Читать книгу Vitamin D in Clinical Medicine - Группа авторов - Страница 30

The 3 main steps in vitamin D metabolism, 25-hydroxylation, 1α-hydroxylation, and 24-hydroxylation are all performed by cytochrome P450 mixed function oxidases (CYPs) located either in the endoplasmic reticulum (e.g., CYP2R1) or in the mitochondrion (e.g., CYP27A1, CYP27B1, and CYP24A1).

25-Hydroxylase. The liver has been established as the major if not sole source of 25(OH)D production from vitamin D. Initial studies of the hepatic 25-hydroxlase found activity in both the mitochondrial and microsomal (endoplasmic reticulum) fractions. Subsequent studies demonstrated a number of CYPs with 25-hydroxylase activity. CYP27A1 is the only mitochondrial 25-hydroxylase. It was initially identified as a sterol 27-hydroxylase involved in bile acid synthesis. This CYP is widely distributed in the body, not just in the liver. It hydroxylates D₃ but not D₂. Moreover, its relevance to vitamin D metabolism has been questioned when its deletion in mice actually resulted in increased blood levels of 25(OH)D [9]. Moreover, inactivating mutations of CYP27A1 in humans cause cerebrotendinous xanthomatosis with abnormal bile and cholesterol metabolism but not rickets [10]. More recently, CYP2R1 was identified in the microsomal fraction of mouse liver [11]. This enzyme 25-hydroxylates both D₂ and D₃ with comparable kinetics, unlike CYP27A1. Its expression is primarily in the liver and testes. When CYP2R1 is deleted from mice, blood levels of 25(OH)D fall over 50% but not to zero [9]. Even the double deletion of CYP2R1 and CYP27A1 does not reduce the blood level of 25(OH)D to zero, and actually has little impact on blood levels of calcium and phosphate [9] suggesting compensation by other enzymes with 25-hydroxylase activity. However, mutations in CYP2R1 have been found in humans presenting with rickets, and these mutations decrease 25-hydroxylase activity when tested in vitro [12]. Although other enzymes including the drug metabolizing enzyme CYP3A4 have 25-hydroxylase activity and may have roles in different tissues or in different clinical conditions, CYP2R1 appears to be the major 25-hydroxylase contributing to circulating levels 25(OH)D. Regulation of vitamin D 25-hydroxylation is modest at best with production being primarily substrate dependent such that circulating levels of 25(OH)D are a useful marker of vitamin D nutrition. Moreover, it circulates in concentrations well above that of other metabolites facilitating its measurement.

1α-Hydroxylase (CYP27B1). Unlike 25-hydroxylation, there is only one enzyme recognized to have 25-OHD 1α-hydroxylase activity, and that is CYP27B1. Although the kidney is the main source of circulating 1,25-dihydroxyvitamin D (1,25[OH]₂D), a number of other tissues also express the enzyme, and the regulation of the extrarenal CYP27B1 differs from that of the renal CYP27B1(review in [13]). The renal 1α-hydroxylase is tightly regulated primarily by 3 hormones: parathyroid hormone (PTH), FGF23, and 1,25(OH₂D itself. PTH stimulates, whereas FGF23 and 1,25(OH)₂D inhibit CYP27B1. Elevated calcium suppresses CYP27B1 primarily through the suppression of PTH; elevated phosphate suppresses CYP27B1 primarily by stimulating FGF23, although these ions can have direct effects on renal CYP27B1 [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