Identification of small intestinal bacterial overgrowth (SIBO)

Abstract

Polyfenol(s) and / or metabolite(s) thereof, and / or precursor(s) to trimethylamine, and / or trimethylamine-N-oxide (TMAO), or a composition comprising polyfenol(s) and / or metabolite(s) thereof and / or precursor(s) to trimethylamine, and / or 5 trimethylamine-N-oxide, for use in a method of identifying whether or not an individual has small intestinal bacterial overgrowth (SIBO), is / are disclosed.

Description

[0001]

[0002] IDENTIFICATION OF SMALL INTESTINAL BACTERIAL OVERGROWTH (SIBO)

[0003] Field of the Invention

[0004] The present invention relates to identification / determination / diagnosis whether or not an individual has small intestinal bacterial overgrowth (SIBO).

[0005] Background of the Invention

[0006] Small intestinal bacterial overgrowth (SIBO) is defined as the presence of excessive (exceeding 105- 106organisms / ml) bacteria in the small intestine. It is an abnormal condition in which both aerobic and anaerobic bacteria, mainly from the colon but occasionally also from the mouth or oesophagus, proliferate in the small intestine.

[0007] The small intestine is sometimes called "the little brain" because of the large number of nerve cells that are present along the wall of the small intestine. A large part of the body's immune system is also located along the permeable small intestine. With high amounts of bacteria in the small intestine, the intestinal wall can be damaged and allow the passage of bacteria and bacterial toxins into the bloodstream and upset the nerve and immune cells in the wall of the small intestine.

[0008] The symptoms associated with SIBO are non-specific and include e.g., bloating, abdominal distension, abdominal pain or abdominal discomfort, diarrhea, fatigue, and weakness. The frequency and severity of the symptoms may reflect both the degree of the bacterial overgrowth in the intestine as well as the extent of mucosal inflammation. Other symptoms that may be seen may reflect complications of SIBO, including nutritional deficiencies, malabsorption, etc. It may be difficult to distinguish SIBO from other stomach problems, such as lactose intolerance and irritable bowel syndrome, and it may therefore easily be misdiagnosed / incorrectly identified as SIBO or not SIBO.

[0009] SIBO is common in patients who are immunosuppressed, such as cancer patients receiving therapy. It has been associated with e.g., chronic fatique syndrome, fibromyalgia, depression, and rosacea. Two tests for diagnosing whether or not an individual has SIBO are commonly employed: bacterial culture and breath tests. The bacterial culture test is rather expensive and complicated, while breath testing is the predominant method to evaluate patients for SIBO because of its simplicity and lack of invasiveness compared to bacterial culture test. When using breath tests, detection of SIBO is typically achieved by detecting hydrogen and / or methane exhaled in the breath after providing an oral substrate such as glucose or lactulose. The breath tests rely on the recovery and quantification of an exhaled gas produced by the bacterial metabolism of the ingested substrate.

[0010] WO2021 / 127027 describes a system and method for implementation of a breath testing system for convenient sampling of intestinal gases exhaled from a patient's breath. The system may give an indication whether a patient has SIBO.

[0011] There are several problems associated also with the breath tests. For instance, differences in bacterial flora among patients can determine the response to the breath test. About 10% of adults and 15% of children may not be colonized with bacteria capable of producing hydrogen. Further, the optimum protocol for the administration, timing and collection of breath specimens is not determined, and the proper interpretation of results is unclear. Thus, although easy to perform, the results may be difficult to interpret. Further, since gas-producing bacteria comprise only a part of the total gut flora content, breath tests do not provide a comprehensive analysis of the entire gut microbiota.

[0012] Thus, there is a need for a non-invasive, effective way of detecting SIBO, which is easy to perform, covers a large proportion of the gut microbiota, and gives an adequate result at a reasonable cost.

[0013] Summary of the Invention

[0014] It is an object of certain aspects of the present invention to provide an improvement over the above described methods and known prior art; particularly to achieve a method for identifying whether or not an individual has SIBO.

[0015] According to the present invention this is achieved by polyfenol(s) and / or metabolite(s) thereof, and / or precursor(s) to trimethylamine (TMA), and / or trimethylamine-N-oxide (TMAO), or a composition comprising polyfenol(s) and / or metabolite(s) thereof, and / or precursor(s) to trimethylamine (TMA), and / or trimethylamine-N-oxide (TMAO), for use in a method of identifying whether or not an individual has small intestinal bacterial overgrowth (SIBO).

[0016] In one aspect, the method is performed in vitro.

[0017] In another aspect, said polyfenol(s) is / are selected from resveratrol and / or curcumin.

[0018] According to one aspect, said metabolite(s) is / are selected from dihydroresveratrol (DHR), dihydrocurcumin (DHC), tetrahydrocurcumin (THC), hexahydrocurcumin (HHC), hexahydro-didemethyl-curcumin (HHDEC), hexahydro- didemethyl-curcumin (HHDDEC) and / or octahydrocurcumin (OHC).

[0019] In one aspect, said precursor(s) to trimethylamin (TMA) is / are selected from choline, L-carnitine, betaine, phosphatidylcholine, lecithin, and / or dimethylglycine.

