability are becoming available.

Intestinal Permeability Tests

Urine
Lactulose Mannitol Test: The use of sugar molecules was one of the first non-invasive laboratory techniques for assessing intestinal permeability. The lactulose-mannitol, or differential sugar test, requires administering an equal, simultaneous oral dose of both a disaccharide (lactulose), and a monosaccharide (mannitol).⁵ The basis for this test is that mannitol, the smaller of the two sugar molecules, freely travels through the transcellular pathway of the mucosal layer, while the larger lactulose is typically restricted from paracellular absorption by tight junctions. Urinary elimination of these molecules is expressed as a ratio of the percentage of the ingested doses found in the urine, known as the LMR (lactulose mannitol ratio).⁵ The ratio identifies increased intestinal permeability.

A drawback of this test is its low specificity and high rate of false positives. Also lacking is information on the intestinal barrier’s permeability to larger molecules.⁶

Serum
Lipopolysaccharide: The presence of specific antibodies in serum can also be a sign of intestinal permeability. Lipopolysaccharide(LPS) is a naturally occurring endotoxin found in the gut, genitourinal, and respiratory tracts. As part of the cell wall of Gram-negative organisms, it is expressed when the organism’s cell membrane is shed or ruptured.⁷ A healthy mucosal layer with intact tight junctions prevents the paracellular translocation of LPS. The presence of LPS and LPS IgA, IgG, and IgM antibodies in the blood has been discovered to be clinically relevant when attempting to identify the degree of intestinal barrier permeability.⁶

Zonulin/Occludin and Actin-myosin: Serum testing for zonulin-occludin and actin-myosin antibodies also provides evidence of intestinal barrier changes. These proteins are responsible for maintaining the integrity and strength of the epithelial cells and tight junction connections. Detection of actomyosin network IgA or occluden/zonulin IgG, IgM, and IgA antibodies may be clinically valuable in understanding these structures.⁴

Zonulin: The protein zonulin by itself has been studied as another useful blood marker for identifying intestinal barrier function.² Enterocytes release zonulin in response to dietary gliadin proteins and pathogenic bacteria and its uncleaved form has been shown to increase intestinal permeability, allowing it, and other bacteria to be translocated into the bloodstream.^2,8

Citrulline: Most often used to assess mucosal integrity after small bowel transplantation, low plasma citrulline is emerging as a reliable biomarker for intestinal epithelial function.⁹ Enterocytes synthesize this amino acid from glutamine or arginine and low circulating levels can be used to reveal changes in villous structure, enterocyte mass and barrier function.¹⁰

Fecal
Secretory IgA: As a component of the gut-associated lymphoid tissue (GALT), secretory IgA or sIgA, is a major player in the body’s defense of its mucosal surfaces. Produced by stimulated B cells, sIgA inhibits the adhesion of intestinal antigens to the epithelium and induces other responses that promote mucosal homeostasis.¹¹ Secretory IgA levels are affected by poor nutrition, antigenic load, stress, immune responses, and some pharmaceuticals. While not a direct marker of intestinal permeability, sIgA levels are useful to assess an increased risk of mucosal damage and disruption of barrier integrity, and are best viewed together with other direct laboratory markers. Saliva and serum tests are also available for measuring sIgA.

Increased intestinal permeability can lead to poor absorption of nutrients and is being progressively recognized as a clinical phenomenon related to other systems.As a practitioner, access to and use of a constellation of non-invasive intestinal permeability laboratory tests provides enhanced assessment, treatment, follow-up, and ultimately better patient outcomes.

REFERENCES

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4. Viggiano, Ianir et al. Eur Rev Med Pharmacolo Sci. 2015. 1077-85.
5. Mishra, A et al. J NeurogastroenterolMotil. October 2012. 18:4.
6. Vojdani, Aristo, PhD.Altern Ther Health Med. Jan/Feb 2013. 19:1. 12-24.
7. Guo, Al-Sadi et al. The American Journal of Pathology. February 2013. 182:2.
8. Lamprecht et al. J Int Soc Sports Nutri. 2012. 9:45.
9. Barzal et al. Acta Biochimica Polonica. 2014. 61:4. 615-31
10. Semba et al. Scientific Reports. 2016. 6:28009.
11. Campos-Rodriguez et al.Frontiers in Integrative Neuroscience. December 2013. 7:86.