REVISIÓN

Intestinal permeability measurements: general aspects and possible pitfalls

Mediciones de permeabilidad intestinal: aspectos generales y posibles riesgos

Tatiana Fiche Salles Teixeira¹, Ana Paula Boroni Moreira¹, Nilian Carla Silva Souza², Rafael Frias³ and Maria do Carmo Gouveia Peluzio¹

¹Department of Nutrition and Health. Federal University of Viçosa. Brazil.
²National Institute of Cancer. Rio de Janeiro. Brazil. ]]> ³Central Animal Laboratory. University of Turku. Finland.

Correspondence

ABSTRACT

Introduction: Disturbances of the gut barrier function have been related to a variety of diseases, including intestinal and extra-intestinal diseases. The intestinal permeability tests are considered useful tools for evaluating disease severity and to follow-up patients after a therapeutic intervention and indirectly assess barrier function.
Objective: The aims of this review were to highlight the possible factors underlying higher intestinal permeability and the clinical conditions that have been associated with this in different age range; and also provide some insight into methodological aspects.
Results and discussion: Abnormal regulation of tight junction function is the main cause of altered intestinal barrier. The impaired barrier function results in higher permeation rates of administered probes through the intestinal mucosa. Lactulose and mannitol are one of the most commonly used probes. The innocuousness and easiness of intestinal permeability tests can be explored to expand the knowledge about the clinical situations in which intestinal barrier dysfunction can be an important feature. Many factors may influence the results of the test. Researchers and healthcare professionals should try to circumvent the possible pitfalls of the intestinal permeability tests to produce consistent evidences. The use of others markers of intestinal physiology may also contribute to understand the role of barrier function in different diseases.

Key words: Intestinal permeability. Gut barrier. Lactulose. Mannitol.

RESUMEN

Palabras clave: Permeabilidad intestinal. Barrera intestinal. Lactulosa. Manitol. Fisiología intestinal.

Abbreviations
BMI: body mass index.
IP: intestinal permeability.
GFR: glomerular filtration rate.
L/M: lactulose/mannitol ratio.
TJ: tight junctions.

Introduction

The gastrointestinal tract has the complex task of absorbing nutrients while excluding the uptake of dietary antigens, luminal microbes and their products. The intestinal mucosa exhibit a selectively permeable barrier property, which supports this task. The histological organization of the gastrointestinal tract mucosa and the interaction between cellular (polarized epithelial cell membrane, tight junctions (TJ), lymphocytes) and extracellular components (mucin, unstirred layer of fluid)^1-4 are essential for the gut barrier function. Homeostasis of gut barrier function is critical for the ability of gastrointestinal tract to articulate aggressive reactions against enteric microbes while developing oral tolerance for food antigens and commensal bacteria⁵.

Disturbances of the gut barrier function have been related to a variety of clinical conditions in different age range (Tables I and II)^2,6. The investigation of gut barrier dysfunction and other intestinal abnormalities (such as polyps, tumors) can be done through methods such as collection of a biopsy sample using surgical and/or endoscopic procedures. However, these procedures are invasive, often inconvenient to the patient and usually imply high healthcare costs⁷. This has led to the development of alternative methods to assess gut barrier function while preventing patients from undergoing such kind of invasive methods.

Intestinal permeability (IP) tests represent one alternative method. The concept of intestinal epithelial batrier function is tightly related to the concept of permeability, which is the property of the membrane to allow non-mediated solute diffusion^8-9. When the barrier is intact, the permeability of substances is highly selective and controlled. Disturbances in gut barrier function can affect the control of permeating substances^9,10. Based on these principles the oral administration of specific probes has been commonly used to indirectly assess gut barrier dysfunction and measure IP. These probes are subsequently quantified in blood or more frequently in urine¹¹. In a simplistic way, injuries in the intestinal mucosa can impair its barrier function. The impaired barrier function results in higher permeation rate of probes and intact proteins through the intestinal mucosa^12,13.

Intestinal permeability tests are not widely used in clinical practice. Their use has been usually restricted for scientific purposes. However, evaluation of IP can be a useful tool in screening for small intestinal disease, in assessing the response in the follow-up period after a therapeutic intervention and in predicting the prognosis, especially in celiac disease^14,15. The majority of probes used have been shown to be non-toxic to patients and relatively easy to quantify. These characteristics can be explored by medical professionals to expand the knowledge about the clinical situations in which intestinal barrier dysfunction can be an important feature.

