or 24-hydroxylated. Like the lactones, 1,24,25(OH)3D has substantial affinity for the VDR and so has biological activity. There may also be a physiologic role for 24,25(OH)2D in the growth plate in that both 1,25(OH)2D and 24,25(OH)2D appear to be required for optimal endochondral bone formation [26]. Inactivating mutations in CYP24A1 have been found in children with idiopathic infantile hypercalcemia and more recently in adults who present with severe hypercalcemia, hypercalciuria, and nephrocalcinosis with decreased PTH, low 24,25(OH)2D, and inappropriately normal to high 1,25(OH)2D [27, 28]. Measuring the ratio of 24,25(OH)2D:25(OH)D has proven useful in diagnosing these cases. Regulation of CYP24A1is the reciprocal of that of CYP27B1 at least in the kidney in that PTH inhibits but FGF23 stimulates its expression. However, in osteoblasts, PTH enhances 1,25(OH)2D induction of CYP24A1 transcription [29], illustrating the fact that regulation of these vitamin D metabolizing enzymes is cell specific. That said in essentially all cells in which it is expressed, CYP24A1 is strongly induced by 1,25(OH)2D, and often serves as a marker of 1,25(OH)2D response in that cell.
3-Epimerase. 3-Epimerase activity was first identified in the keratinocyte, which produces large amounts of the C-3-epi form of 1,25(OH)2D [30]. It has also been identified in a number of other cells but not in the kidney [31]. The enzyme per se has not yet been purified and sequenced, so it is not clear that one gene product is involved. The 3-epimerase isomerizes the C-3 hydroxy group of the A ring from the alpha to beta orientation of all natural vitamin D metabolites. This does not restrict the action of CYP27B1 or CYP24A1. However, the C-3 beta epimer of 25(OH)D has reduced binding to DBP relative to 25(OH)D, and the C-3 beta epimer of 1,25(OH)2D has reduced affinity for the VDR relative to 1,25(OH)2D [32], thus reducing its transcriptional activity and most biologic effects [32]. Surprisingly, however, it is equipotent to 1,25(OH)2D with respect to PTH suppression [33]. Clinically, interest in the C-3 epimerase arises because the C-3 beta epimer of the vitamin D metabolites is not readily distinguished from their more biologically active alpha epimers by LC/mass spectrometry unless special chromatographic methods to separate the epimers prior to mass spectrometry are employed. Thus, the measurement of these metabolites using standard LC/mass spectroscopic procedures results in a value increased above true levels of the C-3 alpha epimers to the extent that the sample contains the C-3 beta epimer. Immunoassays by and large do not recognize the C-3 beta epimer and so are not affected [34]. This issue is particularly important in assessing 25(OH)D levels in infants where levels of the C-3 beta epimer of 25(OH)D can equal or exceed that of the C-3 alpha epimer of 25(OH)D [31]. However, levels in adults can also be substantial [31]. Given that the C-3 beta epimer does have biologic activity and that the epimers can be separated prior to mass spectrometry, there may be justification for measuring both epimers to provide a more complete picture of vitamin D status at least in future research protocols or when assessing infant samples. At this point it is not clear whether the cost/benefit of such additional effort justifies its application to adult samples measured routinely. Unless otherwise stated, reference to the vitamin D metabolites without stipulating which epimer implies the C-3 alpha epimer.
CYP11A1. Recently an alternative pathway for vitamin D metabolism at least in keratinocytes has been identified, namely, 20-hydroxylation of vitamin D by CYP11A1, the side chain cleavage enzyme essential for steroidogenesis [35]. The product, 20OHD, or its metabolite 20,23(OH)2D, appears to have activity similar to that of 1,25(OH)2D at least for some functions [35]. At this point, the measurement of these metabolites is not commercially available and not further discussed in this review
Assays for Vitamin D and Its Metabolites
Vitamin D
Vitamin D is seldom measured in the blood clinically, but methods for its analysis have been developed for the food industry. Vitamin D is the most lipophilic of the compounds we will consider. Relative to 25(OH)D it has reduced affinity for DBP, and is cleared within hours from the blood, presumably deposited into fat tissues. Typical methods include saponification of the sample with organic extraction and high performance liquid chromatography (HPLC) to separate D2 and D3. These peaks are detected and quantitated by UV absorption with a diode array detector (LC-UV) or mass spectrometry (LC-MS) [36]. The latter detection system is more specific and less sensitive to interfering substances, but ionization of vitamin D required for MS is limited thus affecting precision. This plus lack of a universal standard has resulted in wide variation between laboratories in measuring vitamin D. More recently, however, the situation has improved with the use of a triple quadrupole tandem mass spectrometer equipped with an atmospheric pressure photo ionization source that enhances the ionization of D versus earlier chemical methods (atmospheric pressure chemical ionization or APCI and electrospray ionization [ESI]) [37]. This method was used to measure D2, D3, 25(OH)D2, and 25(OH)D3 in the same sample with limits of quantitation (LOQ) for each D of 2 ng/mL and for each 25(OH)D of 1 ng/mL [38].
25-Hydroxyvitamin D
The accurate measurement of 25(OH)D for the assessment of vitamin D status has been the major goal for most commercial laboratories measuring vitamin D metabolites for a good reason. Levels of 25(OH)D in the blood are higher than those of any other vitamin D metabolite, and most of the 25(OH)D in the body is found in the blood stream with limited distribution into less accessible depots like fat (unlike vitamin D). Its level in blood is the best indicator of vitamin D nutritional status because of its relatively long half life in the blood stream and first-order kinetics in which the rate of 25(OH)D production is dependent on vitamin D levels. Testing for 25(OH)D levels has soared over the past several years [39] driven by the appreciation that vitamin D deficiency may be contributing to a number of disease states [1]. This has generated the need for high throughput assays done in specialized laboratories. However, the disparity of results from one laboratory to the next, or one method to another has been a problem. But now many laboratories are part of the Vitamin D External Quality Assessment Scheme in which these laboratories report their results using defined standards from the National Institute of Standards and Technology (NIST). These standards currently include known concentrations of 25(OH)D2, 25(OH)D3, 3-epi 25(OH)D3, 24S,25(OH)2D3, and 24R,25(OH)2D3 [34]. There are 3 general types of assays currently in use today: competitive protein binding assay (CPBA) and immunoassays, LC-UV and LC-MS. LC-MS is becoming the gold standard and is gradually replacing the CPBA and immunoassays, although immunoassays remain the dominant method in use today [40]. However, each method has its advantages and disadvantages.
CPBA. This was the first