Vitamin D3-25-Hydroxylase

Akiyoshi-Shibata, M., E. Usui, T. Sakaki, Y. Yabusaki, M. Noshiro, K. Okuda et al. (1991). Expression of rat liver vitamin D3 25-hydroxylase cDNA in Saccharomyces cerevisiae. FEBS Lett. 280, 367-370. 

Russell and his associates [1183] first cloned mouse P450 2R1 in a search for a liver microsomal vitamin D3 25-hydroxylase. The mRNA is abundant in liver and testis of mice and was also identified (mice) in kidney, brain, epididymis, skin, heart, muscle, and spleen [1183]. In humans, a similar mRNA profile was reported [1186], with the highest levels in testis, followed by pancreas, and then the tissues reported by Cheng et al. [1183], including liver. Thus, P450 2R1 mRNA is expressed in many tissues. Protein detection has not been reported. 

Cloning, structure, and expression of a cDNA encoding vitamin D3 25-hydroxylase. Biochem Bio- 2553. phys Res Commun 241 491-497 Hayashi S, Noshiro M, Okuda K (1984) Purification of cytochrome P-450 catalyzing 25-hydrox-ylation of vitamin Dj from rat liver microsomes. 

Attempts to demonstrate 25-hydroxy-vitamin D3-l-hydroxylase activity in vitro with rat kidney homogenates have been unsuccessful, although chick kidney preparations exhibit such activity. A heat-labile and very potent inhibitor of the hydroxylase has now been found in the rat preparation 322 all fractions of the kidney homogenate contained the factor, but the microsomes were the richest source, and they released the inhibitor during incubation. A similar inhibitor is also present in rat intestine and serum and in pig kidney, and it may well play a regulatory role in the synthesis of 1,25-dihydroxy-vitamin D3.323 Direct spectroscopic and inhibitory evidence for the presence of cytochrome P450 in kidney mitochondria and of its... 

OH Vitamin D3-24-hydroxylase gene structure Rat gene 15kb, 12 exons... 

Vitamin D2 and vitamin D3 are metabolized to 2 5-hydroxy-vitamin D [25(OH)D] by vitamin D-25-hydroxylase, a cytochrome P450 enzyme, in liver (Figure 49. 

Derived from cholesterol, vitamin D is biosynthesized from its prohormone cholecalciferol (Ds), the product of solar ultraviolet irradiation of 7-dehydrocholesterol in the skin (2). In 1966, it was first recognized that vitamin D must undergo activation via two oxidative metabolic steps (Fig. 35.2). The first oxidation to 25-hydroxycholecalciferol (25(OH)D3 calcifediol Calderol) occurs in the endoplasmic reticulum of the liver and is catalyzed by vitamin D 25-hydroxylase. This activation step is not... 

The enzyme is localized in liver mitochondria. Some confusion existed in the early literature because some animal species have liver microsomal vitamin Dj 25-hydroxylases (e.g., hog hver and kidney P450 2D25 [2540, 2541]), but not humans [2542]. The rat and human hver mitochondrial P450 27A1 recombinant enzymes were clearly shown to catalyze both vitamin D3 25-hydrojgrlation and the 27-hydroxylation of the side chains of cholesterol and several derivatives [2543, 2544]. 

Figure 4 A schematic representation of the origin of vitamin D3 and its metabolism in the liver by the hepatic vitamin D-25-hydroxylase. Once formed, the 25-hydroxyvitamin D3 (25(OH)D3> is metabolized by either a 25(OH)D-1a-hydroxylase or a 25(OH)D-24-hydroxylase. 1,25-Dihydroxyvitamin D3 (1,25(OH)2D3) can either go to its target tissues to carry out its biologic functbn(s), or it can be metabolized in its side-chain and degraded to calcitroic add. (Reproduced with permissbn from Holick MF (1995) Vitamin D Photobiology, Metabolism, and Clinical Applications. In DeGroot U etal. (eds.) Endocrinology, 3rd edn, pp. 990-1013. Philadelphia W.B. Saunders.)...

CYP24 is a 25-hydroxyvitamin D3 24-hydroxylase that degrades vitamin D metabolites. 

When 1,25 DHCC provision is adequate or when plasma calcium concentration is above approximately 2.20mmol/l, la hydroxylase activity is suppressed and 25-hydroxy vitamin D3 is converted by 24-hydroxylase into 24,25 dihydroxy vitamin D3 a metabolite whose true role is uncertain but one which seems to have little if any physiological activity. Renal 24-hydroxylase does have a role to play in the deactivation of 1,25 dihydroxy vitamin D the major metabolite of the vitamin being 1,24,25 trihydroxy vitamin D. 

Danan JL, Delorme AC, Mathieu H. 1982. Presence of 25-hydroxyvitamin D3 and 1,25-dihydroxyvitamin D3 24-hydroxylase in vitamin D target cells of rat yolk sac. J Biol Chem 257 10715-10721. 

Matkovits T, Christakos S. 1995. Variable in vivo regulation of rat vitamin D-dependent genes (osteopontin, Ca, Mg-adenosine triphosphatase, and 25-hydroxyvitamin D3 24-hydroxylase) implications for differing mechanisms of regulation and involvement of multiple factors. Endocrinology 136 3971-3982. 