In this context, the aims of this review were to highlight the possible factors underlying higher IP and the clinical conditions that have been associated with this in different age range; and also provide some insight into methodological aspects to be considered in future studies.

Methods

Medline/Pubmed, Scielo and Lilacs were used to search for articles accomplishing the following terms (alone or associated): intestinal or gut permeability, intestinal or gut barrier, lactulose, mannitol, tight junctions. Review and original articles were selected and read critically.

Factors underlying increased intestinal permeability

The intestinal epithelium is a single layer of columnar epithelial cells that separates the intestinal lumen from the underlying lamina propria. It is believed that there are two routes for substances permeation through the intestinal epithelial cells: transcellular (across the cells, both by active and passive processes), and paracellular (between adjacent cells, by a passive process)^16,17. The epithelial cells are tightly bound together by intercellular junctional complexes. They are formed by TJ, gap junctions, adherens junctions and desmosomes. The space between cells is called paracellular space. The permeability of molecules through this space is under control of the junctional complexes, which are crucial for the integrity of the epithelial barrier¹⁷.

Tight junctions are complex structures comprising over 50 types of proteins (claudin, occludin, zonulin, junctional adhesion molecules). They form a continuous, circumferential seal around cells through the interaction with the perijunctional actomyosin ring of epithelial cells¹⁷. It has been observed that TJ have a central role in processes that regulate epithelial proliferation and differentiation¹⁸.

Regulation of the assembly, disassembly and maintenance of TJ structure is influenced by various physiological and pathological stimuli. The knowledge of how TJ are modified in response to signals that alter their functional properties is of great importance in the context of diseases associated with altered IP^16,19-21. Experimental studies using animal and cell culture models or human studies have shown that deregulated TJ are the main cause of altered intestinal barrier. This alteration can be induced by endogenous and exogenous factors (Table III).

Recently, it has been demonstrated that increased IP can occur due to discontinuities in the epithelial cell layer in the gut. These discontinuities are called gaps and have been identified in the mouse and humans. They are formed when epithelial cells leave the epithelium. These gaps have the diameter of an epithelial cell and are devoid of cellular contents, but filled with an unknown substance that maintains local barrier function. The rate at which cells leave may have implications for the permeability of the epithelium as a unit. The processes that control the rate of cell egress have not been well defined. This mechanism of increased permeability may be important in human diseases^22,23.

As summarized by Teshima and Meddings²² "simply measuring an increase in permeability provides no information to the physician about the mechanisms underlying the abnormality. However, an understanding of these mechanisms may prove valuable in designing interventions". Thus the main causes of increased IP that should guide the development of efficacious intervention are: genetic alterations of TJ proteins, abnormal microbiota, abnormal regulation of TJ function (increased zonulin release), mucosal inflammation and abnormal epithelial dynamics²².

General aspects of intestinal permeability tests

Intestinal permeability tests are based on probes of different molecular weight, which determines the route of permeation (Table IV). Smaller molecules usually permeate through membrane pores. They are expected to be present in urine in higher proportion (10 to 30% of an orally ingested dose)²⁴. Less than 1% of higher molecular weight molecules are expected to be recovered in urine after an oral dose²⁵. These molecules need to cross the barrier through the paracellular route, which is more tightly regulated by protein complexes.

The choice of probes depends on the intention of what part of intestine is meant to be assessed. Usually, recovery of sucrose in the urine reflects gastroduodenal permeability²⁶, since sucrose is rapidly hydrolyzed by sucrase-isomaltase upon entering the duodenum and reflects absorption only in the most proximal portion of the gut²⁷. Lactulose and mannitol, which are one of the most commonly used probes, are destroyed in the caecum and provide information regarding the small intestinal epithelium¹⁶. Sucralose is an artificial sweetener with similar molecular weight of lactulose and is resistant to bacterial fermentation²⁸. It spends most of a 24 hour exposure period in the large intestine¹⁶.Therefore, sucralose has been suggested as better suitable sugar for whole gut permeability assessment²⁹.

