Using mass spectrometry to profile sex steroids in metabolic diseases
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Date
30/11/2020Author
Faqehi, Abdullah Mossa M
Metadata
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 (LCMS). 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 postmenopausal 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 postmenopausal 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 chromatographic separation had not been achieved
and was not ideal. Thus, methods had not progressed to full validation or
biological application. We aimed to overcome the chromatographic challenges
faced with the hydrazine derivatives and validated an analytical approach to
detect low abundance 5α-dihydrotestosterone by derivatisation in plasma,
comparing several different reagents.
Derivatisation to form HMP androgens, in conjunction with LC-MS/MS,
proved highly suitable for quantitative analysis of androgens in low
abundance in human plasma with low volumes (10-200µL), offering clear
advantages in sensitivity over analysis of underivatised steroids. The
approach allows concomitant analysis of testosterone, androstenedione and
5α-dihydrotestosterone and this method could be expanded to include further
ketone containing steroids. Robust measurement was achieved across typical
physiological ranges found in post-menopausal women and men, comparing
to immunoassays.
Next the sensitivity of analysis for estrogens was enhanced using a
methylpyridinium derivative to detect these steroids in low abundance in
human plasma and, in particular, to evaluate if the approach could quantify
circulating steroids in post-menopausal females. A highly sensitive method
with lower limits of quantitation (LOQs) than previous methods was validated
using ion-exchange solid phase extraction in conjunction with LC-MS/MS.
Derivatisation by reaction of 2-fluoro-1-methylpyridinium-p-toluenesulfonate
(FMP-TS) with phenolic estrogens, in conjunction with LC-MS/MS, was found
to be suitable for quantitative analysis of E1 and E2 in low abundance in
plasma. The derivatisation reaction may be applicable to other naturally
occurring phenolic steroids.
Finally, using these novel analytical approaches sex steroid profiles were
investigated in relation to metabolic parameters in a cohort of healthy men
across the age range 20-80years, using correlation and multiple regression
statistics. Generally, results show a stronger correlation of androgens than
estrogens to metabolic parameters such as glucose disposal and adiposity.
In conclusion, sensitive measurements of sex steroids were achieved using
solid phase extraction and enhanced with derivatisation, in conjunction with
LC-MS/MS. The approaches developed allow robust quantitative analysis of
androgens and estrogens in low abundance in biological fluids across typical
physiological ranges found in pre- and post-menopausal women and men,
overcoming concerns over use of non-specific immunoassays. These methods
developed may also be used in applied to rodent models with low plasma
volumes. The derivatisation methods could be further applied to other ketone
and phenol containing steroids. Use of this analytical method development
approach has allowed insight into relationships between sex steroids, adipose
tissue and metabolic disease.