Diet 3 HF-disrupted : This diet is also high in fibre but with disrupted cellular structures. It is the same as the HF-whole diet, but foods will be milled or blended to disrupt the cellular structure. However, the quality of carbohydrate present is significantly different. For example, the HF-whole and HF-disrupted diets contain The dietary plan for each diet is outlined in Table 2. E, energy, HF, high fibre; LF, low fibre. Diet preparation. For the LF-refined diet, breakfast will be prepared the night before serving but the bread will be toasted and the milk will be added to the cornflakes immediately before serving.
For the HF-whole diet, the breakfast will be prepared the day before serving and the oats will be soaked in milk from approximately the evening before. The lunch will be prepared in the hour before serving; the apples will be cut into chunks that are easier to eat and the soup will be weighed out and microwaved just before serving. The dinner will be prepared in the hour before serving. Both will be drained and weighed after cooking.
All other foods will be served cold. The carrot, beetroot and tomatoes will be cut up into smaller pieces that are easier to eat.
The evening snack will be prepared at dinner-time. For the HF-disrupted diet, the oats will be blended in advance into a flour-like consistency. The rye bread and humous will be made in advance in-house, frozen and defrosted as needed. The breakfast will be prepared the day before serving. The oranges will be blended to a smoothie. The oats will be soaked in milk from approximately the evening before and heated in the microwave immediately before serving.
The lunch will be prepared in the hour before serving. The apples will be blended to a puree. The can of soup will be blended, the required portion will be weighed out and microwaved just before serving. The beetroot and tomatoes will be blended to a smoothie. The vegetables will then be drained, blended and weighed. The potato, carrot and peas will then be re-heated in the microwave to comply with food safety standards. The snack is prepared at dinner-time and the bananas and oranges will be blended. All foods that are heated or cooked by the study team will be temperature probed before serving.
An overview of the study visit protocol is outlined in Figure 1. Participants will attend their first study visit within 12 weeks of their screening visit. There will be a minimum wash out period of 7 days between study visits. Day 1: Nasoenteric tube insertion. Participants will be asked to arrive at the Imaging Department in Charing Cross Hospital, London, having fasted overnight and having avoided intense exercise, caffeine and alcohol the day before their study visit.
Females of child-bearing age will be asked to provide a urine specimen in order to perform a pregnancy test. A custom-made nasoenteric tube Figure 2 will then inserted through the nose. Once it has passed the stomach and the first part of the small intestine, the balloon at the terminal end will be inflated to approximately 6 ml and will be used to carry the tube through the small intestine by peristalsis. The tube position will be confirmed by fluoroscopy throughout the tube insertion process.
Dilute barium sulphate may also be administered in order to help confirm the positioning. Once the tube has reached the terminal ileum, the balloon will be deflated and the tube will be restrained from additional movement for the rest of the 4-day visit.
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Participants will be provided with food and drinks during the tube insertion process. However, the food provided will not be standardised to allow for flexibility to aid tube insertion. Day 1 — Start of dietary intervention. Participants will be fed one of the three diets over the 4-day study period according to their randomisation. The dietary intervention will start with dinner on Day 1 and end with lunch on Day 4.
Meals will be provided at set times; breakfast at , lunch at , dinner at and an evening snack at During their stay, participants will be asked to eat all of the food that is provided and to not eat any other food including chewing gum. Participants will have free access to water, except for during sampling periods on Day 3 and 4 where water will be limited. Participants will be asked to collect a stool sample each time they pass stool during their inpatient stay for metabolomic and microbiological analysis.
Day 2 — Acclimatisation day. Day 2 will allow participants to acclimatise to their new diet and environment. Meals will be provided at set meal times and participants will have free access to water. The only samples collected on Day 2 will be stool samples. Day 3 — Ileal sampling. On day 3, ileal samples will be collected via the nasoenteric tube for metabolomic and microbiological analysis. The time at which each participant starts eating breakfast will be considered to be 0 min. Further ileal samples will be collected every 60 min for min.
Lunch will be served directly after the min sample with ml water. Dinner will be served at , after the min sample and participants will have free access to water for the rest of the day. Day 4 — Ileal and blood sampling. On the morning of day 4, participants will have an intravenous cannula inserted to allow for blood sampling and body composition will be assessed by bio-impedance analysis. Participants will be asked to draw a vertical line on the VAS depending on how intensely they are experiencing each feeling.
