Using Mass Spectrometry to Profile Sex Steroids in Metabolic Diseases

Abstract

Abstract Sex hormones are steroids that are key co-ordinators of sexual development and are principally formed in endocrine glands. The main types of sex hormones in females are estrogens: notably estrone (E1) and estradiol (E2), whereas in males, androgens, in particular testosterone, androstenedione and 5α-dihydrotestosterone are the most prevalent. Obesity is one of the most important risk factors in development of metabolic diseases. Therefore, it is important to study factors that influence body weight in order to develop preventative and curative approaches. Many studies have investigated the role of sex hormones in the accumulation of body fat, given the differences in adiposity between sexes and, in particular, following the menopause. In general, abdominal fat has a stronger relationship than total body fat in the development of metabolic diseases, suggesting factors underlying these sex differences in fat distribution may be important in pathology. Investigations of sex hormone signalling in metabolic disease may therefore improve strategies to prevent abdominal fat accumulation and reduce the adverse impact of obesity on lifelong health. At the outset of this thesis, few studies existed assessing relationships between metabolic risk factors and exposure to sex steroids. Moreover, the data from cross-sectional studies were not consistent and some concerns raised over the validity of data due to the analytical methods used, often immunoassays in the older literature. An understanding of the effect of sex steroids on metabolism was needed but researchers emphasised the need for better profiling of androgens and estrogens to gain a clearer picture, recognising none of the commercially existing automatic immunoassay methods had satisfactory sensitivity and selectivity.

Analysis of sex steroids is challenging due to their extremely low concentrations in different biological samples, dependent on age, sex and disease. Sex steroids suffer from interference from substances in the biological matrix such as endogenous isomers. This raises the importance of alternative analytical methods such as liquid chromatography mass spectrometry (LC-MS). LC-MS is a gold standard analytical technique that is capable of measuring multiple analytes in a single sample. This thesis aimed to develop LC-MS approaches to allow sensitive measurements of estrogens and androgens to investigate the role of sex steroids in the distribution and function of adipose tissue and its association with metabolic diseases. In the first chapter, an initial analytical approach to quantify androgens in human and murine biological fluids by tandem mass spectrometry (MS/MS) was established, and a particular goal was to evaluate if the approach could quantify circulating steroids in samples with very low levels; including post-menopausal females and small volume pre-clinical samples. Extraction was validated using solid phase extraction, in conjunction with LC–MS/MS analysis and the benefits and limitations of the method were assessed. This approach allowed robust measurement of testosterone and androstenedione across typical physiological ranges in plasma found in pre- and post-menopausal women and men. The method was also found suitable for animal models to quantify testosterone in small volumes (30-50µL) of plasma. However, for the detection of 5α-dihydrotestosterone this method required much large volumes (>500µL), due to inefficient ionisation and therefore further steps were taken to improve the sensitivity of the method.

The next Chapter describes investigations of approaches to derivatisation of androgens - aiming to improve sensitivity by endowing the analyte with a chargeable or permanently charged group for MS ionisation. Prior to this thesis, 2-hydrazino-1-methylpyridine (HMP) had been applied for this purpose but the necessary chromatogr