C Probert

Intestinal transit time in the population calculated from self made observations of defecation.

STUDY OBJECTIVES--To assess the feasibility of estimating intestinal transit time in the general population using self recorded data on stool form, frequency of defecation, and the interdefecatory time interval. DESIGN--Prospective measurement of bowel function. SETTING--Bristol, Avon, UK between 1987 and 1989. SUBJECTS--Subjects were drawn from 1897 people who comprised 72.2% of a stratified random sample of all men aged 40-69 years and women aged 25-69 years on the lists of 19 general medical practitioners. Altogether 1561 subjects (59.4%) recorded bowel function and a subsample of 98 (50 women and 48 men) had intestinal transit time measured. MEASUREMENTS AND MAIN RESULTS--The interdefecatory time interval and stool form (on a validated 1-6 scale sensitive to transit time) were recorded prospectively from three consecutive defecations. In the subsample the mean intestinal transit time was measured simultaneously using a four marker, two stool x ray technique. Multiple regression analysis was used to assess the extent to which intestinal transit time could be predicted from the defecatory data. The formulas obtained were then applied to the whole study population. In women, intestinal transit time was best predicted by the formula 103-1.23 (DF)--4.69 (SFS)+0.638 (IDTI), where DF is the stated defecation frequency per week, IDTI is the interdefecatory time interval, and SFS is the sum of the three stool form scores, for which the correlation coefficient r = 0.736. For men the intestinal transit time = 79-1.33 (DF)--1.88 (SFS)+0.329 (IDTI), for which the correlation coefficient r = 0.541. The predicted intestinal transit time was longer in women than men at equivalent ages. Women of childbearing age had longer transit times than older women. CONCLUSIONS--Observations made by untrained subjects can be used to estimate intestinal transit time in epidemiological studies. A gender related difference in transit time exists.

PWE-173 Investigation of volatile organic compounds emitted from faeces for the diagnosis of giardiasis

Introduction Giardiasis is a common but neglected intestinal infection worldwide, caused by the flagellated intestinal protozoanGiardia duodenalis(syn. lamblia/intestinalis). Several methods are available for the laboratory diagnosis of Giardia. It has unique metabolic pathways related to its lack of mitochondria, making it an ideal target for volatile organic compound profiling. Method Faecal gas headspace extraction and Gas Chromatography-Mass Spectrometry (GCMS) were used to extract and identify VOCs. Analytical tools including Metab,1R and Metaboanalyst2were subsequently used. Stool was obtained from patients with confirmed isolated Giardiainfection, together with stool from subjects with diarrhoea but no identifiable infection, including Giardia, these acted as controls. Significant differences between the VOCs were then analysed. Results The composition of these compounds was dominated by Esters, Acids and Alcohols, respectively. With an absence of Amides, Alicyclic compounds, Ether compounds and Nitrogen Containing compounds. >100 VOCs were identified across both the control and Giardiagroups. Of these 10 were noted to be significantly different between the two groups (p < 0.01). Three VOCs were identified with a significant predominance to the Giardiasamples, 2,2,4,4-tetramethyloctan, acetic acid and 2,2,4,6,6-pentamethylhepatance (p < 0.0001). 10 VOCs were found to be significantly different in terms of abundance (p < 0.01), acetone and 2-butanone were the two most significantly different compounds, both demonstrating a p value of <0.001 and both having down regulation in the Giardia samples. PLS-DA showed a good degree of separation. AUROC analysis gave 5 VOCs with an AUC between 0.8 and 0.93. Conclusion There is a significant difference in the VOC profile of stool from subjects infected with Giardia spp, when compared to non-infected controls, both in terms of prevalence and abundance. There is also a defined metabolic reason for this difference originating from the unique metabolic pathways seen in Giardia. Disclosure of interest None Declared. References Aggio R, Villas-Bôas SG, Ruggiero K. Metab: an R package for high-throughput analysis of metabolomics data generated by GC-MS. Bioinformatics (Oxford, England)2011;27:2316–2318. doi:10.1093/bioinformatics/btr379 Xia J, Psychogios N, Young N, and Wishart DS. MetaboAnalyst: a web server for metabolomic data analysis and interpretation. Nucl Acids Res. 2009;37:W652–660.

