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The Purine and Pyrimidine Metabolism in Lactating Dairy Cows

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The Purine and Pyrimidine Metabolism in Lactating Dairy Cows THE PURINE AND PYRIMIDINE METABOLISM IN LACTATING DAIRY COWS CHARLOTTE STENTOFT NIELSEN Ph.D. THESIS ∙ SCIENCE AND TECHNOLOGY ∙ 2014 Aarhus University Faculty of Science and Technology Department of Animal Science Blichers Allé 20 P.O. Box 50 DK-8830 Tjele Supervisors and Ph.D. assessment committee Supervisors Head of Research Unit, Ph.D., Mogens Vestergaard Aarhus University, Faculty of Science and Technology, Department of Animal Science, Denmark Senior Scientist, Ph.D., Søren Krogh Jensen Aarhus University, Faculty of Science and Technology, Department of Animal Science, Denmark Assistant professor, Ph.D., Mogens Larsen Aarhus University, Faculty of Science and Technology, Department of Animal Science, Denmark Senior Scientist, Ph.D., Torben Larsen Aarhus University, Faculty of Science and Technology, Department of Animal Science, Denmark Consultant / Project leader, Ph.D., Niels Bastian Kristensen Knowledge Centre for Agriculture, Aarhus, Denmark Ph.D. assessment committee Senior Scientist, Ph.D., Stig Purup (Chair) Aarhus University, Faculty of Science and Technology, Department of Animal Science, Denmark Associate Professor, Ph.D., Kristian Fog Nielsen Technical University of Denmark, Department of Systems Biology, Denmark Professor, Ph.D., Richard Dewhurst Scotland’s Rural College, Beef and Sheep Research Centre, Midlothian, EH25 9RG, Scotland, United Kingdom I Preface The nitrogen efficiency of dairy cows is generally low due to the inherent characteristics of the ru- minant digestive system and to the feedstuffs and rations used. Any attempt to optimize the diet is fundamental for improving nitrogen efficiency and utilization. The search for quantitative im- provements in nitrogen utilization has mainly focused on feed nitrogen and ration formulation. However, a better understanding of the quantitative absorption and intermediary metabolism of the nitrogenous purine and pyrimidine metabolites, the main constituents of nucleic acids, could most likely contribute to uncover new ways to improve dairy cow nitrogen utilization. So far, the possi- ble significance of microbial nucleic acids in the nutritional physiology of ruminants has sparsely been investigated, regardless of the fact that they correspond to approximately 20% of the total mi- crobial nitrogen supply. One reason for not including the nucleic acid metabolism in the search for improved nitrogen utilization can partly be ascribed to the lack of reliable methods for quantitative measurements of purine and pyrimidine metabolites in bovine blood plasma. The aim of the Ph.D. study was to improve our knowledge about the quantitative absorption and intermediary metabolism of purine and pyrimidine metabolites in lactating dairy cows. Therefore, a high performance liquid chromatography-based technique coupled to electrospray ionization tan- dem mass spectrometry, to quantify key purine and pyrimidine metabolites in plasma, was devel- oped and combined with individual matrix-matched calibration standards and isotopically labelled reference components. Results from the development and employment of this technique in experi- ments with lactating dairy cows are presented herein. Valuable insight into the mechanisms of the purine and pyrimidine metabolism was obtained, which adds significantly to the present knowledge of the nitrogen metabolism in dairy cows. In addition, these results may in the future be used to im- prove nitrogen utilization through reformation of feeding plans and strategies. The PhD program and the experimental work were carried out at the Department of Animal Sci- ence, Faculty of Science and Technology, Aarhus University from February 1st 2011 until Novem- ber 30th 2014. There has been collaboration with Dr. Jon M. Moorby, Institute of Biological, Envi- ronmental and Rural Sciences, University of Aberystwyth (UK) and Professor Christopher K. Reynolds, School of Agriculture, Policy and Development, University of Reading (UK). The Ph.D. scholarship was financed by the Faculty of Science and Technology and the Danish Milk Levy Board, c/o Food and Agriculture, Aarhus N, Denmark. Funding for the cow animal experiments were partly provided by the Commission of the European Communities (Brussels, Belgium; Rednex project FP7, KBBE-2007-1) and the Department of Animal Science, Aarhus University. Foulum, November 2014, Charlotte Stentoft Nielsen II Acknowledgements I would like to express my sincere gratitude towards my main supervisors Mogens Vestergaard, Søren Krogh Jensen and Mogens Larsen for their competent and encouraging supervision, construc- tive criticism on my work, and continuous collaboration during this project. The hard work and ef- fort would not