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Before discussing the different assays, it is important to review both the production of vitamin D and its subsequent metabolism to its active metabolites, the measurement of which is the major focus of this review.
Vitamin D Production
Vitamin D3 (D3) (cholecalciferol) is produced from 7-dehydrocholesterol through a 2 step process in which the B ring is broken by ultraviolet light (UVB spectrum 280–320 nm) in sunlight, forming pre-D3 that isomerizes to D3 in a thermo-sensitive but a noncatalytic process. Both UVB intensity and skin pigmentation level contribute to the rate of D3 formation [4]. Melanin in the skin blocks UVB from reaching 7-dehydrocholesterol in the lower portions of the epidermis, thus limiting D3 production, as do clothing and sunscreen. The intensity of UVB from sunlight varies according to season and latitude, so the further one lives from the equator, the less time of the year one can rely on solar exposure to produce D3[5]. Vitamin D is also obtained from the food consumed. Most foods with the exception of fatty fish contain little vitamin D unless fortified. The vitamin D in fish is D3, whereas that used for fortification is often D2 (ergocalciferol). D2 is produced by UVB irradiation of ergosterol in plants and fungi (e.g., mushrooms). It differs from D3 in having a double bond between C22-C23 and a methyl group at C24 in the side chain. These differences from D3 in the side chain lower its affinity for DBP resulting in faster clearance from the circulation, alter its conversion to 25-hydroxyvitamin D (25[OH]D) by at least some of the 25-hydroxylases to be described, and alter its catabolism by the 24-hydroxyase (CYP24A1) [6–8]. Moreover, a number of immunoassays do not recognize the D2 metabolites as well as the D3 metabolites. However, the biologic activities of D2 and D3 metabolites are comparable, and if no subscript is used, both forms are meant.
Vitamin D Metabolism
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