Endocrinology; Endocrinology, Diabetes and Metabolism; Internal Medicine
Abstract :
[en] The measurement of vitamin D metabolites aids in assessing vitamin D status and in diagnosing disorders of calcium homeostasis. Most laboratories measure total 25(OH)D, while others have taken the extra effort to measure 25(OH)D2 and 25(OH)D3 separately and additional metabolites such as 1,25-dihydroxyvitamin D and 24,25-dihydroxyvitamin D. The aim of this review is to provide an updated overview of the main markers of vitamin D metabolism, define the intended measurands and discuss the advantages and disadvantages of the two most widely used assays, automated assays and LC-MS/MS. Whether using the easy and fast automated assays, or the more complex LC-MS/MS, one should know the pitfalls of the used technique in order to interpret the measurements. In conclusion, automated assays are unable to accurately measure 25(OH)D in all patient groups, including persons using D2. In these cases, a LC-MS/MS method, when appropriately developed and standardized, produces a more reliable measurement.
Disciplines :
Laboratory medicine & medical technology
Author, co-author :
Dirks, Niek F; N Dirks, Atalmedial Diagnostics Centre, Spaarne Gasthuis, Haarlem, Netherlands
Cavalier, Etienne ; Centre Hospitalier Universitaire de Liège - CHU > > Service de chimie clinique
Heijboer, Annemieke C; A Heijboer , Department of Clinical Chemistry, Endocrine Laboratory, Amsterdam UMC Locatie VUmc, Amsterdam, Netherlands
Amrein K, Scherkl M, Hoffmann M, Neuwersch-Sommeregger S, Köstenberger M, Tmava Berisha A, Martucci G, Pilz S & Malle O. Vitamin D deficiency 2.0: an update on the current status worldwide. European Journal of Clinical Nutrition 2020 74 1498–1513. (https://doi.org/10.1038/s41430-020-0558-y)
Christakos S, Li S, De La Cruz J & Bikle DD. New developments in our understanding of vitamin metabolism, action and treatment. Metabolism: Clinical and Experimental 2019 98 112–120. (https://doi.org/10.1016/j.metabol.2019.06.010)
Zhu JG, Ochalek JT, Kaufmann M, Jones G & Deluca HF. CYP2R1 is a major, but not exclusive, contributor to 25-hydroxyvitamin D production in vivo. PNAS 2013 110 15650–15655. (https://doi.org/10.1073/pnas.1315006110)
Shi M, Grabner A & Wolf M. Importance of extra-renal CYP24A1 expression for maintaining mineral homeostasis. Journal of the Endocrine Society 2021 5 A234–A234. (https://doi.org/10.1210/jendso/bvab048.476)
Lensmeyer G, Poquette M, Wiebe D & Binkley N. The C-3 epimer of 25-hydroxyvitamin D(3) is present in adult serum. Journal of Clinical Endocrinology and Metabolism 2012 97 163–168. (https://doi.org/10.1210/jc.2011-0584)
van den Ouweland JMW, Beijers AM & van Daal H. Overestimation of 25-hydroxyvitamin D3 by increased ionization efficiency of 3-epi-25-hydroxyvitamin D3. In LC-MS/MS methods not separating both metabolites as determined by an LC-MS/MS method for separate quantification of 25-hydroxyvitamin D3, 3-epi-25-hydroxyvitamin D3 and 25-hydroxyvitamin D2 in human serum. Journal of Chromatography. Part B 2014 967 195–202. (https://doi.org/10.1016/j.jchromb.2014.07.021)
Singh RJ, Taylor RL, Reddy GS & Grebe SKG. C-3 epimers can account for a significant proportion of total circulating 25-hydroxyvitamin D in infants, complicating accurate measurement and interpretation of vitamin D status. Journal of Clinical Endocrinology and Metabolism 2006 91 3055–3061. (https://doi.org/10.1210/jc.2006-0710)
Bailey D, Veljkovic K, Yazdanpanah M & Adeli K. Analytical measurement and clinical relevance of vitamin D(3) C3-epimer. Clinical Biochemistry 2013 46 190–196. (https://doi.org/10.1016/j.clinbiochem.2012.10.037)
Alonso N, Zelzer S, Eibinger G & Herrmann M. Vitamin D metabolites: analytical challenges and clinical relevance. Calcified Tissue International 2023 112 158–177. (https://doi.org/10.1007/s00223-022-00961-5)
