Mass Spectrometric Profiling of Vitamin D Metabolites beyond 25-Hydroxyvitamin Dby M. J. Muller, D. A. Volmer

Clinical Chemistry

About

Year
2015
DOI
10.1373/clinchem.2015.241430
Subject
Clinical Biochemistry / Biochemistry, medical

Similar

Changes in circulating 25-hydroxyvitamin D according to vitamin D binding protein genotypes after vitamin D3 or D2 supplementation

Authors:
Hataikarn Nimitphong, Sunee Saetung, Suwannee Chanprasertyotin, La-or Chailurkit, Boonsong Ongphiphadhanakul
2013

Measurement of 25-Hydroxyvitamin D Revisited

Authors:
J. A. Schmidt
2006

Meal conditions affect the absorption of supplemental vitamin D 3 but not the plasma 25-hydroxyvitamin D response to supplementation

Authors:
Bess Dawson-Hughes, Susan S Harris, Nancy J Palermo, Lisa Ceglia, Helen Rasmussen
2013

Text

Mass Spectrometric Profiling of Vitamin D Metabolites beyond 25-Hydroxyvitamin D

Miriam J. Mu¨ller1 and Dietrich A. Volmer1*

BACKGROUND: The frequency of measurements of vitamin

D in the human population has significantly increased over the last decade because vitamin D has now been linked to many diseases, in addition to its established role in bone health. Usually, serum 25-hydroxyvitamin D concentrations are measured to assess the vitaminD status of individuals. Unfortunately, many studies investigating links between vitamin D and disease also use only this single metabolite. Intricate correlations with other vitamin Dmetabolites or dynamic effects of downstreammetabolitesmay therefore be overlooked. Fortunately, powerful LC-MS/MS approaches have recently become available that can simultaneously quantify the concentrations ofmultiple vitaminD metabolites.These approaches are challenging, however, because of inherent instrumental problems with detection of vitamin D compounds and the low concentrations of the metabolites in biological fluids.

CONTENT: This review summarizes recent mass spectrometry assays for the quantitative measurement of multiple vitamin D metabolites and their application in clinical research, with a particular focus on the lowabundance downstreammetabolic species generated after the initial hydroxylation to 25-hydroxyvitamin D.

SUMMARY: To study the pathobiological effects and function of vitamin D metabolites in disease, in particular in low-abundance species beyond 25-hydroxyvitamin D, we need to know their concentrations. Although detection of these vitamin D species is challenging, a number of recent mass spectrometry assays have successfully demonstrated that LC-MS/MS methods can quantify multiple vitamin D compounds over a wide dynamic range individually or as part of multimetabolite assays. © 2015 American Association for Clinical Chemistry

The term vitaminD describes a large class of secosteroids, of which the 2 most important representatives are vitamin D2 and D3. While chemically very similar, both vitamers have entirely different biological origins; whereas D3 is photosynthesized in mammals in their skin (1 ), D2 mainly occurs in mushrooms (1 ). Here we use the generic term vitamin D to describe the “human species” D3. D2 is, of course, also active in humans and is sometimes used as food supplement and, therefore, D2 is always specifically labeled in this review. Vitamin D is best known for its vital role in bone health (1 ), but it has been linked to a much wider range of diseases, including diabetes, cancer, multiple sclerosis, depression, and cardiovascular diseases (2 ).

Traditionally, the measurement of vitamin D compounds has been performed using methods such as immunoassays (3 ) or liquid chromatography (4 ); in fact, 90% of routine analyses today are performed by immunoassay techniques. In recent years, LC-MS/MS has been established as the gold standard technique for vitamin D analysis because of the technique’s inherent analytical specificity and sensitivity (5 ). Measurements of vitamin

D species from biological fluids such as plasma or serum using mass spectrometry are not without challenges, however, for several reasons, including the lipophilic nature of the analytes, their tight binding to the carrier protein (e.g., vitamin D binding protein [DBP]2), abundant isobaric and isomeric interferences in serum/plasma (6 ), and the low ionization efficiencies for mass spectrometric analyses (7 ). As a result, LC-MS/MS assays described in the literature for vitamin D compounds often permit only quantitative analysis of the main metabolite, 25-hydroxyvitamin D [25(OH)D], whereas detection capability is too low to access the required physiological concentrations of low-abundance metabolites such as 1 Institute of Bioanalytical Chemistry, Saarland University, Saarbru¨cken, Germany. * Address correspondence to this author at: Saarland University, Institute of Bioanalytical

Chemistry, D-66123 Saarbru¨cken, Germany. Fax +49-681-302-2963; e-mail dietrich.volmer@mx.uni-saarland.de.

Received March 24, 2015; accepted May 19, 2015.

Previously published online at DOI: 10.1373/clinchem.2015.241430 © 2015 American Association for Clinical Chemistry 2 Nonstandard abbreviations: DBP, vitamin D binding protein; 25(OH)D, 25hydroxyvitamin D; 1,25(OH)2D, active 1,25-dihydroxyvitamin D; 1-OHase, 25hydroxylase D-1-hydroxylase; FGF-23; fibroblast growth factor 23; 24-OHase, 25-hydroxyvitaminD-24hydroxylase; PP, protein precipitation; LLE, liquid–liquid extraction; SPE, solid phase extraction; MRM, multiple reaction monitoring; ESI, electrospray ionization; APCI, atmospheric-pressure chemical ionization; PTAD, 4-phenyl-1,2,4-triazoline-3,5-dione; 3-epi-1,25(OH)2D, C-3 epimer of 1,25(OH)2D; LLOQ, lower limit of quantification; UHPLC, ultra–high-performance liquid chromatography; c, Lin concordance correlation coefficient; LOD, limit of detection; C-18, cctadecyl; CN, cyano; IDS,

Immunodiagnostic Systems; PFP, pentafluorophenyl; HL-60, human promyelocytic leukemia cells; 25(OH)DS, vitaminD-3-sulfate; CKD, chronic kidney disease; FGF23, fibroblast growth factor 23; QqQ, triple quadrupole; DAPTAD, 4-(4’-dimethylaminophenyl)1,2,4-triazoline-3,5-dione; d-DAPTAD, d6- 4-(4’-dimethylaminophenyl)-1,2,4-triazoline3,5-dione; PBMC, peripheral blood mononuclear cell; APPI, atmospheric pressure photoionization.

Clinical Chemistry 61:8 1033–1048 (2015) Review 1033 1,25-dihydroxyvitamin D [1,25(OH)2D] in human serum or plasma.

Ideally, a true gold standard method for all vitamin

D metabolites would cover all relevant high- and lowabundance species, using a universal and standardized analytical method. Although no simple assay solution for this purpose presently exists, several promising techniques have been reported over the last few years that demonstrate the future potential of mass spectrometry for comprehensive profiling of vitamin D metabolites (8, 9 ). The role of LC-MS/MS in the determination of 25(OH)D has recently been reviewed by us (7 ). The present review focuses on the mass spectrometric analysis of low-abundance vitamin D species beyond 25(OH)D and its future role in profiling and applications as well as clinical metabolomics.