Although some biological trace elements, such as phosphorus and calcium are involved in important physiological processes, their concentrations may differ significantly between healthy and diseased individuals . The knowledge of changes in calcium (Ca) and phosphorus (P) concentrations is pivotal to understand both normal metabolic processes and pathophysiology in a tissue like the bone [2, 3], and these may be affected by both biochemical and mechanical stimuli.
However, the investigation of the role of each element considered in isolation has been questioned since it ignores potential interactions between them . For all that, even when concentrations of several elements are obtained in the same study, comparisons between healthy and diseased tissues, or correlations between the various elements, both intrinsically multivariate, are often implemented with univariate methods, which may result in observed effects or the inability to detect such effects [4, 5].
Diabetes, a growing global epidemic, is a biochemical stimulus that can be harmful to bone health . Low bone mineral density is associated with type 1 diabetes (T1DM), while in type 2 diabetes (T2DM) bone mineral density is comparable to or greater than in non-diabetic subjects. Nevertheless, latest cohort studies and meta-analyses corroborate that T1DM and T2DM have an increased risk of osteoporotic fracture as a chronic complication , with various factors seemingly affecting the likelihood of fractures .
Other studies also show that bone plays an essential part in regulating intermediary metabolism, acting as pathophysiological factor in the disease itself . Because of its ability to secrete osteocalcin, bone may be considered an endocrine organ . The connection between osteocalcin and metabolic factors, namely glycemia, β-cell proliferation, insulin secretion, and lipid profile, unravelled the possible effect of the skeleton on energy control and glucose metabolism .
Similarly, infrasound, a physical stimulus defined by acoustic vibrations whose frequency is below the low frequency limit of normally audible sound, is ubiquitously present . In fact, many studies suggest that infrasound pollution is on the rise worldwide, particularly in industrial and urban settings . Osteoblasts and osteoclasts functioning are affected by vibrations, suppressing bone resorption, and encouraging bone formation .
Furthermore, bone remodelling is apparently promoted by this type of stimulation, which indicates that it has an impact on bone mineralization . Additionally, it was demonstrated that infrasonic exposures may possibly stimulate osteoblast-like cells growth and secretion activity in vitro . There also evidence that that infrasound may possibly promote osteogenesis and fracture healing in vivo . It was also shown that infrasound and low frequency noise induce alterations in various animal tissues, such as cardiac atrium , salivary glands , periodontium  and liver .
So, little is known regarding the effects of both diabetes and infrasound on elemental bone composition, and even less regarding the effects of the potential interaction of both. For major human pathologies, such as diabetes, a full variety of well-described induced models is accessible, namely in rats. .
X-ray fluorescence spectroscopy (XRF) has been applied in determining the concentration of major and vestigial elements in biological tissues, namely humans and animals, and presents important methodological advantages for this type of studies compared to other techniques [4, 5, 8, 21]. The main methodological advantages of XRF spectroscopy compared to other techniques are the minimum preparation of the sample, eliminating the risk of contamination or loss of elements of interest, and the simultaneous determination of the concentration of several elements, providing a fast, accurate and sensitive elemental analysis. This technique has been applied in studies of histological alterations in metabolic disorders such as diabetes and osteoporosis, which have demonstrated its validity, when complemented with a correct data analysis, to distinguish between normal and altered tissues [1, 2, 22, 23]. Moreover, animal models have been shown to be an apt model for investigating the relationships between Ca and P concentration in bone .
The methodologies used in this study provide an important contribute to fill existing gaps in current knowledge of the role of specific chemical elements, such as for e.g. Ca and P, in such metabolic pathways. Thus, the aim of this study was to evaluate the elemental composition of calcium (Ca) and phosphorus (P) in the bone of rats subjected to glucose intolerance and/or infrasound.