Method for determining intestinal permeability

Abstract

The present invention relates to an in vitro method for assessing the state of intestinal permeability in a subject and thus for diagnosing a disease or dysfunction associated with hyperintestinal permeability. More specifically, the procedure allows the amount of dietary antigens that can pass through the dysfunctional intestinal tract to be measured using common food components. The program allows for the development of analytical products and processes within the framework of the medical instrument industry.

Description

Technical Field

[0001] This invention pertains to the medical field, clinical analysis, production and commercialization of health products and services, and basic and clinical research on the digestive system. In particular, this invention relates to an in vitro method for assessing the intestinal permeability status of a subject, and thus for diagnosing diseases or functional disorders associated with or potentially associated with increased intestinal permeability, such as (not an exhaustive list): celiac disease, Crohn's disease, irritable bowel syndrome, microscopic colitis, functional dyspepsia, chronic / acute villous atrophy due to long-term treatment with certain medications, and / or non-IgE-mediated food hypersensitivity. Background Technology

[0002] The intestinal barrier is fundamental to human health, forming the interface between the body's external and internal environments. A functional intestinal barrier allows for the absorption of nutrients and fluids while preventing harmful substances (such as toxins, bacteria, or viruses) from crossing intestinal epithelial cells and causing damage to the organism. Altered intestinal permeability has been associated with a variety of chronic conditions, such as inflammatory bowel disease, irritable bowel syndrome, celiac disease, and other pathologies originating from the gastrointestinal tract, as well as diseases like Alzheimer's and Parkinson's.

[0003] The permeation pathways of incompletely digested antigens, or even bacteria and / or viruses, are either paracellular (through the intercellular spaces between cells) or transcellular (through the epithelial cells of the intestine). Increased intestinal permeability to macromolecules is associated with a variety of inflammatory conditions.

[0004] The lumen of the intestine is lined with epithelial cells that facilitate the absorption of water and nutrients; these cells also provide a dynamic and semi-permeable barrier between the luminal microbiota and the host. This barrier is formed by the membranes of individual epithelial cells and tight junction proteins that seal the pericellular space between adjacent cells. Therefore, the permeability of this barrier is regulated by the integrity of the cytoplasmic membrane and tight junctions, as well as by epithelial cell-mediated processes of secretion and absorption. Small molecules (<300 Da) and electrolytes passively cross the tight junction barrier.

[0005] Membrane permeability can be altered by a number of physiological and pathological stimuli. During homeostasis, intestinal epithelial cells absorb nutrients while effectively preventing the translocation of luminal bacteria. However, certain pathological conditions (e.g., intestinal toxins or inflammation) can increase paracellular transport and intestinal barrier permeability, leading to ineffective nutrient absorption and a risk of failing to prevent the translocation of luminal bacteria and their products. This can result in chronic intestinal disease and affect distal organs that excrete and filter translocated bacteria and related products.

[0006] Increased intestinal permeability can lead to systemic inflammation, which is associated with a variety of clinical conditions, including:

[0007] • Food allergies or sensitivities.

[0008] • Gluten sensitivity and celiac disease.

[0009] • Inflammatory bowel disease (Crohn's disease or ulcerative colitis).

[0010] • Autoimmune diseases (rheumatoid arthritis, psoriasis, type I diabetes, spondylitis, etc.).

[0011] • Cognitive impairment (anxiety, depression, schizophrenia, etc.).

[0012] • Neurological conditions (Guillain-Barré syndrome, multiple sclerosis, etc.).

[0013] Known methods for determining intestinal permeability include:

[0014] 1. The lactulose / mannitol or lactitol / mannitol test, which has been available for over 40 years, measures the passage of two sugar molecules or derivatives across intestinal epithelial cells: lactulose (an isomer of lactose) and mannitol. Mannitol is a small molecule sugar that is readily absorbed, while lactulose is a large molecule sugar that is not effectively absorbed. After collecting a baseline urine sample, the patient ingests lactulose and mannitol. The concentrations of lactulose and mannitol in urine collected over the following 6 hours are then quantified. This is an economical and easy-to-perform method because urine samples can be collected at the patient's home. However, this method has the drawback of measuring the permeability of small sugar molecules that are not immunogenic. Furthermore, it cannot analyze the ability of antigenic macromolecules to cross the epithelial barrier, which can contribute to and exacerbate underlying inflammatory conditions and autoimmune diseases. Lactulose also has a low molecular weight, and its translocation across the intestinal barrier does not reflect the translocation of dietary proteins and the overall immune response. Furthermore, lactulose intake can exacerbate symptoms associated with small intestinal bacterial overgrowth (SIBO) and is also considered a prebiotic. Additionally, this approach requires technical expertise and / or expensive equipment, such as high-performance liquid chromatography (HPLC), gas chromatography, or tandem mass spectrometry (MS / MS). Finally, due to the high levels of mannitol naturally present in plant-based foods (fruits, vegetables, legumes, etc.), patients may need to follow a complex diet.

