In this pilot study we describe LC–MS metabolomics and lipidomics approaches to identify changes in the plasma metabolome and lipidome of OVX and OVXG sheep as a large animal model for postmenopausal osteoporosis. This is the first longitudinal study that presents results on the plasma metabolite and lipid profiles of OVX sheep treated with glucocorticoids as an animal model of osteoporosis. The results obtained demonstrate metabolite and lipid changes in response to hormonal interventions over time: either OVX alone or OVX in combination with glucocorticoid treatments. Results of both the short-term and long-term statistical analysis approach from the linear mixed models highlights mass spectral features that change over the two- or five-month study period.
Effect of OVX and OVXG on the plasma Metabolome of sheep
Osteoporosis in this model was associated with early changes in the plasma metabolite profile, including amino acids and other metabolites. Overall, circulating metabolites were elevated in plasma after OVX in sheep. In this study, compared to the OVX animals, OVXG2 animals could reverse the impacts of glucocorticoids on the plasma metabolites at month four and month five. These results suggest that short-term administration of glucocorticoid might be reversible as the relative intensities of the metabolites in the OVXG2 group showed a slight return on the abundance levels similar to those ones observed in the OVX group. Further, in the OVXG5 group longer administration of glucocorticoid treatment altered their metabolite profile. The OVX sheep is an established model for osteoporosis [
38]. The changes observed in the metabolite profile suggested that OVX alone or when combined with glucocorticoid treatments affects plasma metabolites during the progression of postmenopausal osteoporosis in sheep. The dynamic change observed in the plasma metabolite profile of the OVX sheep may reflect systemic changes related to the increased bone turnover observed in estrogen deficiency models, where an imbalance within the bone remodelling process suggests enhanced bone resorption and a reduced bone formation [
32]. In addition, due to uncoupling of the bone turnover cycle in the presence of low estrogen, bone resorption increases at basic multicellular units of bone, and the rate of bone loss is greater than the formation of new bone [
39,
40]. In the glucocorticoid-induced osteoporosis model, bone resorption markers have been associated with a decreased BMD as a result of an excessive bone resorption rate and reduced osteoblast activity [
41].
Amino acids are not only the building blocks of proteins but are also substrates for secondary metabolism and are involved in the process of cell signalling and signal transduction of key metabolic pathways. Amino acids enhance immunity by upregulating pro-inflammatory and downregulating anti-inflammatory cytokines [
42]. Menopause is associated with the risk of developing several chronic diseases including cardiovascular disease, diabetes and osteoporosis, among others. Osteoporosis is accompanied by oxidative stress and inflammation, and changes of amino acid levels may indicate which key metabolic pathways may be altered in postmenopausal women [
43‐
46].
Although this is the first study that reports changes in the plasma metabolite profile of OVX and OVXG sheep, previous metabolomic analyses have shown altered metabolism in OVX rats, another common animal model for postmenopausal osteoporosis. A previous study using nuclear magnetic resonance reported that six months after OVX, serum levels of alanine, asparagine, glutamine, isoleucine, leucine, valine, tryptophan, lysine, proline, serine and threonine increased, and glutamate, glycine, phenylalanine and tyrosine were decreased [
47]. Another study reported increased serum concentrations of isoleucine, valine and leucine after OVX using gas chromatography time-of-flight mass spectrometry [
48]. Similarly, branched-chain amino acids (valine, leucine and isoleucine), homocysteine, hydroxyproline and ketone bodies (3-hydroxybutyric acid) have been observed to be elevated, while levels of lysine decreased in the OVX group compared to the sham group [
17].
In this study, plasma methionine levels were increased in the OVXG group. Methionine plays key roles in cell metabolism as one of the essential amino acids required for cellular proliferation, protein synthesis, RNA synthesis and transamination. Further, methionine can be catabolized into homocysteine and cysteine [
49,
50]. A previous study on methionine reported changes in bone turnover in hyperhomocysteinemia aged rats, where osteocalcin (OC) decreased and urine N-terminal type 1 collagen, a marker of bone resorption, increased [
51]. Hermann et al. [
52] reported a positive correlation between hyperhomocysteinemia and urinary deoxypyridinoline crosslinks in peri- and postmenopausal women; however, no significant correlations were observed between hyperhomocysteinemia and OC. In another study, hyperhomocysteinemia levels were associated with higher bone turnover and lower BMD [
53]. This suggests that circulating levels of methionine may alter bone remodelling and could possible explain the reduced BMD observed in treated ewes by upregulating osteoclast development induced by OVX and glucocorticoid treatments.
