Impact of non-digestible carbohydrates on biomarkers of GI health: a human intervention study

What we eat affects the health of all parts of the body including the gut. Symptoms, disorders and diseases of the large bowel are major causes of anxiety, visits to general practitioners and medical treatment. In particular, the large bowel is one of the commonest sites for cancer development. Large scale observational studies of dietary practices and associated incidence of cancer provide very strong evidence that dietary choices and nutritional status (e.g. obesity) influence risk of cancer in the large bowel (colorectal cancer; CRC). Such evidence is very encouraging because it suggests that many cases of bowel cancer could be avoided by appropriate dietary choices and/or by the development of novel foods or dietary agents with anti-cancer properties. Identification of beneficial dietary agents requires intervention studies i.e. carefully controlled experiments in which volunteers are given known amounts of the test agent. For both practical and ethical reasons, it is seldom appropriate to use the development of cancer as the endpoint in such experiments and there is a need to use surrogate outcome measures. This is analogous to using blood pressure or blood cholesterol concentration as surrogate outcome measures (or biomarkers) in studies of diet and heart disease risk. Unfortunately, in the area of diet and gut health, progress is hampered by the lack of robust biomarkers of CRC risk for use as surrogate endpoints. To address this gap, we have developed a number of novel biomarkers of diet-related CRC risk which can be measured in small samples (biopsies) taken during clinical examination of the large bowel. We have shown that these biomarkers can be detected BEFORE the development of CRC and so may be a useful tool to identify those at higher risk of the disease. In our on-going work, we are investigating relationships between what people eat (and other aspects of lifestyle) and these biomarkers in a cross-sectional study. The next logical step is to test how the most promising biomarkers respond to dietary intervention to determine how useful they will be as biomarkers of gut health. We will do this by carrying out a carefully controlled experiment in which volunteers will be given food supplements of resistant starch (RS) and polydextrose (PD) - both are carbohydrates with special properties. They are widely used food ingredients for which there is already evidence that they may help reduce CRC risk. Both food agents show bioactivity in the large bowel where they appear to have beneficial effects on gut physiology and immune function including anti-inflammatory effects. In our human intervention study, 70 healthy volunteers will be given RS and/or PD or another carbohydrate with no effects on the large bowel (a placebo) for 7 weeks. We will collect tiny pinch samples of the lining of the gut (mucosal biopsies) before and after the intervention for biomarker measurement. These biomarker studies will include measurement of genes which are known to be involved in the early stages of the development of cancer and which may be modifiable by changing diet. Dietary components such as RS and PD may influence how genes are switched on and off by affecting regulatory marks on DNA known as DNA methylation so we will quantify DNA methylation for a panel of key cancer-related genes. We will also measure the rates at which cells are been produced (cell proliferation) in the gut lining because faster cell proliferation appears to indicate higher CRC risk. In addition we will collect blood and stool for measurements of markers of inflammation. There is growing evidence that poor diet and obesity can lead to the development of a chronic inflammatory state and that this may predispose to CRC. Through their fermentation by bacteria in the large bowel, RS and PD may help reduce inflammation and so protect gut health.

Epidemiological evidence shows that dietary choices influence risk of colorectal cancer (CRC). Identification of beneficial dietary agents requires intervention studies but progress in this area is hampered by the lack of robust risk biomarkers for use as surrogate endpoints. To address this gap, we have developed a number of novel biomarkers of diet-related CRC risk measured in colo-rectal mucosal biopsies. These biomarkers include protein biomarkers, DNA methylation markers and inflammation markers. In on-going work, we are investigating relationships between dietary exposure and these biomarkers in a cross-sectional study. The next logical step is to test responses of a panel of the most promising biomarkers to dietary intervention to determine their utility as biomarkers of GI health and potential in future studies. We propose to use resistant starch (RS) and polydextrose (PD) as our model non-digestible carbohydrate (NDC) intervention agents. Both agents show bioactivity in the large bowel where they are fermented (to a greater or lesser extent) producing short-chain fatty acids (SCFA) including butyrate. Butyrate has beneficial effects on gut physiology and immune function including anti-inflammatory effects. It is probable that NDC exert anti-neoplastic effects via butyrate. We will carry out a human intervention study with healthy volunteers using a 2*2 factorial design. 70 volunteers will consume RS and/or PD or placebo for 50d. Bioactivity of the NDC in the large bowel will be monitored by measurement of SCFA in stool and urine. We will collect colorectal mucosal biopsies before and after the intervention for biomarker measurement including methylation of tumour-related genes, crypt cell kinetics, expression of cell cycle regulatory genes CDK4 and GADD45A and protein biomarkers (CK8 a diet-related biomarker of CRC risk). In addition we will collect blood and stool for measurements of inflammatory markers viz. CRP and calprotectin respectively.

1. Who will benefit from this research? This project is designed to enhance understanding of the links between diet and function and health of the gut in the context of the BBSRC DRINC initiative. We anticipate that this research will have a wide range of beneficiaries (in addition to the academic community) including: Industrial members of the BBSRC DRINC initiative; The wider food industry including those developing novel foods or food ingredients aimed at improved gut health and those advising consumers; Regulators with responsibilities in the area of food and health e.g. the European Food Safety Authority and the UK's Advisory Committee on Novel Foods and Processes; Manufacturers of diagnostic and other tests for gut function and bowel cancer risk; The general public. 2. How will they benefit from this research? Industrial members of the BBSRC DRINC initiative will have access to emerging data and new ideas in respect of both biomarkers of gut function and bowel cancer risk and the impact of diet on these processes in advance of publication. Outcomes from this research will provide the wider food industry with more robust outcome measures and tools for R&D in the area of functional foods for improved gut health. The lack of reliable measures of gut health is a major impediment for regulators assessing requests from industry for authorisation of new foods to be placed in the European market and for claims about new or existing food products. This project will provide objective outcome measures which can be used directly in humans (the most critical type of evidence necessary for regulators). The testing of novel biomarkers within this project will provide manufacturers of diagnostic and other tests with objective evidence of the utility of putative biomarkers in a well-designed human intervention study. Often such manufacturers have excellent laboratory facilities but restricted access to studies in humans. The general public are expected to benefit from this project in at least 3 ways i) through provision of objective information about the effects of two widely used food components on gut function and health; ii) by enhancing the quality and robustness of evidence which will be required to make claims about new (or existing) food products and iii) encouraging the food industry to produce additional food products or ingredients which are aimed to improve gut function and to reduce bowel cancer risk. 3. What will be done to ensure that they have the opportunity to benefit from this research? We will work towards timely dissemination of the outcomes from the research project via the conventional route of academic publications using open access journals where possible. This will ensure widespread access by all stakeholders to the project outcomes following the important quality assurance step of peer review. In addition, we will present findings from the project on an on-going basis via conference presentations and we will target conferences and workshops attended by diverse user groups including industry and regulators. As an integral part of the DRINC initiative, we will participate in the twice yearly dissemination events which will provide opportunities for one-to-one meetings with food industry representatives to discuss emerging findings and to identify opportunities for further developments. We do not anticipate that this project will lead to exploitable intellectual property but we will take appropriate action should commercialisation opportunities be identified. The Institute for Ageing and Health (Newcastle University) has a Technology Transfer Officer to support such actions, and the Regional Development Agency has established a Centre for Excellence in the Life Sciences which is developing a major programme of activity aimed at exploitation and dissemination of research. Dissemination to the public and other stakeholders will be via the University and IFR websites