[0015] 2. Macromolecular indicators of intestinal permeability, which can assess molecules that may be antigenic. The presence of specific antibodies against these molecules can reveal increased intestinal permeability in some past events. These indicators are as follows:

[0016] a. Lipopolysaccharide (LPS) is a large molecule found in the outer membrane of Gram-negative bacteria. These LPS are endotoxins, and if absorbed, they trigger a strong immune response. Detection of LPS antibodies indicates that the endotoxin has crossed the intestinal barrier and entered the systemic circulation.

[0017] b. Closure proteins are key components of proteins that maintain tight junctions between intestinal cells. Detection of closure proteins with antibodies indicates disruption of tight junctions (paracellular pathway).

[0018] c. Connexin is considered a protein that regulates intestinal permeability. Detection of connexin with antibodies indicates impaired regulation of tight junctions (paracellular pathway).

[0019] d. The actomyosin network is a protein complex that regulates intestinal barrier function by maintaining the plasticity of tight junctions. Antibodies against actomyosin are biomarkers of intestinal barrier dysregulation via cell infiltration (a transcellular pathway).

[0020] e.LBP (lipopolysaccharide-binding protein). This protein has been identified as binding to LPS and can be measured in blood.

[0021] These indicators are suggested to be able to identify the level of permeable gut and its triggers (LPS or bacterial overgrowth vs. stress or food sensitivity).

[0022] Generally, the larger the molecule, the less likely it is to enter the circulatory system. Antibody measurements are affected by steroid use. Therefore, patients must discontinue oral or topical steroid use for at least 60 days before analysis. Reference values ​​are also based only on adults and are difficult to apply to pediatric populations because there are no known normal ranges.

[0023] 3. Direct measurement of serum or blood conjugates. Circulating conjugates (vs. antibodies against conjugates) do not show false negatives when using oral or topical steroids, but circulating conjugate levels may increase when using corticosteroids, leading to possible false positives due to intestinal permeability. Conjugate levels are higher in obese individuals and those with glucose intolerance, indicating a possible false positive due to intestinal permeability.

[0024] Therefore, in order to report the most important information, a simple method for assessing intestinal permeability status should be based on immunogenic proteins or peptides, monitoring diseases associated with changes in permeability from normally ingested foods.

[0025] The present invention aims to address these technical problems associated with existing methods that cannot measure the entry capacity of food antigenic peptides, are more invasive, expensive, indirect and / or laborious, and proposes a novel strategy for assessing the intestinal permeability status of subjects. Summary of the Invention

[0026] This invention relates to an in vitro method for assessing the intestinal permeability status of a subject, and thus for diagnosing diseases or functional disorders associated with or manifesting as increased intestinal permeability, including, for example, celiac disease, Crohn's disease, irritable bowel syndrome, microscopic colitis, functional dyspepsia, chronic / acute villous atrophy due to long-term treatment with certain medications, diseases of non-IgE-mediated food hypersensitivity, and also as risk factors for some neurodegenerative diseases such as Alzheimer's disease, Parkinson's disease, or multiple sclerosis. Currently, for macromolecules such as immunogenic proteins or large food peptides, individual reference values ​​and variations are not satisfactorily determined. Therefore, this invention relates to an in vitro method for determining the degree of intestinal permeability through the following general sequential procedure:

[0027] Step 1: Maintain a gluten-free diet for at least 16 hours, preferably 32 hours, and then fast for at least 8 hours.

[0028] • Step 2: Ingest a limited amount (at least 0.5g, preferably 10g) of gluten powder in a liquid medium (water, milkshake, yogurt, etc.) in suspension or hydrolyzed form.

[0029] • Step 3: Continue fasting for at least 4 hours during the period of collecting all urine for at least 6 hours, and maintain a gluten-free diet throughout the urine collection period.

[0030] • Step 4: Use a procedure to detect less than 15 ng / ml gliadin (the protein used as the analytical standard for gluten) in urine to measure the volume of collected urine and the amount of gluten in the collected urine.

[0031] • Step 5: Compare the excreted gluten value to the internal reference value (for the same individual) or the mean for healthy individuals and other reference values ​​for altered gluten peptide permeability. If the urinary gluten peptide level is higher than the internal reference value, it means the individual's intestinal permeability has not improved. If the urinary gluten peptide level is higher than the normal range compared to the corresponding mean of their reference study group (age, sex), the individual will have increased intestinal permeability. If the urinary gluten peptide level is within the reference range, the intestine does not show any signs of increased permeability.

