Vitamin D is derived from dietary sources or from the action of ultraviolet light on 7-dehydrocholesterol and undergoes a number of enzymatic modifications that lead to the synthesis of active vitamin D metabolites or metabolites with reduced biological activity. Among these, epimerization at the 3-hydroxyl group leads to the synthesis of 3-epimer 25-hydroxyvitamin D (3EVD). Described first in biological system experiments using in vitro incubation of vitamin D in cell culture, this molecule has been reported as having distinct activities when compared with 25-hydroxy vitamin D (25OHVD). Measurements of vitamin D have been conducted using a variety of methodologies and have led to conflicting assessments of the quantities of 3EVD3 that are measured.
Method
The present article describes the development and use of a simple liquid chromatography–tandem mass spectrometry method validated by the Clinical Laboratory Improvement Amendments to quantitate 3EVD3 in 3528 subjects, including 309 children (162 are <2 years) and 232 pregnant women.
Results
Our findings demonstrate that, although 3EVD3 constitutes a significant proportion of measureable 25OHVD3 in subjects younger than 1 year, 3EVD3 levels are negligible in most subjects older than 1 year.
Conclusions
It is important to choose the correct 25OHVD assay dependent on the age of the patient. Patients younger than 1 year should be run on a liquid chromatography–tandem mass spectrometry assay proven to not have potential contributions from any 3EVD present in the sample.
HolickMF.Vitamin D deficiency. N Engl J Med. 2007; 357: 266–281.
2.
HolickMF.The vitamin D deficiency pandemic and consequences for nonskeletal health: mechanisms of action. Mol Aspects Med. 2008; 29: 361–368.
3.
HolickMF.Vitamin D: a d-lightful solution for health. J Investig Med. 2011; 59: 872–880.
4.
AdoriniL, PennaG.Control of autoimmune diseases by the vitamin D endocrine system. Nat Clin Pract Rheumatol. 2008; 4: 404–412.
5.
BouillonR, EelenG, VerlindenL, . Vitamin D and cancer. J Steroid Biochem Mol Biol. 2006; 102: 156–162.
6.
PlumLA, DeLucaHF.Vitamin D, disease and therapeutic opportunities. Nat Rev Drug Discov. 2010; 9: 941–955.
7.
BattaultS, WhitingSJ, PeltierSL, . Vitamin D metabolism, functions and needs: from science to health claims. Eur J Nutr. 2013; 52: 429–441.
8.
AranowC.Vitamin D and the immune system. J Investig Med. 2011; 59: 881–886.
9.
LiYC.Molecular mechanism of vitamin D in the cardiovascular system. J Investig Med. 2011; 59: 868–871.
10.
ChengJB, LevineMA, BellNH, . Genetic evidence that the human CYP2R1 enzyme is a key vitamin D 25-hydroxylase. Proc Nat Acad Sci U S A. 2004; 101: 7711–7715.
11.
LevineMA, DangA, DingC, . Tropical rickets in Nigeria: mutation of the CYP2R1 gene encoding vitamin D 25-hydroxylase as a cause of vitamin D dependent rickets. Bone. 2007; 40: S60–S61.
Al MutairA, NasratGH, RussellDW. Mutation of the CYP2R1 vitamin D 25-hydroxylase in a Saudi Arabian family with severe vitamin D deficiency. J Clin Endocrin Metab. 2012; 97: E2022–E2025.
14.
KatoS, YanagisawaJ, MurayamaA.The importance of 25-hydroxyvitamin D3 1α-hydroxylase gene in vitamin D-dependent rickets. Curr Opin Nephrol Hypertens. 1998; 7: 377–383.
15.
CarlbergC, CampbellMJ.Vitamin D receptor signaling mechanisms: integrated actions of a well-defined transcription factor. Steroids. 2013; 78: 127–136.
16.
MakinG, LohnesD, ByfordV, . Target cell metabolism of 1,25-dihydroxyvitamin D3 to calcitroic acid: evidence for a pathway in kidney and bone involving 24-oxidation. J Biochem. 1989; 262: 173–180.
17.
ReddyGS, TserngKY.Calcitroic acid, end product of renal metabolism of 1,25-dihydroxyvitamin D3 through C-24 oxidation pathway. Biochemistry. 1989; 28: 1763–1769.
18.
ReddyGS, MuralidharanKR, OkamuraWH, . Metabolism of 1a, 25-dihydroxyvitamin D3 and one of its A-ring diastereomer 1a, 25-dihydroxy-3-epivitamin D3 in neonatal human keratinocytes. In:NormanAW, BouillonR, ThomassetM, eds.Vitamin D A Pluripotent Steroid Hormone: Structural Studies, Molecular Endocrinology and Clinical Applications. New York, NY: Walter de Gruyter; 1994: 172–173.
19.
ReddyGS, Siu-CalderaM-L, SchusterI, . Target tissue specific metabolism of 1a, 25-dihydroxyvitamin D3 through A-ring modification. In:NormanAW, BouillonR, ThomassetM, eds.Vitamin D, Chemistry, Biology and Clinical Applications of the Steroid Hormone. Riverside, CA: University of California; 1997: 139–146.
20.
ReddyGS, MuralidharanKR, OkamuraWH, . Metabolism of 1alpha, 25-dihydroxyvitamin D(3) and its C-3 epimer 1alpha, 25-dihydroxy-3-epi-vitamin D(3) in neonatal human keratinocytes. Steroids. 2001; 66: 441–450.