have been as easy to manage without their invaluable support and not half as exhila- rating without our inspiring discussions. I would also like to thank Niels Bastian Kristensen for ini- tiating and getting the project funded and for his constructive support throughout the project. I would also like to thank Jon Moorby for professional and organizational support during my stay at IBERS (UK) and his warm and kind manner towards me. Also, thanks goes to Felicity Crotty and Alejandro Belanche Gracia for making my stays in Wales more than just work. Warm thoughts also go to Chris Reynolds, Cassie Barratt and Les Compton at the University of Reading. Our col- laboration on manuscript III has been invaluable to this project. My deepest thanks go to Peter Løvendahl for introducing me to the vast world of experimental sta- tistics and SAS. Without his sustained technical assistance, this project could not have been con- ducted. For advice concerning handling of milk samples and for his time analysing milk samples, a special thanks go to Torben Larsen and his technical staff. For skilled assistance and essential advice during the experimental work I wish to give a special thanks to Lis Sidelmann, Birgit Hørdum Løth and Anne Krustrup. I really can never thank this team of technicians enough, none of this research would have seen the day without their assistance. Also, special thanks go to members of the Department of Animal Science – Integrative Physiology group; Adam Storm and Bettina Røjen and especially Vibeke Bjerre-Harpøth for indispensable sparring during the entire project. The atmosphere in the office, in the laboratory, in the barn, at the halls, and at breaks has been pleasant and fun and their everyday good spirits and cheers have made many a bad day into a good one. Warm thanks go to my family and friends for their indefinite love, for their support and for their interest in my work. A special thanks to my parents for their faith in me and continuous support. Finally, thank you Jakob, Mia and Mads for supporting me and bearing with my lack of presence in the final hours. You are my love and my life. III Contents Supervisors and Ph.D. assessment committee ________________________________________________________I Preface _______________________________________________________________________________________ II Acknowledgements ____________________________________________________________________________ III Contents _____________________________________________________________________________________ VI Summary _____________________________________________________________________________________ 1 Sammendrag (summary in Danish) _______________________________________________________________ 3 List of scientific papers and manuscripts included in the Ph.D. thesis ___________________________________ 5 List of other scientific contributions from the Ph.D. program __________________________________________ 6 Abbreviations _________________________________________________________________________________ 7 1. Introduction _______________________________________________________________________________ 11 2. Background ________________________________________________________________________________ 14 2.1 Nitrogen metabolism in dairy cattle ___________________________________________________________ 14 2.2 The nucleic acid metabolism ________________________________________________________________ 16 2.2.1 Bases, nucleosides, nucleotides, nucleic acids, and DNA/RNA __________________________________ 16 2.2.2 Purine and pyrimidine nucleotide biosynthesis, regulation, salvage, and catabolism __________________ 17 2.3 The purine and pyrimidine metabolism in dairy cattle _____________________________________________ 20 2.3.1 Degradation of dietary nucleic acids and re-synthesis of microbial nucleic acids ____________________ 20 2.3.2 Degradation of microbial nucleic acids in the small intestine ____________________________________ 21 2.3.3 Absorption and intermediary metabolism of purine and pyrimidine metabolites _____________________ 22 2.3.4 Endogenous purine and pyrimidine metabolites ______________________________________________ 23 2.3.5 Renal clearance of purine and pyrimidine metabolites _________________________________________ 23 3. Hypotheses and objectives ____________________________________________________________________ 25 4. Methods ___________________________________________________________________________________ 27 4.1 The multicatheterized cow model ____________________________________________________________ 27 4.1.1 Blood plasma flow ____________________________________________________________________ 28 4.1.2 Net flux _____________________________________________________________________________ 29 4.1.3 Animals and experimental designs ________________________________________________________
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