Analytical challenges of measring sex steroids in mouse tissues

Abstract

Sex steroid hormones such as estrogens and androgens play a critical role in many areas of biology. Disruption to the balance of sex steroids can lead to metabolic disorders, cerebrovascular and cardiovascular diseases, and cancer. Men with estrogen insufficiency suffer from insulin resistance (IR) and increased visceral fat; while in women estradiol deficiency results in increased risk of metabolic syndrome, lipodystrophy, IR, and hyperandrogenism impairs insulin secretion and leads to type 2 diabetes. Recent observations of a higher incidence of mortality in men with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) suggests there may also be a role of sex hormones in this disease. To investigate the role of sex steroids in diseases then knock out mouse models are used. It is important that sex steroids are accurately measured in these mouse models, in both circulation and in tissues as steroids are known to have systemic but also local action – referred to as intracrinology. Measuring sex steroids accurately and comprehensively in tissues is critical to understanding their role, but this presents an analytical challenge as handling and extracting steroids from tissue is not a simple process and different tissues have different compositions. Historically, sex steroids have been measured by immunoassays in plasma or serum. However, this only allows measurement of one or two steroids in a single sample. There are also concerns over poor sensitivity and cross reactivity of immunoassays for sex steroid measurement. More recently mass spectrometry (MS) coupled with either liquid chromatography (LC) or gas chromatography (GC) have become the methods of choice, and in fact since 2014 this has been a requirement in clinical situations for plasma and serum sex steroid measurement. Mass Spectrometry has the added benefit of being able to profile multiple steroids in a single sample. This review aims 1to assess current published Mass Spectrometry methods used to measure sex hormones within mouse tissue. 14 articles were identified in the past ten years that used LC-MS/MS or GC- MS/MS to measure quantities of sex steroids in mouse tissues indicating that the technology is being used for sex steroid profiling in pre-clinical research tissue samples. The specifics of the methods were compared. Overall, several analytical challenges have been identified in murine tissue sex steroid analysis and need to be overcome in order for preclinical research studies to fully profile sex steroids in tissue. This review highlights that chromatographic MS methods often do not include all method details needed to repeat the method and that tissue homogenisation protocols need to match the tissue type, before extraction of the steroid for analysis. Thus, harmonisation of methods is needed across laboratories to be confident in the results of sex steroid analysis in mouse tissues. Mass spectrometry imaging (MSI) is emerging as an additional bioanalytical tool that allows mapping of sex steroids in tissues. MSI does not result in a concentration; it gives molecular histology of tissues which is important in understanding of intracrinology and regionalisation of steroids and their metabolites. This promises an additional dimension to understanding the role of sex steroids in mouse models.Overall the review indicates that mass spectrometry methods are being used to assess sex steroids in tissues from preclinical mouse models. Challenges of standardisation of methods for homogenisation, extraction and sufficient sensitivity for sex steroids are still to be overcome. Methods would benefit from expansion to profile more steroids, while standardisation of homogenisation and extraction techniques and continued innovation to improve method sensitivity so that more steroids could be profiled in small tissue samples would improve our understanding of the role of sex steroids in preclinical models of disease.