WO2009098354A1

WO2009098354A1 - Method of diagnosing irritable bowel syndrome ibs

Info

Publication number: WO2009098354A1
Application number: PCT/FI2009/050089
Authority: WO
Inventors: Kajsa Kajander; Riitta Korpela
Original assignee: Valio Ltd
Priority date: 2008-02-05
Filing date: 2009-02-04
Publication date: 2009-08-13
Also published as: FI20085101A0; FI20085101L

Abstract

Novel biomarkers associated with irritable bowel syndrome (IBS) have been found. The invention provides methods of diagnosing IBS by determining said biomarkers in samples taken from subjects suffering from the disease. Appropriate biomarkers includes certain lipids and/or certain non-lipid metabolites. Particularly suitable biomarkers are lipids involved in the metabolism of acylglycerol and sphingolipids, such as acylglycerols e.g. triacylglycerols (TG), and diacylglycerols (DG), glycerophospholipids, especially lysophospholipids such as lysophos- phatidylcholines (LysoPC) and lysophosphatidylethanolamines (LysoPE), and sphingolipids, especially ceramides (Cer) and glycosphingolipids (GlycoSL). A great number of lipids and/or water-soluble other metabolites may be determined simultaneously to obtain a metabolite profile of the subject.

Description

Method of diagnosing irritable bowel syndrome IBS

Field of the Invention

Novel biomarkers associated with irritable bowel syndrome (IBS) have been found. The invention provides methods of diagnosing IBS by de- termining said biomarkers in samples taken from subjects suspected to suffer from the syndrome.

Background of the Invention

Irritable bowel syndrome (IBS) is a functional bowel disorder characterized by abdominal pain or discomfort and an irregular bowel habit. The prevalence is up to 20% in adult westerners, which makes IBS the most common diagnosis in gastroenterology. The precise aetiology and pathophysiology of IBS are still incompletely understood and largely unknown. The current knowledge does, however, suggest that changed gut motility, visceral hyperalgesia and dysregulation of the brain-gut axis are essential in IBS. IBS is diagnosed by the presence of symptoms according to the

Rome criteria together with concomitant exclusion of organic diseases, and hence there is no specific biological, radiographic, endoscopic or physiological marker for IBS. The most studied and proposed putative biomarkers in IBS are immune markers such as increased mast cell counts, mast cells in close prox- imity to nerves, and mast cell mediators that are able to stimulate murine visceral sensory nerves, elevated levels of plasma pro-inflammatory interieuktn (IL)-6 and IL-8, high amounts of tumor necrosis factor (TNF)-α, IL-1β, IL-6 and IL-12 in peripheral blood mononuclear cells, and increased numbers of immu- nocytes in mucosa. These findings are supported by genotyping studies indi- eating that IBS patients are predisposed towards a pro-inflammatory cytokine profile.

