Effects of Combined Administration of Calcium and L-tryptophan on Gut Functions and Blood Glucose in Healthy Humans

The intervention in this study consists of 75-minute intraduodenal infusions of isosmotic solutions containing either saline (control), L-tryptophan, or calcium combined with L-tryptophan. Participants enrolled into the study will receive, in a randomised, double-blind fashion either (i) saline (control), (ii) L-tryptophan at a rate of 0.1 kcal/minute, (iii) combination of L-tryptophan (0.1 kcal/minute) + 500 mg calcium, or (iv) combination of L-tryptophan (0.1 kcal/minute) + 1000 mg calcium in four separate sessions, each of which will be separated by at least 4 (and up to 10) days. Each study session will be 4-6 hours. Studies will be carried out in the Clinical Research Facility of the Adelaide Medical School, the University of Adelaide, by staff and students trained in the required techniques.

Each participant will be required to consume a standardised dinner meal (beef lasagne; total energy content: 602 kcal; McCain Food, Wendouree, Victoria, Australia) on the night before each visit by 7 pm. After fasting overnight for \~ 13.5 hours and refraining from exercise and alcohol during the previous 24 hours, the participant will arrive in the laboratory at 8.30 am. After confirming adherence to the study requirements, vital signs ( blood pressure, heart rate and body temperature) will be measured to establish baseline values for safety, and they will be monitored throughout the study. Then, a manometric catheter will be inserted through an anaesthetised nostril and allowed to pass through the stomach and into the duodenum by peristalsis. The infusion port will be positioned \~ 14 cm distal to the pylorus. The correct positioning of the catheter will be monitored continuously by measurement of the transmucosal potential difference in the stomach (\~ -40 mV) and the duodenum (\~ 0 mV). Once the catheter has been positioned correctly, an intravenous cannula will be placed into a forearm vein for regular blood sampling. At time = -30 minute, a baseline blood sample (6 ml), visual analogue scale (VAS) questionnaire to assess gastrointestinal (GI) symptoms, and breath sample to assess gastric emptying will be collected. Then the infusion of one of the study treatments, i.e. (i) saline, (ii) L-tryptophan, (iii) L-tryptophan + 500 mg calcium, or (iv) L-tryptophan + 1000 mg calcium, will commence and continue for 75 minutes (times = -30 to 45 minutes). At time = -1 minute, participants will consume, within 1 minute, a mixed-nutrient drink (350 mL, containing 500 kcal, 74 g carbohydrates) labelled with 100 mg 13C-acetate for measurement of gastric emptying by non-invasive breath sampling at regular intervals, for subsequent analysis of 13CO2 levels. Blood samples for the measurement of plasma glucose and hormone concentrations, and VAS questionnaires, will be collected before and after the mixed-nutrient drink. At time = 240 minute, after collecting final blood and breath samples and VAS questionnaire, the intravenous cannula will be removed and the participant will be served a light lunch, after which they will be allowed to leave the laboratory. A total of 84 mL of blood (14 sampling time points, 6 mL each) will be taken on each study day (study total of 344 mL, including screening).

Inclusion Criteria: * Healthy, lean males (only men will be included in the study to avoid the confounding effects of the menstrual cycle on gastric emptying). * BMI: 19-25 kg/m2, * Weight-stable (i.e. \<5% fluctuation) at study entry, which will be ascertained by a stable body weight in the preceding 3 months. Exclusion Criteria: * Significant GI symptoms, or history of GI disease or surgery * Current gallbladder or pancreatic disease * Cardiovascular or respiratory diseases * Any other illnesses as assessed by the investigator (including chronic illnesses not explicitly listed above) * Use of prescribed or non-prescribed medications (including vitamins and herbal supplements) which may affect energy metabolism, GI function, body weight or appetite (e.g. domperidone, cisapride, anticholinergic drugs (e.g. atropine), metoclopramide, erythromycin, hyoscine, orlistat, green tea extracts, Astragalus, St Johns Wort etc.) * Lactose intolerance/other food allergy(ies) * Individuals with low ferritin levels (\<30 ng/mL), or who have donated blood in the 12 weeks prior to taking part in the study * High performance athletes * Current intake of \> 2 standard drinks on \> 5 days per week (\>140g/week) * Current smokers of tobacco (cigarettes, cigars, pipes, sheesha, chewing, vaping etc.) * Current use of recreational drugs, e.g. marijuana * Current intake of any illicit substance * Vegetarians * Inability to tolerate nasoduodenal tube * Inability to comprehend study protocol * Restrained eaters (score \>12 on the 3-factor eating questionnaire)