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Small, Alexandra C. (2011) Complex Metaprobe Production and Characterisation for Metabolic Studies in Relation to Bowel Health

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Small, Alexandra C. (2011) Complex Metaprobe Production and Characterisation for Metabolic Studies in Relation to Bowel Health Small, Alexandra C. (2011) Complex metaprobe production and characterisation for metabolic studies in relation to bowel health. MSc(R) thesis. http://theses.gla.ac.uk/2446/ Copyright and moral rights for this thesis are retained by the author A copy can be downloaded for personal non-commercial research or study, without prior permission or charge This thesis cannot be reproduced or quoted extensively from without first obtaining permission in writing from the Author The content must not be changed in any way or sold commercially in any format or medium without the formal permission of the Author When referring to this work, full bibliographic details including the author, title, awarding institution and date of the thesis must be given Glasgow Theses Service http://theses.gla.ac.uk/ [email protected] Complex Metaprobe Production and Characterisation for Metabolic Studies in Relation to Bowel Health ALEXANDRA C. SMALL BSc (Hons) Thesis submitted for MSc (Med Sci) To College of Medical, Veterinary & Life Sciences University of Glasgow September 2010 Research Conducted at Stable Isotope Biochemistry Laboratory, S.U.E.R.C. University of Glasgow Colon Cancer Cells Wellcome Trust Medical Photographic Library. This Thesis is dedicated to my Husband Keith and my children, Laurence & Juliet 2 Barley grain sown, labelled, harvested & photographed by the author “More than 40% of some cancers can be prevented by a mixture of healthy eating and exercise. Cutting down on red meat and alcohol whilst increasing intake of fruit and vegetables, could prevent thousands of cancer cases each year.” A quotation from a report entitled “Policy and Action for Cancer Prevention” funded by World Cancer Research Fund (2009) 3 Abstract Complex Metaprobe Production and Characterisation for Metabolic Studies in Relation to Bowel Health Introduction Colorectal cancer is the third most common cancer in the UK and the second most common cause of cancer mortality. There is increasing evidence that the presence of a systemic inflammatory response plays an important role in predicting survival in patients with colorectal cancer. The Glasgow Prognostic Score includes a measure of systemic inflammatory response and has been used successfully as a predictor of survival rate in cancer patients. Butyric acid is one of the short chain fatty acids (SCFA) produced by colonic bacteria through fermentation of dietary non-digestible carbohydrate (NDC). This simple organic acid is known to have anti-inflammatory and possibly direct anti-neoplastic effects. The inaccessibility of the human colon means that little is known of the in vivo rate of butyrate production, its metabolic fate or the foodstuffs that may maximise its production. Butyrate may be produced in greater quantities from other SCFA such as acetate and lactate rather than by direct production by saccharolytic bacteria. Specialist organisms may be responsible for producing butyrate through conversion of other SCFA which are produced by fermentation in the colon. The distal site of butyrate production may be a very important feature and the implication that slowly fermentable NDC, such as resistant starch, may be significant. Previous Work The European Union project EUROSTARCH (www.eurostarch.org) investigated the way in which different starchy foods are digested and metabolised within the body. The results of this project have informed the development of low GI foods. Results have also increased our knowledge of NDC fermentation and promise to lead to improvements in human bowel health. We pioneered the measurement of starch digestion and fermentation during this project. 4 What are Metaprobes? The term metaprobe is used to describe stable isotope labelled tracers. Complex metaprobes are produced by biological synthesis e.g. 13 the introduction of C enriched CO2 gas during the production of crops. Simple metaprobes are produced by chemical synthesis e.g. urea. We can produce complex metaprobes by labelling staple foods such as cereals using the stable 13 13 isotope C incorporated from enriched CO2 during anthesis of the crops. Complex metaprobes are unique tools with which to study human metabolism in health and disease and they can be used in several ways. The labelled cereals allow in vivo measurement of butyrate production from dietary NDC and to permit its production to be monitored. Hypothesis 30% of cancer deaths in the UK are bowel related. Butyric acid produced naturally through fermentation of dietary carbohydrates, is a potent and natural anti-inflammatory agent which can protect against cancer. We urgently need to develop methods to