Furthermore, other markers, such as serotonin (5-hydroxytrypt- amine, 5-HT) and gut hormones (disturbed levels of plasma cholecystokinin and plasma motilin, lower levels of colonic peptide YY) have been associated with IBS. Numbers of enterochramaffin cells, the source of 5-HT in the bowel, have been shown to be increased especially in post-infectious IBS (Spider RC, Jenkins D₁ Thornley JP, Hebden JM, Wright T, Skinner M, Neal KR, Increased rectal mucosal enteroendocrine cells, T lymphocytes, and increased gut permeability following acute Campylobacter enteritis and in post-dysenteric irrita- ble bowel syndrome. Gut 2000;47:804-11 ; Dunlop SP, Jenkins D, Neal KR₁ Spiller RC, Relative importance of enterochromaffin cell hyperplasia, anxiety, and depression in postinfectious IBS. Gastroenterology 2003;125:1651-9). Elevated plasma 5-HT concentrations have been observed in a mixed IBS population and in post-infectious IBS, while the opposite was demonstrated in constipation IBS (Bearcroft CP, Perrett D, Farthing MJ. Postprandial plasma 5- hydroxytryptamine in diarrhoea predominant irritable bowel syndrome: a pilot study. Gut 1998;42:42-6; Dunlop SP, Coleman NS, Blackshaw E, Perkins AC, Singh G, Marsden CA, Spiller RC, Abnormalities of 5-hydroxytryptamine metabolism in irritable bowel syndrome. Clin Gastroenterol Hepatol 2005;3:349- 57). Moreover, decreased 5-HT levels and turnover, lower 5-HT transporter mRNA concentration, and increased expression of p11 , a protein affecting serotonin metabolism, have been proven in IBS (Dunlop et al., 2005; Coates MD, Mahoney CR, Linden DR, Sampson JE, Chen J, Blaszyk H, Crowell MD, Sharkey KA, Gershon MD, Mawe GM, Moses PL, Molecular defects in muco- sal serotonin content and decreased serotonin reuptake transporter in ulcerative colitis and irritable bowel syndrome. Gastroenterology 2004;126:1657-64; Camilleri M, Andrews CN, Bharucha AE, Carlson PJ, Ferber I, Stephens D, Smyrk TC, Urrutia R, Aerssens J, Thielemans L, Gδhlmann H, van den Wyn- gaert I, Coulie B, Alterations in expression of p11 and SERT in mucosal biopsy specimens of patients with irritable bowel syndrome. Gastroenterology 2007;132:17-25.

A recent review of Camilleri and Gorman concludes that there appears to be at least an IBS subgroup with increased gut permeability, although the role of permeability defects in IBS is not fully elucidated (Camilleri, M. and Gorman H., Intestinal permeability and irritable bowel syndrome, Neurogastro- enterol Motil, 2007, 19: 545-52). Furthermore, a raised number of inflammatory cells in mucosal biopsies and an abnormal interleukin (IL)-10/IL-12 ratio suggest the presence of slight/low-grade inflammation in some IBS patients.

Despite extensive interest and investigation, the understanding at a molecular level of the pathophysiology of IBS is still insufficient, and one object of the present invention is to throw light on the metabolic events associated with IBS, with special emphasis on developing diagnostic methods for the disease. Another object of the invention is to find biomarkers of IBS with the aid of novel applications and powerful tools for simultaneous measurement and modeling of huge numbers of molecular metabolites in a short period of time. Since no appropriate biomarkers for the complex syndrome IBS exist, there is a need for efficient and reliable in vitro methods for diagnosing the disease, and there is also a continuous and evident need for finding specific bio- markers for this syndrome. The present invention meets these needs.

The present invention is based on metabolomic, and especially on lipidomic, studies of patients suffering from IBS. Metabolomics has been utilised in the investigation of the pathophysiology of inflammatory bowel diseases (IBD)₁ obesity, and cancer. IBD is a type of bowel disease other than IBS, and it is characterized by chronic inflammation in the intestines. Ulcerative colitis (UC) and Crohn's disease (CD) are the most common diseases belonging to IBD. Marchesi et al. found that the faecal extracts of Crohn's disease (CD) and ulcerative colitis (UC) patients contained reduced levels of short chain fatty acids such as butyrate and acetate, methylamine, and trimethylamine, suggesting obvious changes in the gut microbial community due to the inflammation. They also found elevated quantities of amino acids in the feces from both CD and UC patients. (Marchesi J.R., Holmes E., Khan F., Kochhar, S., Scanlan, P., Shahahan, F., Wilson, I. D., Wang, Y., Rapid and noninvasive metabonomic characterization of inflammatory bowel disease, J Proteome Res, 2007, 6:546- 51 ). The metabolic background of IBS is less investigated than that of IBD, and still remains to be clarified. The present invention contributes to solving this is- sue.

Brief Description of the Invention

It has now been found that IBS is associated with abnormal levels of specific groups of metabolites, and especially particular lipid metabolites.

The present invention relates to a method of diagnosing irritable bowel syndrome (IBS) in a subject, wherein said method comprises determining the amount of at least one iipid involved in lipid metabolism in a body sample taken from said subject, whereby an abnormal amount of said Iipid indicates IBS.