[0020] In a further aspect, said polyfenol(s) and / or precursor(s) to trimethylamine (TMA), and / or composition comprising polyfenol(s) and / or precursor(s) to trimethylamine (TMA), has / have been orally administered earlier to the individual.

[0021] In another aspect, the concentration(s) of said metabolite(s) of said polyfenol(s), and / or the concentration of trimethylamine-N-oxide (TMAO, in a urinary sample earlier obtained from the individual, is / are measured.

[0022] The TMA produced from the TMA precursor(s) is oxidized in the liver into TMAO, thus it is the TMAO that is detected in the urine and not the TMA.According to one aspect, said urinary sample preferably is obtained at least about 1 hour, preferably at least about 1.5 hours, after said oral aministration of said polyfenol(s) and / or precursor(s) to trimethylamine.

[0023] According to another aspect, provided is also a method for in vitro identifying whether or not an individual has SIBO, which method comprises measuring a concentration of metabolite(s) of polyfenol(s) and / or a concentration of trimethylamine-N-oxide in a urine sample taken from the individual, wherein said polyfenol(s) and / or optionally at least one precursor to trimethylamine has / have earlier been orally administered to said individual, and thereby determining whether or not the individual has SIBO.

[0024] This means that the polyfenol(s) may be administered alone, i.e., without the at least one precursor to trimethylamine, or together with the at least one precursor to trimethylamine, and further that at least one precursor to trimethylamine may be administered alone, i.e., without polyfenol(s). The polyfenol(s) may, in one aspect, be selected from resveratrol and / or curcumin.

[0025] In one aspect, the metabolite(s) may be selected from dihydroresveratrol (DHR), dihydrocurcumin (DHC), tetrahydrocurcumin (THC), hexahydrocurcumin (HHC), hexahydro-didemethyl-curcumin (HHDEC), hexahydro-didemethyl-curcumin (HHDDEC) and / or octahydrocurcumin (OHC).

[0026] According to one aspect, said precursor(s) to trimethylamine is / are selected from choline, L-carnitine, betaine, phosphatidylcholine, lecithin, and / or dimethylglycine.

[0027] The urine sample is preferably obtained at least about 1 hour, preferably at least about 1.5 hours, after said oral administration of said polyfenol(s) and / or optionally said precursor(s) to trimethylamine.

[0028] According to a further aspect, the method further comprises measuring a concentration of lactulose (LAC) in the urine sample taken from the individual.

[0029] In one aspect, said lactulose is administered orally to the individual together with the oral administration of said polyfenol(s) and / or said precursor(s) to trimethylamine.

[0030] According to one aspect, the concentration of metabolite(s) and / or trimethylamine-N-oxide, in nanogram / ml, in the urine sample lower than or equal to:

[0031] DHR: < 50;

[0032] DHC: < 0.7;

[0033] THC: < 10;

[0034] HHC: < 10;

[0035] HHDEC: < 10;

[0036] HHDDEC: < 2;

[0037] OHC: < 5, and / or

[0038] TMAO: < 50 000 are indicative that the individual does not have SIBO, and the concentration of metabolite(s) and / or trimethylamine-N-oxide higher than the concentration(s) above are indicative that the individual has SIBO.

[0039] In one aspect, the individual is suspected of having SIBO.

[0040] In one aspect, the individual is a human. According to a further aspect, disclosed is also use of polyfenol(s), and / or metabolite(s) thereof, and / or trimethylamine and / or precursors to trimethylamine, and / or trimethylamine-N-oxide, in identifying whether or not an individual has SIBO.

[0041] In one aspect, the identifying is in vitro.

[0042] Further, according to another aspect, provided is also a kit for determining the presence of SIBO, said kit comprising:

[0043] (a) polyfenol(s) and / or optionally precursor(s) to trimetylamine, or a composition comprising polyfenol(s) and / or optionally precursor(s) to trimethylamine;

[0044] (b) instructions for performing the method as defined according to the present invention.

[0045] TMA produced in the intestines from TMA precursor(s) is oxidized by a flavin- containing monooxygenase enzyme in the liver into trimethylamine-N-oxide (TMAO), whereafter it is efficiently excreted in urine where the TMAO can be detected. Thus, when collecting urine samples from an individual to analyze and determine whether or not the individual has SIBO, it is the concentration of TMAO and not the concentration of TMA that is analyzed in the sample.

[0046] Brief Description of the Drawings

[0047] These and other aspects, features, and advantages of which embodiments of the invention are capable of, will be apparent and elucidated from the following description of embodiments and aspects of the present invention, reference being made to the accompanying drawings, in which

[0048] Fig. 1 A-C shows the metabolite profiles in urine samples taken about 1.5 hours after oral administration of resveratrol and curcumin in five individuals (PVL, TGR, SIB, FB, and ALX) with SIBO symptoms as described above, and one individual without SIBO symptoms. The concentrations shown are in nanogram / ml urine.