[0032] Performing the gluten-free diet in step 1 beforehand ensures that all gluten peptides originate from the gluten ingested in suspension in step 2, rather than from food consumed in the hours preceding the intake. Fasting for 8 hours before intake also ensures no food residue remains in the stomach and the portion of the small intestine that connects to the jejunum (i.e., the duodenum). Extending the fasting period beyond 8 hours reduces the likelihood of interference from other foods in the metabolism of the gluten ingested in step 2. Eliminating gluten from the diet at least 24 hours before intake reduces the chance of residual gluten in other previously ingested foods, although a 16-hour restriction is generally sufficient to eliminate all gluten detectable in urine by commercially available methods (e.g., Gluten Detect by Biomedal, Seville).

[0033] The applied gluten is provided in an amount of at least 0.5 g to ensure its detection in urine, and preferably, gluten is purified in powder or hydrolyzed form (>80%) to increase the speed and efficiency of intestinal digestion and absorption due to the high contact area between gluten and the hydrolysant in the digestive system. To facilitate intake and enhance individual comfort, gluten suspensions can be prepared in water or other fluids (e.g., smoothies, yogurt, etc.) using dispersants and flavorings. Gluten can be hydrolyzed in pepsin-trypsin enzymatic hydrolysis or in enzymatic digestion that dissolves gluten without destroying most immunogenic gluten peptides (GIPs) detectable by commercially available immunological methods. Preferably, 4g to 40g of gluten product should be used to make the level of gluten peptides in the urine high enough to quantify GIP using commercially available methods (e.g., quantitative GIP urine lateral flow strips (LFIA) iVYCHECK read by LFIAreader (Biomedal, Spain)) and other methods that can be applied in small clinical laboratories or even in places close to the patient (point of care).

[0034] After gluten intake, patients are advised to fast for 2 to 9 hours to facilitate rapid digestion of the ingested gluten and produce fragmented gluten peptides that will translocate through the intestines.

[0035] While maintaining a gluten-free diet, collect all urine samples over a fixed period of at least 8 hours after gluten consumption. However, if it is necessary to collect most of the gluten peptides excreted in the urine, this period can be extended to up to 16 hours to ensure that subsequent excretion is of the residual amount. Ideally, the collection period after ingestion should be urine collected between 6:00 AM and 8:00 AM and between 10:00 PM and 12:00 AM, following a gluten-free diet for 24 to 36 hours and fasting for at least 8 hours. Measure the volume of excreted urine during this period using a graduated urine collection container or graduated cylinder. Determine the concentration of gluten peptides in the urine collected from 8 to 16 hours using quantitative methods.

[0036] In a preferred embodiment of the invention, the method for determining gluten peptides can use specific antibodies against gluten proteins that have been described, particularly those specific antibodies that have been shown to quantify gluten in food with high sensitivity (capable of detecting less than 15 ng / ml of gliadin) and have demonstrated their ability to detect gluten peptides in urine, such as G12, A1 (Biomedal, Spain) and R5 (r-Biumma, Germany, or Eurofins Ingenasa, Spain). These can also be obtained by performing routine procedures for obtaining antibodies, i.e., immunizing animals with peptides having the most abundant sequences found when analyzing gluten peptides in urine by liquid chromatography and mass spectrometry (LC / MS) (Table 1).

[0037] Table 1

[0038]

[0039]

[0040] The antibody is effective as long as the quantitation limit of the analytical procedure is preferably below 15 ng / ml for gliadin, although the extremely low concentration of gluten peptides in urine requires a detection limit below 6 ng / ml for peptide SEQ ID No. 1 (33-mer of α-gliadin) to be able to detect the level of specific gluten peptides in urine after ingestion of 0.5 g to 5 g of gluten.

[0041] To prepare urine samples and improve detection sensitivity, a peptide concentration system in urine can also be used in one form of this invention. Some proven effective methods for preparing and concentrating gluten peptides include solid-phase extraction using a C18 matrix, size exclusion chromatography, solvent evaporation, affinity chromatography, etc.

[0042] Preferred methods for detecting gluten in urine include sandwich ELISA, which quantifies the amount of antigen-antibody complexes by coupling one of the specific antibodies to an enzyme capable of using colorimetric, fluorescent, or chemiluminescent reagents that deliver a measurable signal proportional to the formation of immune complexes with gluten peptides. Fluorescent or chemiluminescent methods are practically more convenient to use due to their wider dynamic range and generally do not require dilution to the input range as is the case with colorimetric methods.

[0043] In a preferred embodiment of the invention, lateral flow immunochromatographic strips are also considered, which can be quantified by image analysis using gold particles or colored polystyrene particles with a strip reader, and by quantification using classical or time-resolved fluorescent probes (e.g., those obtained with europium particles) via a suitable fluorescence device. Other options such as Heatsens or quantum dots can be used as signal quantification options by conjugating suitable probes to antibodies.

[0044] In an alternative embodiment of the invention, in addition to antibodies, aptamers developed for recognizing gluten in biological matrices, such as those already developed in food, may be used.