An inconvenience of IP tests is the prolonged period of urine collection, usually 5 to 6 hours. The introduction of sucralose has been suggested as better suitable sugar troduction of sucralose into permeability measurements might extend the test period up to 24 hours, making it less convenient in clinical practice. McOmber and co-workers recommend reexamining the usual 5 to 6 hours collection times to compare healthy individuals to those with abnormal permeability, because this period of time might not include the point of maximal urinary recovery. They studied the recovery of sucrose, lactulose, mannitol and sucralose over a 24 hours period in healthy adults and children³⁰. It was suggested that by using different collection periods greater differences may be seen between groups with less interindividual variation: 4 to 6 hours for sucrose, 13 to 15 hours for lactulose, mannitol and sucralose. If sucralose/lactulose ratio is to be measured, collection time might be extended to 16 to 18 hours³⁰. However, Akram and co-workers³¹ have compared different urine times collection and their results suggest that the use of Lactulose/Mannitol (L/M) ratio to assess IP could be simplified by shortening the time of urine collection³¹. The reduction of the time can also be achieved by measuring the probes in blood 60-90 min post-ingestion of solution^32,33. More studies are needed to confirm that prolonged time collection is not needed.

The calculation of the ratio between sugar probes used (such as L/M) is considered a good marker of small intestinal permeation⁹. It is meant to circumvent confounding factors as interindividual variation of gastric emptying, intestinal transit and transport, blood distribution and renal clearance³⁴.

In general, the integrity of intestinal barrier function is dependent on healthy epithelial cells and on the proper functioning of the paracellular route⁹. Theoretically, an increase in the sugar probes ratio -for example L/M ratio- would indicate altered IP. This alteration may reflect a decrease in smaller probes (e.g. mannitol) absorption and/or an increase in the absorption of higher weight probes (e.g. lactulose). Decreased small weight probes absorption can be the result of a diminished absorptive area. Increased permeation of higher weight probes may be due to a facilitated diffusion of this marker into the crypt region as a consequence of decreased villous height or TJ loosening³⁵.

The results of IP tests are usually expressed as percentage of excretion of probes (Table V). Other units can be also found (mg/mL, mmol/L, mg) ^{11,31,32,36,37}.

Possible pitfalls in intestinal permeability tests

Previous orientation of individuals to avoid -few days before the test- the use of non-steroidal inflammatory drug^38,39, acute alcohol ingestion^32,40,41, psychological and physical stressful situations^42-44 should be given as part of the protocol. Considering that some genetic background may exert negative influence on barrier function, family history of inflammatory bowel diseases should be considered before inclusion of patients in a study. Regarding the personal medical history some clinical factors influencing IP such as food allergy, human immunodeficiency virus, diabetes, starvation, iron deficiency, diarrhea, viral gastroenteritis, smoking^45-48 should be an exclusion criteria, except if this is the topic under investigation. Additionally, search for evidence of endoparasite infection in the stools should be ideally performed before inclusion of individuals in the study⁴⁹.

Usually, all tests are performed under overnight fast (8 to 10 hours). Few authors mention the instruction of individuals to follow a diet free of the sugars used as probes in the test at least 24 hours before it^13,32,50. Lactulose, mannitol and sucralose are commonly used in IP tests and can be present in some common foods (Table VI). An important issue mentioned in some protocols to circumvent the possible influence of the intake of the same sugars that will be used in the IP test is the collection of a urine sample before the administration of the sugar probes. The amount of sugar quantified in this sample should be subtracted from the results in the urine collected after the ingestion of the probes^13,28,33,50. Avoidance of some foods should be also advised when they contain other sugars that can imply in methodological difficulties to properly quantify the probes. Farhadi and co-workers recommend subjects to avoid consumption of dairy products on the previous day of the test since lactose peak tend to overlap that of lactulose⁵¹. During the IP test- in some studies it is mentioned that subjects are encouraged to drink water and/or to have a snack after 1 to 2 hours of probes administration^11-13,37. It is not clear if this can affect the results. However- an important detail of this practice is to standardize the type of food and the volume of liquid offered to all individuals. Mattioli and co-workers⁵² found that the L/M ratio was significantly lower in subjects that excreted more than 500 mL of urine. The greater urine volume was associated with a higher mannitol recovery. Thus- they emphasized that urine volume may influence urinary excretion of sugar probes and intake of liquids should be carefully monitored before and during the test⁵².

It is noteworthy that Camilleri and co-workers question the concept that lactulose and mannitol in urine collected between 0 to 6 hours reflect small intestine permeability. They have investigated the administration of these probes (radiolabelled) in a liquid formulation or in a delayed-release methacrylatecoated capsule. It was showed that after 2 h of liquid formulation intake around 50% of the probes was in the colon- suggesting that sugars may not be absorbed exclusively in the small intestine. Thus- they suggest that the interpretation of the 0 to 6 hours differential two sugar urine excretion as an exclusive marker of small IP should be done cautiously²⁴.