The present invention further relates to a method of diagnosing irri- table bowel syndrome (IBS) in a subject, wherein said method comprises determining the amount of at least one non-lipid metabolite selected from the group consisting of 2(3H)-furanone, ribitol, heptan, L-mannose, creatinine, do- decane, decanoic acid, dodecanoic acid, n-butylamine, D-ribose, gluco- pyranose, azelaic acid and adipic acid in a body sample taken from said sub- ject, whereby an abnormal amount of said non-lipid metabolite indicates IBS. Other objects, details and advantages of the present invention will become apparent from the following drawings, detailed description and example.

Brief Description of the Drawings Figure 1 shows concentrations (mean ± SD) of selected lipids in mucosal biopsies from IBS patients (n=15) and healthy controls (n=9) as measured by UPLC-MS. (A) shows lysophospholipids; LysoPE = lysophos- phatidylethanolamine; LysoPC = fysophosphatidylcholine, and (B) shows dia- cylglycerol (DG) and ceramides (Cer). Patients and controls differ significantly from each other for all presented lysophospholipids, as well as for diacylglyc- erol and ceramides. ** indicates p<0.01 and *** p<0.001 , where the P values are based on Wilcoxon rank sum test.

Figure 2 shows a partial least squares discriminant analysis (PLS/DA) of non-lipid metabolite profiles for IBS patients (n=15; α) and healthy controls (n=9; 0). Two latent variables (LVs) were used (Q2=61 %).

Detailed Description of the Invention

For the first time significant differences have been found in the global mucosal metabolic profile between subjects with irritable bowel syndrome IBS and healthy controls. Colonic mucosal metabolites typical of IBS were characterized by employing a high-throughput metabolomic approach.

Metabofomic profiling is a large-scale study of non-water-soluble metabolites (in practice lipids) and water-soluble metabolites, i.e. non-lipids obtainable e.g. by technologies including electrospray ionization (ESI(+/-)), mass spectrometry (MS), liquid chromatography coupled to mass spectrometry (LC/MS), and comprehensive two-dimensional gas chromatography coupled to a high speed time-of-flight mass spectrometry (GCxGC-TOF). Water-soluble has a broad meaning in this context meaning soluble in a polar solvent. Relationships between the metabolites may be characterized by multivariate methods. This enables an analysis of several or even a huge number of metabolites simultaneously from a single sample to obtain a "metabolic profile" such as a "lipid profile", a "non-lipid metabolite profile" or a "metabolomic profile" (i.e. a combination of lipid and non-lipid metabolites). These results may then be used to identify a metabolic profile typical to IBS using statistical modeling methods. A "metabolite" is an intermediate or a product of metabolism, usually restricted to small molecules. According to one embodiment of the invention, the metabolite is a "primary metabolite" which is a metabolite that is directly involved in the normal growth, development or reproduction. Preferably, the pri- mary metabolite is one that is involved in an energy metabolism pathway, like the citric acid (TCA) cycle, the pentose phosphate pathway, or lipid metabolism.

It has now been found that certain lipids or non-lipid metabolites show systematic up- or down-regulation in IBS patients compared to healthy controls. Such lipids or other metabolites may serve as biomarkers for diagnosing the disease. Appropriate lipids are those involved in the lipid metabolism, and especially in the metabolism of acylglycerols and/or sphingofipids. These lipids include glycerolipids e.g. acylglycerols such as triacylglycerols (TG) and diacylglycerols (DG), and glycerophospholipids, especially lysophos- pholipids such as lysophosphatidylcholine (LysoPC) and lysophosphatidyl- ethanolamine (LysoPE). Other suitable lipids are those taking part in the cera- mide/sphingomyelin pathway, such as sphinoglipids, especially ceramides (Cer) and glycosphingolipids (GlycoSL).