In this study, tryptophan levels were increased in the OVX group. Tryptophan and its metabolites impact bone formation, stimulating the proliferation and differentiation of bone marrow-derived mesenchymal stem cells [
54]. The possible mechanism of altered tryptophan in bone loss may be linked to enhanced osteoclastogenesis. Studies have indicated that tryptophan may play a role in osteoclastogenesis via the kynurenine pathway of tryptophan catabolism, as interferon
y (IFN-
y) is formed via the kynurenine pathway [
55]. IFN-
y is a cytokine that is produced in the bone microenvironment by cells, and it promotes osteoclast activity. During menopause, estrogen deficiency upregulates the pro-inflammatory cytokines, as IFN-
y increases the bone loss in postmenopausal women [
56,
57]. The results in this study provide evidence that OVX and glucocorticoid treatment perturbed plasma tryptophan levels in both OVX and OVXG sheep. Further studies in animal models are needed to determine whether tryptophan metabolites could be utilized as novel biomarkers for bone loss in human osteoporosis.
Plasma concentrations of hydroxylysine were decreased in the OVXG group when compared with the OVX control group. Hydroxylysine is a urinary marker of bone resorption derived from collagen crosslinks [
58]. In bone tissue, bone collagen proteins comprise about 90% of the organic matrix of the bone. Bone collagen supports properties of bone such as tissue mass, micro- and macro-architecture and material properties, which all contribute to bone strength [
59]. Bone collagen is rich in proline, hydroxyproline and glycine. Proline and lysine residues are hydroxylated to hydroxyproline and hydroxylysine, respectively, which contribute to the formation of collagen crosslinks and to bone matrix quality [
60‐
62]. Circulating levels of hydroxylysine as biomarker have been reported in human osteoporotic bone matrix [
63]. Although the hydroxylysine urinary marker was not measured here, an decreased in the bone resorption marker C-terminal telopeptide of type I collagen was seen in the OVXG sheep plasma [
32]. Thus, the plasma levels of hydroxylysine in OVXG sheep may reflect the increased bone remodelling as a response to estrogen deficiency and glucocorticoid intervention. Functional studies will be required to determine whether the identified metabolites had a direct effect on bone remodelling.
Effect of OVX and OVXG on the plasma Lipidome of sheep
The results revealed differences in the lipidome in both short-term and long-term approaches, which indicates effects on circulating plasma lipids. The dynamic changes of the lipidome of OVX sheep, along with OVXG2 and OVXG5 were evaluated.
The short-term statistical approach revealed OVX increased CL (76:7) and PI species at month one. In contrast, in the OVXG group, a decrease in levels of all identified lipids was observed at month one, and the same effect was observed at month two. In general, this study had a small sample size for a lipidomic analysis; however, the short-term approach provided a better probability to improve the characterization of the lipidome in OVX sheep due to it having more animals in each treatment group (control group n = 10; OVX n = 12; and OVXG n = 6).
The long-term statistical approach highlighted lipid profile changes after OVX or OVXG2/OVXG5 treatments. Relative intensities of lipids were increased at months one and three and remained relatively stable until the end of the study in the OVX group. In contrast, in the OVXG2, a dynamic lipid compositional change could be observed from month one. These relative intensities decreased steadily until the end of this study, with the exception of CL (72:6), CerP (39:1) and PI (31:5); while in the OVXG5 treatment, the relative intensities of the all lipids decreased after five months.
The OVX sheep model was selected because it mimics postmenopausal bone loss and it is recommended by the WHO as a large species for evaluating skeletal osteoporosis therapies [
64]. Lipids are involved in several cellular functions and physiological conditions associated with energy storage, structure, apoptosis and signalling, and these have an effect on skeletal metabolism and bone health [
65]. In this study, lipid profiles were affected, with various relative intensities of lipids changing during the early stage of estrogen withdrawal in the OVX sheep. Although basic lipid clinical markers such as triglycerides, total cholesterol, low density lipoprotein cholesterol or high-density lipoprotein cholesterol were not measured, these findings coincide with previous results, where it was reported that perturbed levels of lipid metabolism are associated with osteoporosis [
41,
66‐
68]. Further studies are required to validate the findings presented in this study; however, the lipids identified provide an overview of the lipidomic changes during postmenopausal osteoporosis in OVX sheep.
