Effect of Diet on Intestinal Gas Production and Evacuation in Healthy Subjects and Flatulent Patients

Background Some patients complain of excessive evacuation, which may become socially disabling (Azpiroz F \& Malagelada J-R, 2005). However, the physiology and pathophysiology of flatulence remains poorly understood (Azpiroz F \& Levitt DG, 2009).

Gas evacuated by anus originates by-and-large in the colon, where unabsorbed meal residues are fermented by colonic bacteria (Anderson et al., 1981;Levitt et al., 1987;Flourie et al., 1988). Indeed, it has been shown that some food components are incompletely absorbed in the small bowel and enter the colon (Wolever \& Robb, 1992;Grimble, 1989;Steggerda FR, 1968;Wagner et al., 1977;Steggerda F.R. \& Dimmick, 1966;Stone-Dorshow \& Levitt, 1987;Wursch et al., 1989) and that other intraluminal substrates interfere with the absorption of nutrients (Boibin M et al., 1998;Hamberg et al., 1989;Brugge \& Rosenfeld, 1987;Taylor et al., 1986;Layer et al., 1986). Hence, the volume of gas production and anal evacuation is determined by two main factors: the diet, particularly the amount of fermentable residues, and the individual composition of colonic microflora. Within subjects, gas output varies in relation to the diet (Steggerda FR, 1968;Kirk, 1949). However, there is a great interindividual variability, and gas evacuation in subjects maintained on a similar diet may differ substantially.

It has been shown that healthy subjects on a normal diet containing 200 g of beans evacuate 705 mL gas per 24 hours, whereas with a fiber the diet gas evacuation was 214 mL (Tomlin et al., 1991). In healthy subjects the frequency of gas evacuation is variable, usually around 10 evacuation per day (Furne \& Levitt, 1996). Based on observations on a patient that who passed large amounts of flatus, a classification of foodstuffs depending on their gas producing capacity was elaborated (Sutalf LO \& Levitt MD, 1979). Few studies on specific foodstuffs have been later added to complement this information (Wolever \& Robb, 1992;Wagner et al., 1977;Hickey C et al., 1972). However, there no systematic investigation on the range of gas evacuation in healthy subjects and in subjects complaining of flatulence under basal conditions and in response to a high-flatulogenic diet.

Procedures Recruitment questionnaire A questionnaire will be used to evaluate gas evacuation, bowel habit and functional gut symptoms.

Daily measurement of the number of anal gas evacuations and symptoms The number of gas evacuations per anus will be measured using an event marker during daytime. At the end of the day the following parameters will be recorded on a questionnaire: discomfort related to flatulence and evaluation of abdominal discomfort/pain (using 0-10 analogue scales); the number of bowel movements and form (using the Bristol scale; (Heaton \& O'Donnell, 1994) and gastrointestinal well-being (satisfaction/dissatisfaction using a 10 point scale graded from +5 to -5).

Analysis of colonic flora The composition of the intestinal flora will be evaluated measuring flora composition in stools. Subjects will be supplied with two Stool Collecting Kits (each containing two weighed sampling tubes containing RNA synthesis stabilization buffers (RNA later, Ambion), one of which is a spare) into which they will provide a small sample of their stool (at least 30g). After mixing the stool with the buffer in the supplied tube, subjects will store the tubes in the box at home until they next visit at the hospital, where it will be centrifuged at 12000rpm for 5 minutes, supernatant removed and fecal pellet stored at - 80oC in the Gastroenterology Research Unit. Samples will be transported to Danone Research, France where they will be analysed for composition of intestinal flora (bacteria).

Using molecular based methods, the concentration of dominant and some sub-dominant bacterial groups of intestinal flora will be measured. These include bacteria that belong to Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria, Proteobacteria, Euryarcheota groups.

Continuous recording of anal gas evacuation Participants will be studied in the laboratory. After a standard breakfast, rectal gas will be collected via a rectal cannula. Anal gas will be collected via a leak proof, low resistance collection line, using a barostat and the volume will be continuously recorded for either 3 or 8 hours (see study design). The composition of the gas collected will be analyzed.

Once the subjects are positioned in bed, a non-stretch belt, 48 mm wide, with a metric scale will be adjusted around the abdomen by means of two elastic bands. All girth changes will be measured while the subjects are breathing relaxed, and will be referenced to the mid point of respiratory displacements.

A graded questionnaire will be used to measure the intensity and the type of sensations perceived, and an anatomical questionnaire to measure the location and extension of the perceived sensations. The graded questionnaire includes five visual analogue scales graded from 0 (no perception) to 6 (pain), specifically for scoring: a) sensation of increased abdominal pressure, b) sensation of abdominal distension, c) colicky/rumbling sensation, d) puncture/stinging sensation, and d) other type of sensation (to be specified by the participant in the questionnaire), respectively. The questionnaire includes two additional scales for scoring feeling of difficult evacuation of gas per anus, and belching, respectively, that will be analyzed separately. Subjects will be asked to score any perceived sensation (one or more perceived simultaneously) on the scales. The anatomical questionnaire incorporates a diagram of the abdomen divided in nine regions corresponding to epigastrium, periumbilical area, hypogastrium, both hypochondria, flanks, and ileal fossae. Subjects will be instructed to mark the location, i.e., abdominal region (s) or extraabdominal, where the sensations are perceived.

Segmental gas distribution in the gut In patients complaining of bloating, an abdominal CT scan will be taken. This scan will be used to rule out organic disorders. Furthermore, using an original program, abdominal morpho-volumetric analysis will be performed to measure gas volumes in different gut. All female participants with childbearing potential will undergo a urine pregnancy test 24 hours prior to the scan.

Hyperflatulogenic diet challenge For 3 days participants will be asked to consume a diet rich in fermentable residues to increase their gas production rate.

Low-flatulogenic diet At the end of the study, patients will be put on a low residue diet for 7 days; this will give an indication of the potentially expected response to treatment.

Working Plan Each subject will attend the following visits; the first recruitment visit will be performed by the attending physician in the outpatient clinic, and the rest, including the physiological measurements in the Digestive System Research Unit; CT imaging will be performed in the Department of Radiology.

Visit 1. Recruitment: entry questionnaires. Visit 2. Before basal period: instructions, informed consent, and delivery of event marker, questionnaires, and material for fecal sampling.

Visit 3. Last day of basal phase. Continuous recording of anal gas evacuation. Delivery of dietary instructions (high-flatulogenic diet) and questionnaires. In 8 patients segmental gas distribution will be also measured during the basal phase.

Visit 4. Last day of challenge phase. Continuous recording of anal gas evacuation. CT for evaluation of segmental gas distribution in the gut. In patients only: delivery of dietary instructions (low-flatulogenic diet) and questionnaires.

Visit 5. Last day of correction phase. In patients only: collection of data and materials.