Dirks NF, Ackermans MT, Lips P, Jongh RT de, Vervloet MG, Jonge R de & Heijboer AC. The when, what & how of measuring vitamin D metabolism in clinical medicine. Nutrients 2018 10 482. (https://doi.org/10.3390/nu10040482)
Holick MF. Vitamin D status: measurement, interpretation, and clinical application. Annals of Epidemiology 2009 19 73–78. (https://doi.org/10.1016/j.annepidem.2007.12.001)
Holick MF, Binkley NC, Bischoff-Ferrari HA, Gordon CM, Hanley DA, Heaney RP, Murad MH, Weaver CM & Endocrine Society. Evaluation, treatment, and prevention of vitamin D deficiency: an Endocrine Society clinical practice guideline. Journal of Clinical Endocrinology and Metabolism 2011 96 1911–1930. (https://doi.org/10.1210/jc.2011-0385)
Ross AC. The 2011 report on dietary reference intakes for calcium and vitamin D. Public Health Nutrition 2011 14 938–939. (https://doi.org/10.1017/S1368980011000565)
Makris K, Bhattoa HP, Cavalier E, Phinney K, Sempos CT, Ulmer CZ, Vasikaran SD, Vesper H & Heijboer AC. Recommendations on the measurement and the clinical use of vitamin D metabolites and vitamin D binding protein-A position paper from the IFCC Committee on bone metabolism. Clinica Chimica Acta; International Journal of Clinical Chemistry 2021 517 171–197. (https://doi.org/10.1016/j.cca.2021.03.002)
Dirks NF, Martens F, Vanderschueren D, Billen J, Pauwels S, Ackermans MT, Endert E, Heijer MD den, Blankenstein MA & Heijboer AC. Determination of human reference values for serum total 1,25-dihydroxyvitamin D using an extensively validated 2D ID-UPLC-MS/MS method. Journal of Steroid Biochemistry and Molecular Biology 2016 164 127–133. (https://doi.org/10.1016/j.jsbmb.2015.12.003)
Ginsberg C, Hoofnagle AN, Katz R, Becker JO, Kritchevsky SB, Shlipak MG, Sarnak MJ & Ix JH. The vitamin D metabolite ratio is independent of vitamin D binding protein concentration. Clinical Chemistry 2021 67 385–393. (https://doi.org/10.1093/clinchem/hvaa238)
Francic V, Ursem SR, Dirks NF, Keppel MH, Theiler-Schwetz V, Trummer C, Pandis M, Borzan V, Grübler MR, Verheyen ND, et al. The effect of vitamin D supplementation on its metabolism and the vitamin D metabolite ratio. Nutrients 2019 11 2539. (https://doi.org/10.3390/nu11102539)
Kaufmann M, Morse N, Molloy BJ, Cooper DP, Schlingmann KP, Molin A, Kottler ML, Gallagher JC, Armas L & Jones G. Improved screening test for idiopathic infantile hypercalcemia confirms residual levels of serum 24,25-(OH)2 D3 in Affected Patients. In Journal of Bone and Mineral Research 2017 32 1589–1596. (https://doi.org/10.1002/jbmr.3135)
Cavalier E, Huyghebaert L, Rousselle O, Bekaert AC, Kovacs S, Vranken L, Peeters S, Le Goff C & Ladang A. Simultaneous measurement of 25(OH)-vitamin D and 24,25(OH)2-vitamin D to define cut-offs for CYP24A1 mutation and vitamin D deficiency in a population of 1200 young subjects. Clinical Chemistry and Laboratory Medicine 2020 58 197–201. (https://doi.org/10.1515/cclm-2019-0996)
Schlingmann KP, Kaufmann M, Weber S, Irwin A, Goos C, John U, Misselwitz J, Klaus G, Kuwertz-Bröking E, Fehrenbach H, et al. Mutations in CYP24A1 and idiopathic infantile hypercalcemia. New England Journal of Medicine 2011 365 410–421. (https://doi.org/10.1056/NEJMoa1103864)
Bikle DD, Gee E, Halloran B, Kowalski MA, Ryzen E & Haddad JG. Assessment of the free fraction of 25-hydroxyvitamin D in serum and its regulation by albumin and the vitamin D-binding protein. Journal of Clinical Endocrinology and Metabolism 1986 63 954–959. (https://doi.org/10.1210/jcem-63-4-954)
Chun RF, Peercy BE, Orwoll ES, Nielson CM, Adams JS & Hewison M. Vitamin D and DBP: the free hormone hypothesis revisited. Journal of Steroid Biochemistry and Molecular Biology 2014 144 132–137. (https://doi.org/10.1016/j.jsbmb.2013.09.012)