Osmolarity of test solutions should be mentioned in every study- since stress induced by high osmolarity can stimulate intestinal motility⁵³ and change the rate of sugars permeation⁸. The amount of sugar administered and the volume of solutions vary between studies (see Tables I and II). In addition, the volume of solution administered is fixed for all subjects. Exception is observed in some studies with children, that use body weight to calculate the volume of solution to be administered individually^50,54. This might have been proposed based on pharmacokinetics studies. At least for children, drugs dosages are based on body weight or body surface area since body size, proportion, organ development and function affect the pharmacokinetic behavior of many drugs⁵⁵. It should be further discussed the possibility of using weight to calculate the volume of solution to be administered also to adult subjects. The body weight or body mass index (BMI) of subjects included in the majority of studies is not mentioned. Could this make any difference for the interpretation of IP results?

A higher BMI is associated with higher filtration fraction. This means that there is a higher glomerular filtration rate (GFR) relative to effective renal plasma flow, suggesting an altered afferent/efferent balance and higher glomerular pressure⁵⁶. In obese subjects, the values for GFR exceeded by 61% the values for GFR of the control group and by 32% the value of renal plasma flow, suggestive of glomerular hyperfiltration. The obesity-related glomerular hyperfiltration ameliorates after weight loss⁵⁷. It is a possible pitfall when subjects with excess of weight are included in studies: could a higher amount of excreted sugar be a consequence of higher intestinal absorption (due to higher IP) or of a higher glomerular hyperfiltration? This has not been investigated in humans. Whenever overweight and obese subjects are submitted to IP test it should be investigated if they present normal renal function (impaired renal function should be adopted as exclusion criteria).

Choosing the best method to assess renal function should consider population characteristics such as age and BMI. Serum creatinine levels, anthropometric and clinical characteristics of patients are often used to estimate GFR. Body weight is an imperfect reflection of creatinine generation because increased body weight is associated more commonly with an increase in body fat or body water, edematous disorders, rather than an increase in muscle mass^58,59. Creatinine clearance is not recommended when obese subjects are involved, but would be advised to exclude individuals that present creatinine level higher than 250 mmol/l¹⁴. A decline in renal function (creatinine clearance) occurs with advancing aging. Interestingly, L/M ratio did not change with aging due to a parallel progressive decline in the ability to excrete both lactulose and mannitol with increasing age⁶⁰.

The use of the ratio L/M may not detect differences in IP between groups if one considers the possibility that an individual may be absorbing and excreting proportionally higher quantities of both mannitol and lactulose. Although this is only a hypothesis, obese women showed higher lactulose excretion, a tendency to higher mannitol excretion, while L/M ratio was not significantly different from lean women⁶¹. It is critical to assess the L/M ratio, as well as lactulose and mannitol recoveries separately, when interpreting test results⁶². Ferraris & Vinnakota⁶³ showed in animal model that genetic obesity is associated with increased intestinal growth, which augments absorption of all types of nutrients. Obese men with chronic hyperglycemia showed evidence of increased small intestinal enterocyte mass (higher plasma citrulline) and increased enterocyte loss (higher plasma intestinal fatty acid binding proteins, I-FABP), but IP was not assessed⁶⁴. Circulating levels of insulin which is a hormone usually increased in obese subjects⁶⁵, may also influence IP. The addition of insulin in a cell culture showed that the insulin-induced decline in transcellular resistance is receptor-mediated and that receptors are localized in the basolateral membrane. Increased mannitol flux was an observed effect paralleled to this altered paracellular permeability⁶⁶.

Barrier dysfunction may not be expressed all the time in particular conditions. It can range from mild to severe dysfunction (manifesting continuously) or intermittent dysfunction (manifesting only when the intestine is challenged). This susceptibility to barrier dysfunction can be detected using a 'challenge' test, as established by Hilsden and co-workers using aspirin⁶⁷. Accordingly, subjects are given 1300 mg of aspirin (four 325 mg tablets) the night before the test and again on the morning of ingestion of the probe mixture. The use of the aspirin challenge showed that patients with non-alcoholic steatohepatitis do not have abnormal IP all the time, but they could easily develop gut leakiness when they are exposed to intestinal barrier stressors such as aspirin⁶⁸.

Of note is the discussion presented recently by Vojdani⁶⁹ in his review entitled "For assessment of intestinal permeability, size matters". Mannitol and lactulose are considered small molecules. Their use for IP assessment will not necessarily indicates structural damage in the TJ barrier, which would in turn allow penetration of large molecules. The use of probes of higher size (polysugars of 12,000- to -15,000 Da) may be more suitable to extrapolate if IP is higher enough to allow macromolecules such as bacterial toxins (such as lipopolysaccharides) and food antigens to permeate. Small inert markers may not mimic large molecules because of the size selectivity of TJ⁶⁹.