PWE-169 Analysis of faecal volatiles from young children infected with and without rotavirus

Introduction Rotavirus is the most common cause of severe diarrhoea among infants and young children. Rotavirus is usually an easily managed disease of childhood, but worldwide nearly 500 000 children under 5 years of age still die from rotavirus infection each year and almost two million more become severely ill. Rotavirus A (responsible for about 90% of infections) is typically diagnosed by finding the virus in the childs stool by enzyme immunoassay. This study was undertaken to expand our knowledge of VOCs from the stool of children and assess whether rotavirus causes easily measurable changes in the gut chemistry of infected young children. Methods The volatile organic compounds (VOCs) from the stool of 53 children from Malawi (26 non-infected children with an unspecified GI problem and 27 rotavirus children diagnosed with the virus) were analysed using Headspace Trap-GC/MS. The faecal samples were placed in headspace vials and were heated from frozen to 90°C. The VOCs were preconcentrated and focused prior to GC/MS analysis. Those VOCs were identified by comparing their mass spectra with those contained in the NIST/EPA/NIH Mass Spectral Database. Results A total of 186 different compounds have so far been identified. Of the 53 stool samples ethanol was found in 69% and 63% samples respectively between the non and infected classes, which contrasts with previous work where ethanol was found in all healthy adult and all healthy neonate stool. This could be due to the more dilute stool due to diarrhoea. Carbon disulphide has previously been found to be ubiquitous in healthy adult stool, again the frequency was much less at 27% and 22% respectively. In contrast the majority of samples contained ethanoic acid, with more samples in the rotavirus group. There were very little differences in the frequency (ca. 92%) and abundance of ethanol in both sample classes, curiously ethanol has been previously shown to be present in all adult samples and absent in neonates. In contrast 2,3-butanedione and other aldehydes were significantly present both at greater frequency and typically at higher concentrations in rotavirus samples compared to non-rota samples, see Abstract PWE-169 figure 1.Abstract PWE-169 Figure 1 Combined peak area (arbitrary units) of selected carbonyl compounds (namely; 2,3-butanedione/propanal/2-heptenal, (Z)-/octanal/nonanal combined) present in faecal samples analysed. Conclusion Very little work has been published on volatile compounds from stool in particular from the stool of children, this work adds to this knowledge. Some compounds such as ethanol were found in approx. equal amounts in the diarrhoea of infected and non-infected stool, however in general there was a greater frequency and abundance of VOCs in the rota infected samples, particularly of aldehydes and 2,3-butandione. Competing interests None declared.

PWE-287 Breath volatile analysis for the recognition of harmful drinking, cirrhosis and hepatic encephalopathy

Introduction Hepatic encephalopathy (HE) is a neuropsychiatric state which may complicate cirrhosis following the accumulation of toxic substances that cross the blood-brain barrier and affect brain function. Cirrhosis or portal-systemic shunting results in the accumulation of substances in the blood that may undergo alveolar gas exchange to be excreted in the breath. The aim of this work was to investigate the use of breath analysis as a non-invasive and simpler means of diagnosing HE, cirrhosis and harmful drinking. Methods A bespoke breath-sampling device was used to sample one litre of breath through adsorbent tubes from patients with alcohol-related cirrhosis with (n=11) and without HE (n=23), non-alcoholic cirrhosis without HE (n=19), harmful drinkers without cirrhosis (n=7), inflammatory respiratory disease (n=18), and healthy controls (n=15). Compounds trapped on these tubes were released via thermal desorption and analysed by gas chromatography mass spectrometry for separation and detection. Multivariate discriminant analysis was used to identify volatile organic compounds to differentiate patients according to disease status and build models for disease classification. Results Models based on the presence or absence of volatiles were tested in the patient groups. HE was correctly classified in 91.0% of patients with alcoholic cirrhosis. Patients with cirrhosis could be discriminated from those without cirrhosis with 100% accuracy in drinkers. In patients without clinical signs of HE, alcohol was correctly predicted as the underlying cause of cirrhosis in 82.6% of patients and non-alcoholic causes of cirrhosis were correctly determined in 84.2% of patients. Non-alcoholic cirrhosis, alcoholic cirrhosis, and harmful drinking could also be discriminated from healthy controls with a sensitivity of 89.5%, 97.1% and 100%, respectively. Conclusion Breath volatiles can be used to aid the diagnosis of HE, cirrhosis, and harmful levels of drinking, therefore breath testing may offer a means to detect liver conditions non-invasively at earlier and more treatable stages. Competing interests None declared.