Nielson CM, Jones KS, Chun RF, Jacobs JM, Wang Y, Hewison M, Adams JS, Swanson CM, Lee CG, Vanderschueren D, et al. Free 25-hydroxyvitamin D: impact of vitamin D binding protein assays on racial-genotypic associations. Journal of Clinical Endocrinology and Metabolism 2016 101 2226–2234. (https://doi.org/10.1210/jc.2016-1104)
Thienpont LM, Stepman HCM & Vesper HW. Standardization of measurements of 25-hydroxyvitamin D3 and D2. Scandinavian Journal of Clinical and Laboratory Investigation. Supplementum 2012 243 41–49. (https://doi.org/10.3109/00365513.2012.681950)
van der Vorm LN, Le Goff C, Peeters S, Makris K, Cavalier E & Heijboer AC. 25-OH vitamin D concentrations measured by LC-MS/ MS are equivalent in serum and EDTA plasma. Steroids 2022 187 109096. (https://doi.org/10.1016/j.steroids.2022.109096)
Wielders JPM & Wijnberg FA. Preanalytical stability of 25(OH)-vitamin D3 in human blood or serum at room temperature: solid as a rock. Clinical Chemistry 2009 55 1584–1585. (https://doi.org/10.1373/clinchem.2008.117366)
Colak A, Toprak B, Dogan N & Ustuner F. Effect of sample type, centrifugation and storage conditions on vitamin D concentration. Biochemia Medica 2013 23 321–325. (https://doi.org/10.11613/bm.2013.039)
Cavalier E. Long-term stability of 25-hydroxyvitamin D: importance of the analytical method and of the patient matrix. Clinical Chemistry and Laboratory Medicine 2021 59 e389–e391. (https://doi.org/10.1515/cclm-2021-0382)
Borai A, Khalil H, Alghamdi B, Alhamdi R, Ali N, Bahijri S & Ferns G. The pre-analytical stability of 25-hydroxyvitamin D: storage and mixing effects. Journal of Clinical Laboratory Analysis 2020 34 e23037. (https://doi.org/10.1002/jcla.23037)
Wyness SP & Straseski JA. Performance characteristics of six automated 25-hydroxyvitamin D assays: mind your 3s and 2s. Clinical Biochemistry 2015 48 1089–1096. (https://doi.org/10.1016/j.clinbiochem.2015.08.005)
Nguyen VTQ, Li X, Castellanos KJ, Fantuzzi G, Mazzone T & Braunschweig CA. The accuracy of vitamin D assays of circulating 25-hydroxyvitamin D values: influence of 25-hydroxylated ergocalciferol concentration. Journal of AOAC International 2014 97 1048–1055. (https://doi.org/10.5740/jaoacint.13-305)
Wise SA, Camara JE, Burdette CQ, Hahm G, Nalin F, Kuszak AJ, Merkel J, Durazo-Arvizu RA, Williams EL, Popp C, et al. Interlaboratory comparison of 25-hydroxyvitamin D assays: vitamin D standardization program (VDSP) intercomparison study 2 – part 2 immunoassays – impact of 25 hydroxyvitamin D2 and 24R,25-dihydroxyvitamin D3 on assay performance. Analytical and Bioanalytical Chemistry 2022 414 351–366. (https://doi.org/10.1007/s00216-021-03577-0)
Shu I, Pina-Oviedo S, Quiroga-Garza G, Meng QH & Wang P. Influence of vitamin D2 percentage on accuracy of 4 commercial total 25-hydroxyvitamin D assays. Clinical Chemistry 2013 59 1273–1275. (https://doi.org/10.1373/clinchem.2013.206128)
Carter GD, Jones JC, Shannon J, Williams EL, Jones G, Kaufmann M & Sempos C. 25-Hydroxyvitamin D assays: potential interference from other circulating vitamin D metabolites. Journal of Steroid Biochemistry and Molecular Biology 2016 164 134–138. (https://doi.org/10.1016/j.jsbmb.2015.12.018)
Heijboer AC, Blankenstein MA, Kema IP & Buijs MM. Accuracy of 6 routine 25-hydroxyvitamin D assays: influence of vitamin D binding protein concentration. Clinical Chemistry 2012 58 543–548. (https://doi.org/10.1373/clinchem.2011.176545)
Depreter B, Heijboer AC & Langlois MR. Accuracy of three automated 25-hydroxyvitamin D assays in hemodialysis patients. Clinica Chimica Acta; International Journal of Clinical Chemistry 2013 415 255–260. (https://doi.org/10.1016/j.cca.2012.10.056)