[0049] Fig. 2 A-E shows correlations between urinary HHC / OHC and LAC and TMAO levels. The scatter plots show the relationship after loglO transformation and outlier removal. Each point represents an individual sample. The dark line indicates the fitted linear regression with a 95% confidence interval (shaded area). Positive correlations were observed in all cases (Pearson's correlation test, p< 0.001). Detailed Description

[0050] Specific embodiments of the invention will now be described. This invention may, however, be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of the invention to those skilled in the art. The terminology used in the detailed description of the embodiments is not intended to be limiting of the invention. The terminology used herein is for the purpose of describing particular aspects of the disclosure only, and is not intended to limit the disclosure. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, unless the context clearly indicates otherwise.

[0051] It should be noted that the word "comprising" does not necessarily exclude the presence of other elements or steps than those listed and the words "a" or "an" preceding an element do not exclude the presence of a plurality of such elements.

[0052] The different aspects, alternatives and embodiments of the invention disclosed herein can be combined with one or more of the other aspects, alternatives and embodiments described herein. Two or more aspects can be combined.

[0053] By "diagnosis" is included determining the presence or absence of a state / disease state in an individual (e.g., identifying whether or not an individual is suffering from SIBO).

[0054] Resveratrol (RSV) is a natural polyphenol, found e.g., in the skin of red grapes, in peanuts and in berries, and is commercially available as a powder.

[0055] Curcumin is a bright yellow chemical produced by plants of the Curcuma longa species. Chemically, curcumin is a diarylheptanoid, belonging to the group of curcuminoids, which are phenolic pigments responsible for the yellow color of turmeric.

[0056] Trimethylamine (TMA) is a downstream metabolite which may be derived from microbial processing of precursors such as choline, L-carnitine, betaine, phosphatidylcholine, lecithin, and / or dimethylglycine.

[0057] The human gut microbiota is predominantly composed of Clostridia and Bacteroidia, accounting for more than 90% of the bacterial population in the human gut. Bacteria within the classes Clostridia and Bacteroides, prominent members of the human gut microbiota, possess enzymatic capabilities that enable them to transform resveratrol, curcumin and TMA precursors into various metabolites, where reduction, demethoxylation and demethylation reactions lead to the formation of metabolites, Li F, Han Y, Wu X, et al. Gut Microbiota-Derived Resveratrol Metabolites, Dihydroresveratrol and Lunularin, Significantly Contribute to the Biological Activities of Resveratrol. Front Nutr. 2022;9:912591. published 2022 May 11. Scazzocchio B, Minghetti L, D'Archivio M. Interaction between Gut Microbiota and Curcumin: A New Key of Understanding for the Health Effects of Curcumin Nutrients, 2020;12(9):2499. published 2020 Aug 19. Wang, Z. et al. Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease. Nature, 472(7341), 57-63, published 2011. The TMA-forming step in the intestine occurs through microbial TMA lyases, enzymes that cleave certain trimethylamine-containing nutrients.

[0058] Since Clostridia and Bacteroides comprise about 90% of the total human gut flora bacteria and use enzymes required for metabolizing resveratrol, curcumin and precursors to TMA, such as choline, L-carnitine, betaine, phosphatidylcholine, lecithin, and / or dimethylglycine, the present technique provides a new and powerful approach for detecting a broad range of bacteria in the small intestine.

[0059] Resveratrol (RSV) may be metabolized into dihydroresveratrol (DHR), a transformation that is carried out exclusively by bacteria, not by human somatic cells. In the case of SIBO, a broad range of bacteria located in the small intestine may reduce resveratrol into dihydroresveratrol during the about 1.5 hour long passage through the small intestine, and the metabolite is transported via the blood to the urine. The urine may thereafter be analyzed and the metabolite detected and its presence used as an indication of SIBO.

[0060] TMA precursors such as choline, L-carnitine, betaine, phosphatidylcholine, lecithin, and / or dimethylglycine may be metabolized into TMA during the about 1.5 hour long passage through the small intestine, whereafter the TMA is oxidized by a flavin-containing monooxygenase enzyme in the liver into trimethylamine-N-oxide (TMAO) and efficiently excreted in urine. The urine may thereafter be analyzed and TMAO and its presence used as an indication of SIBO. Both RSV and DHR are absorbed by the cells lining the small intestine, and are conjugated to glucuronide and / or sulphated RSV and DHR in order for their water solubility to be increased so that they may be excreted via the kidneys into the urine. Epithelial cells can also absorb TMA precursors and the unabsorbed portion is readily converted to TMA by the microbes in the small gut.

[0061] Resveratrol Dihydroresveratrol

[0062] Reduction of curcumin into metabolites like dihydrocurcumin (DHC), tetrahydrocurcumin (THC), hexahydrocurcumin (HHC), hexahydro-didemethyl- curcumin (HHDEC), hexahydro-didemethyl-curcumin (HHDDEC) and octahydrocurcumin (OHC) is a process facilitated by the above-mentioned gut microbiota, which possess the anaerobic reductase enzymes required for this transformation. These bacterial enzymes reduce curcumin's double bonds, resulting in the above hydrogenated metabolites. Human somatic cells cannot reduce, demethylate or hydroxylate curcumin into the above-mentioned metabolites. Thus, in the case of SIBO, bacteria located in the small intestine can metabolize curcumin into DHC, THC, HHC, HHDEC, HHDDEC and OHC during the about 1.5 hour long passage through the small intestine, and the metabolites are transported via the blood to the urine. The urine may thereafter be analyzed and the metabolites detected and their presence used as an indication of SIBO.

