Introduction

​Volatile Organic Compounds (VOC’s) are produced in response to endogenous metabolic processes within the body and as a result of functional activation of the trillions of microbes within the gut (exogenous VOC’s). VOC’s pass from the gut lumen into the blood stream and can be detected on exhaled breath. To date, microbiome research has mainly relied on stool collection and analysis which has both spatial and temporal limitations in that stool represents the end of the metabolic process and sampling is mostly constrained to spontaneous bowel movements. Breath can be assessed repeatedly in real time and, following ingestion of carbohydrate probes, samples can be taken during periods when the microbiome is known to be functionally active. In this study we used state of the art collection and detection technology to fully characterise both endogenous and exogenously produced VOC’s in healthy subjects for the first time.

Aims & Methods

In 45 healthy subjects, baseline breath samples were taken after a 24-hour low fermentable diet and overnight fast. Subjects then ingested 10g of lactulose mixed with 200ml water and subsequent breath samples were taken at 45, 90 and 180-mins following ingestion. VOC breath samples were acquired using the Breath Biopsy ® collection system over 10-minutes whilst subjects breathed filtered air and VOC’s were collected onto sorbent tubes which concentrated the compounds for later analysis. Analysis was then performed using Gas Chromatography Mass Spectrometry (GC-MS).
The exhaled breath samples were evaluated in targeted (i.e. absolute amounts of 42 compounds of interest) and untargeted manner (i.e. gathering as many compounds in the sample as possible).

Results

600 distinct VOC’s were detected at baseline and these represent the endogenous healthy human VOCome. Targeted analysis indicated that levels of isoprene, 3-methylthiophene, phenol and propionic acid showed differences in exhaled breath between baseline and post-lactulose challenged individuals (p < 0.05). Moreover, the untargeted analysis identified 14 additional compounds in exhaled breath with significant increases following lactulose ingestion (p-value<0.05). All those compounds were reliably measured only on breath and appeared in at least 85% of individuals.

Conclusion

This study provides the first comparative profile of the entire healthy human VOCome in relation to endogenous and exogenously (microbiome related) VOC’s. We identified several changes in the composition of exhaled breath during functional activation of the microbiome following ingestion of a carbohydrate probe. This fundamental work describes a unique experimental model and provides normative reference ranges that will form the foundation for future novel microbiome, nutritional and gut biomarker research.

Disclosure

A Hobson is a shareholder in the Functional Gut Clinic
S Bloor is an employee of he Functional Gut Clinic
N Nagalingam, A Somlinska, B O'Brien, R Stollard, T Woodland, A Tawfike, M Allsworth and B Boyle are employees of Owlstone Medical

Introduction

Recent societal guidelines suggest there is no role for hydrogen and methane breath testing (HMBT) to exclude small intestinal bacterial overgrowth (SIBO) in patients with diarrhoea predominant irritable bowel syndrome (IBS) and to treat symptoms with rifaximin empirically^1,2. However, a positive result for SIBO with lactulose HMBT has been shown to predict more favourable outcomes to rifaximin in patients with diarrhoea predominant IBS³. We wanted to investigate if symptoms and medical history can predict lactulose HMBT outcome hence justifying treatment of SIBO without a formal diagnosis.

Aims & Methods

A large questionnaire-based study was conducted in over 1000 patients of which 731 completed lactulose HMBT. The 26-item questionnaire included questions about medical history, symptoms, and pattern of symptoms, and we used this to identify any positive predictors for SIBO on HMBT. A positive lactulose HMBT was determined by a rise of ≥20 ppm in breath hydrogen from baseline within 90 minutes³. Breath samples were analysed using gas chromatography. Associations and predictors were calculated using chi square tests and logistic regression, respectively.

Results

Bloating (93.6%), abdominal pain (87.3%), and diarrhoea (80.3%) were the most common symptoms reported by those with a positive result for SIBO, but their duration, frequency and onset were no different to patients with a negative result (P > 0.05). There were no positive predictors for SIBO on lactulose HMBT based on symptoms, medications or medical history except for chronic low ferritin (P = 0.025).

Conclusion

This large questionnaire study has demonstrated that symptoms and medical history cannot predict HMBT outcomes for SIBO. Therefore, treatment of SIBO without a diagnosis confirmed on HMBT may lead to unnecessary antibiotic use and costs.