[0045] A preferred form of the procedure of this invention can be facilitated using a kit containing gluten powder in the form of gelatin capsules or sachets with dispersants and / or flavoring agents, containing at least 0.5 g of gluten, preferably 4 g to 40 g of gluten, more preferably 8 g to 10 g of gluten. With such amounts of gluten, gluten excretion can be detected by at least one commercially available method. For example, using the LFIA method (iVYCHECK GIP Urine) already marketed by Biomedal, based on an antibody with sensitivity and specificity against the 33-mer of α-gliadin, most individuals (87%) achieved levels above the detection limit in their urine after ingesting 0.5 g. As described, 0.5g of gluten does not cause symptoms in most patients with celiac disease (Burger, JPW; van Lochem, EG; Rovers, EA; Drenth, JPH; Wahab, PJ). Dose-Escalating (50–500mg) Gluten Administration Leads to Detectable Gluten-Immunogenic-Peptides in Urine of Patients with Coeliac Disease Which Is Unrelated to Symptoms, a Placebo Controlled Trial. Nutrients 2022, 14, 1771. https: / / doi.org / 10.3390 / nu14091771. Therefore, these amounts can be used for celiac patients concerned about developing symptoms. In any case, for the maximum efficacy of the procedure of this invention, regardless of the degree of intestinal permeability, due to the need for sensitivity in detecting gluten peptides in urine, it is recommended to use at least 4g to produce a sufficient amount of gluten peptides for detection in most individuals. Population studies of celiac disease have shown that their risk of death is not higher than that of the general population. Even in most patients with celiac disease, gluten intake is frequent, as 67% to 89% of patients with celiac disease consume gluten at least once a week or once a month [Silvester JA, Comino I, Rigaux LN et al. Exposure sources, amounts and time course of gluten ingestion and excretion in patients with coeliac disease on a gluten-free diet. 2020;00:1–11. https: / / doi.org / 10.1111 / apt.16075].Occasional gluten consumption appears to be less harmful than small but more frequent consumption [Garzón-Benavides M, Ruiz-Carnicer M, Segura V, Fombuena B, García-Fernandez F, Sobrino Rodriguez S et al. Clinical utility of urinary gluten immunogenic peptides in the follow-up of patients with coeliac disease. Aliment Pharmacol Ther. 2023;00:1–11. https: / / doi.org / 10.1111 / apt.17417].

[0046] Other optional components of the kit may be at least one urine collection container with a capacity of up to 2 L for at least the following 8 hours, preferably 9 to 16 hours, after gluten ingestion. Generally, a total capacity of only 1.2 L for all containers is sufficient for this period (up to 12 hours from gluten ingestion). Gluten peptides are stable in urine and can be delivered to an analytical laboratory at room temperature. Finally, another component of the kit is the method for determining gluten peptides. The possibility of quantification using LFIA allows it to be performed as a point-of-care (POC) device in a healthcare center near the patient equipped with a reader (which may be added in a preferred form of the invention), although quantitative laboratory methods such as ELISA can be used. CLIA (chemiluminescent immunoassay), biosensors, etc.

[0047] In a more advanced form of the present invention, gluten peptides in urine can be concentrated by solid-phase extraction, precipitation / centrifugation using a specific formulation, or size exclusion chromatography. For example, gluten peptides can be concentrated by size exclusion chromatography (SEC) or by using a molecular size filter (e.g., Fractionation can be performed using any device that can separate peptides corresponding to two molecular sizes. Preferably, the procedure will have a range of gliadin sizes from a minimum of 1 kDa (detectable by immunological methods and containing two epitopes) to a maximum of 30 kDa. The ratio between total gluten peptides in the urine and total gluten peptides in each fraction will be a factor that allows us to know the amount of high molecular size peptides in the urine, and thus the degree of intestinal permeability to peptides of a particular size. These additional detailed studies can be performed on individuals who require more information to determine the extent of their intestinal disease or progression of intestinal permeability.

[0048] The amount of gluten peptides in the urine of these individuals will be measured using an immunological method with a specific antibody that quantifies gluten peptides resistant to intestinal digestion via epitopes detectable in the urine of these individuals. The amount of gluten peptides excreted in the urine can be compared to reference values. These reference values ​​will be generated from tests performed on a population without gastrointestinal tract diseases or conditions that impair their intestinal permeability. Alternatively, the values ​​obtained in the first determination of individuals undergoing the procedures of this invention can be personalized reference values ​​considered when assessing their intestinal permeability as a treatment or chronic disease being evaluated or monitored progresses. A larger amount of gluten peptides detected compared to baseline or reference values ​​will indicate increased intestinal permeability, while a decrease in these values ​​will indicate normalization of intestinal permeability.