21.
BrownAJ, RitterC, SlatopolskyE, . 1a, 25-Dihydroxy-3-epi-vitamin D3, a natural metabolite of 1a, 25-dihydroxyvitamin D3, is a potent suppressor of parathyroid hormone secretion. J Cell Biochem. 1999; 73: 106–113.
22.
Siu-CalderaML, SekimotoH, WeiskopfA, . Production of 1a, 25-dihydroxy-3-epi-vitamin D3 in two rat osteosarcoma cell lines (UMR 106 and ROS 17/2.8): existence of the C-3 epimerization pathway in ROS 17/2.8 cells in which the C-24 oxidation pathway is not expressed. Bone. 1999; 24: 457–463.
23.
BischofMG, Siu-CalderaM-L, WeiskopfA, . Differentiation-related pathways 1α,25-dihydroxycholecalciferol metabolism in human colon adenocarcinoma-derived caco-2 cells: production of 1α-25-dihydroxy-3-epi-cholecalciferol. Exp Cell Res. 1998; 241: 194–201.
24.
Siu-CalderaM-L, SekimotoH, BremAS, . Metabolism of 1a, 25-dihydroxyvitamin D3 into 1a, 25-dihydroxy-3-epi-vitamin D3 in rat osteosarcoma cells (UMR 106 and ROS 17/2.8) and rat vascular smooth muscle cells. J Bone Min Res. 1998; 25 (suppl): S426.
25.
KamaoM, TatematsuS, HatakeyamaS, . C-3 epimerization of vitamin D3 metabolites and further metabolism of C-3 epimers: 25-hydroxyvitamin D3 is metabolized to 3-epi-25-hydroxyvitamin D3 and subsequently metabolized through C-1alpha or C-24 hydroxylation. J Biol Chem. 2004; 279: 15897–15907.
26.
KamaoM, TatematsuS, SawadaN, . Cell specificity and properties of the C-3 epimerization of vitamin D3 metabolites. J Steroid Biochem Mol Biol. 2004; 89–9039–42.
27.
NormanAW, BouillonR, Farach-CarsonMC, . Demonstration that 1 beta, 25-dihydroxyvitamin D3 is an antagonist of the nongenomic but not genomic biological responses and biological profile of the three A-ring diastereomers of 1 alpha, 25-dihydroxyvitamin D3. J Biol Chem. 1993; 268: 20022–20030.
28.
MolnárF, SigüeiroR, SatoY, . 1 a, 25(OH)2-3-epi-vitamin D3, a natural physiological metabolite of vitamin D3: its synthesis, biological activity and crystal structure with its receptor. PLoS One. 2011; 6: e18124.
29.
RollinsR.Vitamin D testing—what's the right answer?Clin Lab News. 2009; 35 (7): 1.
30.
FraserWD, MilanAM.Vitamin d assays: past and present debates, difficulties, and developments. Calcif Tissue Int. 2013; 92: 118–127.
31.
CouchmanL, BentonCM, MonizCF.Variability in the analysis of 25-hydroxyvitamin D by liquid chromatography-tandem mass spectrometry: the devil is in the detail. Clin Chim Acta. 2013; 413: 1239–1243.
32.
PhinneyKW, BednerM, TaiSS, . Development and certification of a standard reference material for vitamin D metabolites in human serum. Anal Chem. 2012; 84: 956–962.
33.
SinghRJ, TaylorRL, SatyanarayanaR, . 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. J Clin Endocrinol Metab. 2006; 91: 3055–3061.
34.
StepmanHC, VanderroostA, StöcklD, . Full-scan mass spectral evidence for 3-epi-25-hydroxyvitamin D in serum of infants and adults. Clin Chem Lab Med. 2011; 49: 253–256.
35.
van den OuwelancJM, BeijersAM, van DaalH. Fast separation of 25-hydroxyvitamin D3 from 3-epi-25-hydroxyvitamin D3 in human serum by liquid chromatography-tandem mass spectrometry: variable prevalence of 3-epi-25-hydroxyvitamin D3 in infants, children, and adults. Clin Chem. 2011; 57: 1618–1619.
36.
SchleicherRL, EnciscoSE, Chaudhary-WebbM, . Isotope dilution ultra performance liquid chromatography-tandem mass spectrometry method for simultaneous measurement of 25-hydroxyvitamin D2, 25-hydroxyvitamin D3 and 3-epi-25-hydroxyvitamin D3 in human serum. Clin Chim Acta. 2011; 412: 1594–1599.
37.
StrathmannFG, SadilkovaK, LahaT, . 3-epi-25 hydroxyvitamin D concentrations are not correlated with age in a cohort of infants and adults. Clin Chim Acta. 2012; 413: 203–206.
38.
LensmeyerG, PoquetteM, WiebeD, . The C-3 epimer of 25-hydroxyvitamin D(3) is present in adult serum. J Clin Endocrinol Metab. 2012; 97: 163–168.
39.
BaecherS, LeinenbachA, WrightJA, . Simultaneous quantification of four vitamin D metabolites in human serum using high performance liquid chromatography tandem mass spectrometry for vitamin D profiling. Clin Biochem. 2012; 45: 1491–1496.
40.
BaileyD, VeljkovicK, YazdanpanahM, . Analytical measurement and clinical relevance of vitamin D(3) C3-epimer. Clin Biochem. 2013; 46: 190–196.
