= Emerging. More than 5 years before clinical availability. (19.79%)
= Expected to be clinically available in 1 to 4 years. (37.97%)
= Clinically available now. (42.25%)
MSACL 2022 : Kaufmann

MSACL 2022 Abstract

Self-Classified Topic Area(s): Metabolomics > Tox / TDM / Endocrine > Cases in Clinical MS

Podium Presentation in De Anza 1 on Thursday at 15:55 (Chair: Julie Ray / Amol Bajaj)

Clinical Utility of Measuring the Vitamin D Metabolome including 24,25-(OH)2D3 by LC-MS/MS

Martin Kaufmann and Glenville Jones
Queen's University, Kingston ON Canada

Martin Kaufmann (Presenter)
Queen’s University

Presenter Bio: Martin is a research associate at Queen’s University in Kingston, Ontario, Canada. Working with a multidisciplinary team of clinicians and basic scientists, Martin’s research involves the application of LC-MS/MS to the study of vitamin D metabolism, and role of in-born errors of vitamin D metabolism in hypercalcemic disorders. Other research interests include the use of REIMS and DESI to support tumor profiling studies, towards the development of diagnostic tools in the operating room and pathology lab. Martin has played an important role in establishing metabolomics facilities at Queen’s, and has been involved in the training of over 30 students in project-based courses involving mass spectrometry.


The advent of modern LC-MS/MS with its improved sensitivity, specificity and versatility has made it possible to simultaneously measure the levels of many more components of the vitamin D metabolome than just the hormone, 1,25-(OH)2D3 and its circulating precursor, 25-OH-D3. The addition of novel extraction procedures such an immuno-purification or selective solvent extraction, the use of specific cis-triene derivatizing agents such as DMEQ-TAD and the inclusion of special columns has made the study of vitamin metabolism much more sensitive down into the low picogram/mL range and use of 100-200 microlitres serum. We have used this technique on serum from a variety of mammalian species to study the vitamin D metabolome in human disease as well as knockout mice lacking specific vitamin D related genes. Furthermore, it is now possible to assay other metabolites than those judged to be biologically active, and thus clinically important.

We set out to monitor the full vitamin D metabolome including metabolites retaining potential biological activity such as 3-epi-25-OH-D3 and 1,24,25-(OH)3D3, as well as inactive products of the catabolic enzymes CYP24A1 (formerly 25-OH-D-24-hydroxylase) and CYP3A4 (non-specific drug-metabolizing P450) such as 24,25-(OH)2D3 or 4beta,25-(OH)2D3.

CYP24A1-derived metabolites including 24,25-(OH)2D3, 1,24,25-(OH)3D3 and 25-OH-D3-26,23-lactone are absent in idiopathic infantile hypercalcemia (IIH), due to mutations of CYP24A1 which results in hypercalcemia, nephrocalcinosis and renal stones in adults, as well as children. We have now studied hundreds of individuals worldwide for CYP24A1 mutations using a simple LC-MS/MS serum screening test and found it to be sensitive and 100% accurate. It uses the ratio of 25-OH-D3:24,25-(OH)2D3 to test for IIH, where those with biallelic mutations in CYP24A1 have ratios over 80, where the normal range in this ratio is 5-25.

Chronic Kidney Disease:
In our studies, we have found that CKD & vitamin D also cause decreases in serum 24,25-(OH)2D3 and moderate increases in the ratio of 25-OH-D3:24,25-(OH)2D3 (also known as VMR) which make the assay of clinical value in a variety of other conditions in addition to IIH. Nephrologists have recently recognized the potential of this ratio to follow the decline in renal function as develops through CKD Stages 1-5

Hypercalcemic conditions:
Serum elevations of CYP24A1-derived metabolites including 24,25-(OH)2D3, 1,24,25-(OH)3D3 and 25-OH-D3-26,23-lactone also occur in patients with a wide range of other non-IIH hypercalcemic conditions. The best known of these is hypervitaminosis D where toxic doses of vitamin D have been administered accidentally and hypercalcemia results. With the recent surge in the awareness of the possible beneficial effects of vitamin D supplements, over-consumption has become a problem in the general population. Studies of the complete vitamin D metabolome make it possible to try to pinpoint the toxic metabolite responsible for the hypercalcemia in hypervitaminosis D. This is likely not 1,25-(OH)2D3 which is suppressed. However, we note that some of these CYP24A1 catabolites increase significantly when vitamin D dependent-VDR-mediated gene transcription is upregulated making them very useful clinical biomarkers for the cause of hypercalcemia. It makes them of potential use in diseases involving calcium/phosphate metabolism such as Williams Syndrome, osteopenia and in any condition where high doses of vitamin D have been employed in therapy.

In summary, LC-MS/MS of the vitamin metabolome has advanced to the point that 24,25-(OH)2D3 can now be easily added to those routinely measured in clinically relevant situations. The added methodological requirements for assaying 24,25-(OH)2D3 include use of a commercially available, deuterated internal standard; using NIST reference materials; and employing external quality assessment through DEQAS. Some laboratories have been posting results from 24,25-(OH)2D3 assays quarterly on DEQAS over the past few years and analysts can now compare their results to posted target values determined by CDC-Atlanta. We encourage others interested in the vitamin D metabolome to explore the added clinical utility of assaying a wider range of vitamin D metabolites particularly 24,25-(OH)2D3 to aid in their diagnosis and help monitor therapy of patients.

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