Sex and estrogen metabolism in Pulmonary Arterial Hypertension (PAH)

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 (<15%). Estrogen-MPPZ derivatives also demonstrated minimal degradation upon short-term storage at 15°C (auto-sampler) and longer-term at -20°C (