Development and Application of Mass Spectrometry Methods for Analysis of Optimal Vitamin D

Project Information

OPTIMAL-D

Grant agreement ID: 840567

DOI

10.3030/840567

Project closed

EC signature date 11 April 2019

Start date 15 October 2019

End date 15 April 2023

Funded under

• EXCELLENT SCIENCE - Marie Skłodowska-Curie Actions

Total cost

€ 277 940,16

EU contribution

€ 277 940,16

Coordinated by

THE UNIVERSITY OF BIRMINGHAM
United Kingdom

Periodic Reporting for period 2 - OPTIMAL-D (Development and Application of Mass Spectrometry Methods for Analysis of Optimal Vitamin D)

Reporting period: 2021-10-15 to 2022-10-14

Summary of the context and overall objectives of the project

Vitamin D deficiency is highly prevalent across European communities and globally. Deficiency of vitamin D is linked with multiple health and social issues across all communities, including bone health and immune functions. Despite the clear importance of optimal vitamin D levels, analysis of vitamin D status and deficiency has continued to rely on measurements of a single inactive vitamin D precursor metabolite; 25-hydroxyvitamin D as the sole biomarker. However, vitamin D undergoes complex metabolism, transport and distribution with many metabolites formed throughout its metabolic pathways that have varying biological actions, or unknown actions. OPTIMAL-D aims to establish state-of-the art mass spectrometry analytical methods to characterise vitamin D status in human populations across different health settings, and identify unique biomarkers of vitamin D linked with health and disease. The project outcomes are expected to lead to important benefits for society by reducing the possible health implications of vitamin D deficiency.

The outputs from OPTIMAL-D include the development of novel liquid chromatography tandem mass spectrometry methods to simultaneously measure multiple vitamin D metabolites across different biological sample matrices. Translational applications of these methods to health and population studies have achieved novel reference levels and provided context to under investigated vitamin D pathways. This includes analysis revealing that conjugated forms of vitamin D, including 25-hydroxyvitamin D circulate at high concentrations that may have important biological storage and reservoir functions. The application of mass spectrometry methods to observational cohorts have revealed differences in circulating vitamin D metabolism following supplementation of vitamin D. Furthermore, different associations have been found between individual vitamin D metabolites and skeletal health during ageing.

The overall conclusions from these findings have highlighted the complex metabolic pathways and the impact of vitamin D sufficiency and deficiency across different population groups and health settings. Further conclusions are that the biological impact of vitamin D can be more accurately assessed by integrating multiple metabolite measurements beyond 25-hydroxyvitamin D. The developed and validated analytical methods established from this project are expected to contribute towards future healthcare tools to monitor vitamin D and guide future policies for supplementation strategies for reducing deficiency and to optimise vitamin D treatment in individuals.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Multiple outcomes have been achieved throughout the project, including novel analytical method developments to measure multiple phase I and phase II vitamin D metabolites, alongside their translational applications for the analysis of vitamin D metabolism in human observational studies. Liquid chromatography tandem mass spectrometry (LC-MS/MS) methods have been developed and validated, including a method for the simultaneous quantitation of 13 mono- and di-hydroxy metabolites across different metabolic pathways in the circulation. A further LC-MS/MS method combined with enzyme hydrolysis has been developed for the quantitation of four phase II conjugated metabolite forms in the circulation. These methods have been used to perform vitamin D metabolite measurements in different biological settings in order to compare circulating, excretory (urine and bile) and tissue metabolism. LC-MS/MS method parameters have also been established for the quantitation of the vitamin D transporter protein (vitamin D binding protein).

The developed LC-MS/MS methods have been applied to the analysis of samples from multiple observational studies to investigate vitamin D metabolism beyond 25-hydroxyvitamin D measurements in different population groups. A key cohort incorporated into this analysis has been samples from the Concord Health and Ageing in Men Project (CHAMP) cohort that consists of males aged over 70. This analysis has revealed important metabolism profiles of vitamin D in older men that have not previously been reported, including the measurement of circulating metabolites derived from the CYP11A1 metabolism pathway of vitamin D. Different biomarkers were linked with markers of bone health across the entire body, highlighting that 1,25-dihydroxyvitamin D3 is the most significant biomarker associated with bone health in this cohort.

Analysis of phase II conjugated vitamin D metabolites in the circulation has revealed that sulfate conjugated vitamin D metabolites circulate in high abundance and at similar levels to their unconjugated counterparts with minimal amounts excreted in urine and bile. This analysis highlighted differences in the abundance of conjugated forms between individuals and their vitamin D supplementation status. A further observational study investigated changes in the metabolism of vitamin D during and after exercise, revealing short and long term differences between metabolites. These results may have significant implications for the assessment of vitamin D status as current methods do not incorporate these high abundant conjugate forms.

The project findings have been disseminated across multiple platforms including two open access journal publications in the Journal of Clinical Endocrinology and Metabolism, and Clinical Chemistry and Laboratory Medicine. Project results have also been disseminated through presentations at multiple conferences including the British Endocrine Society meeting. Ongoing exploitation of these findings include application notes describing the developed analytical methods and metabolism pathways, and how these findings can contribute to future policies for the assessing vitamin D status.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The overall results at the end of the project have significantly progressed analytical chemistry and vitamin D research. State of the art analytical methods have been described for multiple metabolite measurements of vitamin D, including reporting on novel sample preparation, separation and mass spectrometry methods. The project has characterised novel metabolism of vitamin D in the circulation, particularly in elderly populations, pregnancy and exercise samples. These results have greatly advanced our understanding of vitamin D by characterising metabolic profiles that have not previously been observed. Additionally, the impact of vitamin D supplementation in individuals on their overall vitamin D metabolome has provided important context for future vitamin D supplementation trials. The results from the project have also defined metabolite measurements in serum, urine and bile samples that have enabled comparisons to be made between circulating and excretory metabolism profiles.

The findings from the project have significant health and social impacts for the general public. A detailed characterisation of vitamin D metabolism and reference range concentrations can be widely incorporated by other vitamin D research and healthcare groups in order to establish novel policy and supplementation strategies for optimal vitamin D levels for health. This will have wide societal implications as improving vitamin D levels in population groups, in particular for vulnerable groups for vitamin D deficiency, will reduce the overall implications for health burden and healthcare costs associated with vitamin D deficiency.

LC-MS/MS chromatogram showing the separation of 13 vitamin D metabolite forms