[0049] Therefore, a first embodiment of the present invention relates to an in vitro method for assessing the intestinal permeability status of a subject, comprising: a) orally administering to the subject at least 0.5 g of gluten (preferably 0.5 g to 40 g of gluten) or a source thereof after a fasting period of at least 8 hours and after a gluten-free diet of at least 16 hours; b) determining the total amount of gluten peptides present in a total urine sample obtained from the subject during a period of at least 6 hours (preferably 6 hours to 16 hours) following the administration in step a); and c) wherein if the amount of gluten determined in step b) is significantly higher than a pre-established reference value, it indicates excessive intestinal permeability.

[0050] A second embodiment of the invention relates to an in vitro method for diagnosing, predicting, or monitoring a patient with a disease associated with excessive intestinal permeability, the disease being selected from: leaky gut syndrome, celiac disease, Crohn's disease, irritable bowel syndrome, microscopic colitis, functional dyspepsia, chronic / acute villous atrophy due to long-term treatment with certain drugs, systemic inflammation, autoimmune diseases, and / or non-IgE-mediated food hypersensitivity, the method comprising measuring the intestinal permeability of the subject according to the method of the first embodiment of the invention.

[0051] In a preferred embodiment of the invention, the gluten source has been purified and suspended in an aqueous fluid medium.

[0052] In a preferred embodiment of the invention, the amount of gluten peptides is determined directly by an immunoassay used to detect the peptides themselves.

[0053] In a preferred embodiment of the invention, the immunoassay includes an antibody against a gluten peptide comprising the following sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and / or derivatives thereof.

[0054] In a preferred embodiment of the invention, the amount of gluten peptides is determined by an immunoassay selected from the following: LFIA, ELISA, CLIA, SIMOA, Luminex, NULISA, turbidimetry, immunochromatography, or biosensors.

[0055] In a preferred embodiment of the invention, gluten peptides are concentrated by one of the following types of processes: solid-phase extraction with hydrophobic resin, size exclusion chromatography, precipitation or affinity purification.

[0056] A second embodiment of the invention relates to a component kit suitable for carrying out the method of the invention, comprising: a) a formulation having at least 0.5 g (preferably 0.5 g to 40 g) of purified gluten (preferably whole or partially hydrolyzed gluten powder for suspension, or ready-to-use solution or suspension), b) a urine collection container, and c) an immunological assay comprising antibodies capable of forming complexes with immunogenic gluten peptides and detectable by turbidimetry, visual inspection, colorimetry, fluorescence, chemiluminescence, and / or thermal detection.

[0057] In a preferred embodiment of the invention, the immunological test consists of a lateral flow strip having an antibody against immunogenic gluten peptides.

[0058] In a preferred embodiment of the present invention, the immunological test consists of ELISA, LFIA, CLIA, or magnetic particles, fluorescent particles, or phosphorescent particles.

[0059] A third embodiment of the present invention relates to a gluten peptide or a reactive antibody against said peptide or a derivative thereof for use in an in vitro method for assessing the intestinal permeability status of a subject, characterized in that the gluten peptide comprises or is composed of the following sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and / or derivatives thereof. Alternatively, the present invention relates to the use of a gluten peptide or a reactive antibody against said peptide or a derivative thereof for in vitro assessment of the intestinal permeability status of a subject, characterized in that the gluten peptide comprises or is composed of the following sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and / or derivatives thereof.

[0060] In a preferred embodiment of the invention, a gluten peptide or antibody is involved for use in an in vitro method for assessing the intestinal permeability status of a subject, the method comprising: (a) orally administering to the subject at least 0.5 g of gluten (preferably 0.5 g to 40 g of gluten) or a source thereof after a fasting period of at least 8 hours and after a gluten-free diet of at least 16 hours; and (b) determining the amount of gluten peptide present in a urine sample obtained from the subject during a period of at least 6 hours (preferably 6 hours to 16 hours) following gluten intake, wherein a significantly higher level of gluten presence as determined in step (b) than a pre-established reference value indicates excessive intestinal permeability. Alternatively, the present invention relates to the use of gluten peptides or reactive antibodies against said peptides or derivatives thereof for in vitro assessment of the intestinal permeability status of a subject, characterized in that the gluten peptide comprises or is composed of the following sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and / or derivatives thereof, the use comprising: (a) orally administering to a subject at least 0.5 g of gluten (preferably 0.5 g to 40 g of gluten) or a source thereof after a fasting period of at least 8 hours and after a gluten-free diet of at least 16 hours; and (b) determining the amount of gluten peptide present in a urine sample obtained from the subject during at least 6 hours (preferably 6 hours to 16 hours) following gluten ingestion, wherein a significantly higher amount of gluten present as determined in step (b) than a pre-established reference value indicates excessive intestinal permeability.

[0061] In a preferred embodiment, the invention includes a concentration step by size exclusion chromatography, wherein an increase in the average size of urinary gluten peptides indicates excessive intestinal permeability.