quantify butyrate production in vivo. Aims Three aims are recognised: optimising the preparation of complex metaprobes of barley and wheat grain; optimising yield and 13C-enrichment of whole grain and its starch and protein fractions; using complex metaprobes to compare SCFA production in vivo. Plan of Work This project will include a review of complex metaprobe production for human nutrition and health studies in relation to bowel health. The practical work will include production and characterisation of complex metaprobes (13C-labelled cereals) and their application to study butyrate production in a pilot study on volunteer human subjects. I have successfully grown cereal crops with different varieties of barley and wheat in glasshouse conditions. During the anthesis phase, the plants were placed in growth cabinets at 18oC with 18 hours of light in every 24 hours. The 13 plants were labelled using C enriched CO2 gas and are incubated for a further 24 hours and returned to the glasshouse. Examples of the harvest are Wheat var Paragon produced 1.7 Kg labelled grain at 0.278 ape 13C enrichment and Barley var Cellar produced 3.4 Kg labelled grain at 0.198 ape 13C enrichment. Some of the 13C labelled grain has already been used for human nutrition studies. The 5 13C-enrichment of starch glucose has been measured by liquid chromatography isotope ratio mass spectrometry (LC-IRMS) analysis. These cereal grains can be incorporated into common foodstuffs for in vivo studies of the production of butyrate and other SCFA. Volunteers were fed two meals made from 13C-enriched barley grain: porridge and whole grain barley. Whole grains being rich in resistant starch. Samples of urine will be used to measure butyrate production. Breath samples will be used to measure oxidation of the 13C-enriched barley, which is a global measure of digestion and fermentation. Laboratory procedures such as ultrafiltration and solid phase extraction will be applied to urine samples. These sample preparation procedures will be followed by SCFA analysis by GC-IRMS. Future Direction Future clinical trials will be considered with Prof Donald C McMillan, University Dept of Surgery, Glasgow Royal Infirmary as a means of identifying the role of butyrate in colonic cancer. The current screening process recognises three phases: early stage diagnosis; polyp development; diagnosis and treatment of advance stage colonic cancer. We wish to study the role of butyrate and its production using our newly developed complex metaprobe tools. 6 Table of Content Chapter 1 Literature Review 1 Introduction………………………………….........................................15 1.1 Carcinogensis & Colorectal Cancer …………………………………………………..16 1.1.2 Carcinogenic Stages …………………………………………………………………………. 16 1.1.3 C-Reactive Protein ……………………………………………………………………………17 1.2 Diet & Lifestyle ………………………………………………………………………………….19 1.2.1 A Healthy Diet …………………………………………………………………………………..20 1.3 Digestive Enzymes & Gastrointestinal Tract …………………………………….23 1.3.1 Mouth & Saliva …………………………………………………………………………………..24 1.3.2 Stomach ………………………………………………………………………………………….… 25 1.3.3 Small Intestine …………………………………………………………………………………. 25 1.3.4 Large Intestine …………………………………………………………………………………. 27 1.4 Dietary Carbohydrates …………………………………………………………………….. 27 1.4.1 Polysaccharides ……………………………………………………………………………….. 28 1.4.2 Non-Starch Polysaccharides ……………………………………………………………… 29 1.4.3 glucans ………………………………………………………………………………………....29 1.4.4 Oligosaccharides …………………………………………………………………………..... 30 1.4.5 Fermentation of Micronutrients ………………………………………………....... 31 1.4.6. Carbohydrate & Protein …………………………………………………………….......31 1.5 Short Chain Fatty Acids…………………………………………………………………..… 33 1.5.1 Butyrate ……………………………………………………………………………………………. 35 1.5.2 Biological Effects of SCFA ………………………………………………………………… 37 1.5.3 Measurement of SCFA Production ……………………………………………………. 39 1.6 Stable Isotopes as Tracers ………………………………………………………………..41 1.7 Mass Spectrometry ………………………………………………………………………….…43 1.7.1 Metaprobes: Complex & Simple ………………………………………………………. 44 1.8 Staple Cereals as Complex Metaprobes …………………………………………….46 1.8.1 Barley ……………………………………………………………………………………………….. 46 1.8.2 Wheat ………………………………………………………………………………………………..47 1.8.3 Varieties of Wheat ………………….. …………………………………………………….. 49 1.8.4 Phytochemicals ………………………………………………………………………………...51 1.9 Plant Morphology & Growth Cycle ………………………………………………..…52 1.9.1 Grain Filling ……………………………………………………………………………………...53 1.9.2 Pollination ………………………………………………………………………………………….53 1.9.3 Photosynthesis …………………………………………………………………………………..56 1.10 Production of Complex Metaprobes ………………………………………………….57 Chapter 2 Materials and Methods 2 Wheat & Barley & Hay Crops ……………………………………………………………65 2.1 Pilot Study ………………………………………………………………………………………… 65 13 2.1.1 Initial Preparation for Labelling Plants with CO2...................... 65 2.1.2 Labelling Trials: Sowing ………………………………………………………………..….67 2.1.3 Labelling of
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