References

1. Vasant DH, Paine PA, Black CJ, Houghton LA, Everitt HA, Corsetti M, Agrawal A, Aziz I, Farmer AD, Eugenicos MP, Moss-Morris R, Yiannakou Y, Ford AC. British Society of Gastroenterology guidelines on the management of irritable bowel syndrome. Gut. 2021 Jul;70(7):1214-1240.
2. Savarino E, Zingone F, Barberio B, Marasco G, Akyuz F, Akpinar H, Barboi O, Bodini G, Bor S, Chiarioni G, Cristian G, Corsetti M, Di Sabatino A, Dimitriu AM, Drug V, Dumitrascu DL, Ford AC, Hauser G, Nakov R, Patel N, Pohl D, Sfarti C, Serra J, Simrén M, Suciu A, Tack J, Toruner M, Walters J, Cremon C, Barbara G. Functional bowel disorders with diarrhoea: Clinical guidelines of the United European Gastroenterology and European Society for Neurogastroenterology and Motility. United European Gastroenterol J. 2022 Jul;10(6):556-584.
3. Rezaie A, Heimanson Z, McCallum R, Pimentel M. Lactulose Breath Testing as a Predictor of Response to Rifaximin in Patients With Irritable Bowel Syndrome With Diarrhea. Am J Gastroenterol. 2019 Dec;114(12):1886-1893.

Introduction

Irritable bowel syndrome (IBS) is a complex multifactorial disorder characterized by recurrent abdominal pain associated with defecation or a change in bowel habits. Patients with IBS often complain of extra-intestinal symptoms resulting in impaired related quality of life. Growing evidence support the hypothesis that intestinal barrier dysfunction has a central role in the pathophysiology of IBS being the interface between the gut lumen and the deeper intestinal layers as well as the brain.

Aims & Methods

The aims of this study were: to characterize the epithelial and vascular barriers; to correlate molecular, morphological and functional aspects of epithelial and vascular barriers with IBS symptoms. 66 healthy subjects (AC) and 223 patients with IBS (112 IBS-D, 58 IBS-C and 53 IBS-M) were enrolled in the study. Rome IV criteria, Bowel disease questionnaire, Short Form 36, Hospital Anxiety Depression Scale were used to phenotype patients. Sugar test was used to evaluate in vivo permeability. The vascular barrier was characterized by using immunohistochemistry, western blot and electron microscopy. Vascular permeability was evaluated by assessing two markers: plasmalemma vesicle–associated protein-1 (PV1) and vascular endothelial cadherin (VEC). A translational model based on Caco-2 cells or HUVEC was used to evaluate permeability changes induced by mediators spontaneously released by mucosal biopsies and to highlight the underlying molecular mechanisms. Correlation analyses have been performed among experimental and clinical data.

Results

Compared to AC, lactulose (P<0.05), sucralose (P<0.01), sucrose (P<0.01) excretion and the L/M ratio (P<0.01) were significantly increased in IBS patients. Soluble mediators released by mucosal biopsies of IBS patients increased Caco-2 permeability compared to AC mediators (P<0.05) via a down-regulation of tight junction gene expression. Blood vessels were significantly increased by 1.5 fold in the colonic mucosa of IBS patients compared to AC (P<0.01). PV1 expression was increased (P<0.01) while VEC expression was decreased in the colonic mucosa of IBS group compared to AC (P<0.05). IBS mucosal mediators increased HUVEC permeability compared to AC mediators. The HUVEC changes associated with IBS samples were mediated by a protease-activated receptor (PAR)2 dependent mechanism resulting in VEC down-regulation. Significant correlations emerged among permeability changes and intestinal, extra-intestinal symptoms and health-related quality of life reported by IBS patients.

Conclusion

Epithelial and vascular barriers are compromised in IBS and are likely involved in symptom development.

Disclosure

Nothing to disclose

Introduction

Patients with inflammatory bowel disease (IBD) have an increased risk of developing intestinal and extra-intestinal cancers. Specific therapies used to control inflammation in IBD may have a different impact on cancer risk. However, there are no long-term studies investigating cancer risk in patients with IBD.

Aims & Methods

In the present study we aimed to quantify the overall risk for of developing cancer and to identify specific risk factors for the occurrence of intestinal and extra-intestinal cancers in 560 consecutive patients with Crohn's disease (CD, N=310) or ulcerative colitis (UC, N=250) referred to our outpatient department between January 2021 and February 2022. Demographic data, smoking status, alcohol consumption, age at IBD diagnosis, disease location and course, therapies, and cancer development after IBD diagnosis were recorded. Odds ratios (OR) were estimated by logistic regression analysis. Standardized Incidence Ratios (SIRs) were estimated compared with the general population.