"Lipid involved in the lipid metabolism" encompasses any lipid that participates in the lipid metabolism of the body. Especially the lipid is involved in the metabolism of acylglycerol, in particular of glycerophospholipids, or in the metabolism of sphingolipids. Lipid metabolism is described e.g. by Mayes P. and Botham K. 2003 In: Harper's Illustrated Biochemistry, 26^th edition, Murray, R., Granner D., Mayes P. and Rodwell V., eds. Lange Medical Books, chapters 14 and 24. The chemical names of the lipids used in the present application follow the nomenclature of Mayes and Botham 2003 supra. Lipids constitute a heterogeneous group of compounds that are relatively insoluble in water, and soluble in non-polar solvents. Simple lipids are esters of fatty acids with various alcohols, and complex lipids are esters of fatty acids containing groups in addition to fatty acids and alcohol, such as e.g. phospholipids, which contain phosphoric acid residues or glycolipids, which contain carbohydrate. Lipids are important constituents in the cell membranes and mithochondria, and they play a significant role in energy transport and storage. Acylglycerols constitute a majority of lipids in the body. Diacylglycerols, phosphatidylcholine, and phophatidylethanolamine are important substances involved in the major pathways of triacylglycerof and phosphoglycerol biosynthesis and in glyc- erophospholipid metabolism. Ceramide is synthesized from serine and it is a combination of a complex amino alcohol sphingosine and fatty acid. When ce- ramides react with phosphatidylcholine, they form sphingomyelin and diacyl- glycerol. Ceramides may also react with sugars to form glycosphingolipids. In addition to the differences found in the lipid levels between samples from IBS patients and controls, a global analysis of non-lipid metabolites revealed further differences between the two test groups. These further differences were seen in basic metabolites, such as 2(3H)-furanone (also known as lactone), ribitol, heptan, L-mannose, creatinine, dodecane, decanoic acid {also known as capric acid), dodecanoic acid (also known as lauric acid), n- butylamine, D-ribose, glucopyranose, azelaic acid, and adipic acid, all participating in common biochemical pathways in cells.

The groups of molecules, such as certain lipids that show systematic up- or down-regulation, are a clear indication of biologically relevant me- tabolite types, and highly relevant biomarkers, and provide a way of standardised metabolite libraries. Fat-soluble lysophosphatidylcholines (LysoPCs) and lipids of the ceramide/sphingomyelin pathway are the most up-regulated lipids in IBS, and provide relevant biomarkers of the pathophysiology of IBS. No earlier indications of ceramides or LysoPCs being involved in the pathology of IBS are available.

The methods of the invention may be used for diagnosing IBS in a subject having symptoms associated with the disease. A sample is taken from the subject's body, and analyzed by determining the metabolic profile thereof, or simply by determining the amount of one or a few certain lipids and/or non- lipid metabolites. The sample may be a biopsy sample, preferably taken from the mucosa of the colon. Alternatively, the sample is taken from non-invasive tissues, such as fecal material or blood. Thus, a more easily obtained matrix in clinical settings is possible.

The lipids are conveniently analyzed by liquid chromatography, coupled to mass spectrometry (LC/MS; LC-MS/MS). Alternatively, gas chromatography coupled to mass spectrometry may be used. These techniques enable analysis of several lipids or even a huge number of lipids simultaneously from a single sample to obtain a lipid profile. These results may then be used to identify a metabolic profile typical to IBS using statistical modeling methods. Preferably, a reversed phase liquid chromatography is used to separate the lip- ids, which are then identified by a hybrid quadrupole time of flight (Q/ToF) mass spectrometer with tandem mass spectrometry (MS/MS) capability.

The non-lipid metabolites are conveniently analyzed by gas chromatography (GC), especially comprehensive two-dimensional gas ehromatog- raphy coupled to a high-speed time-of-flight mass spectrometry (GCxGC- TOF).

According to one embodiment of the invention the metabolites are established by collecting a lipid profile and an optional non-lipid metabolite profile using liquid or gas chromatography coupled to mass spectrometry, and sta- tistical modeling methods are used on the collected profiles to identify abnormal amounts of the lipids or non-lipid metabolites.