PTH-051 Influence of Iron Supplementation on The Natural History of Colitis

Introduction Iron deficiency anaemia is common in IBD. Iron supplementation may induce or exacerbate colitis in rats (APT 2001;15:1989–99).Dysbiosis of the microbiota is common in IBD and iron contributes to this as it is a growth factor for pathogenic bacteria.We investigated the effect of dietary iron supplementation and/or reduction on the severity of chronic colitis in a murine model using clinical, histological and biochemical parameters. Methods Studies were performed on 6 groups of 8 wild type (WT) C57BL/6 mice.Chronic colitis was induced with 1.25% Dextran Sodium Sulphate (DSS) for 5 days,followed by16 further days on water [for 3 consecutive cycles]. DSS-treated mice were fed one of three diets:low iron [LI] (100 ppm), normal iron [NI] (200 ppm) and high iron[HI] (400 ppm) supplemented chow. Also, 3 non-DSS-treated groups were studied and fed similarly. Half of the mice in each control group were treated with 1 cycle of acute 2% DSS for 5 days at day 53, followed by 5 further days on water. All mice were sacrificed at day 63. Daily weights and clinical features were recorded. Histological colitis was scored using the Bauer score(Gut 2010; 59:1192–99).Faecal calprotectin was measured by ELISA and faecal iron by immunoassay at various time points [day (d) 1, 21, 42 & 63]. Statistical analyses used the Kruskal–Wallis test with post-hoc analysis. Results Oral DSS administration induced colitis in all treated mice. While chronic DSS colitis was not associated with weight loss, there was severe weight loss in acute DSS mice which was greatest in the low iron diet group (p = 0.001 LI vs. HI; p = 0.01 for LI vs. NI). Histologically, the colitis features were more prominent in acute DSS-treated mice ingesting low and high iron diets, with median colitis scores 6 & 5.5 respectively.Cyclic administration of DSS in drinking water resulted in a significant rise in faecal calprotectin, from baseline to d63 in LI (p = 0.05) and for HI (p = 0.01), but this was not significant in the NI group. In acute DSS, the rise was greater in LI and HI (p = 0.001) and less in NI (p = 0.01).Total faecal iron was increased in a dose-dependent manner within 9 weeks in all non-DSS groups. Nevertheless, in chronic DSS groups, p = 0.001 at d1 vs. d63 for all groups [382% change for LI, 331% for NI and 355% for HI].However, in acute DSS p = 0.05 for LI, p = 0.001 NI & HI (d1 vs. d10). Conclusion Changes in nutritional luminal iron exacerbate colitis.Oral administration of DSS causes a reproducible acute colitis, followed by a slow recovery phase with a concomitant chronic inflammation. Chronic colitis was worse in mice fed low or high iron diets, as shown by elevated calprotectin. Faecal iron rose equally in all 3 groups: with iron increases likely arising from diet and bleeding during colitis. Dysbiosis may be a consequence of this change in luminal iron. Disclosure of Interest None Declared

OC-030 Investigation of faecal volatile organic compounds as biomarkers for the diagnosis of necrotising entercolitis

Introduction Necrotising enterocolitis (NEC) is the most common serious gastrointestinal complication of preterm birth. Clinical signs and symptoms are often non-specific and may only become apparent once disease is established. A biomarker to indicate early disease would allow earlier treatment and may improve outcome. We have reported a pilot study showing changes in faecal volatile organic compounds (VOCs) profile in the prodromal period of NEC. Here, we report a much larger series with more comprehensive clinical and laboratory data. Method Daily faecal samples were collected prospectively from 1375 apparently healthy preterm babies, born between 23 and 34 weeks gestation, in 8 NICUs over a 2-year period. Over 13,000 samples were catalogued. Samples from 70 healthy preterm babies and 34 preterm babies who developed NEC (Bell’s stage II and III) were analysed. In short, 50–100 mg of faeces underwent headspace gas extraction using solid phase micro-extraction followed by gas chromatography–mass spectrometry using our optimised method. Each NEC patient was matched with 1 to 3 healthy control patients. Samples from NEC patients between 1 to 6 days prior to diagnosis and matched samples from controls were selected. Compounds were identified using an in-house developed pipeline involving the AMDIS software, the NIST database and the R packages Metab and XCMS. Univariate and multivariate analysis were performed on the clinical and VOC data. The features best describing the differences between NEC and control samples were used to build a classifier for the diagnosis of NEC. Results Six VOCs and birthweight were significant in multivariable analysis. Birthweight was the strongest predictor. Of the VOCs, 3 were positively associated with NEC and 3 negatively associated. A logistic regression model was developed to diagnose NEC overall, then for each of the 6 days that preceded the clinical diagnosis. Overall the model had an area under the ROC (AUROC) curve of 0.83. When estimated for each day, the AUROC ranged from 0.78 to 0.9. Conclusion To the best of our knowledge, this is the first large scale metabolomics study of faecal VOCs aiming at classifying NEC patients. The data indicate the VOCs may be used to predict NEC up to 6 days before the condition is diagnosed using current methods. This may have an important impact on reducing the mortality of this devastating condition. Further investigation of VOCs may also elucidate the pathogenesis of NEC. Disclosure of interest None Declared.