Baseline blood samples will be collected to assess blood hormones and metabolites and ileal samples will be collected via the nasoenteric tube as on Day 3. Ileal samples will be collected across both Day 3 and 4 in order to gain a full understanding of the dynamic change of the ileum environment over the study period. The time at which the participant starts eating breakfast will be considered to be 0 min. Further samples will be collected every 60 min for min.
Following the final sample at min, the cannula and nasoenteric tube will be removed and participants will be discharged.
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Stool samples. Ileal samples. In order to collect ileal samples, the aspiration channel of the tube will first be flushed with a volume of water that is equal to the deadspace leading to the lumen. The air channel will be opened and the volume of water that the tube was flushed with will be taken back using a 50 ml syringe. Once the flush has been collected, the aspiration channel will be clamped using a medical clamp and the flush will be discarded.
Up to 5 ml ileal sample will be collected at each sampling timepoint. Following sampling, the aspiration channel of the tube will be flushed with a volume of water that is equal to the deadspace and both the aspiration and air channels will be closed until the next sample. Blood samples. Serum tubes will be allowed to clot before centrifugation. Samples will be analysed upon completion of the study.
Metabolic and microbiological profiling of ileal and faecal samples will be performed using 1 H-NMR spectroscopy and 16S sequencing, respectively. Subjective appetite will be assessed using visual analogue scales VAS and breath H 2 will be measured in real-time using a handheld gastrolyser. A one-stage continuous fermentation model system will be established using ileal samples from the human study to monitor in vitro the effect of carbohydrate quality on ileal microbiota composition and their subsequent metabolites.
The system will be adapted from the three-stage colonic model described by Gibson et al. Ileal samples will be collected via a nasoenteric tube Figure 2 as previously described and anaerobically transferred into hungate tubes, which will then be used to inoculate the in vitro system. The diets from the human study will then be tested in the in vitro system. Diets will be digested by an in vitro simulation of upper gut digestion as per Mills et al. The products will be freeze-dried before their addition to the in vitro system.
Participants will be given an anonymised personal study code number once randomised, which will be used throughout the study and in the analysis of data. The study codes will be kept on departmental databases. All personal data will be stored in locked filing cabinets in the Section of Investigative Medicine, Imperial College London. Only members of the Section of Investigative Medicine will have access to these.
The Principal Investigator and the study team will have access to the final trial dataset. A formal data analysis plan will be drawn up upon completion of the study. However, assuming the data is parametric and conforms to similar studies we have conducted, we would expect our outcome measures to be analysed using a variety of statistical methods. As this is a pilot study, there are too few participants to have a data monitoring committee. In addition, the results of the study will be presented at scientific meetings and conferences, and published in relevant high-impact journals.
Research papers will be written by the study team. An anonymised dataset will be made available to the scientific community. This study will be conducted in accordance with the recommendations for physicians involved in research on human subjects adopted by the 18 th World Medical Assembly, Helsinki and later revisions. Once these changes have been approved, these modifcations will be explained to participants and implemented with their consent.
Participants will have the choice during the consent process to agree to their samples being stored and used for future analyses. Samples will be kept in the Section of Investigative Medicine and will only be used for other research purposes that have been ethically approved. Any significant adverse event as assessed by the researchers will halt the study and the research ethics committee and sponsor will be informed as per standard protocol. All adverse events will be recorded and investigators will review each adverse event as it arises. Imperial College London holds negligent harm and non-negligent harm insurance policies, which apply to this study.
The maintenance of body weight throughout life is important for metabolic homeostasis. Over the last 15 years, both epidemiological and experimental evidence have highlighted the importance of carbohydrate fermentation within the gut to appetite regulation. However, our current investigative tools are not capable of characterising the complex signalling that mediates these effects in the gut. In the current study, we will investigate how the quality of dietary carbohydrate consumed affects the form and type of carbohydrate in the ileum.
This will allow us to gain a deeper understanding of the composition of the digesta reaching the colon from the ileum following such diets.
In addition, we will determine how this subsequently affects subjective appetite measures and gut hormone secretion. We will use an in vitro model, inoculated with ileal fluid, to provide a deeper understanding of how ileum contents, following diets differing in carbohydrate quality, affects the colonic gut microbiota and the production of SCFAs in the colon.
This is important in identifying how dietary carbohydrate drives colonic gut hormone release and is critical in understanding the relationship between the colon and the maintenance of energy homeostasis. This knowledge may facilitate the development of food products designed for body weight maintenance. The first study visit was completed in October Eight participants have successfully completed the study so far. Figshare: A study protocol for a randomised crossover study evaluating the effect of diets differing in carbohydrate quality on ileal content and appetite regulation in healthy humans.