Results

During a median follow-up interval or time of 10 years (range 1-26 years), 6.6% of patients (37/550, female 65 %, mean age 51 years) developed cancer (SIR 1.94, 95%CI 1.4-2.6). The most common cancers were colorectal (12.3%), skin (8.2%), and breast cancer (7.2%). Female patients had an increased risk for all cancers (SIR 3.11, 95%CI 2.06-4.3), melanoma (SIR 5.6, 95%CI 1.14-16.2) and colorectal cancer (SIR 7.59, 95%CI 3-15.4). Male patients had an increased risk of lymphoma (SIR 26.15, 95%CI 3.2-95.7). Younger age at diagnosis of CD (A1 Montreal classification, OR 37.9, 95%CI 10-14), immunosuppressive monotherapy (OR 5.5, 95%CI 2.65- 11) or in combination with anti-TNFα (OR 9.9, 95%CI 3.3-29.09) were associated with an increased risk of cancer in the study population.

Conclusion

We have identified subgroups of IBD patients who would particularly benefit from screening programs. Our findings need to be validated in independent cohorts.

Introduction

Gastro-oesophageal reflux disease (GERD), caused by reflux of stomach contents into the oesophagus, results in discomfort and pain. Evidence suggests reflux- induced oxidative stress in oesophageal epithelial cells may have a role in GERD pathogenesis and symptom development. High mobility group box 1 (HMGB1), a nuclear protein found in most cell types, is actively released in response to oxidative stress. HMGB1 binds to TLR2, TLR4, and RAGE, leading to downstream inflammatory signalling.

Aims & Methods

Our aim was to investigate the potential role of epithelial-derived HMGB1 in the context of GERD.
Oesophageal biopsy tissue was obtained from consented healthy volunteers (N=10) and GERD patients categorised as functional heartburn (FH; N=7), non-erosive reflux disease (NERD; N=13), and erosive reflux disease (ERD; N=11) based on clinical assessment. Oesophageal HMGB1 mRNA was assessed by RT-qPCR. Tissue was used to assess protein expression of HMGB1, TLR2, TLR4, and RAGE using immunohistochemistry (IHC). Healthy oesophageal biopsies were used to assess HMGB1 release ex vivo following synthetic refluxate challenge (pH2, 1mM deoxycholate (DCA), 1mg/mL pepsin). Cellular translocation of HMGB1 was assessed using immunofluorescence in NE-1 cells in response to acidic bile salts (pH4, 1mM DCA).

Results

HMGB1 mRNA and epithelial protein expression were consistent between healthy control and GERD patients. TLR2 and TLR4 were expressed primarily on epithelial cells with cell quantification revealing TLR4 to be the predominantly expressed epithelial receptor. The proportion of TLR4+ cells was higher in ERD compared to healthy controls (1.4% ± 0.43 vs 7.2% ± 2.567; p<0.05). In contrast, RAGE was expressed exclusively on CD45+ leukocytes – predominantly in GERD phenotypes. Many RAGE+ immune cells in GERD tissue were in close apposition to sensory CGRP+ nerve fibres. Incubating NE-1 cells with acidic bile salts (10mins), but not acid alone, induced HMGB1 translocation to the cytosol (nuclear:cytosolic HMGB1 ratios: pH7 = 2.42 ± 0.19; pH4 = 2.66 ± 0.07; pH4 + DCA = 1.416 ± 0.06; p=0.0003). Challenging oesophageal biopsies with synthetic refluxate (5mins) led to release of HMGB1 over 18 hours compared to paired biopsies (218 ± 57 vs 586 ± 220 pg/mL/mg; p<0.05).

Conclusion

HMGB1 released by oesophageal epithelial cells following reflux may contribute to inflammatory pathways in GERD, particularly via TLR4 and RAGE on epithelial and immune cells, respectively. This is of particular interest in PPI-refractory GERD, due to the exaggerated role of bile salts in HMGB1 release. Further, the close proximity of RAGE+ leukocytes and CGRP+ nerve fibres may represent a cross-talk between epithelial cells, immune cells, and sensory nerves. HMGB1 may therefore be a potential new treatment target for GERD.