Additional markers to indicate alteration in barrier function

There are other markers that could be associated to IP tests to improve the interpretation of dysfunctions of gut barrier. D-lactate is produced from carbohydrate fermentation by abnormal microbiota or when the number of bacteria elevates rapidly (bacterial overgrowth and short bowel syndrome)^70-72. Plasma D-lactate had the lowest false-negative rate among C-reactive protein level and leukocyte counts to diagnose appendicitis, and acute inflammatory disorder⁷³.

Circulating citrulline is an amino acid produced from glutamine by differentiated small intestinal enterocytes. Citrulline is a non-protein amino acid that seems to exert an important role in preserving gut barrier function and reducing bacterial translocation⁷⁴. The circulating levels are dependent only on de novo synthesis from intestinal metabolic activity. It reflects the functional enterocyte mass and can be used as a biological tool to quantitatively investigate epithelial integrity and follow intestinal adaptation (i.e., post-surgical) at the enterocyte level. Loss of small bowel epithelial cell mass results in declined circulating levels of citrulline, such as for short bowel syndrome, chronic villous atrophy and chemotherapy⁷⁵. Another situation in which the citrulline availability is decreased was shown to be during the course of induced endotoxemia in rats⁷⁶. There some studies using animal models that show an association between endotoxemia and increased IP^77-79. As citrulline is metabolized into arginine by kidney cells, the interpretation of its levels in patients with compromised renal function should not be reliable⁸⁰.

The quantification of claudin-3 in the urine showed that its rapid appearance in this fluid correlated with immunohistochemically visualized loss of claudin-3, which is a major sealing TJ protein. Measurement of urinary claudin-3 can be used as noninvasive marker for intestinal TJ loss⁸¹.

The assessment of urinary concentration of endogenous cytosolic enterocyte proteins such as I-FABP and liver FABP (L-FABP) are potentially useful in reflecting enterocyte damage. Pelsers and co-workers investigated the distribution of these proteins in segments of human intestine⁸². They showed similar pattern of tissue distribution along the duodenal to colonal axis, being the jejunum the segment with highest content. In each intestinal segment it is observed a more than 40fold higher content of L-FABP than I-FABP. Elevated plasma levels of both proteins were found in patients with intestinal diseases⁸². Since FABP are small, water-soluble cytosolic proteins, the loss of enterocyte membrane integrity will lead to release of these proteins into the circulation^71,83. FABP are expressed in cells on the upper part of the villi. Thus, destruction of these cells can lead to increased release of these proteins to the circulation. Results from a pilot study with celiac patients showed that circulating levels of FABP are significantly elevated in untreated patients with biopsy proven celiac disease compared with healthy controls⁸⁴.

Local inflammation is associated with increased IP. An increased migration of granulocytes into the intestinal mucosa, usually due to conditions of inflammation, might result in the degranulation of their secondary granules, resulting in an increase in their proteins in feces⁸⁵. Neutrophil derived proteins such as calprotectin, lactoferrin^85-88 and elastase⁸⁹ can be present in stool and also in plasma as a marker of inflammation⁹⁰.

Finally, zonulin is a protein that exhibits the ability to reversibly modulate intercellular TJ similar to the toxin from Vibrio cholera known as zonula occluden toxin^91,92. Proteomic analyses characterized zonulin as pre-haptoglobulin-2 (pre-HP2), a multifunctional protein that contains growth factor-like repeats. In its single-chain form, zonulin has the molecular conformation required to induce TJ disassembly by indirect transactivation via proteinase-activated receptor-2⁹². Higher levels of zonulin are associated with disorders such as celiac disease and type 1 diabetes, and positive correlation between zonulin and IP has been demonstrated^92,93.

Conclusion

Acknowledgements

Coordenação de Aperfeiçoamento de Pessoal de Nivel Superior (CAPES), Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG) and Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq).

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Correspondence:
Tatiana Fiche Salles Teixeira.
Departamento de Nutrição e Saúde.
Universidade Federal de Viçosa.
Av. Ph. Rolfs. Campus Universitário.
36570-000 Viçosa. Minas Gerais. Brasil.
E-mail: tatifichee@hotmail.com

Recibido: 19-VIII-2013.
1.^a Revisión: 25-X-2013.
Aceptado: 15-XI-2013.