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High Resolution Mass Spectrometry to enhance hormonal profiling across the lifespan.

Reference	BB/V019066/1
Principal Investigator / Supervisor	Professor Ruth Andrew
Co-Investigators / Co-Supervisors	Mr Scott Denham, Dr Natalie Homer, Professor Margaret MacLean, Dr Richard Mellanby, Professor Rebecca Reynolds
Institution	University of Edinburgh
Department	Centre for Cardiovascular Science
Funding type	Research
Value (£)	749,033
Status	Completed
Type	Research Grant
Start date	15/08/2021
End date	31/12/2022
Duration	17 months

Abstract

Steroid hormones are vital for health and underpin disease. Glucocorticoids modulate inflammation and stress. Sex hormones fine-tune reproduction and drive sex differences in health. Vitamin D maintains healthy bones and growth. Here we introduce high resolution mass spectrometry (MS) to improve specificity and throughput of hormone profiling. MS offers gold-standard steroid measures allowing absolute quantitation, high sensitivity and confident identification. Increasingly targeted, multi-hormone profiles are preferred to single hormone assays and we are globally recognised for bespoke profiles of adrenal and gonadal steroids, and Vitamin D. We aim to profile in high-throughput for population studies and also in low sample volume in model organisms. However, as profiles build and enrich, we face significant challenges in retaining analytical specificity with fast turnover. Overlap of mass spectra of active, inactive and isobaric hormones (e.g. natural stable isotopes) must be avoided. Our expertise ensures highly robust analysis but at the expense of time. Sometimes we must conduct 2 different analyses, using double sample volume, limiting in neonates and rodents, or apply arithmetic corrections. We increasingly support biobanks (e.g. COVID Recovery Trial), exceeding realistic throughput. We must attenuate the risk of error as we widen our use of profiling. High resolution MS offers orthogonal specificity, increasing confidence in identification within complex mixes. We will also operate faster by linking to multiplex chromatography. Thus we will improve throughput, reduce sample volumes, avoid multiple analyses and increase specificity against unknown confounders. New workflows will expedite and enhance internationally leading research into adrenal and gonadal steroids and Vitamin D in healthy ageing and the interface with lifestyle and diet in humans, animals and model organisms. We will no longer compromise breadth of profiling versus speed and sample size.

Summary

Hormones formed from cholesterol, known as "steroids", play critical roles in regulating stress, inflammation, metabolism, muscle and bone strength and fertility. Understanding how hormones control body functions is key to supporting life-long health, e.g. do hormones during pregnancy determine wellbeing during life and do they determine physical and mental frailty with age? Our team investigate nutrition and stress during life-stages e.g. pregnancy, childhood, menopause, ageing and study the differences in health of women vs men with age, e.g. protection against heart and lung disease and dementia. To gain insight into hormonal signalling, researchers must assess a wide array of molecules from large ones (like DNA and proteins) to small molecules, known as "metabolites", each with their own role to play. To measure small metabolites, we use machines called "mass spectrometers", which weigh molecules and can identify them. In the Edinburgh Mass Spectrometry Core, we focus on "targeted hormonal profiles" to allow researchers to investigate amounts and turnover of sets of closely related hormones in humans and animal models. Researchers interrogate hormone families of interest and our particular expertise lies in profiling steroid hormones. "Targeted profiling" of all members of the steroid hormone family, in excess of 40, offers a road map of how these hormones are activated and inactivated, in other words a "fingerprint" of health status. When a particular hormonal pathway is earmarked as vital to a body response, we can measure the building bricks used to create the hormones, the amounts of active hormones and the breakdown products all together, letting us know how quickly the body is making and removing essential hormones. We have gradually expanded the technology in our lab to encompass a hand-picked suite of instruments well matched to targeted hormonal profiling. We have refined pertinent hormonal screens and adapted measurements in adult humans to smaller volume samples to allow us to study babies and animal models with the same fidelity. However, in widening our view, we have rendered the profiles increasingly complex, bringing with it challenges. It is becoming harder for us to be sure we are not inadvertently measuring two or more closely related hormones at the same time by mistake. We can use a technique called "chromatography" to spread out the steroids to limit interference. Indeed we are skilled at this, but as complexity rises this approach causes longer analysis times and slows down lab productivity. In the current era of large population studies, we must achieve highly specific readouts but with faster throughput. Therefore we are applying to install a new "high resolution" mass spectrometer to support high-turnaround and high quality hormonal profiling. This system will allow us to measure weights of hormones more accurately so we can confidently work in a faster timeframe. We will also upgrade to include automated sample preparation. We currently inject a sample into the instrument, perform our analysis and then wait while the system stabilises before moving onto the next sample. With the new instrument we will operate two injectors in tandem, which will allow us to switch between them and thus run twice as fast. One will operate while the other stabilises and resets and vice versa. We will be able to measure more complex mixtures in single experiments, faster. This will be a big step forward from our currently set-up where we have to analyse the sample several times, often consuming more sample than is routinely available from infants and animals. We will initially roll-out projects related to early-life stress in babies, and effects of gender on lung health and growth in humans and animals, including mice, sheep and quail. We will make the approaches we develop available to support many University researchers and industries and we will communicate our findings quickly to scientists and the public.

Committee	Not funded via Committee
Research Topics	Animal Health, The 3 Rs (Replacement, Reduction and Refinement of animals in research)
Research Priority	X – Research Priority information not available
Research Initiative	Advanced Life Sciences Research Technology Initiative (ALERT) [2013-2014]
Funding Scheme	X – not Funded via a specific Funding Scheme

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