Overall, lipid levels are increased in patients with IBS. The most significant upregulation is seen in pro-inflammatory iysophosphatidylcholines. Other lipid groups that are significantly upregulated in IBS patients include gly- cosphingolipids, di- and triacylglycerols, and lipotoxic ceramides. It may thus be concluded that IBS mucosa is characterized by a distinct pro-inflammatory and lipotoxic metabolic profile, specifically with an increase in several lipid species, such as lysophospholipids and ceramides. Among the non-lipid metabolites, the cyclic ester 2(3H)-furanone was the most upregulated. According to one particular embodiment of the invention, a lipid profile is determined which contains at least one of the following lipids: triacylglycerols (TG), diacylglycerols (DG), Iysophosphatidylcholines (LysoPC), lyso- phosphatidylethanolamines (LysoPE), ceramides (Cer), and glycosphingolipids (GlycoSL), or preferably all of them. According to another embodiment of the invention only one or a few lipids may be analyzed, separately or simultaneously. The lipid analysis may also be combined with an analysis of non-lipid metabolites.

An abnormal amount of one or several of the selected lipids or other metabolites in the analyzed body sample indicates that the subject from whom the sample is taken suffers from IBS. An "abnormal amount" means that the amount is increased or decreased as compared to the amount of the same compound or compounds in healthy controls.

A "subject" as used herein refers mainly to a human being, but it may also be an animal. The following example illustrates the present invention. The example is not to be construed to limit the claims in any manner whatsoever. Example

Metabolomic profiling of mucosal biopsies

Sixteen adult IBS patients (mean age 42 years, SD 15; 6 male) fulfilling the Rome Il criteria (Thompson, W.G., Longstreth, G. F., Drossman, D.A., Heaton, K.W., Irvine, EJ, Mϋller-Lissner, SA₁ Gut, 1999,45 Suppl 2:43-7), and devoid of organic intestinal diseases were recruited to participate in the study. Nine healthy subjects (mean age 49 years, SD 14; 4 male) devoid of organic intestinal diseases or gastrointestinal symptoms consistent with IBS, and undergoing colonoscopy for clinical reasons were recruited as controls. Inclu- sion criteria for all subjects were: an age between 20 and 65 years; normal blood count (erythrocytes, haemoglobin, haematocrit, MCV, MCH, MCHC, thrombocytes, leukocytes), within reference values for serum creatinine, ALT and ALP₁ and a normal gut histology as evaluated by an experienced pathologist. Subjects were excluded if they had a history of major or complicated gas- trointestinal surgery, severe endometriosis, complicated abdominal adhesions, malignant tumours, were pregnant or lactating, or had received antimicrobials during the previous month. Patients with lactose intolerance were allowed to participate if they followed a continuous low-lactose or lactose-free diet. Sample collection and preparation. Mucosal biopsies (mean weight 5.2 mg/sample; SD 1.5) from the ascending colon were obtained from each subject during colonoscopy after bowel cleansing. The samples were immediately frozen and transferred into - 2O⁰C. Samples were then moved into -70°C until required for analysis. For lipi- domics samples were weighed into Eppendorf-tubes and 10 μl of 0.9% sodium chloride and 10 μl of an internal standard mixture (11 lipid compounds, 0.1 μg each) were added. The samples were extracted with 100 μl of chloro- form:methanol (2:1 ; 2 min vortexing, 2 h extraction time) and centrifuged (10000 rpm, 3 min). Of the lower organic phase, 60 μl aliquots were taken into vial inserts and 10 μl of a standard mixture containing 3 labelled lipid com- pounds was added. For non-lipid compounds samples were weighed into Eppendorf-tubes and 10μI of 1000 ppm (mg/ml) labelled palmitic acid (16:0- 16,16,16d₃) was added as internal standard. The samples were extracted with 500 μf methanol (2 min vortexing, 0.5 h extraction time) and centrifuged (10000 rpm, 3 min). The separated supernatants were evaporated into dryness under nitrogen and the residues were derivatized with 2% methoxyamine HCI in pyridine (MOX; 25 μl, 90 min at 3O⁰C) and N-Methyl-N- trimethylsilyltrifluoroacetamide (MSTFA; 50 μl, 30 min at 37°C). All samples were run in duplicates.

