The growing interest in vitamin D has stimulated intensive research activities aiming to address unresolved analytical, clinical and physiological aspects of vitamin D [1], [2], [3], [4]. This work has led to an increasing awareness that our knowledge about vitamin D metabolism and its assessment in clinical practice harbours substantial limitations. For example, Blacks have a markedly lower average 25(OH)D concentration than Whites [2], [5], [6], but exhibit higher bone mineral density (BMD) and a lower risk of fragility fracture [7], [8], [9]. Also, the relationship between 25(OH)D and parathyroid hormone (PTH) seems to differ between races [2]. These findings have led researchers to look for other markers that are capable of providing more accurate information about the adequacy of patients’ vitamin D supply. Several studies suggested that free and bioavailable 25(OH)D reflect vitamin D metabolism better than 25(OH)D [2], [10], [11]. However, both markers require the measurement of vitamin D binding protein (VDBP). Early studies quantified VDBP with either monoclonal or polyclonal immunoassays. However, later studies that employed LC-MS/MS based methods have demonstrated that these immunoassay are strongly biased due to common genetic polymorphisms [4]. The limited number of laboratories that offer VDBP measurement by LC-MS/MS and the lack of a reference measurement procedure hamper a wider use of free and bioavailable 25(OH)D in clinical studies. Another potential surrogate marker of vitamin D metabolism is 24,25(OH)2D, the major product of 25(OH)D catabolism. The circulating concentrations of both metabolites are strongly correlated [12] and can reliably be measured by LC-MS/MS [13], [14], [15], [16]. The simultaneous quantitation of 24,25(OH)2D and 25(OH)D has been proposed as a dynamic measure of vitamin D metabolism that allows distinguishing CYP24A1 deficiency from vitamin D intoxication and granulomatous disease. However, the interpretation of 25(OH)D and 24,25(OH)2D results is still a matter of intensive debate. Previous studies have established reference intervals [17], [18] and clinical cut-offs [19], [20], [21], [22]. However, the close relationship between 25(OH)D and 24,25(OH)2D implies that a meaningful interpretation is only possible when both metabolites are considered together. This has led to the idea of a ratio between 24,25(OH)2D and 25(OH)D, also known as vitamin D metabolite ratio (VMR) [23]. Theoretically, a higher VMR indicates better supply with vitamin D so that excessive 25(OH)D is catabolized to 24,25(OH)2D. Several studies have investigated the clinical utility of VMR, but results are inconclusive [3], [24], [25], [26]. In addition, the VMR cannot be calculated when 24,25(OH)2D is below the limit of quantitation. When one measurand has a much lower concentration than the other, calculating the ratio between the two enhances the intrinsic measurement uncertainty. In this issue of Clinical Chemistry and Laboratory Medicine (CCLM) a study by Cavalier et al. has analyzed 24,25(OH)2D and 25(OH)D simultaneously in 1200 samples from children, adolescents and young adults [27]. Instead of calculating the VMR the authors propose to compare 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. They assume that a low or undetectable 24,25(OH)2D concentration has a different meaning in the context of high or low 25(OH)D. Theoretically, a vitamin D-deficient patient cannot afford to waste 25(OH)D and CYP24A1 is down regulated. Consequently, little or no 24,25(OH)2D is produced. According to Cavalier et al., 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. Cavalier et al. suggest that in clinical practice the concentrations of 24,25(OH)2D and 25(OH)D should be reported together with the probability that this constellation occurs in healthy subjects. This information would help physicians judging their patients’ metabolic status in a more dynamic fashion and leave the historical concept of vitamin D deficiency on the basis of a universal 25(OH)D cut-off [19], [20], [21], [22]. With the established 25(OH)D cut-offs a large portion of the population has vitamin D deficiency or at least insufficiency, which, in many cases, would trigger vitamin D supplementation even in the absence of risk factors for metabolic bone disease or manifest osteoporosis [20], [28], [29]. Although robust evidence is lacking, some clinicians and researchers believe that every person has an individual set point above which vitamin D supplementation has no beneficial effects. In many persons this set point is probably below the commonly used cut-off of 50 nmol/L for sufficiency [28]. The interpretation of 24,25(OH)2D and 25(OH)D proposed by Cavalier et al. allows an individual evaluation of patients’ metabolic situation.
The data presented by Cavalier show that amongst individuals with a 25(OH)D concentration above 52 nmol/L over 99% exhibit detectable amounts of 24,25(OH)2D and thus are probably vitamin D sufficient. This finding supports the 50 nmol/L cut-off recommended by the IOF [30]. Finally, this study represents a valuable data set that provides a robust overview about the vitamin D status in Belgian infants, children, adolescents and adults. Of note, more than 50% of these individuals have 25(OH)D concentrations <50 nmol/L. This finding is critical as vitamin D deficiency at this age may interfere with bone growth and mineralization. A particular strength of this study is that analyses have been performed with a VDSP certified LC-MS/MS method.
In summary, the study by Cavalier et al. is a nice example of personalized medicine and may trigger similar approaches for other analytes, such as B-vitamins and homocysteine. It also highlights the valuable contribution that laboratory doctors can provide for patient care.
Author contributions: The author has accepted responsibility for the entire content of this submitted manuscript and approved submission.
Research funding: None declared.
Employment or leadership: None declared.
Honorarium: None declared.
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