PWE-078 Mean Corpuscular Volume But Not Lymphocyte Count Is A Predictor Of Thiopurine Dose Adequacy And Toxicity

Introduction The thiopurines, azathioprine (AZA) and mercaptopurine (MP), commonly used in the treatment of inflammatory bowel diseases (IBD), are typically dosed according to patient’s body weight. A previous meta-analysis showed higher remission rates in patients with “therapeutic” levels of 6-thioguanine (6-TGN), but weight based dosing correlates poorly with 6-TGN levels (1). 6-TGN testing is not universally available, results are not available immediately and repeated measurements are necessary to ensure dose adequacy and adherence to therapy. Proxy measures such as mean corpuscular volume (MCV) and lymphocyte count (LC) have been advocated as markers of dose adequacy. We aimed to analyse the relationship between 6-TGN levels, MCV, LC and other putative surrogate markers of therapeutic 6-TGN levels. Methods This retrospective study was conducted at the Royal Liverpool University Hospital. All patients who had concurrent measurements of 6-TGN and full blood count were included in the analysis. 6-TGN levels were classed as sub-therapeutic (<230), therapeutic (230–450) or supra-therapeutic (>450). The association between 6-TGN, patient demographics, MCV, LC and other putative surrogate markers was estimated using a multivariable linear regression model for continuous 6-TGN and a proportional odds logistic regression model for the ordered 6TGN levels. All results were declared statistically significant if p < 0.05. Results A total of 106 patients (48 male, 58 female) were included and contributed 133 measurements. Of these patients 58 (55%) had Crohn’s disease and 47 (44%) had ulcerative colitis. The mean azathioprine dose was 123.5 mg (SD 73.8) or 1.70 mg/kg (SD 0.67). After adjusting for other variables, a one unit increase in MCV, was associated with a 10.88 unit increase in 6TGN levels, Figure 1 (95% CI: 7.63 and 14.014, p < 0.0001) and a one unit increase in ALT was associated with a 2.67 unit decrease in 6TGN levels (95% CI: 0.36 to 4.97 p = 0.0237). There was no correlation between LC, NC, WCC or ALKPHOS and 6-TGN levels. Conclusion MCV and 6-TGN nucleotide levels increase together. If 6-TGN levels are not available, MCV can be used as a crude but imperfect surrogate marker of dose adequacy and toxicity. Reference Osterman MT, Kundu R, Lichtenstein GR, Lewis JD. Association of 6-thioguanine nucleotide levels and inflammatory bowel disease activity: a meta-analysis. Gastroenterology 2006;130:1047–53 Disclosure of Interest None Declared.

PTH-023 Paediatric Faecal Voc Analysis: Method Optimisation

Introduction Faecal Volatile Organic Compounds (VOCs) analysis is an emerging diagnostic tool for gastrointestinal conditions because of its sampling ease and non-invasive characteristics. Solid phase micro-extraction (SPME) is often used with gas chromatography–mass spectrometry for the analysis of VOCs; however no procedure has been standardised for an application in faecal analysis with the potential for on–site utilisation. Several aspects of the sampling preparation applied to neonatal faecal samples were studied to improve the robustness of the analytical process for paediatric studies. Methods Thirty-three faecal neonate samples of weight 50–700 mg were analysed. The results produced by SPME coatings CAR/PDMS and DVB/CAR/PDMS were compared (n = 5/variable), as were vial volumes of 2 and 10 ml (n = 4/variable; N=3) and the addition of 0.5 and 1 ml (n = 3/variable) of a saturated NaCl solution prior to GC-MS analysis. In addition, the influence of leaving the samples for 14 h at 1°C instead of -20°C was studied (n = 3/condition). Finally, the reproducibility of the method was tested by looking at the internal variation of 10 sets of triplicate; furthermore, 3 sets were reanalysed 4 times in order to characterise the repeatability across GC-MS runs. Independent samples t-test, one–way ANOVA and Tukey’s HSD test were performed to test differences between data classes. Final p-values were adjusted by Bonferroni and p-values < 0.05 were considered significant. Results Twenty (±2) VOCs were identified using samples of 100 mg, while 28 (±1) and 25 (±2) VOCs were identified in samples of 450 respectively 700 mg. There were no significant differences in VOCs intensities between samples of 450 and 700 mg as between 50 and 100 mg. However, all VOCs intensities were significantly higher in samples of 450 and 700 mg when compared to 100 mg. No differences were observed between the two SPME coatings, and the addition of salt did not improve results quantitatively or qualitatively. Keeping samples at 1°C instead of -20°C and/or varying the volume of the vial did not influence the results significantly. Finally, for 7 sets of triplicates out of 10, more than 90% of the ion intensities varied less than 30% but multiple runs of GC-MS resulted in significant changes in intensity of 40% or more of the VOCs identified. Conclusion Several parameters were studied to optimise a method for paediatric faecal VOCs analysis and a robust method has now been developed and detailed. Samples of 50–100mg may be studied. This weight gives reproducible results, but samples should not be reanalysed as headspace VOCs are altered by the procedure. Other changes to sample processing had little impact on the results. A chilled auto sampling rack may be safely used. Disclosure of Interest None Declared.

