This post is part of a series on bone markers standardization from the Committee on Bone Metabolism of the International Federation of Clinical Chemistry.

Vitamin D represents a group of over 50 cholesterol derived secosteroid species that has an essential role in calcium homoeostasis and is also involved in many other physiological processes, such as immune function, cell proliferation and differentiation.

(1). In recent years, the growing awareness that vitamin D deficiency is a highly prevalent condition has stimulated enormous interest in the measurement of 25(OH)D as a surrogate marker of vitamin D status. However, our knowledge about vitamin D metabolism and its assessment in clinical practice still harbours substantial limitations

(2). For example, significant correlations between serum 25(OH)D concentrations, bone mineral density (BMD), bone loss and fracture risk have been shown by some but not all studies (3-5). These inconsistencies have prompted researchers to look for alternative markers that provide additional information about patients’ vitamin D status. 24,25(OH)2D, the major catabolite of 25(OH)D, has emerged as a particularly promising candidate. Both metabolites, 25(OH)D and 24,25(OH)2D are strongly correlated [12] and can reliably be measured by LC-MS/MS (6-11). Recently, the National Institute of Standardization (NIST) has developed a candidate reference measurement procedure for 24,25(OH)2D based on isotope dilution LC-MS/MS, which is recognized as reference measurement procedure by the Joint Committee for Traceability in Laboratory Medicine (JCTLM). In addition, 24,25(OH)2D is included in the DEQAS program, which allows laboratories to assess the accuracy of their results.

Due to the tight relationship between 25(OH)D and 24,25(OH)2D the isolated measurement of 24,25(OH)2D does not add clinical value beyond 25(OH)D. However, the simultaneous assessment of both metabolites with the results expressed as a ratio between 24,25(OH)2D and 25(OH)D (known as vitamin D metabolite ratio; VMR) seems to provide a dynamic measure of vitamin D metabolism that allows distinguishing CYP24A1 deficiency or mutations (12, 13) from vitamin D intoxication and granulomatous disease (14). It has also been speculated that the VMR might aid the detection of vitamin D deficiency in individuals without the aforementioned conditions and is not influenced by ethnicity (15) . Theoretically, a higher VMR should indicate a better supply with vitamin D so that excessive 25(OH)D is catabolized to 24,25(OH)2D. However, this concept is hampered by some technical issues. When 24,25(OH)2D is below the limit of quantitation the VMR cannot be calculated. Furthermore, calculating the ratio of two measurands with substantially different concentrations enhances the intrinsic measurement uncertainty. Previous studies that have investigated the clinical utility of VMR yielded inconclusive results (14, 16-18). Recently, Cavalier and colleagues proposed a new way of interpreting 24,25(OH)2D results (19). Instead of calculating the VMR they compared the 24,25(OH)2D and 25(OH)D concentrations of patients with those of healthy subjects classified according to their 25(OH)D concentration. The principal assumption of Cavalier et al. is that low 24,25(OH)2D concentrations have a different meaning in the context of high or low 25(OH)D. In the context of vitamin D deficiency CYP24A1 is expected to be down regulated so that 25(OH)D is not wasted. This concept implies that with lower 25(OH)D concentrations undetectable

24,25(OH)2D concentrations are increasingly likely and most probably indicate functional vitamin D deficiency. In turn, when 25(OH)D is high, the organism aims to protect itself against hypercalcemia by eliminating excessive amounts of 25(OH)D through 24-hydroxylation. As a result, undetectable 24,25(OH)2D concentrations are highly unlikely in this context and would rather suggest an enzyme defect than vitamin D deficiency. Reporting both metabolites together with the probability that this constellation occurs in healthy subjects might facilitate a better judgement of patient’s vitamin D status and enable physicians to target vitamin D supplementation to those who are expected to benefit most. This approach would also support the concept that every person has an individual set point above which vitamin D supplementation has no beneficial effects. So far, the possibility to identify individuals with functional vitamin D deficiency on the basis of probable and unprobable constellations of 24,25(OH)2D and 25(OH)D has only been shown in one study that includes 1200 Belgian children and adolescents (19). Therefore, additional studies are needed to confirm these result and to extend them to adults and elderly individuals.


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15. Berg AH, Powe CE, Evans MK, Wenger J, Ortiz G, Zonderman AB, Suntharalingam P, Lucchesi K, Powe NR, Karumanchi SA, Thadhani RI. 24,25-Dihydroxyvitamin D3and Vitamin D Status of Community-Dwelling Black and White Americans. Clin Chem 2015;61(6):877-84.

16. Kaufmann M, Gallagher JC, Peacock M, Schlingmann KP, Konrad M, DeLuca HF, Sigueiro R, Lopez B, Mourino A, Maestro M, St-Arnaud R, Finkelstein JS, Cooper DP, Jones G. Clinical utility of simultaneous quantitation of 25-hydroxyvitamin D and 24,25-dihydroxyvitamin D by LC-MS/MS involving derivatization with DMEQ-TAD. J Clin Endocrinol Metab 2014;99:2567–74.

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19. 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. Clin Chem Lab Med. 2020;58(2):197-201.