Profiling Pathways of Estrogen Metabolism in Patients with Pulmonary Arterial Hypertension: Development of a Novel Assay for Estrogen Metabolites by LC-MS/MS Nina Denver (Presenter) University of Glasgow Presenter Bio: Nina Denver is a PhD Student working within the lab of Professor Mandy MacLean at the University of Glasgow collaborating with the mass spectrometry core lead by Professor Ruth Andrew at the Queen’s Medical Research Institute, University of Edinburgh. Her research focus is on the role of estrogen and its metabolites in the onset and progression of pulmonary arterial hypertension (PAH). Prior to this, she obtained an international Erasmus mundus master degree in chemistry from Paris Sud University, Paris, France and Adam Mickiewicz University, Poznan, Poland and received her undergraduate degree from Glasgow Caledonian University with first class honours in Forensic Investigation.

Authors: Nina Denver1, Ruth Andrew2, Natalie ZM Homer2, Shazia Khan2, Colin Church3, Margaret R MacLean1.
(1) Institute of Cardiovascular and Medical Sciences, University of Glasgow, G12 8QQ. (2) Mass Spectrometry Core, Queen’s Medical Research Facility, University of Edinburgh, EH16 4TJ (3) Golden Jubilee Hospital, Agamemnon St, Clydebank, Glasgow, G81 4DY

Pulmonary arterial hypertension (PAH) is a debilitating, life limiting, commonly misdiagnosed disease, which ultimately leads to right heart failure and death. Estrogen metabolism plays a key role in disease onset and progression. To simultaneously quantify circulating estrogen levels in human plasma a sensitive liquid chromatography mass spectrometry (LC-MS/MS) was developed to compare plasma from healthy controls and PAH patients. Initial studies show increases in metabolites involved in PAH pathophysiology prompting further investigation for potential diagnostic and therapeutic strategies. Furthermore, implementation of this method will be clinically relevant to a number of estrogen sensitive diseases.

Introduction

Pulmonary arterial hypertension (PAH) is a progressive, life limiting disease, which without treatment ultimately leads to right heart failure and death. A strong gender bias in this disease, 4:1 female to male ratio, implicates sex hormones as playing a pivotal role in both development and progression of PAH. Therefore, comparison of circulating bioactive estrogens and their metabolite levels in PAH patients in comparison to healthy subjects may elucidate the estrogen paradox in PAH. Current methods to quantify estrogen and its metabolites include immunoassays, a technique limited by only measuring one metabolite at a time, being expensive, and using cross-reacting antibodies. As such, an improved assay to analyse a panel of estrogen metabolites would be beneficial for quantification of their low circulating levels for clinical applications. Our aim was to quantify multiple estrogens simultaneously by Liquid chromatography tandem mass spectrometry (LC-MS/MS).

Methods

Plasma from Male and Female PAH patients was obtained with ethical approval (Golden Jubilee, Glasgow) to distinguish estrogen profiles of PAH patients whilst control plasma was purchased from TCS Biosciences (Buckingham, UK) as collection of healthy individuals remains ongoing. Extraction of estrogen from 0.5 mL human plasma was performed using solid phase extraction cartridges under gravity. Derivatisation to permanently charged molecules by formation of methylpiperazine (MPPZ) derivatives enhanced ionisation of all estrogen metabolites and thus boosted efficiency and sensitivity. Chromatographic separation of derivatives was achieved on a Shimadzu Nexera X2 system using a C18-PFP column with a gradient elution. This coupled to a Sciex QTRAP 6500+ in positive electrospray ionization mode (ESI-MS) allowed specific detection of each estrogen metabolite derivatives

Results

We have developed an assay to quantify estrogen metabolites in 500 µL human plasma using LC-MS/MS. Derivatisation of estrogens with methylpiperazine (MPPZ) allows detection by formation of charged molecules. The reaction progressed using sodium bicarbonate as a base catalyst for 9 of 13 metabolites although formation of 2, 4-hydroxyestrogen-MPPZ derivatives was more efficient using an alternative base catalyst. Solid phase extraction of estrogen metabolites from plasma gave extraction efficiencies of 93 – 111%. Optimisation of elution and wash steps reduced ion suppression (1 – 30%) permitting typical limits of detection in the range of 0.43 - 2.17 pg on column for estrone, estradiol, 16-hydroxyestrogen and methoxyestrogen metabolites. The 2, 4-hydroxyestrogen metabolites however remain problematic with average recoveries of 64 -73% and significant ion suppression (68-80%) even with optimisation of wash and elution steps. As a result, a more stringent extraction protocol or an alternative approach may be applicable for these catechol estrogens. Using the developed method for 9 metabolites, initial screening of human plasma detected increased metabolite presence the PAH patient samples analysed. Most notably, 16-alpha-hydroxyestradiol (16αOHE2) and 2-methoxyestrone (2MeOE1) metabolites were the most abundant metabolites in the majority of female PAH patient samples.

Conclusions & Discussion

We have demonstrated that MPPZ-derivatisation with LC-MS/MS analysis allows detection of estrogen metabolites in this disease cohort and in healthy controls. Our initial analysis suggests an elevated presence of estrogen metabolites in PAH patients. For analysis of the 2, 4- hydroxyestrogen metabolites, an alternative extraction approach may be necessary. Thus, it may be necessary to split plasma samples prior to processing for two methods of extraction, derivatisation and LC-MS/MS analysis, allowing quantification of 13 estrogen metabolites of interest.