[0063] The excretion of water-soluble DHR, DHC, THC, HHC, HHDEC, HHDDEC, and OHC by bacteria located in the small intestine occurs via the urine after consumption of resveratrol and curcumin. By analyzing the urine for metabolites of resveratrol and / or curcumin produced by bacteria during the about 1.5 hour passage through the small intestine, the present inventors have surprisingly found that bacterial overgrowth in the small intestine, SIBO, can indeed be identified / diagnosed. It is not necessary to use both resveratrol and curcumin for identifying whether or not an individual has SIBO / diagnosing SIBO, they can be used separately, and their metabolites be analyzed separately, but the use of a combination of both resveratrol and curcumin increases the sensitivity of the test for identification / diagnosis of SIBO.

[0064] Further, the excretion of TMAO (after oxidation of TMA into TMAO in the liver) by bacteria located in the small intestine occurs via the urine after consumption of TMA precursors, such as those selected from choline, L-carnitine, betaine, phosphatidylcholine, lecithin, and / or dimethylglycine. The precursors may be administered as such to the individual to be tested for SIBO, although the precursors may be present in food and thereby naturally ingested by the individual. Thus, it may not be necessary to administer TMA precursors to an individual to be tested for SIBO as they may be comprised in the food ingested, but the precursors may be administered to increase the significance of the result of the test, as the amount of TMA produced by the bacteria in the intestine (and thus the amount of TMAO after oxidation of the TMA in the liver) may be increased and thus more TMAO can be detected. By analyzing the urine for TMAO (after oxidation of TMA into TMAO in the liver) produced by bacteria during the about 1.5 hour passage through the small intestine, the present inventors have surprisingly found that bacterial overgrowth in the small intestine, SIBO, can indeed be identified / diagnosed.

[0065] It is not necessary to use both resveratrol and / or curcumin together with precursors to TMA, i.e. it is not necessary to analyze the urine for metabolites of resveratrol and / or curcumin together with TMAO (as TMA has been oxidized in the liver into TMAO) for identifying whether or not an individual has SIBO / diagnosing SIBO. They can be used and analyzed separately, but the use of a combination of resveratrol and / or curcumin together with TMA precursors, and analysis in the urine for metabolites of resveratrol and / or curcumin together with TMAO, may increase the sensitivity of the test for identification / diagnosis of SIBO.

[0066] If no SIBO is present in the small intestine, the amounts of TMAO and / or metabolites of resveratrol and / or curcumin found in a urine sample taken at least about 1 hour, preferably at least about 1.5 hours, after oral administration of resveratrol and curcumin, and possibly also administration of a precursor of TMA (not mandatory to administer since precursors to TMA may be comprised in food ingested by an individual and therefore not always necessary to actively administer), will be very low, while a significant increase in TMAO and / or metabolites can be seen in the urine in case of SIBO, where the case for metabolites of resveratrol and curcumin is seen in Fig. 1 A-C.

[0067] Resveratrol and / or curcumin and / or optionally precursors of TMA to be used for identifying whether or not an individual has SIBO is / are preferably orally administered on an empty stomach. Further, no food or drinks are preferably to be administered / swallowed within a period of approximately 20 minutes after said oral administration.

[0068] The resveratrol and / or curcumin and / or TMA precursors may e.g., be orally administered together with prebiotic fibers, i.e. substrates that cater to a broad range of bacteria, inducing bacterial metabolism and priming metabolic functions of a large range of bacteria in the small intestine, including the ability to reduce resveratrol and / or curcumin to their metabolites.

[0069] The metabolites produced by the bacteria in the small intestine, together with the TMAO achieved after oxidation in the liver of the TMA produced in the small intestine, will accumulate in the bladder during about 1.5 hours after oral administration. Preferably, the individual to be tested regarding possible presence of SIBO avoids going to the toilet to urinate after the oral administration of resveratrol and / or curcumin and / or TMA precursors in order to let the metabolites and / or TMAO accumulate in the urine in the bladder. In that way, the accumulation in the bladder increases the probability of detecting significant levels of resveratrol and / or curcumin metabolites and / or TMAO. This is especially relevant, since SIBO has been shown to affect the duodenum / jejunum, which is at the very beginning of the small intestine, directly under the stomach / ventricle.

[0070] For an individual to be classified as not having SIBO, the concentrations of metabolites of resveratrol and / or curcumin and / or TMAO in a urine sample obtained from said individual about 1.5 hrs after oral administration of resveratrol and / or curcumin and / or TMA precursors, measured in nanogram / ml, should be lower than or equal to the following values: for DHR: < 50, DHC: < 0.7, THC: < 10, HHC: < 10, HHDEC: < 10, and HHDDEC : <2, OHC: < 5, and / or TMAO: < 50 000, which concentrations levels may be seen in healthy individuals. Concentrations higher than those now mentioned are indicative that the individual may be affected by SIBO.

