rel="nofollow" href="#ulink_8453b57e-20a8-5862-9e05-830b82ef3c83">14, 15]. One major extrarenal location of CYP27B1 is in epithelia including epithelial cells of the epidermis, intestine, mammary gland, lung, and prostate [16]. In epidermal keratinocytes tumor necrosis factor-α [17] and interferon-γ [18 ]are the major inducers of CYP27B1 activity although PTH also stimulates CYP27B1 but not through cAMP mediated mechanisms as in the kidney [19]. Immune cells likewise express CYP27B1 especially when activated, and like the keratinocyte CYP27B1 is induced by tumor necrosis factor-α and interferon-γ [20]. In these cells, PTH and calcium have little impact on CYP27B1 activity, and feedback regulation by 1,25(OH)2D is mediated indirectly by induction of CYP24A1 expression [21], a mechanism that is blunted in macrophages [22]. Thus, the measurement of 1,25(OH)2D is useful not only in renal disease and in diseases associated with too little or too much PTH, FGF23, calcium and phosphate but also in identifying diseases of extrarenal tissues in which production of 1,25(OH)2D is activated.
24-Hydroxylase. CYP24A1 is the only established 24-hydroxylase involved with vitamin D metabolism. This enzyme has both 24-hydroxylase and 23-hydroxylase activity, the ratio of which is species dependent [23]. The enzyme in humans has both capabilities, but the rat enzyme is primarily a 24-hydroxylase [24]. Mutating ala 326 to gly 326 in the human CYP24A1 shifts the profile from one favoring 24-hydroxylation to one favoring 23-hydroxylation [25]. The 24-hydroxylase pathway results in the biologically inactive calcitroic acid, whereas the 23-hydroxylase pathway produces the biologically active 25(OH)D-26,23-lactone and 1,25(OH)2D-26,23 lactone. All steps are performed by one enzyme [24]. 1,25(OH)2D is the preferred substrate relative to 25(OH)D, but both are 23 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