Participant Information Sheet - Version 5 — Data are available under the terms of the Creative Commons Attribution 4. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. They can now be found at the top of the panel on the right, linked from the box entitled Open Peer Review. Choose the reviewer report you wish to read and click the 'read' link. You can also read all the peer review reports by downloading the PDF.
Is the rationale for, and objectives of, the study clearly described? Are sufficient details of the methods provided to allow replication by others? Are the datasets clearly presented in a useable and accessible format? Competing Interests: No competing interests were disclosed. Alongside their report, reviewers assign a status to the article:. All Comments 0. Provide sufficient details of any financial or non-financial competing interests to enable users to assess whether your comments might lead a reasonable person to question your impartiality.
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Close Copy Citation Details. A study protocol for a randomised crossover study evaluating the effect of diets differing in carbohydrate quality on ileal content and appetite regulation in healthy humans [version 1; peer review: 1 approved with reservations]. Introduction: A major component of the digesta reaching the colon from the distal ileum is carbohydrate. This carbohydrate is subject to microbial fermentation and can radically change bacterial populations in the colon and the metabolites they produce, particularly short-chain fatty acids SCFA. However, very little is currently known about the forms and levels of carbohydrate in the ileum and the composition of the ileal microbiota in humans.
Most of our current understanding of carbohydrate that is not absorbed by the small intestine comes from ileostomy models, which may not reflect the physiology of an intact gastrointestinal tract. Methods: We will investigate how ileal content changes depending on diet using a randomised crossover study in healthy humans. Participants will be inpatients at the research facility for three separate 4-day visits.
During each visit, participants will consume one of three diets, which differ in carbohydrate quality: 1 low-fibre refined diet; 2 high-fibre diet with intact cellular structures; 3 high-fibre diet where the cellular structures have been disrupted e. On day 1, a nasoenteric tube will be placed into the distal ileum and its position confirmed under fluoroscopy. Ileal samples will be collected via the nasoenteric tube and metabolically profiled, which will determine the amount and type of carbohydrate present, and the composition of the ileal microbiota will be measured.
Blood samples will be collected to assess circulating hormones and metabolites. Stool samples will be collected to assess faecal microbiota composition. Subjective appetite measures will be collected using visual analogue scales. Breath hydrogen will be measured in real-time as a marker of intestinal fermentation. Finally, an in vitro continuous fermentation model will be inoculated with ileal fluid in order to understand the shift in microbial composition and SCFA produced in the colon following the different diets. Corresponding Author s.
Introduction Obesity Obesity is a chronic health problem that has reached epidemic proportions globally 1. The gastrointestinal tract The GI tract is the largest endocrine organ in the body and is responsible for digesting and absorbing dietary components. Dietary fibre There is an increasing amount of evidence to suggest that consumption of dietary fibre is beneficial to human health.
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Importance of the cellular structure of foods The cellular structure of foods can also be an important determinant of the subsequent impact of a food on appetite. Study objectives Hypothesis We hypothesise that the consumption of minimally-processed high-fibre foods will result in the presence of a greater number of intact cellular structures in the ileum, which will subsequently be fermented in the colon resulting in the production of SCFAs and greater release of PYY and GLP Objectives There are a number of objectives to the present study: to identify the impact of dietary carbohydrate on 1 the forms and levels of carbohydrates in, and 2 the microbiological profile of, the ileum, and to 3 to determine whether the carbohydrate content of the ileum relates to appetite responses and gut hormone release.
Primary and secondary outcome measures The co-primary outcome measures for this study are: 1. Metabolic profiling of ileal samples 2. Microbiological profiling of ileal samples. Microscopy of ileal samples for plant structures, cellular structures and starch granules 2. Metabolic and hormonal profiling of blood samples 3. Metabolic and microbiological profiling of faecal samples 4. Subjective appetite measurements 5.
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Breath H 2 concentrations 6. Health screening Healthy humans will be recruited using existing healthy volunteer databases e. Study population Healthy humans aged between 18—65 years inclusive with a body mass index BMI of Randomisation Following the health screening, eligible participants will be randomised into the study. Sample size We aim to recruit 15 subjects. Dietary intervention Dietary calculations. Table 1. BMR, basal metabolic rate. PAL, physical activity level.
TEE, total energy expenditure. Table 2. Dietary plan for intervention diets. Study visit protocol An overview of the study visit protocol is outlined in Figure 1.
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