[0062] In the context of this invention, the following terms or expressions are defined for better understanding of its scope:

[0063] The term "including" means, but is not limited to, the content following the word "including". Therefore, the use of the term "including" indicates that the listed elements are required or mandatory, but other elements are optional and may or may not be present.

[0064] The term "composed of" refers to and is limited to the content included in the phrase "composed of". Therefore, the phrase "composed of" indicates that the listed elements are necessary or mandatory, and no other elements may exist.

[0065] The term "pre-established reference value," when referring to the quantification of gluten measured in this invention, refers to the quantification observed in healthy individuals (i.e., subjects without excessive intestinal permeability), particularly in the initial state of the same individual before treatment. In a preferred aspect, the reference value is measured in the initial measurement of subjects based on results from a group without intestinal disease, or based on results from a group without intestinal disease within the same age range as the individual whose intestinal permeability is being analyzed. If the quantification of gluten measured in the subject's urine is higher than the "pre-established reference level," the subject may have increased intestinal permeability, along with a certain degree of sensitivity and specificity. The "reference" value can be a threshold or cutoff value that can be determined experimentally, empirically, or theoretically. As known to those skilled in the art, the threshold can also be arbitrarily chosen based on existing experimental and / or clinical conditions. The threshold must be determined based on the test functionality and the benefit / risk balance (clinical consequences of false positives and false negatives) to obtain optimal sensitivity and specificity. Typically, optimal sensitivity and specificity (and the threshold) can be determined by using ROC (subject operating characteristic) curves based on experimental data. Attached Figure Description

[0066] Figure 1 Classification of intestinal villus atrophy status. VH:CD (villus height, crypt depth) is the average ratio between the height of the intestinal villi and the depth of the crypts. A ratio of 2 or higher is considered a Marsh classification of 0 to 2, indicating no or mild intestinal atrophy. A ratio below 2 is considered Marsh 3, ranging from 3a to 3c, signifying severe intestinal atrophy.

[0067] Figure 2 The dependence of urinary immunogenic gluten peptide concentration on the degree of villus atrophy. All patients ingested 3g of gluten from an energy bar. VH:CD is the ratio between villus height and crypt depth. LOQ = Limit of Quantification (2.5ng / ml).

[0068] Figure 3 The mean excretion of GIP in celiac disease individuals was compared based on the degree of villous atrophy observed in biopsies taken after a daily intake of 3g of gluten for at least 2 months.

[0069] Figure 4 Gluten peptide excretion kinetics in individuals without known intestinal diseases after ingestion of 8g of powdered purified gluten under fasting conditions. Each color represents a different volunteer.

[0070] Figure 5 Lactulose excretion kinetics from total intake (10 g) in individuals without known intestinal diseases (n=15), expressed in ppm. Each color represents a different volunteer.

[0071] Figure 6 The correlation between GIP excretion and lactulose / mannitol excretion ratio in total urine collection 15 hours after ingestion of 8 g of purified gluten suspension in 15 volunteers under prior fasting conditions. 2 =0.51. Detailed Implementation

[0072] The present invention is illustrated by the following embodiments, but is not intended to limit its scope of protection.

[0073] Example 1. Changes in gluten peptide concentration in urine of celiac disease individuals with varying degrees of villonodular atrophy.

[0074] This example illustrates that clinical study results indicate that, for continuous intake, increased villous atrophy in celiac patients increases the value of urinary GIP excretion (u-GIP), even without controlling other food intake or the duration of urine collection. Statistically significant changes in peptide concentrations can be observed based on the degree of duodenal villous atrophy. In this study, the same amount of gluten was administered several days before or after duodenal biopsy, and data on the degree of villous atrophy in celiac individuals were obtained by measuring the mean ratio of villus height to crypt depth (Vh:Cd). The subjects analyzed (celiac patients) received the same amount of gluten (3g, in bar form) daily for 6 weeks after duodenal biopsy, and data on the degree of villous atrophy were obtained by measuring the mean ratio of villus height to crypt depth. Significantly higher concentrations of gluten immunogenic peptides detectable by immunoassay were observed in more severe intestinal mucosal atrophy. Therefore, for the same amount of gluten intake, the most decisive factor in the concentration of gluten peptides excreted in urine is the increased intestinal permeability caused by the progressive deterioration of intestinal epithelial cells due to gluten intake. Figure 2 The mean gluten concentration in urine of patients with celiac disease atrophy grades 0 to 2 was 23.7 ng / ml, with a median of 11.8 ng / ml. For patients with Marsh 3, the mean was 45.7 ng / ml, with a median of 29.9 ng / ml. Although the mean deviations were high in both cases (21 and 33, respectively), the trend was significant, indicating higher permeability in Marsh 3 patients. Mean and median values ​​were observed for Marsh 3a, Marsh 3b, and Marsh 3c patients within the Marsh 3 group. These results are shown in Table 2 and... Figure 3 It is displayed in the middle.