In addition to the endogenous metabolites, some exogenous sterols possess biological activity similar to that of D3. Ergocalciferol (vitamin D2) is derived from the plant sterol ergosterol and may act as a substrate for both the 25-hydroxylase and the 1-hydroxylase enzyme systems of the liver and kidney to form 25-(OH)D2 and 1,25-(0H)2 D2, respectively. Ergocalciferol (vitamin D2) is the form used in commercial vitamins and supplemented dairy products. Dihydrotachysterol, another sterol that is used as a therapeutic agent, also functions as a substrate for the hydroxylase enzymes in the liver and kidney. 

Formation of 1,25-diOH D3 Vitamins D2 and D3 are not biologically active, but are converted in vivo to the active form of the D vitamin by two sequential hydroxylation reactions (Figure 28.23). The first hydroxylation occurs at the 25-position, and is catalyzed by a specific hydroxylase in the liver. The product of the reaction, 25-hydroxycholecalciferol (25-OH D3), is the predominant form of vitamin D in the plasma and the major storage form of the vitamin. 25-OH D3 is further hydroxylated at the one position by a specific 25-hydroxycholecalciferol 1 -hydroxylase found primarily in the kidney, resulting in the formation of 1,25-dihydroxycholecalciferol j (1,25-diOH D3). [Note This hydroxylase, as well as the iver 25-hydroxylase, employ cytochrome P450, molecular oxygen, and NADPH.]... 

Uptake of Ca2+ from the intestine is stimulated by vitamin D.447 Vitamin D3 is converted to the 25-hydroxy derivative in the liver (equation 11) by a two component mixed-function hydroxylase.448 The metabolically active 1,25-dihydroxy form is synthesized by further hydroxylation in the kidney. This latter stage involves the renal 25-hydroxyvitamin D3- 1-hydroxylase in a reaction which is controlled by Ca2+, parathyroid hormone and phosphate. This renal hydroxylase contains a flavoprotein, an iron-sulfur protein (with an Fe2S2 cluster) and cytochrome P-450.447... 

In the absence of inadequate endogenous synthesis, vitamin D must be obtained from dietary sources or from supplements. Few foods contain vitamin D except for the flesh of fatty fish (salmon, mackerel, sardines), fish liver oils, and eggs from hens fed feed enriched with vitamin D. In the United States, all commercially produced milk is fortified with vitamin D2 at a level of 400 IU/L (1 IU = 0.025 fig of vitamin D3). Therefore, in the United States (and other economically advanced countries) most dietary vitamin D is obtained from milk and other vitamin D2-fortified foods. Both vitamin D2 and vitamin D3 are converted at the same rate to 25-hydroxyvitamin D by a hydroxylase in the liver and are equally active as a prohormone. Because dietary uptake of vitamin D is dependent on normal fat absorption, conditions in which fat malabsorption is present can result in vitamin D deficiency. Because breast milk contains little vitamin D, vitamin D deficiency can occur in infants who are solely breastfed, are not exposed to adequate sunlight, and are not receiving vitamin D supplements. The adequate intake of vitamin D for children is 5 pg/day (200 IU/day) (Table 30-2). 

Critical to vitamin D3 action is its further metabolic conversion to more active compounds (Figure 1.3). Via its transport by DBP, vitamin D3 accumulates in the liver [48]. In rats, as much as 60-80% of an injected or oral dose of vitamin D3 locates to the liver [49-51], Intestinal absorption of vitamin D3 is in association with the chylomicron fraction via the lymphatic system. Vitamin D3 is delivered to the liver in blood from the thoracic duct only a few hours post ingestion [44], A specific portion of hepatic vitamin D3 in the rat is converted to 25-OH-D3 by a 25-hydroxylase system in the endoplasmic reticulum of hepatocytes [52, 53]. This enzyme (Km 10"8 M) is regulated to an extent by 25-OH-D3 and its metabolites. Higher concentrations of vitamin D3 are handled by a second 25-hydroxylase located in liver mitochondria [54], This enzyme, also known as CYP27, 27-hydroxylates cholesterol and thus appears less discriminating than the microsomal 25-OHase which does not use cholesterol as substrate [55, 56]. In humans, however,... 

This hydroxylase carries out the 1-hydroxylation of the compound 25-hydroxyvitamin D3 (25-OH-D3) to form the product 1,25-dihydroxy-vitamin D3 [l,25-(OH)2-D3]. The HPLC assay developed replaces those using radiolabeled substrates. 

Vitamin D3 is converted to 1,25-dihydroxyvitamin D3 by a series of two hydroxyl a tion reactions. The 25-hydjroxylation, which occurs first, is catalyzed by an enzyme of the liver. The subsequent conversion of 25 hydroxyvitamin Dj to 1,25-dihydroxyvitamin Dj i.s catalyzed by an enzyme of the kidney The enzyme that catalyzes this latter step is called "1-hydroxylase" in this text. Figure 9.50 indicates the positions of the 1-carbon and 25-carbon of the steroid ring. Vitamin Dean also be converted to 24,25-dihydroxyvitamin Dj, as well as a number of other metabolites. Although there is much interest in the possible physiological value of these compounds, they seem generally to be inactive catabolites of the vitamin. 1,25-Di-hydroxyvitamin Dj is also call calcitrol. 25-Hydroxyvitamm Dg is also called calcidiol. 

Zou, A., Elgort, M. G., and Allegretto, E. A. (1997). Retinoid X receptor (RXR) ligands activate the human 25-hydroxyvitamin D3 24 hydroxylase promoter via RXR heterodimer binding to two vitamin D-responsive elements and elicit additive effects with 1,25-dihydroxyvitamin D3. /. Biol Chem. 272,19027-19034. 

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