Analysis of lipids bv UPLC/MS.

Characterisation of lipid molecular species in colonic mucosa was performed by a lipidomics strategy using an ultra performance liquid chromatography coupled to mass spectrometry (UPLC-MS). The column (50⁰C) was an Acquity UPLC™ BEH C18 10 x 50 mm with 1.7 μm particles. The solvent system included A. ultra pure water (1% 1M NH₄Ac, 0.1% HCOOH) and B. LC/MS grade acetonitrile/isopropanol (5:2, 1% 1M NH₄Ac, 0.1% HCOOH). The gradient started from 65% A/ 35% B, reached 100% B in 6 min and remained there for the next 7 min. There was a 5 min re-equilibration step before the next run. The flow rate was 0.200 ml/min and the injected amount 1.0 μl. The lipid profiling was carried out using ESI+ mode and the data was collected at mass range of m/z 300-2000 with a scan duration of 0.08 s. Lipids were identified using an internal spectral library or tandem mass spectrometry. The normalization of lipidomics data was performed as follows: all monoacyl lipids except cholesterol esters, such as monoacylglycerols and monoacyl-glycerophospholipids, were calibrated with a lysophosphatidyl- choline (LysoPC) (17:0/0:0) internal standard, all diacyl lipids except etna- nolamine phospholipids were normalized with phosphatidylcholine (LysoPC) (17:0/17:0), the diacyl ethanolamine phospholipids were calibrated with phos- phatidylethanolamine (LysoPE) (17:0/17:0), and the triacylglycerols and cholesterol esters with triacylglycerol TG (17:0/17:0/17:0). Other molecular species were normalized by (LysoPC) (17:0/0:0) for retention time <310 s, (LysoPC) (17:0/17:0) for retention time between 310 s and 450 s, and TG (17:0/17:0/17:0) for higher retention times. Data was processed using the MZmine software version 0.60 (Katajamaa, M., and Oresic, M., 2005, Processing methods for differential analysis of LC/MS profile data, BMC Bioinformatics 2005: 6:179), and metabolites were identified using an internal spectral library or tandem mass spectrometry.

Analysis of non-lipid metabolites bv GCxGC-TQF A broad screening of non-lipid metabolites was conducted by a comprehensive two-dimensional gas chromatography coupled to a high speed Time-of-Flight mass spectrometry (GCxGC-TOF). The instrument used was a Leco Pegasus 4D GCxGC-TOF with Agilent 6890N GC and CombiPAL auto- sampler. The GC was operated in split mode (1 :20) using helium as carrier gas at 1.5 ml/min constant flow. The first GC column was a relatively non-polar RTX-5 column, 10 m x 0.18 mm x 0.20 μm and the second was a polar BPX- 50, 1.10 m x 0,10 mm x 0.10 μm. The temperature programme was as follows: Initial 5O⁰C, 1 min → 280⁰C, 7°C7min, 1 min. The secondary oven was set to +3O⁰C above the primary oven temperature. The second dimension separation time was set to 3 seconds. The mass range used was 40-600 amu and the data collection speed was 100 spectra/second. A commercial mass spectral library, Palisade Complete 600K, was used for identifying metabolites. Statistics A partial least squares discriminant analysis (PLS/DA) was utilized as a supervised modeling method using an SIMPLS algorithm to calculate the model. A contiguous blocks cross-validation method and Q scores were used to develop the models. Top loadings for latent variables associated with drug specific effects were reported. The VIP {variable importance in the projection) values were calculated to identify the most important molecular species for the clustering of specific groups. Multivariate analyses were performed using Mat- lab version 7.2 (Mathworks, Inc.) and the PLS Toolbox version 4.0 Matlab package (Eigenvector Research, Inc.). One statistical outlier IBS patient was left out of the analyses after initial quality check of the results. Univariate com- parisons for individual metabolites between the groups were performed using the Wilcoxon rank-sum test.