[0075] Table 2: GIP concentration in samples from patients with varying degrees of intestinal atrophy after consuming 3g of gluten. VH:CD is the villus height / crypt depth ratio.

[0076] Table 2

[0077]

[0078] Higher degrees of atrophy were associated with higher concentrations of GIP in urine, with the differences being particularly pronounced in severe atrophy (Marsh 3b and Marsh 3c). The most plausible explanation is that daily gluten intake causes damage to the intestinal mucosa, leading to increased pericellular intestinal permeability.

[0079] Example 2. Metabolic changes among individuals.

[0080] This embodiment of the invention demonstrates that the values ​​of metabolized and excreted gluten on different dates in two different individuals yielded similar u-GIP values. For this purpose, Individual 1 and Individual 2 followed a gluten-free diet for 36 hours and fasted for at least 9 hours before consuming a packet of gluten (8g) suspended in 250ml of water. Before gluten ingestion, a urine GIP test was performed to confirm a negative result. After ingestion, they maintained a gluten-free diet for at least 8 hours, collected urine for 9 hours, and then quantified the urinary GIP concentration using a lateral flow test and an iVYCHECK Reader, calculating the corresponding volume. In the two individuals analyzed, the standard deviation of GIP excretion between measurements performed on different dates was less than 20% (Table 3), therefore the values ​​for each individual can be used as internal reference values ​​prior to treatment to improve intestinal permeability in a given individual, as the changes are not significant in the absence of pathology.

[0081] Table 3. Quantitative determination of gluten peptide excretion in two volunteers on different dates after fasting and ingestion of 8 g gluten suspension (μg of gluten excreted in urine during 9 hours after ingestion).

[0082] Table 3

[0083] Day 1 Day 5 Day 10 average value Standard deviation Volunteer 1 16.3 12.0 14.9 14.4 2.2 Volunteer 2 26.0 21.9 21.3 23.1 2.5

[0084] The ratio of GIP in urine to ingested amounts was 14 μg to 23 μg for every 8g of gluten ingested. Using a specific anti-33-mer immunoassay, approximately 8g of gluten intake was equivalent to approximately 1g to 1.33g of GIP measured using iVYCHECK urine (Biomedal, Spain). Therefore, excretion accounts for only 0.001% to 0.002% of ingested amounts, which is why specific and highly sensitive techniques are needed to detect these peptides in urine.

[0085] Example 3. Amount of gluten peptides excreted in different individuals compared to the permeability of lactulose / mannitol.

[0086] In this embodiment, it was observed that within several hours of consuming a fixed amount of powdered gluten after fasting, almost all detectable gluten was excreted in the urine, and its correlation with lactulose excretion was better than its correlation with small molecules (such as mannitol), suggesting that we were estimating paracellular osmotic pressure (laculose) vs. intracellular osmotic pressure (mannitol). Furthermore, it was observed that the majority (>80%) of detectable gluten excretion occurred within the first 9 to 15 hours, reducing the degree of intra-individual variability in lactulose. Specifically, after at least 36 hours of gluten-free diet in individuals without a diagnosed celiac disease, subjects (n=15) fasted for at least 8 hours and then received 8g of gluten. The gluten, consisting of powdered gluten and flavorings (sweeteners and food coloring), was contained in a single-use heat-sealed pouch. Simultaneously, volunteers ingested 1g of mannitol and 10g of lactulose. Subsequently, urine samples were collected in separate containers to allow for individualized measurements of the concentration and total volume of urine in each excretion. The amount of gluten peptides present in urine excreted 15 hours after gluten intake was determined using VYCHECK urine products and a calibrated lateral flow strip reader, the iVYCHECK Reader (Biomedal, Spain). Ingested gluten was mixed with 1 g mannitol and 10 g lactulose as a control. Mannitol is used to estimate intracellular permeability, and lactulose to estimate paracellular permeability; these are reference values ​​widely described in the biomedical literature. The amount of gluten peptides is directly related to intestinal permeability. Although factors such as the rate of gluten digestion may have an effect, it should be understood that reference values ​​can be constant within the same body, as seen in previous examples. Given the variability in gluten metabolism and its excretion kinetics, it is recommended that intake be performed after the subject urinates to empty the bladder as much as possible (early morning), preferably maintaining a fasting period of at least 8 hours to accelerate the metabolism of ingested gluten until its excretion, unaffected by other concurrently ingested foods. The values ​​obtained can be compared with reference values ​​obtained by performing the same procedure on individuals with or without intestinal diseases related to intestinal permeability, or these values ​​can be used on their own as a reference for subsequent measurements, indicating progress toward higher gluten peptide excretion values ​​(higher permeability) or lower gluten peptide excretion values ​​(lower permeability).

