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The diagnosis of inflammatory bowel diseases (IBDs) and differentiation between Crohn’s disease (CD) and ulcerative colitis (UC) requires a multimodal approach involving clinical, endoscopic, histologic, serologic, and radiologic modalities.1, 2 IBD patients commonly suffer from a lag in time from first occurrence of symptoms to diagnosis, with a median diagnostic delay of 9 months.3 The length of diagnostic delay positively correlates with the later occurrence of bowel stenosis and need for intestinal surgery. Hence, the diagnostic delay may hinder our ability to alter the progression of disease.3 In addition, once the diagnosis of IBD is made, its subcategorization into CD or UC is critical to determine the optimal treatment strategy. It remains unclear which patients with an initially inflammatory disease classification will develop into a more severe vs. benign disease phenotype. Thus, it would be beneficial to identify at-risk populations that could benefit from a tailored therapeutic approach. The use of biologic and/or immunomodulator agents for therapy might be justified early in the disease course for patients at risk for rapid disease progression.4, 5 Metabolomics are defined as the investigation of a group of intermediate or end-point metabolites of a physiologic or pathophysiologic process,6, 7 which have the potential to provide a signature pattern for specific disease conditions. Metabolomic studies have entered the field of IBD, derived from serum, urine, or tissue samples in humans and IBD animal models.7 Recent technical advances now allow the measurement of some metabolites in the form of volatile organic compounds (VOCs) in the breath.7 This is important because certain VOC patterns are linked to disease inside and outside of the intestine, such as asthma, chronic obstructive pulmonary disease, chronic kidney disease, heart failure, alcoholic hepatitis, colon cancer, and others.8, 9, 10, 11, 12, 13, 14, 15 In the past, volatility and very low concentrations of breath components as well as difficulties with standardization and normalization have limited our ability to analyze them. However, these challenges have been largely overcome with advanced analysis techniques such as selective ion flow tube mass spectrometry (SIFT-MS). This technique has already shown a high discriminatory capability analyzing breath of pediatric IBD patients and controls.16 Limited data are available using this technique in adult IBD patients for diagnosis and differentiation, and most studies focus on single or only few VOCs.17, 18, 19, 20, 21 Information is missing about a more comprehensive evaluation of multiple VOCs at the same time and a link between the breath metabolome and disease phenotypes. In addition, the origin of the VOC changes remains to be defined. This study was designed to fill these knowledge gaps. As a comparator, we also performed an analysis on a group of patients with ileal pouch anal anastomosis (IPAA). The inclusion criteria for IPAA patients were: patients who had IPAA for refractory UC, UC-associated dysplasia or cancer, familial adenomatosis polyposis, and pouchoscopy to document endoscopic findings at the time of breath sample procurement. Exclusion criteria were age younger than 18 years or older than 85 years, closure of diverting ileostomy <3 months from the time of sample collection, subjects refusing to sign informed consent, subjects not having command over the English language, and who could not be nil orally for 8 h owing to any medical reasons. Also in this group patients who underwent full bowel preparation were excluded. Subjects were recruited into each of the three different groups under the following categories: normal pouch (which included patients with irritable pouch syndrome), refractory pouchitis (RP), and CD of pouch. Subjects were classified into each category after review of in-patient and outpatient medical records, a structured medical interview at the time of sample procurement, and review of endoscopy and biopsy reports performed immediately after recruitment and sample procurement. Irritable pouch syndrome was defined as the presence of abdominal pain, pelvic discomfort and diarrhea with no inflammation of the afferent limb, and pouch or the rectal cuff on endoscopy.22, 23 Pouchitis was defined as a clinical syndrome characterized by the onset of increased stool frequency often with bloody diarrhea, pelvic discomfort, urgency, malaise, and fever.22 RP was defined as the requirement for continuous antibiotic treatment for symptom relief or symptoms