Lipidomic analysis

By applying UPLC-MS altogether 651 lipid peaks were found and 75 of those were identified using the internal spectral library as described by Yetukuri et af. (Yetukuri, L., Katajamaa, M., Medina-Gomez, G., Seppanen- Laakso, T., Vidal-Puig, A., Oresic, M., Bioinformatics strategies for lipidomics analysis: characterization of obesity related hepatic steatosis, BMC Systems Biol., 2007, 1 :12), or with tandem mass spectrometry using UPLC/MS/MS. PLS-DA analysis of lipidomic data revealed significant differences in the muco- sal lipid profiles between IBS patients and healthy controls. Overall, lipid species were up-regulated in biopsies from IBS patients compared those from healthy subjects. The top-15 lipids with the largest differences between the groups by fold change appear in Table 1. A significant up-regulation in the concentrations of typical cell membrane metabolites, lysophospholipids, in IBS patients was among the most obvious findings (Figure 1A). Other lipid groups contributing significantly to the separation between IBS patients and healthy controls were ceramides (Figure 1 B), glycosphingolipids as well as di- and tri- acylglycerols, which all showed up-reguiation in the IBS group.

Metabolomic analysis

A broad metabolite screening by GCxGC-TOF resulted in several hundred mucosal metabolites, of which 107 were identified and kept in analyses. Based on the PLS/DA analysis, a clear separation of IBS cases and controls was surprisingly obtained (Figure 2). Both up-regulation and down- regulation of metabolites were observed in IBS patients versus controls. The top ranked metabolites contributing to separation between the groups appear in Table 2.

Table 1. The top-15 lipids with largest and most significant differences between IBS patients and healthy controls in mucosal concentration as measured by Wilcoxon test P-value (IBS patients/healthy controls). LysoPC = lysophos- phatidylcholine; LysoPE = lysophosphatidylethanolamine; TG = triacylglycerol; GlycoSL = glyco-sphingolipid; DG = diacylglycerol; Cer = ceramide.

Fold

Lipid name (IBS/healthy control) p value*

Cer(d18:1/24:1)

1.30 0.0013

Cer(d18:1/24:2)

1.37 0.000038

DG(36:2)

1.93 0.00000097

GlycoSL(m/z=1199.805)

1.88 0.00097

GlycoSL(m/z=1195.851)

1.95 0.00027

LysoPC(16:0)

2.13 0.000058

LysoPC(18:0)

1.87 0.00016

LysoPC{18:1 )

2.79 0.000023

LysoPE(18:1e)

2.41 0.000018

TG(46:5)

1.37 0.039

TG(48:5)

1.63 0.028

TG(48:6)

1.67 0.032

TG(49:3)

2.10 0.0089

TG(51 :4)

1.55 0.037

TG(51:5)

1.80 0.016

"Wilcoxon rank sum test. Table 2. Major non-lipid metabolites contributing to discrimination between IBS patients (n=15) and healthy controls (n=9). Separation is based on a variable importance projection (VIP) analysis with a cut-off value of 2.

Fold

Metabolite p value* (IBS/healthy control)

13.73

2(3H)-Furanone 0.03

3.63 ns.

Ribitol

2.87 0.02

Heptan

L-Mannose 2.75 ns.

1.70 0.04

Creatinine

1.47

Dodecane ns.

1.26

Decanoic acid ns.

-1.46

Dodecanoic acid ns.

-1.47 0.01 n-Butylamine

-1.51 d-Ribose ns.

-1.58 ns.

Glucopyranose

-1.77 0.02

Azelaic acid

-2.69 0.0008

Adipic acid *Wilcoxon rank sum test. ns.= not significant.