[0071] When using the test according to the present invention, using polyphenols, such as resveratrol and / or curcumin and their metabolites, and / or further using TMA precursors and TMAO, to identify whether or not an individual has SIBO, it is preferred that the individual to be tested does not consume lactic acid bacteria from supplements, fermented vegetables, kombucha as well as tea, fruit, grapes, red wine, nuts, turmeric (e.g., in curry), curcumin (e.g., as a dietary supplement) and dark chocolate at least 48 hours before the test. This is due to the fact that the lactic acid bacteria in the supplements / food may convey metabolic actions in the small intestine and make the results difficult to interpret. Furthermore, the above- mentioned supplements / food contain the substrates resveratrol and curcumin that eventually will reach the heavily colonized large intestine, resulting in massive levels of circulating metabolites which will affect the results of a subsequent SIBO-urine test taken within 48 hours after ingestion of the supplements / food. For the SIBO urine test / method according to the present invention to be as accurate as possible, there should be no added bacteria to the small intestine and no substrate for colon bacteria to produce metabolites during 48 hours before the test.

[0072] The polyphenol(s) and / or TMA precursor(s) are preferably mixed with water and swallowed on an empty stomach and with an empty bladder, preferably in the morning after no eating or drinking during the night. Preferably no food is consumed at least 20 minutes after swallowing the solution comprising the polyphenol(s) and / or TMA precursor(s). Fluid intake should be minimized after intake so that the urine can be held in the urinary bladder for at least about 1 hour, preferably at least about 1.5 hours.

[0073] The test and the method according to the present invention may also further comprise measuring a concentration of lactulose (LAC) in the urine sample taken from the individual. Lactulose permeability is an indicator of intestinal barrier impairment (so called leaky gut), a condition frequently associated with SIBO due to mucosal inflammation and disruption of epithelial tight junctions. Thus, LAC in the urine is a further indicator of SIBO, which can be used to strengthen the test, enabling an even more comprehensive evaluation and possibility to diagnos SIBO in an individual. The lactulose is administered orally, preferably together with the polyphenol(s) and / or TMA precursor(s).

[0074] Example 1

[0075] Substrate

[0076] A powder comprising 0.3 g resveratrol, 0.3 g curcumin, 5 g glucose and 10 g lactulose was dissolved / suspended in about 125 ml of water and orally administered. These doses of resveratrol and curcumin are within the limits accepted for human use, and result in measurable levels of their metabolites in the case of SIBO, and the doses may thus be varied. Urine sampling

[0077] A urine sample of about 1 ml for analysis was taken at about 1.5 hrs after the administration of the substrate solution.

[0078] Urine analysis of curcumin and / or resveratrol and their metabolites

[0079] 20 pl of 0.1 M Sodium acetate buffer (pH 5.0) was added to 30 pl of urine. Thereafter 5 pl of 25 U of beta-glucuronidase (from bovine liver) and 5 pl of sulfatase (from Helix pomata) was added. The samples were incubated at 37° C under mild agitation for 2 hours in order to remove sulphate- and glucoronidase from resveratrol, curcumin and their various metabolites. Subsequently, the samples were treated with 120 pl of acetonitrile, incubated for 30 min at 4° C and centrifuged at 15.000 g for 10 minutes in order to remove proteins in the samples. 100 pl of the supernatant was added to 400 pl water.

[0080] Mass spectrometry

[0081] The samples were chromatographically separated on a standard C18 column and analyzed using a triple-quadropole LC-MS mass spectrometer. The concentrations of curcumin and its metabolites (DHC, THC, HHC, HHDEC, HHDDEC, OHC) as well as resveratrol and its metabolite DHR were quantified using corresponding standards.

[0082] The molecular profiles based on the mass and charge (m / z -H) were: Resveratrol: 227.0715

[0083] DHR: 229.0866

[0084] Curcumin: 367.1207

[0085] DHC: 369.1344

[0086] THC: 371.1498

[0087] HHC: 373.1666

[0088] HHDEC: 359.1507

[0089] HHDDEC: 345.1362

[0090] OHC: 375.1824

[0091] Fig. 1 A-C shows the metabolite profiles in urine samples taken about 1.5 hours after oral administration of resveratrol and curcumin in five individuals (PVL, TGR, SIB, FB, and ALX) with SIBO symptoms as described above, and one individual without SIBO symptoms.

[0092] Whereas the non-SIBO individual had no detectable curcumin or resveratrol metabolites, all SIBO individuals presented one or more of the above metabolites in the urine 1.5 hrs after oral administration of a substrate solution as described above, comprising 0.3 g resveratrol, 0.3 g curcumin, 5 g glucose and 10 g lactulose.

[0093] Example 2

[0094] Substrate

[0095] A powder comprising 1 g of lecithin derived from sunflower was dissolved / suspended in about 125 ml of water and orally administered. This dose is within the limits accepted for human use and resulted in measurable urine levels of TMAO.

