19 As the low FODMAP diet was lower in RS than the high FODMAP diet, wheat muffins containing high amylose maize (high in RS) were added to balance the RS content. An example of the test diets provided on day 2 of the study is shown in Table 1. All food used was purchased from the local supermarket with the exception of the 355 mL can of soda that was sweetened with high fructose corn syrup (imported from the USA) and was

obtained from a specialty supermarket. Samples of food and drinks used in the study were separately analyzed for their content of fructose, glucose, lactose, sugar polyols (sorbitol and mannitol), JQ1 solubility dmso galacto-oligosaccharides (GOS, raffinose and stachyose) by high performance liquid chromatography

(HPLC) as previously described.20 Total fructan content was measured using an enzymatically-based assay kit (Megazyme International Ireland Ltd, Wicklow, Ireland), as per the manufacturer’s instructions.21 Results from laboratory analysis were added to the Foodworks database (Foodworks Professional 2007, Xyris Software, Highgate Hill, QLD, Australia) to enable the complete assessment of the macronutrient Afatinib and FODMAP dietary intake during the study. Breath samples were collected every hour for 14 h into 250 mL sample holding bags (Quintron Instrument Co., Milwaukee, Wisconsin). The first sample of the day was a fasting sample and taken prior to breakfast. Samples were then taken hourly for a total of 14 h. All of the supplied food was to be consumed within the 14-h time period. The exact time each subject consumed their meals varied slightly between individuals but was kept constant within each individual during the two dietary MCE periods (i.e. each person was their own control in the crossover design). Breath samples were analyzed for

hydrogen and methane within 24 h using a Quintron Microlyzer Model DP Plus (Quintron Instrument Co., Milwaukee, WI, USA). Total breath gas production over the 14-h period was then calculated from the graphed area-under-the-curve (AUC) using the trapezium rule and expressed in parts per million over 14 h (ppm.14 h). A subject was considered to produce hydrogen or methane if the AUC was more than 10 ppm.14 h during at least one of the dietary periods. All subjects were asked to complete the gastrointestinal symptom questionnaire at the same time in the evening of each day. The questionnaire comprised five categories for general gastrointestinal symptoms (abdominal pain/discomfort, abdominal bloating/distension, wind, nausea, heartburn and lethargy). Bowel function was noted but not analyzed due to the heterogeneity of bowel habits across the subjects. The symptoms were rated using a Likert scale 0 to 3, where 0 = none, 1 = mild, 2 = moderate and 3 = severe.