Rapid diagnosis of gastro-intestinal infections using faecal odour

This book describes how the analysis of the trace gases in exhaled breath can be used for non-invasive clinical diagnosis of disease and for monitoring the effectiveness of therapy. This approach offers an important addition to the diagnostic techniques available to medicine, having the advantage that on-line breath analysis can provide information to the clinician immediately and thus facilitate rapid diagnosis and treatment. The book is a compilation of contributions to a conference held in Dornbirn, Austria, 23–26 September 2004 on various aspects of this new topic. Written by the foremost workers in the field, it will provide clinicians and others in the medical fraternity with an up-to-date summary of the status of the subject. The wide scope of the chapters ranges from descriptions of the analytical methods that are available, through the use of breath analysis in the study of physiological phenomena, to the identification of biomarkers of particular injury and disease.

PTH-064 Longitudinal investigation of microbiota dynamics in a model of mild chronic dss-induced colitis in wild-type (wt) c57bl/6 mice receiving diets with different iron contents

Introduction Iron deficiency anaemia is common in inflammatory bowel disease (IBD). Iron supplementation may induce or exacerbate colitis in rats (APT 2001;15:1989–99). Dysbiosis is common in IBD and iron contributes to this as it is a growth factor for some bacteria. We examined the long-effect of dietary iron supplementation and iron reduction on the intestinal microbiome in a chronic murine model of colitis. We report results of changes at the phylum level. Method Studies were performed on 6 groups of 8 WT mice. Chronic colitis was induced with 1.25% dextran sodium sulphate (DSS) for 5 days, followed by 16 further days on water [for three consecutive cycles]. DSS-treated mice were fed one of three diets (start from day-1 of experiments): low iron [LI] (100ppm), normal iron [NI] (200ppm) and high iron [HI] (400ppm) supplemented chow. Also, three non-DSS-treated groups were studied and fed similarly. Half of the mice in each control group were treated with one cycle of acute 2% DSS for 5 days at day 53, followed by 5 further days on water.All mice were sacrificed at day 63. Clinical and pathological data were compared at day-1, 21, 42 and 63 (chronic) and day-1 and 10 (acute); bacterial gDNA was extracted from faeces and microbiota composition determined from the sequence of V4 region of 16 S rDNA on the Illumina MiSeq platform. Statistical inferences were made using Kruskal-Wallis H-test with post-hoc analysis (Bioinformatics 2010;26:715–21). Results DSS-induced colitis in all treated mice. Chronic DSS colitis was not associated with significant weight loss, while weight loss in acute DSS mice was greatest in the LI diet group (p<0.001 LI vs. HI; p<0.05 for LI vs. NI). Histologically, the colitis features were worse in LI (p<0.001) than HI and NI (p<0.01 each) and more prominent in acute DSS-treated mice ingesting low and high iron diets, with median colitis scores 6 and 5.5 respectively. However, faecal phyla changes were seen in both LI and HI DSS-treated groups and controls fed an HI diet for chronic experiments only: Proteobacteria were increased significantly at day-63 (p<0.01) in LI, HI DSS groups and HI controls. Actinobacteria were also increased in the latter group whereas, a reduction was observed for Bacteroidetes (p<0.028) in the HI DSS group. Conclusion Changes in nutritional luminal iron exacerbate colitis. Oral administration of DSS causes a reproducible acute colitis, followed by a slow recovery phase with a concomitant chronic inflammation. Chronic colitis was worse in mice fed low or high iron diets. Dysbiosis was found in mice with chronic colitis and altered iron intake as well as controls receiving high iron diets. Iron, therefore, appears to contribute to the dysbiosis associated with IBD. Disclosure of Interest None Declared