refractory to antibiotic treatment for >4 weeks as well as patients needing any additional therapy besides antibiotics.22, 23 CD of the pouch was defined as involvement of the small bowel mucosa proximal to the ileal pouch or the development of perianal complications or pouch fistula more than 3 months after ileostomy closure.23, 24 Mechanical complications of surgery were excluded. Stool studies to rule out infection as a cause for the pouchitis were available. This study was performed with approval from the institutional review board at Cleveland Clinic, Cleveland, Ohio. Seventeen out of 22 VOCs were different compared with all other groups, suggesting an entirely distinct breath metabolome compared with patients with a colon, regardless of normal or diseased (Table 2 and Supplementary Figure 1). We next compared HC vs. IPAA subjects with a normal pouch and found marked differences in VOCs in 18 out of 22 VOCs (Supplementary Table 4). The same was true for 16 out of 22 VOCs when comparing UC patients with a colon in situ with IPAA subjects with a normal pouch (Supplementary Table 5). We again tested for a potential influence of inflammatory activity on the VOC profile. For this purpose, we compared normal, non-inflamed pouches with the most severe inflammatory pouch disorders, namely RP and CD of the pouch. Demographics of the groups can be found in Supplementary Table 6. Patients with CD of the pouch had a shorter time from diagnosis to pouch creation and the RP patients had a higher rate of preoperative immunomodulators. Patients with RP and CD of the pouch had a higher frequency of antibiotics at the time of sample procurement compared with normal pouches and more patients with RP were on 5-aminosalicylic acid compared with the two other groups (data not shown). Only one out of 22 VOCs (acrylonitrile) was higher in CD of the pouch compared with a normal pouch with an AUC of 0.846. All other VOCs remains unchanged (Table 4). We furthermore examined the endoscopic and clinical pouch disease activity index and none of the VOCs correlated with either of the two scores (data not shown). This supports the notion that active inflammation of the pouch does not alter the examined VOCs. We additionally assessed the pouch subjects for an association between antibiotic intake and VOC profile. Pouch patients with any antibiotic intake within the past 3 months had significantly higher acetaldehyde and benzene levels (P<0.05). None of those VOCs was used in the DCA. The main findings of our study are (1) the human breath metabolome can distinguish IBD from non-IBD with high accuracy; (2) the breath VOCs are not different between CD and UC; (3) the changes observed in IBD are not linked to clinical or radiologic disease activity; (4) VOCs do not differ among CD phenotypes; and (5) the breath metabolome is markedly different in the absence of a colon, but is not altered by inflammation of the pouch. Recent technical advances allow for the measurement of metabolites in the form of VOCs in the breath. SIFT-MS technology is a new method allowing the detection of breath gases in complex mixtures regardless of water vapor content in real time. Compounds in concentrations as low as parts per billion can be distinguished from each other on the basis of their unique reaction with precursor ions. Pathologic GI conditions, such as alcoholic hepatitis,12 non-alcoholic fatty liver disease,15 or colorectal cancer9 can lead to a distinct breath pattern of VOCs that can aid in their diagnosis. In IBD most studies used single or a limited number of VOCs. For diagnosis and differentiation of IBD from HC, elevated levels of pentane have been demonstrated with an AUC reported to be 0.927.17, 21 Additional VOCs found to be linked to IBD were NO, ethane, and propane.20, 21, 28 In our own cross-sectional study examining 21 VOCs in the breath of 62 pediatric IBD patients via SIFT-MS, six VOCs differentiated between IBD and HC: 1-octene, 3-methylhexane, and 1-decene were increased and 1-nonene, 2-nonene, and hydrogen sulfide were decreased. The AUC for a discriminant analysis IBD vs. HC was 0.96.16 In one very recent study, a panel of 26 VOCs was analyzed in 56 patients (38 IBD and 18 healthy controls).19 Concentrations of dimethyl sulfide, hydrogen sulfide, butanal, and nonanal were significantly different between CD and HC, ammonia was different in UC compared with HC, and hydrogen cyanide, hydrogen sulfide, and butanal differed in CD vs. UC. The AUC for distinguishing CD from healthy controls was 0.86 for UC vs. controls 0.74 and for CD vs. UC 0.82. In this small study, clinically active disease