title: Sex and estrogen metabolism in Pulmonary Arterial Hypertension (PAH)
Creators: name: Denver, Nina
Publisher: name: University of Glasgow
Year: 2020

description: Pulmonary arterial hypertension (PAH) is a progressive, life-limiting disease, characterised by increased pulmonary vascular resistance and right ventricular hypertrophy leading to right heart failure and death. In PAH, a sexual dimorphism is present with a strong sex bias in disease penetrance (4:1 female to male ratio). Despite being more susceptible to the development of PAH, females display improved right ventricular (RV) function and survival rate in comparison to males. As a result, a sex paradox exists and sex hormones have been implicated in the development and progression of PAH, with numerous studies displaying roles for estrogen, its metabolites and receptors in disease progression. Little is known about the systemic endogenous levels of estrogen and its metabolites, or their relationship to sexual dimorphism in PAH. This is partially due to the fact that current methods to quantify estrogens commonly employ immunoassays, which measure only one metabolite at a time and often over-estimate levels due to cross-reacting antibodies. Therefore, an improved assay to analyse a panel of estrogen metabolites is required to quantify the wide range of endogenous concentrations in females and males. The main aim of this work was to develop, validate and apply a liquid chromatography tandem mass spectrometry (LC-MS/MS) approach to quantify multiple estrogens simultaneously. Estrogens were extracted from serum or plasma by solid phase extraction on Oasis MCX® cartridges followed by derivatisation using 1-(5-fluoro-2, 4-dinitrophenyl)-4-methylpiperazine (MPPZ). Nine derivatives were quantified using a Shimadzu Nexera X2 interfaced with a QTrap 6500+. Recovery was 90–110% and ion suppression minimal (0 - 30%). The limits of quantification were between 2–6 pg/mL with acceptable precision and accuracy (&lt;15%). Estrogen-MPPZ derivatives also demonstrated minimal degradation upon short-term storage at 15°C (auto-sampler) and longer-term at -20°C (&lt;20%). Application of this method to clinical samples from various cohorts of PAH and portopulmonary hypertension (PPHTN) patient samples allowed detection of parent estrogens (estrone (E1), estradiol (E2), and its isomer α-estradiol (17αE2)) alongside specific metabolites (16-hydroxyestrone (16OHE1), 16-hydroxyestradiol (16OHE2), 2-methoxyestrone (2MeOE1), 2-methoxyestradiol (2MeOE2), 4-methoxyestrone (4MeOE1) and 4-methoxyestradiol (4MeOE2)). In summary, sex-dependent differences in estrogen profiles were displayed in comparisons of PAH and non-PAH control samples. In females, E1 and E2 concentrations were reduced in PAH, with an elevation in 16OHE1 and/or 16OHE2, dependent on disease classification. Conversely, in males, E1 and E2 were elevated alongside increased 16OHE1 and, in PPHTN only, 16OHE2. In addition, a proof-of-concept trial of estrogen receptor alpha (ERα) antagonism by fulvestrant administration (intramuscular, 500 mg dose) in five postmenopausal women demonstrated a reduction in 16OHE2 concentrations following 9 weeks. Comparison of estradiol quantification by LC-MS/MS and immunoassays in two studies provided evidence of over-estimation by immunoassay. In the first, a correlation between the two methods was apparent with lower concentrations consistently quantified by LC-MS/MS than by immunoassay. In the second study, elevated estradiol concentrations following fulvestrant treatment were measured by immunoassay but not by LC-MS/MS. This suggests structural similarities between fulvestrant and estradiol causes cross-reactivity during immunoassay detection, a finding stated in a number of research papers. Furthermore, in vitro, 16OHE2 caused proliferation of rat and human pulmonary artery smooth muscle cells (PASMCs). The proliferative phenotype was confined to female rat PASMCs and female human PAH-PASMCs, with no apparent effect in male rats or in female and male control human PASMCs. RNA sequencing also identified potential upstream effects of estradiol and estrogen receptors in female PAH PASMCs. This study is the first to simultaneously quantify estrogen and the bioactive metabolites in PAH patients, providing a sensitive and selective method to quantify endogenous estrogens in human serum or plasma. Its application to a number of PAH cohorts from international collaborators generated evidence of elevated 16-hydroxylation in disease. The novel findings of elevated 16OHE2 in all female patient samples, of reduced concentrations following ERα antagonism in post-menopausal women, and of sex-dependent proliferative effects of this metabolite in PASMCs indicates its potential influence in the pathogenesis of PAH. They also provide evidence that this LC-MS/MS method may be utilised as a clinical tool to monitor estrogen concentrations in patients and for the analysis of estrogen-inhibition therapies in PAH.