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

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. 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.