was not associated with changes in VOC patterns. In the present investigation, 7/22 VOCs discriminated between IBD and HC, namely 2-propanol, acrylonitrile, carbon disulfide, dimethylsulfide, ethanol, isoprene, and trimethylamine. The major metabolic themes arising from the VOC differences between IBD and controls are bacterial fermentation, fatty acid and carbohydrate metabolism, and changes induced by an increase in reactive oxygen species.7 Data suggest that the intestinal microbiota may generate isoprene, dimethylsufilde, and ethanol.29, 30 Isoprenes are also products of cholesterol metabolism.7, 29 The presence of pentane in exhaled breath is considered a result of lipid peroxidation of polyunsaturated fatty acids in cellular membranes, a process mediated by free radicals and oxidative stress.7, 31 Dimethylsulfide has been established as a source of extra oral halitosis, which is thought to be derived from unexplained metabolic processes and is directly derived from the blood stream.32 Endogenous and exogenous sources of sulfur, mucin, or taurocholic acid are usually metabolized by bowel bacteria to produce toxigenic sulfur compounds such as hydrogen sulfide, methanethiol, and dimethylsulfide.33 These compounds have been implicated in the pathogenesis of UC.34 Dietary phosphatidylcholine is degraded by the intestinal microflora to form the volatile compound trimethylamine.7, 35 The influence of IBD on these areas of metabolism has been previously described and fits with the previously published VOC patterns. Considering the fact that we used an identical technical procedure to measure the breath VOCs in adults and pediatric patients in the same center, the pattern of VOCs that differentiate IBD in adult and pediatric populations were found to be different.16 Even though single gases, in which differences were detected, might be different to some prior reported studies, the pathways they belong to are shared among the published studies.7 This finding is in concordance with our observation that there was no difference in the breath pattern between CD and UC, given that metabolic pathways that we found to be altered are presumably shared between both entities of IBD. In addition, the patients in the pediatric study were not nil per os and hence diet could have influenced the expression of the VOCs. Much less information is available in the literature on VOCs and their link with disease activity in CD. Pentane correlated with disease activity as measured by white blood cell scintigraphy in IBD18 and ethane, propane, and isoprene were linked to clinical and/or endoscopic disease activity in UC.20 No data are available on associating the breath metabolome with disease phenotypes, location, or medications and no studies have been performed in subjects without a colon. None of the VOCs in our study was associated with any of the above-mentioned parameters. This was also true for the quality and quantity of inflammation on cross-sectional imaging. This is novel information and suggests that IBD-associated factors other than inflammation could lead to a distinct expression of metabolic pathways measureable in the breath. One such factor could be the intestinal microbiota, known to be distinct in IBD compared with controls.36 We therefore assessed whether the absence of the colon, the site of the largest amount of microbes, influences the breath metabolome. Subjects lacking a colon had a marked alteration of their breath metabolome. The difference between IPAA subjects and all other groups was significantly stronger compared with the difference between IBD patients and all other groups (data not shown). This was true when comparing HC with IPAA patients (normal pouch), and UC with IPAA patients (normal pouch). We again assessed intestinal inflammatory activity and VOCs. Given the fact that no full colon preparation is necessary for pouchoscopy, we were able to compare endoscopic and histologic disease activity with breath VOCs. For this purpose we chose extreme phenotypes, RP and CD of the pouch. The absence of an association of intestinal inflammation with changes in the VOCs was confirmed in this setting. How could the breath VOC differences in IBD and their marked changes after colectomy be explained? The combined findings indicate that colon-derived factors in IBD lead to a distinct and inflammation-independent VOC profile. Given these data, we can speculate that this is either due to colonic microbial factors, changes in diet or an altered metabolism of luminal components (including diet), or all of them combined. The gut microbiome is critical in