[0096] Urine sampling

[0097] A urine sample of about 1 ml for analysis was taken at about 1.5 hrs after the administration of the substrate solution.

[0098] Urine analysis

[0099] 20 pl of 0.1 M Sodium acetate buffer (pH 5.0) was added to 30 pl of urine. Thereafter 5 pl of 25 U of beta-glucuronidase (from bovine liver) and 5 pl of sulfatase (from Helix pomata) was added. The samples were incubated at 37° C under mild agitation for 2 hours in order to remove sulphate- and glucoronidase from resveratrol, curcumin and their various metabolites. Subsequently, the samples were treated with 120 pl of acetonitrile, incubated for 30 min at 4° C and centrifuged at 15.000 g for 10 minutes in order to remove proteins in the samples. 100 pl of the supernatant was added to 400 pl water.

[0100] Mass spectrometry

[0101] The samples were chromatographically separated on an Ultra AQ. C18 column (MZ Analysentechnik) and analyzed using a triple-quadropole LC-MS mass spectrometer using a gradient of water and acetonitril (with 0.5% Acetate added). The concentrations were quantified using corresponding standards.

[0102] Figure 2A illustrates the statistical relationship between two biological markers: (1) HHC, which serves as an indicator of SIBO, and (2) LAC, which reflects the integrity of the small-intestinal epithelial barrier (i.e., "leakiness" of the small intestine). To enable direct comparison, both variables were loglO-transformed to normalize their distributions, and extreme outliers were removed according to predefined criteria. After preprocessing, the association between HHC and LAC was quantified using Pearson's correlation coefficient, yielding a value of r = 0.51. A Pearson correlation of this magnitude indicates a moderate to moderately strong positive relationship. In practical terms, this means that higher levels of HHC tend to occur together with higher levels of LAC within the analyzed sample set. This trend suggests that individuals exhibiting stronger signs of SIBO (high HHC) also tend to show increased compromise of the small-intestinal epithelial lining (high LAC). Overall, the results presented in Figure 2A provide statistical evidence that increased bacterial overgrowth in the small intestine is associated with a worsened epithelial barrier condition, supporting the hypothesis of a functional link between SIBO and small-intestinal permeability.

[0103] Figure 2B presents the statistical relationship between two biological markers: (1) HHC, which functions as an indicator of SIBO, and (2) TMAO, a functional metabolic marker associated with bacterial metabolic activity. As with the prior analysis, both variables were loglO-transformed to reduce skewness and normalize their variance. Outliers were identified and removed according to predefined exclusion criteria. Following these preprocessing steps, the linear association between HHC and TMAO was evaluated using Pearson's correlation coefficient, resulting in a value of r = 0.47. A correlation of this magnitude constitutes a moderate positive association, indicating that higher HHC values generally co-occur with higher TMAO concentrations. This pattern is statistically significant and suggests that individuals with more pronounced evidence of SIBO (elevated HHC) also tend to exhibit increased levels of TMAO, reflecting enhanced bacterial metabolic activity in the small intestine

[0104] Figure 2C illustrates the statistical association between: (1) OHC, a biochemical marker indicative of SIBO, and (2) LAC, a marker reflecting compromised integrity ("leakiness") of the small-intestinal epithelial lining. As with the prior analysis, both variables were loglO-transformed to reduce skewness and normalize their variance. Outliers were identified and removed according to predefined exclusion criteria. After data preprocessing, the linear relationship between OHC and LAC was assessed using Pearson's correlation coefficient, which yielded a value of r = 0.63. A coefficient of this magnitude indicates a strong positive and statistically significant association. In practical terms, this means that higher OHC levels consistently correspond with higher LAC concentrations within the study population. Thus, individuals exhibiting more pronounced signs of SIBO (as indicated by elevated OHC) also tend to show greater disruption of the epithelial barrier in the small intestine. Overall, the results presented in Figure 2C provide strong quantitative evidence supporting a link between small-intestinal bacterial overgrowth and increased epithelial permeability, reinforcing the functional connection between microbial overgrowth and barrier dysfunction in the small gut.

[0105] Figure 2D illustrates the statistical association between: (1) OHC, a biochemical marker indicative of SIBO, and (2) TMAO, a functional metabolite generated by bacterial activity within the small intestine, reflecting microbial overgrowth and microbial metabolic activity in the small intestine, where the TMAO is from TMA that is oxidized in the liver into TMAO. As with the prior analysis, both variables were loglO-transformed to reduce skewness and normalize their variance. Outliers were identified and removed according to predefined exclusion criteria. Following data preprocessing, the linear relationship between OHC and TMAO was evaluated using Pearson's correlation coefficient, yielding r = 0.42. This coefficient reflects a moderate and statistically significant positive association between the two measures. Practically, this means that individuals with higher OHC concentrations - suggestive of more pronounced SIBO - tend to exhibit correspondingly elevated TMAO levels, consistent with increased small-intestinal microbial metabolic activity. Overall, the results presented in Figure 2D provide quantitative evidence supporting a functional connection between bacterial overgrowth in the small intestine and enhanced production of microbially derived metabolites such as TMAO. This relationship further reinforces the link between SIBO and altered metabolic output within the small gut environment.

