Measurement of sex steroids in murine blood and reproductive tissues by liquid chromatography–tandem mass spectrometry

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Abstract

Accurate measurement of sex steroids is essential to evaluate mouse models for human reproductive development and disorders. The recent advent of liquid chromatography–tandem mass spectrometry (LC–MS/MS) assays that match the sensitivity of steroid immunoassay could overcome problems arising from the limited specificity of steroid immunoassay. In this current study we validate a LC–MS/MS assay for the measurement of key sex steroids from murine serum and reproductive tissues. The assay gave excellent dilutional linearity (r2  0.98) and reproducibility (CV  10% of replicate samples) in serum and reproductive tissues with sensitive quantitation limits; testosterone (T; 2 pg), dihydrotestosterone (DHT; 10 pg), 5α-androstane-3α,17β-diol (3αDiol; 40 pg), 5α-androstane-3β,17β-diol (3βDiol; 40 pg), estradiol (E2; 0.5 pg) and estrone (E1; 0.3 pg). Using 0.1 mL sample, T was the only consistently detectable steroid (detection limit 20 pg/ml) in both male and female mouse serum. In the testis, T and DHT were quantifiable as were both diols at relatively high levels. Prostatic T levels were low and DHT was determined to be the most abundant androgen in this tissue. Uterine and ovarian levels of E2, E1 and T were measurable, with levels varying according to estrous cycle stage. Hence, we demonstrate that this LC–MS/MS method has the sensitivity, specificity and multi-analyte capability to offer accurate steroid profiling in mouse serum and reproductive tissues.

Introduction

Steroid hormones are vital for reproductive development, health and hormone-dependent disorders as well as the optimal function of most non-reproductive tissues which express steroidogenic enzymes and steroid receptors. Investigating the pathogenesis of human development or disease is constrained by ethical and practical limitations on clinical research. As a result, mammalian animal models are an indispensable tool for investigating reproductive health, medicine and biology. Among the alternative animal models, the laboratory mouse is the most cost-effective with their unmatched versatility to undergo targeted genetic modification with high fidelity replication of development and/or disease processes involving the highly conserved mammalian reproductive system. Investigating such model systems requires accurate and sensitive measurement of sex steroids in blood and tissues.

Prior to the advent of immunoassay (IA) for steroids in the early 1970s, measurement of sex steroids was largely unavailable as it relied on whole animal bioassays which were laborious, costly and insensitive. Although steroid IA produced a dramatic improvement in sensitivity allowing measurement of steroids to the picogram level, the limitations of specificity were always recognized. Cross-reactivity with structurally related steroids, steroid conjugates and matrix interference required elimination by pre-assay steps of solvent extraction, chromatography and the use of only authentic tritiated steroid tracers. The increasing demand for steroid IA resulted in assay simplification, mostly to allow incorporation of steroid measurements into high throughput immunoassay platforms. This required shedding of the pre-assay steps which safeguarded the specificity of steroid IA. Consequently, it has become evident that steroid IA using unextracted, non-chromatographed samples and the use of bulky non-authentic tracers is susceptible to major limitations in specificity. This has led to apparent method-specific differences in reference ranges, especially at low circulating steroid levels where steroid IA generates unreliable measurements [1], [2], [3].

While mass spectrometry (MS)-based measurements of steroids have always remained the gold standard, available MS methods (using gas chromatography) remained insensitive and largely inaccessible as reference methods. This was largely due to the requirement of large amounts of sample and lengthy sample preparation steps, thereby limiting throughput. Recently, bench-top liquid chromatography–tandem mass spectrometry (LC–MS/MS) instruments have been developed that overcome historical limitations of MS sensitivity for sex steroid measurement while retaining unmatched, reference level specificity. In addition, LC–MS/MS enables the analysis of multiple analytes from a single sample [4], [5], [6], [7], thereby maximizing the information gained from limited amounts of samples. This is especially relevant to material harvested from small animals such as the mouse. Also, it allows for a steroid metabolism profile to be obtained, which is particularly useful when studying steroid dependant and metabolizing tissues, such as the prostate, where significant steroid metabolism occurs [6], [8], [9] and where novel steroids and metabolism pathways are suggested to be functionally relevant [10], [11], [12], [13], [14], [15].

The specificity limitations of steroid IAs developed for use with unprocessed human serum are particularly troublesome when used to measure sex steroids in mouse serum (where a circulating sex hormone binding globulin is absent) or tissues with their different complex matrices. Until recently MS methods were insufficiently sensitive to measure sex steroids directly in mouse samples, although a limited number of LC–MS/MS methods were described that measure steroids in mouse and rat prostate and serum by using derivatized adducts to improve sensitivity [16], [17], [18], [19]. As expected, the few available MS-based studies report differences from IA data especially at low blood steroid levels [6], [18], [20], [21], [22], [23]. Therefore, there is a need for the development of methods capable of accurate, precise and direct (non-derivatized) measurement of steroids in mouse serum and tissues. We recently described a novel LC–MS/MS method to measure biologically active sex steroids and their primary metabolites in human serum [24], including both androgens and estrogens; testosterone (T), dihydrotestosterone (DHT), 5α-androstane-3α,17β-diol (3αDiol), 5α-androstane-3β,17β-diol (3βDiol), estradiol (E2) and estrone (E1) in a single run without derivatization. In this study we report validation of this method for use with murine serum, steroidogenic (ovary, testis) and steroid dependent (prostate, uterus) tissues.

Section snippets

Materials

T, DHT, 3αDiol and 3βDiol were obtained from the National Measurement Institute (NMI; Sydney, Australia). Deuterium labeled internal standards of these steroids were also from the NMI: testosterone-1,2,3-d3 (d3-T), dihydrotestosterone-16,16,17-d3 (d3-DHT), 5α-Androstane-3α,17β-diol-16,16,17-d3 (d3-3αDiol) and 5α-Androstane-3β,17β-diol-16,16,17-d3 (d3-3βDiol). E2 and E1 were from Steraloids (Newport, RI, USA). Deuterium labeled estradiol-2,4,16,16-d4 (d4-E2) was from Cambridge Isotope Laboratory

Serum

T was measured in 100 μL of serum from all intact male and female mice, and was able to be accurately quantified in as little as 25 μL of male mouse serum. Recovery of non-isotopically labeled steroids from a spiked serum pool was between 95 and 120%, both for low and high spiked values (Table 1). For intact wildtype males, serum T displayed wide variability between mice creating a highly skewed distribution (Fig. 1; mean 7.0 ng/mL; median 1.6 ng/mL; range 0.30–39.4 ng/mL). Detectable T levels were

Discussion

Genetic mouse models provide an indispensable tool to investigate mammalian reproductive physiology and pathology. It is essential to be able to acquire accurate measurements of sex steroids from the very limited serum volumes and tissue samples available from mice. Immunoassays optimized for use with unextracted human serum samples have major limitations at low steroid concentrations and when using murine samples. In this paper we report the validation in murine serum and reproductive tissues

Disclosure statement

The authors have nothing to disclose.

Acknowledgements

We would like to thank Dr Charles Allan, Dr Lucy Yang, Jenny Spaliviero, Natalie Farrawell and Ellen Gao for their help with various aspects of the project. We are grateful to Scott Heffernan for providing us with hamadryas baboon serum from the NHMRC national baboon colony [52], Dr Simon de Graaf and Dr Natasha Ellis from the Faculty of Veterinary Science at the University of Sydney for obtaining sheep and horse serums, respectively. Mamdouh Khalil and staff in the Molecular Physiology Unit at

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