The metabolite contributing most to the separation was 2(3H)- furanone, a cyclic ester commonly produced in biochemical pathways, which was almost 14-fold up-regulated in IBS patients compared to healthy subjects (p<0.05). The fold change for other top ranked metabolites was clearly lower (3.7 to -2.7 fold change). Also other basic metabolites frequently found in biochemical pathways, such as the second messenger d-ribose, were among the major factors contributing to the separation between cases and controls based on the PLS/DA analysis, although the result in the Wilcoxon rank sum test was not significant. Compared to healthy controls, organic, carboxylic acids were found to be both slightly down-regulated (dodecanoic, azelaic and adipic acid) as well as slightly up-regulated (decanoic acid) in IBS patients.

Claims

1. A method of diagnosing irritable bowel syndrome (IBS) in a subject, said method comprising determining the amount of at least one lipid in- volved in lipid metabolism selected from a glycerolipid, and a lipid that takes part in the ceramide/sphingomyelin pathway in a body sample taken from said subject, whereby an abnormal amount of said lipid indicates IBS.

2. The method of claim 1 , wherein the glycerolipid is an acylglycerol or a glycerophospholipid.

3. The method of claim 2, wherein the glycerophospholipid is a fysophospholipid.

4. The method of claim 3, wherein the lysophospholipid is a lyso- phosphatidylcholine (LysoPC) or a lysophosphatidyiethanolamine (LysoPE).

5. The method of claim 2, wherein the acylglycerol is triacylglycerol (TG) or diacylglycerol (DG).

6. The method of claim 1 , wherein the lipid is a sphingolipid.

7. The method of claim 6, wherein the sphingolipid is a ceramide (Cer) or a glycosphingolipid (glycoSL).

8. The method of claim 1 , wherein an increased amount of triacyl- glycerol (TG), diacylglycerol (DG), ^phosphatidylcholine (LysoPC), lysophosphatidyiethanolamine (LysoPE), ceramide (Cer), and glycosphingolipid (GlycoSL) indicates IBS.

9. The method of any one of the previous claims, further comprising determining at least one non-lipid metabolite in said body sample, whereby an abnormal amount of said non-lipid metabolite also indicates IBS.

10. The method of claim 9, wherein the non-lipid metabolite is a primary metabolite involved in an energy metabolism pathway, such as in the citric acid cycle or in the pentose phosphate pathway.

11. The method of claim 9, wherein the non-lipid metabolite is se- lected from the group consisting of 2(3H)-furanone, ribitol, heptan, L-mannose, creatinine, dodecane, decanoic acid, dodecanoic acid, n-butylamine, D-ribose, glucopyranose, azelaic acid, and adipic acid.

12. The method of any one of the previous claims, wherein the amounts of several metabolites are determined simultaneously.

13. The method of claim 12, wherein a lipid profile comprising at least one lipid from the group consisting of triacylglycerols (TG), diacylglycerols (DG), lysophosphatidylcholines (LysoPC), lysophosphatidylethanolamines (LysoPE), ceramides (Cer) and glycosphingolipids (GlycoSL) is determined from the sample.

14. The method of claim 12, wherein the metabolites are estab- lished by collecting a lipid profile and an optional non-lipid metabolite profile using liquid or gas chromatography coupled to mass spectrometry, and statistical modeling methods are used on the collected profiles to identify abnormal amounts of lipids or non-lipid other metabolites.

15. A method of diagnosing irritable bowel syndrome (IBS) in a sub- ject, said method comprising determining the amount of a non-lipid metabolite selected from the group consisting of 2(3H)-furanone, ribitol, heptan, L- mannose, creatinine, dodecane, decanoic acid, dodecanoic acid, n-butyl- amine, D-ribose, glucopyranose, azelaic acid and adipic acid in a body sample taken from said subject, whereby an abnormal amount of said non-lipid me- tabolite indicates IBS.

16. The method of claim 1 or 15, wherein the sample is a blood, feces or biopsy sample taken from the subject.