[0106] Figure 2E illustrates the statistical association between: (1) LAC, a biochemical marker indicative of increased permeability of the small-intestinal epithelial lining ("leaky gut"), and (2) TMAO, a functional metabolite produced by the small-intestinal bacteria, where the TMAO is from TMA that is oxidized in the liver into TMAO, reflecting microbial activity associated with SIBO. As with the prior analysis, both variables were loglO-transformed to reduce skewness and normalize their variance. Outliers were identified and removed according to predefined exclusion criteria. After data preprocessing, the linear relationship between LAC and TMAO was assessed using Pearson's correlation coefficient, which yielded r = 0.36. This coefficient indicates a moderate and statistically significant positive association between the two measures. In practical terms, subjects exhibiting higher LAC levels, reflecting greater disruption of the small-intestinal epithelial barrier, tend to have higher TMAO concentrations, consistent with increased bacterial metabolic activity in the small gut. Overall, the results in Figure 2E provide quantitative evidence supporting a link between compromised epithelial integrity, enhanced microbial activity in the small intestine and the detection of higher levels of these molecules in the urine samples.

[0107] Results and Discussion

[0108] The analysis of the metabolites as mentioned throughout the present application and / or TMAO, optionally together with lactulose, and their concentration in urine after the about 1.5 hours long small intestinal passage of curcumin, resveratrol and / or TMA precursor(s) can be used to assess bacterial overgrowth in the small intestine (SIBO). The novel aspect of the present test / method focuses on how levels of metabolites of polyfenols, preferably metabolites of resveratrol and curcumin, and / or TMAO, optionally further together with lactulose, in urine about 1.5 hours post-ingestion of curcumin and / or resveratrol and / or precursor(s) of TMA (not always necessary to actively administer to the individual since they may be comprised in food ingested by the individual) and / or lactulose, can serve as reliable and non-invasive markers for SIBO.

[0109] The practical application of the present method and urine test used in clinical or home settings for SIBO screening is evident, since the methods available at the moment involve costly and time-consuming breath tests or invasive sampling via gastric tubes of bacteria in the small gut, while a urine sample is easy to take and analysis can be made at a low cost.

[0110] The present method / test provides a timely, user-friendly, and cost-effective way to determine if an individual is affected by SIBO or not, with potential applications in regular monitoring for those with recurring symptoms or as a followup for treatment efficacy. In non-SIBO conditions, ingested resveratrol, curcumin, and / or TMA precursor(s) pass through the stomach, where practically no absorption takes place. After about 120 minutes these substances will reach the large intestine, where curcumin, resveratrol and / or TMA precursor(s) undergo extensive metabolism by bacterial reductases and demethylases available in the colon microbiota.

[0111] In individuals with SIBO, this bacterial metabolization of resveratrol, curcumin and / or TMA precursor(s) occurs before these substances reach the colon, i.e. during the about 1.5 hours transition time through the small gut.

[0112] The bacterial metabolism of resveratrol, curcumin and / or TMA precursor(s) yields metabolites, such as dihydroresveratrol (DHR), dihydrocurcumin (DHC), tetrahydrocurcumin (THC), and hexahydrocurcumin (HHC), hexahydroxy-demethyl- curcumin (HHDEC), hexahydroxy-didemethyl-curcumin HHDDEC, octahydrocurcumin (OHC) and / or TMAO, where the TMAO is from TMA that is oxidized in the liver into TMAO (2, 3). These bacterial metabolites readily enter the blood stream and via glucuronidation and sulfation processes (mainly in the liver) these metabolites become sufficiently hydrophilic for urinary elimination. This is a critical factor in using urinary metabolites as biomarkers in diagnostic tests.

Claims

CLAIMS1. Polyfenol(s) and / or metabolite(s) thereof, and / or precursor(s) to trimethylamine (TMA), and / or trimethylamine-N-oxide (TMAO), or a composition comprising polyfenol(s) and / or metabolite(s) thereof, and / or precursor(s) to trimethylamine, and / or trimethylamine-N-oxide, for use in a method of identifying whether or not an individual has small intestinal bacterial overgrowth (SIBO).

2. Polyfenol(s) and / or metabolite(s) thereof, and / or precursor(s) to trimethylamine, and / or trimethylamine-N-oxide, or a composition comprising polyfenol(s) and / or metabolite(s) thereof, and / or precursor(s) to trimethylamine, and / or trimethylamine-N-oxide, for use according to claim 1, wherein said method of identifying is performed in vitro.

3. Polyfenol(s) and / or metabolite(s) thereof, and / or precursor(s) to trimethylamine, and / or trimethylamine-N-oxide, or a composition comprising polyfenol(s) and / or metabolite(s) thereof, and / or precursor(s) to trimethylamine, and / or trimethylamine-N-oxide, for use according to claim 1 or claim 2, wherein said polyfenol(s) is / are selected from resveratrol and / or curcumin.

