Copyright is owned by the Author of the thesis. Permission is given for a copy to be downloaded by an individual for the purpose of research and private study only. The thesis may not be reproduced elsewhere without the permission of the Author. Biomarker development to assess bone health A thesis presented in partial fulfilment of the requirements for the degree of Doctor of Philosophy in Nutritional Science at Massey University, Palmerston North, New Zealand. Diana Leticia Cabrera Amaro 2019 Abstract Postmenopausal women experience an accelerated bone loss with increased fracture risk caused by oestrogen deficiency. Biomarkers of bone turnover assess the changes of bone metabolism in postmenopausal women; however, prediction of bone loss with these common biomarkers cannot be achieved because bone biomarkers might not reflect the bone microenvironment status. Thus, there is a need for discovering new bone biomarkers that can efficiently predict bone loss in postmenopausal women. Previous studies suggest that the ovariectomised sheep in combination with injected glucocorticoids may be a reliable model to evaluate the biological response to oestrogen withdrawal as well as the bone remodelling process. The purpose of this research programme was to test the following hypotheses: 1) ovariectomising sheep in combination with monthly injections of glucocorticoids would result in decreased bone mineral density (BMD) and increased plasma bone remodelling marker concentration over a shorter period of time; 2) the plasma metabolome and lipidome of ovariectomised sheep would be different, and the biochemical changes in plasma and bone remodelling would be associated with bone loss; 3) and finally, there would also be a difference in the plasma metabolome and lipidome of Singaporean–Chinese postmenopausal women according to their bone mineral density status. The hypotheses were evaluated using the OVX sheep in combination with glucocorticoids as a large animal model for postmenopausal osteoporosis, as well as comprehensive LC– MS-based metabolomics as a diagnostic approach to identify lipids and metabolites associated with bone loss in postmenopausal women. i The OVX sheep model was successfully validated over five months of this study period, and bone mineral density was decreased and bone biomarkers increased after five months. Then, plasma samples from this animal model were analysed to measure the metabolome and lipidome of the OVX sheep. In the OVX sheep, metabolite and lipid alterations associated with bone loss included methionine, glutaric acid, tryptophan, 5-methoxytryptophan, CL and CerP, and these correlated with OC, CTx-1, femoral BMD and lumbar spine BMDThese studies revealed dynamic changes of the metabolite and lipid profiles from affected sheep, such as perturbation in multiple amino acids, metabolites, and fatty acid β-oxidation. Additionally, the results from the Singaporean–Chinese postmenopausal women showed alterations in proline, threonine, methionine, 4- aminobutyric acid, aminopropionitrile, phosphatidic acid, diacylglycerol, CerP and phosphatidylinositol correlated with low femoral neck BMD. Methionine and CerP were the common compounds altered in OVX sheep and SC women with low BMD when compared with healthy groups. Those compounds, which are known to be involved in bone remodelling, have the potential for studying early bone loss in postmenopausal women, where identifying new bone-specific biomarkers may aid in clarifying novel molecular mechanisms of bone loss. ii Acknowledgements This PhD would have not been possible without the collaboration, dedication, and friendship of many people that contributed in some way towards this project. I would like to thank my Massey University supervisors, Marlena Kruger and Fran Wolber, and my Agresearch supervisors, Karl Fraser and Nicole Roy, for their guidance and being so generous with their time and sharing their knowledge and expertise. I am lucky to have got an opportunity to work with them. I particularly appreciated their always kept me motivated, supported my ideas and made me question my knowledge. During all these years their kept maintaining their doors open and smiling regardless of how many interruptions were. I would also like to thank the Ministry of Business, Innovation and Employment, New Zealand; Singapore-New Zealand Foods for Health Grant (MAUX1309) and The Mexican National Council for Science and Technology (Scholarship No. 383233) for financially supporting my project and my PhD scholarship. My time living in New Zealand has been one of the greatest experience in my life and I would also like to thank to all the people that made me feel at home. In these years I have crossed paths with wonderful friends with who I shared many laughs and received their support, thank you for expanding my world view. I would like also to thank to all family and friends back home. I would specially like to thank to my father Armando, my mother Leticia and my brother Armando for all the love and support they have given me, and for encouraging me to pursuit all my dreams. iii Table of Contents Abstract ......................................................................................................................... i Acknowledgements ....................................................................................................... iii List of figures .............................................................................................................. viii List of tables ................................................................................................................. ix Structure of the thesis .................................................................................................. xi Abbreviations .............................................................................................................. xiii Chapter 1 ...................................................................................................................... 1 Thesis Introduction ........................................................................................................ 1 1.1 Thesis background .............................................................................................. 2 1.2 Initial research questions ..................................................................................... 3 1.3 Thesis introduction .............................................................................................. 6 Chapter 2 ...................................................................................................................... 7 Literature review ........................................................................................................... 7 2.1 Bone .................................................................................................................... 8 2.1.1 Bone structure ............................................................................................... 8 2.1.2 Bone cells ................................................................................................... 10 2.1.3 Bone remodelling ........................................................................................ 15 2.2 Pathogenesis of osteoporosis ............................................................................ 22 2.2.1 Current assessments of osteoporosis ......................................................... 23 2.2.2 Biochemical markers of bone turnover ........................................................ 25 2.3 The application of metabolomics to the study of postmenopausal osteoporosis 32 2.3.1 Metabolomics .............................................................................................. 32 2.3.2 The workflow for untargeted metabolomics analysis ................................... 35 2.3.3 Metabolomics approaches in bone health ....................................................... 47 2.4 Problem statement ............................................................................................ 56 2.5 Research questions ........................................................................................... 57 2.6 Research hypothesis ......................................................................................... 57 Chapter 3 .................................................................................................................... 59 Glucocorticoids affect bone mineral density and bone remodelling in ovariectomised sheep: a pilot study ..................................................................................................... 59 Abstract ................................................................................................................... 61 3.1 Introduction........................................................................................................ 62 3.2 Materials and methods ...................................................................................... 64 3.2.1 Experimental animals .................................................................................. 64 3.2.2 Blood sampling ........................................................................................... 67 iv 3.2.3 Euthanasia and dissection .......................................................................... 67 3.2.4 Analysis of biochemical markers of bone .................................................... 68 3.2.5 Analysis of bone parameters ......................................................................