engleski

Analytical challenges in determining vitamin D

General acceptance of serum total 25-hydroxyvitamin D (25(OH)D) as the best biomarker of an individual’s vitamin D status has resulted in the development of several specific and sensitive commercial assays over the past 20 years. Its hydrophobic nature, small circulating concentrations, ability to bind to lipids and vitamin D binding proteins (VDBP), and presence of multiple vitamin D metabolites in bloodstream have defined 25(OH)D as a “difficult analyte”. Precise and accurate measurement of 25(OH)D concentrations is challenging, and large variations exist between different assay methodologies. Such variations depend on several factors: different methods of vitamin D extraction and deproteinization, antibody cross-reactivity with epimers and/or other vitamin D metabolites, and presence of isobaric compounds or matrix interferences. Assays based on liquid chromatographic techniques (LC) or mass spectrometry (MS) are more accurate than the automated chemiluminescent immunoassay-based methods. Since the above mentioned methods are usually based on achiral chromatographic techniques, they cannot distinguish between 25(OH)D3 and its 3-epimer, or other isobaric compounds (e.g. 7-α-hydroxy-4-cholesten-3-one, an endogenous precursor of bile acids) resulting in a slight overestimation of concentrations. Currently isotope-dilution LC/MS-MS is considered the gold standard method. The large discrepancies between LC-MS methods and immunoassays, but also among different immunoassay methods, are mainly due to differences in cross-reactivity with various vitamin D metabolites, which accounts for a significant proportion of total 25(OH)D. Although immunoassays do not detect 3-epi-25(OH)D3, generating specific antibodies against small antigenic molecules such as 25(OH)D is difficult, and cross-reactivity with 24, 25(OH)2D3 and other vitamin D metabolites is common. While some immunoassays cannot detect 25(OH)D2, those that can are unable to distinguish between 25(OH)D2 and 25(OH)D3. Strong binding between the lipophilic 25(OH)D and VDBP creates competition with the capturing antibody in the assays where 25(OH)D and VDBP are not completely separated. Furthermore, the discordance between 25(OH)D values from different assays is magnified by differences in standardization of each assay. As the number of 25(OH)D methods increases, it will be essential to carefully standardize and harmonize all available methods to enable the development and use of evidencebased guidelines. Fortunately, the Vitamin D Standardization Program (VDSP) is making substantial progress towards both goals.

25-hydroxyvitamin D ; LC-MS ; Immunoassay ; Cross-reactivity ; Standardization

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