4. Polyfenol(s) and / or metabolite(s) thereof, and / or precursor(s) to trimethylamine, and / or trimethylamine-N-oxide, or a composition comprising polyfenol(s) and / or metabolite(s) thereof, and / or precursor(s) to trimethylamine, and / or trimethylamine-N-oxide, for use according to any one of the previous claims, wherein said metabolite(s) is / are selected from dihydroresveratrol (DHR), dihydrocurcumin (DHC), tetrahydrocurcumin (THC), hexahydrocurcumin (HHC), hexahydro-didemethyl-curcumin (HHDEC), hexahydro-didemethyl-curcumin (HHDDEC) and / or octahydrocurcumin (OHC).

5. Polyfenol(s) and / or metabolite(s) thereof, and / or precursor(s) to trimethylamine, and / or trimethylamine-N-oxide, or a compositioncomprising polyfenol(s) and / or metabolite(s) thereof, and / or precursor(s) to trimethylamine, and / or trimethylamine-N-oxide, for use according to any one of the previous claims, wherein said precursor(s) to trimethylamine-N- oxide is / are selected from choline, L-carnitine, betaine, phosphatidylcholine, lecithin, and / or dimethylglycine.

6. Polyfenol(s) and / or metabolite(s) thereof, and / or precursor(s) to trimethylamine, and / or trimethylamine-N-oxide, or a composition comprising polyfenol(s) and / or metabolite(s) thereof, and / or precursor(s) to trimethylamine, and / or trimethylamine-N-oxide, for use according to any one of the previous claims, wherein said polyfenol(s) and / or precursor(s) to trimethylamine, and / or composition comprising polyfenol(s) and / or precursor(s) to trimethylamine, has / have been orally administered earlier to the individual.

7. Polyfenol(s) and / or metabolite(s) thereof, and / or precursor(s) to trimethylamine, and / or trimethylamine-N-oxide, or a composition comprising polyfenol(s) and / or metabolite(s) thereof, and / or precursor(s) to trimethylamine, and / or trimethylamine-N-oxide, for use according to any one of the previous claims, wherein the concentration(s) of said metabolite(s) of said polyfenol(s) and / or the concentration of trimethylamine-N-oxide, in a urinary sample earlier obtained from the individual, is / are measured.

8. Polyfenol(s) and / or metabolite(s) thereof, and / or precursor(s) to trimethylamine, and / or trimethylamine-N-oxide, or a composition comprising polyfenol(s) and / or metabolite(s) thereof, and / or precursor(s) to trimethylamine, and / or trimethylamine-N-oxide, for use according to claim 7, wherein said urinary sample preferably is obtained at least about 1 hour, preferably at least about 1.5 hours, after said oral aministration of said polyfenol(s) and / or precursor(s) to trimethylamine.

9. A method for in vitro identifying whether or not an individual has SIBO, which method comprises measuring a concentration of metabolite(s) ofpolyf enol (s) and / or a concentration of trimethylamine-N-oxide in a urine sample taken from the individual, wherein said polyfenol(s) and / or optionally at least one precursor to trimethylamine has / have earlier been orally administered to said individual, and thereby determining whether or not the individual has SIBO.

10. The method according to claim 9, wherein the polyfenol(s) is / are selected from resveratrol and / or curcumin.

11. The method according to claim 9 or 10, wherein the metabolite(s) is / are selected from dihydroresveratrol (DHR), dihydrocurcumin (DHC), tetrahydrocurcumin (THC), hexahydrocurcumin (HHC), hexahydro- didemethyl-curcumin (HHDEC), hexahydro-didemethyl-curcumin (HHDDEC) and / or octahydrocurcumin (OHC).

12. The method according to any one of claims 9 to 11, wherein said precursor(s) to trimethylamine is / are selected from choline, L-carnitine, betaine, phosphatidylcholine, lecithin and / or dimethylglycine.

13. The method according to any one of claims 9 to 12, wherein said urine sample has been obtained at least about 1 hour, preferably at least about 1.5 hours, after said oral administration of said polyfenol(s) and / or optionally said precursor(s) to trimethylamine.

14. The method according to any one of claims 9 to 13, wherein the method further comprises measuring a concentration of lactulose in the urine sample taken from the individual.

15. The method according to any one of claims 9 to 14, wherein the concentration of said metabolite(s) and / or TMAO, in nanogram / ml is lower than or equal to:DHR: < 50;DHC: < 0.7;THC: < 10;HHC: < 10;HHDEC: < 10;HHDDEC: < 2;OHC: < 5, and / orTMAO: < 50000 are indicative that the individual does not have SIBO, and the concentration of said metabolite(s) and / or TMAO higher than the concentration(s) specified above are indicative that the individual has SIBO.

16. Use of polyfenol(s), and / or metabolite(s) thereof, and / or trimethylamine and / or precursor(s) to trimethylamine, and / or trimethylamine-N-oxide, in identifying whether or not an individual has SIBO.

17. A kit for determining the presence of SIBO, comprising:(c) Polyfenol(s) and / or optionally precursor(s) to trimethylamine, or a composition comprising polyfenol(s) and / or optionally precursor(s) to trimethylamine;(d) instructions for performing the method as defined in any one of claims 9 to 15.

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