In this study, levels of phospholipids decreased after OVX and increased at month three until the end of the study. Although this is the first study that presents lipid profiles of OVX sheep, previous studies have reported lipidome changes in OVX rats. Zhu, Liu [
69] reported decreased eicosapentaenoic acid, ergocalciferol and cholecalciferol, and increased arachidonic acid, in OVX rats. Conversely, an earlier study reported increased levels of fatty acids (arachidonic acid and octadecadienoic acid) and cholesterol after OVX compared to before surgery [
17,
70]. However, in this study, arachidonic acid was not detected; PCs and PIs are substrates for this fatty acid and may be involved in the bone loss in the OVX sheep [
71]. This implies that altered phospholipids might be involved in osteoclastogenesis promoting loss of BMD, which affects bone remodelling as result of estrogen depletion.
Sphingolipids are a major component of cell membranes and play a role in cell signalling. Ceramides are precursors to many sphingolipids. In blood plasma, they are associated with lipoproteins [
72,
73]. The major subclass identified in this study was two species of ceramide phosphate. Our findings were similar to a previous study that reported that levels of ceramide, ceramide-1-phosphate, sphingomyelin, 1-
O-alkenyl-lysophosphatidylethanolamine and lysophosphatidylethanolamine were elevated in the OVX rats compared to those in the sham-operated rats [
74]. Ceramides have been implicated in increasing the cellular oxidative state, and this has been linked to stress or death signals. Ceramide levels have been reported to affect osteoblast apoptosis [
75,
76]. Thus, perturbed ceramide levels may play a key role in bone loss in postmenopausal women.
Cardiolipins (CL) are phospholipids that are embedded in the inner mitochondrial membrane and are a key factor for energy production. Current evidence highlights that mitochondrial dysfunction by reactive oxygen species leads to the oxidation of mitochondrial cardiolipins, and this has been linked to atherosclerosis [
77,
78]. Although the results presented here are unable to establish a direct role of CL in osteoporosis, it may be related via bone marrow cells, where estrogen withdrawal regulates the complex cell differentiation of osteoblasts and adipocytes [
79].
The data reported here is the outcome from a liner mixed model of a pilot intervention, where we analyzed both the metabolome and lipidome data without adjusting for baseline values. The longitudinal responses for each metabolite and lipid were measured and reported for the same group of ewes repeatedly as the goal was to understand the metabolic and lipidomic alterations that accompany bone loss in osteoporosis development. The result from the linear mixed model specifically indicated no significant different between the baseline group means for the metabolome results. However, for the baseline lipidome results significant differences between-group were noted. In metabolomics studies, both intrinsic and extrinsic factors are responsible for changes occurring in the metabolome and lipidome of biological samples, and therefore the pre-analytical operation procedure including general experimental design and blood collection require careful sample handing to ensure a reproducible extraction is performed. In this pilot study, the ewes (7–9 years) were first fed a basal diet for 10 days (adaption period), then randomly allocated to four treatment groups with limited study size due to some operation constraints. Therefore, it must be noted that the effects reported here may be influenced by individual variation within the OVX sheep and the findings presented should be considered as baseline for follow-up studies.
Strengths of this study are the application of untargeted metabolite profiling using HILIC–MS and lipidomic analysis of plasma samples from OVX sheep to explore the dynamics of disease progression in the metabolite profile, and subsequent identification of plasma biomarkers of osteoporosis. Limitations of this study include the small sample size of treated groups, specifically the ewes that were treated with glucocorticoids, and due to the large variability of the data further validation will be required. In addition, blood samples were collected once a month, as the study design considered both a short- and long-term response to treatments. Larger sample size, and multiple sampling time points from the early stage of estrogen deficiency and glucocorticoid treatment, should be considered in future studies to identify biomarkers that are unique to the early onset of osteoporosis in postmenopausal women. Finally, although a large number of features resulted significant from the interaction treatment and time from the linear mixed model, most of the features remain unidentified. Further, the analysis of other biological matrices such as bone tissues would maximize the identification of molecular changes of the bone microenvironment in osteoporosis. Follow-up studies also should be conducted to examine the effects on the metabolome and lipidome of the microbiome and a low calcium and vitamin D diet on osteoporosis prevention.