maintaining mucosal homeostasis and it is altered in IBD compared with healthy controls, showing reduced diversity.36, 37 The VOCs of fecal matter are distinct in IBD compared with healthy controls38 supporting this link. Walton et al.39 demonstrated that several VOCs in the headspace of feces differ markedly between patients with CD and other gastrointestinal conditions including UC and irritable bowel syndrome. The authors, using gas chromatography-mass spectrometry, showed that patients with CD had significant elevations in the concentrations of ester and alcohol derivatives of short-chain fatty acids and indole compared with patients in the other groups. After therapy, the levels of many of the VOCs significantly decreased and were similar to healthy controls. The authors concluded that intestinal dysbiosis in IBD may contribute to different fecal metabolite profiles. They also concluded that the normalization of the fecal VOC profile following therapy suggests re-establishment of relatively normal microbiota. Interestingly, the intake of probiotics in the preceding 3 months did not have an influence on the VOC profile in our cohort (data not shown). Based on our study protocol, the prior intake of antibiotics was an exclusion criterion with the exception of the pouch patients. In this group, the intake of antibiotics in the preceding 3 months had a minimal effect on the VOC expression profile. Intriguing is the finding of largely increased VOC levels in our pouch subjects, despite the removal of the colon. Our study results cannot explain this finding and this may invite further investigations on the contribution of the intestinal microbiota to breath VOCs. Our study has several limitations. Our population is a single referral center study possibly introducing a referral bias. The VOCs were determined at a single time point and no longitudinal samples are available. Even though utmost care was taken to avoid an immediate influence of dietary factors, we cannot control for other environmental exposures that might influence the exhaled breath collection. This represents a pilot study and patient numbers are limited, which may influence the power to detect differences in phenotypes. The lack of association with disease activity and phenotype, however, is likely robust, given the absence of any statistical trends in the analysis. While we used filtered air in a controlled setting some of the VOCs may be of exogenous origin. Concomitant diagnoses of chronic obstructive pulmonary disease, asthma, or interstitial lung disease may confound the VOC profile, but the number of patients with concomitant lung diseases in our cohort were negligible (<3 per lung disease). The residence time of the chyme in the whole colon varies between 15 and 50 h. An 8 h fasting period may hence not allow normalization of VOC production. Administration of a standardized diet 4–5 days before the test would be optimal. In conclusion, our study shows that exhaled VOC are a promising noninvasive method to discriminate IBD from non-IBD. The breath metabolome could not distinguish CD from UC and was not linked to clinical, radiologic, or endoscopic disease activity or disease phenotypes. The absence of a colon leads to a marked change in the exhaled VOCs, suggesting a critical role of the colon in their generation. This is a pilot study and the results need to be confirmed before they can be applied in clinical practice.  Guarantor of the article: Florian Rieder, MD. Specific author contributions: Study concept and design: FR, SK, CF, RD, RL, NP, NA; acquisition of data: SK, DG, CF, RL, BS, AB, MB; analysis and interpretation of data: FR, SK, DG, FC, RL, MB, CF, RD; drafting of the manuscript and critical revision of the manuscript for important intellectual content: all authors; statistical analysis: RL, DG; obtained funding: FR, CF, RD; administrative, technical, or material support: BS, PN, NA; study supervision: FR, RD. Financial support: N.A. is funded by the American College of Gastroenterology Junior Faculty Development Award. C.F. is funded by NIH DK050984. R.D. is supported by BRCP 08-049 Third Frontier Program grant from the Ohio Department of Development (ODOD) and by NIH 1U01AA021890. F.R. is funded by T32DK083251, 1K08DK110415, and P30DK097948. Potential competing interests: FR is on the advisory board of AbbVie and UCB and on the speakers bureau for AbbVie. Supplementary Information accompanies this paper on the Clinical and Translational Gastroenterology website

A Distinct Colon-Derived Breath Metabolome is Associated with Inflammatory Bowel Disease, but not its Complications