Cavalier E, Lukas P, Bekaert AC, Peeters S, Le Goff C, Yayo E, Delanaye P & Souberbielle JC. Analytical and clinical evaluation of the new Fujirebio Lumipulse®G non-competitive assay for 25(OH)-vitamin D and three immunoassays for 25(OH)D in healthy subjects, osteoporotic patients, third trimester pregnant women, healthy African subjects, hemodialyzed and intensive care patients. Clinical Chemistry and Laboratory Medicine: CCLM / FESCC 2016 54 1347–1355. (https://doi.org/10.1515/cclm-2015-0923)
Hawkes CP, Schnellbacher S, Singh RJ & Levine MA. 25-Hydroxyvitamin D Can interfere with a common assay for 1,25-dihydroxyvitamin D in vitamin D intoxication. Journal of Clinical Endocrinology and Metabolism 2015 100 2883–2889. (https://doi.org/10.1210/jc.2015-2206)
Wright MJP, Halsall DJ & Keevil BG. Removal of 3-epi-25-hydroxyvitamin D(3) interference by liquid chromatography-tandem mass spectrometry is not required for the measurement of 25-hydroxyvitamin D(3) in patients older than 2 years. Clinical Chemistry 2012 58 1719–1720. (https://doi.org/10.1373/clinchem.2012.191460)
Stepman HCM, Vanderroost A, Stöckl D & Thienpont LM. Full-scan mass spectral evidence for 3-epi-25-hydroxyvitamin D₃ in serum of infants and adults. Clinical Chemistry and Laboratory Medicine 2011 49 253–256. (https://doi.org/10.1515/CCLM.2011.050)
Dirks NF, Ackermans MT, Martens F, Cobbaert CM, Jonge R de & Heijboer AC. We need to talk about the analytical performance of our laboratory developed clinical LC-MS/MS tests, and start separating the wheat from the chaff. Clinica Chimica Acta; International Journal of Clinical Chemistry 2021 514 80–83. (https://doi.org/10.1016/j.cca.2020.12.020)
Phinney KW, Tai SS, Bedner M, Camara JE, Chia RRC, Sander LC, Sharpless KE, Wise SA, Yen JH, Schleicher RL, et al. Development of an improved Standard reference material for vitamin D metabolites in human serum. Analytical Chemistry 2017 89 4907–4913. (https://doi.org/10.1021/acs.analchem.6b05168)
Camara JE, Wise SA, Hoofnagle AN, Williams EL, Carter GD, Jones J, Burdette CQ, Hahm G, Nalin F, Kuszak AJ, et al. Assessment of serum total 25-hydroxyvitamin D assay commutability of Standard Reference Materials and College of American Pathologists Accuracy-Based Vitamin D (ABVD) Scheme and vitamin D External Quality Assessment Scheme (DEQAS) materials: Vitamin D Standardization Program (VDSP) Commutability Study 2. Analytical and Bioanalytical Chemistry 2021 413 5067–5084. (https://doi.org/10.1007/s00216-021-03470-w)
Sempos CT, Williams EL, Carter GD, Jones J, Camara JE, Burdette CQ, Hahm G, Nalin F, Duewer DL, Kuszak AJ, et al. Assessment of serum total 25-hydroxyvitamin D assays for vitamin D External Quality Assessment Scheme (DEQAS) materials distributed at ambient and frozen conditions. Analytical and Bioanalytical Chemistry 2022 414 1015–1028. (https://doi.org/10.1007/s00216-021-03742-5)
Benton SC, Tetteh GK, Needham SJ, Mücke J, Sheppard L, Alderson S, Ruppen C, Curti M, Redondo M & Milan AM. Evaluation of the 25-hydroxy vitamin D assay on a fully automated liquid chromatography mass spectrometry system, the Thermo Scientific Cascadion SM Clinical Analyzer with the Cascadion 25-hydroxy vitamin D assay in a routine clinical laboratory. Clinical Chemistry and Laboratory Medicine 2020 58 1010–1017. (https://doi.org/10.1515/cclm-2019-0834)
Jones G & Kaufmann M. Diagnostic aspects of vitamin D: clinical utility of vitamin D metabolite profiling. JBMR Plus 2021 5 e10581. (https://doi.org/10.1002/jbm4.10581)
Kaufmann M, Schlingmann KP, Berezin L, Molin A, Sheftel J, Vig M, Gallagher JC, Nagata A, Masoud SS, Sakamoto R, et al. Differential diagnosis of vitamin D-related hypercalcemia using serum vitamin D metabolite profiling. Journal of Bone and Mineral Research 2021 36 1340–1350. (https://doi.org/10.1002/jbmr.4306)