[0087] In this embodiment, basal urinalysis was performed on 15 volunteers without diagnosed symptoms of digestive system disease. All urine samples were collected in separate containers 15 hours after ingestion of the gluten pouch. The following urine samples were combined: 0h to 6h post-ingestion, and then 2h to 15h post-ingestion. The concentration of GIP in the excreted urine was measured by ELISA and LFIA at 0h to 6h and 2h to 15h post-ingestion, respectively. The GIP concentration was measured for each volunteer per urine sample. Figure 4 ) and lactulose concentration ( Figure 5), and then the percentage of excretion relative to ingested GIP and the lactulose / mannitol ratio were calculated ( Figure 6 The correlation between lactulose, mannitol, and GIP was determined. The kinetics of lactulose excretion differ from individual to individual, with excretion occurring after 15 hours. It is well known that sugars can be reabsorbed by the body. This explains why lactulose can have several excretion peaks. It is estimated that both lactulose and GIP excrete via the same pathway, namely the paracellular pathway. Therefore, the mixture of GIP excretions from 2 hours to 15 hours is more similar to the mixture of lactulose excretions from 0 hours to 6 hours and from 2 hours to 15 hours.

[0088] Further analysis was performed to detect outliers in each group and remove them from the correlation.

Claims

1. An in vitro method for assessing the intestinal permeability status of a subject, comprising: a. After a fasting period of at least 8 hours and after a gluten-free diet of at least 16 hours, administer orally at least 0.5 g of gluten or a source thereof to the subject. b. Determine the total amount of gluten peptides present in a total urine sample obtained from the subject during at least 6 hours following the administration in step a). If the amount of gluten measured in step b) is significantly higher than the pre-established reference value, it indicates that the intestinal permeability is too high.

2. An in vitro method for diagnosing, predicting, or monitoring a patient with a disease associated with excessive intestinal permeability, said disease being selected from: leaky gut syndrome, celiac disease, Crohn's disease, irritable bowel syndrome, microscopic colitis, functional dyspepsia, chronic / acute villous atrophy due to long-term treatment with certain drugs, systemic inflammation, autoimmune diseases, and / or non-IgE-mediated food hypersensitivity, said method comprising measuring the intestinal permeability of the subject as described in claim 1.

3. The in vitro method according to any one of the preceding claims, characterized in that, The gluten source has been purified and suspended in an aqueous fluid medium.

4. The in vitro method according to any one of the preceding claims, characterized in that, The amount of the gluten peptide was determined directly by an immunoassay used to detect the peptide itself.

5. The in vitro method according to any one of the preceding claims, wherein, The immunoassay includes an antibody against a gluten peptide comprising the following sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and / or derivatives thereof.

6. The in vitro method according to any one of the preceding claims, characterized in that, The amount of gluten peptides is determined by an immunoassay selected from the following: LFIA, ELISA, CLIA, SIMOA, Luminex, NULISA, turbidimetric assay, immunochromatography, or biosensor.

7. The in vitro method according to any one of the preceding claims, characterized in that, The gluten peptides are concentrated using one of the following types of processes: solid-phase extraction with hydrophobic resins, size exclusion chromatography, precipitation or affinity purification.

8. The in vitro method according to any one of the preceding claims, wherein, A concentration step performed by size exclusion chromatography, in which an increase in the average size of urinary gluten peptides indicates excessive intestinal permeability.

9. A component kit suitable for implementing the method of claims 1 to 8, comprising: a. A preparation containing at least 0.5g of purified gluten, b. Urine collection container, c. Immunological tests, including antibodies capable of forming complexes with immunogenic gluten peptides and detectable by turbidimetric, visual, colorimetric, fluorescence, chemiluminescent, and / or thermal detection methods.

10. The component kit according to claim 9, wherein, The immunological test consists of a lateral flow strip containing antibodies against immunogenic gluten peptides.

11. The component kit according to any one of claims 9 or 10, wherein, The immunological test consists of ELISA, LFIA, CLIA, or magnetic particles, fluorescent particles, or phosphorescent particles.

12. The use of gluten peptides or reactive antibodies against said peptides or derivatives thereof for in vitro assessment of the intestinal permeability status of a subject, characterized in that, The gluten peptide comprises or consists of the following sequences: SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11 and / or derivatives thereof.

13. The use of the gluten peptide or antibody according to claim 12 for in vitro assessment of the intestinal permeability status of a subject, said use being achieved by: (a) orally administering at least 0.5 g of gluten or a source thereof to the subject after a fasting period of at least 8 hours and after a gluten-free diet of at least 16 hours; and (b) determining the amount of gluten peptide present in a urine sample obtained from the subject during at least 6 hours after gluten intake, wherein a significantly higher level of gluten present as determined in step (b) than a pre-established reference value indicates excessive intestinal permeability.

14. The use according to claim 12 or 13, wherein, A concentration step performed by size exclusion chromatography, in which an increase in the average size of urinary gluten peptides indicates excessive intestinal permeability.

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