Metabolism of 20-hydroxyvitamin D3 and 20,23-dihydroxyvitamin D3 by rat and human CYP24A1.

CYP11A1 hydroxylates vitamin D3 producing 20S-hydroxyvitamin D3 [20(OH)D3] and 20S,23-dihydroxyvitamin D3 [20,23(OH)2D3] as the major and most characterized metabolites. Both display immuno-regulatory and anti-cancer properties while being non-calcemic. A previous study indicated 20(OH)D3 can be metabolized by rat CYP24A1 to products including 20S,24-dihydroxyvitamin D3 [20,24(OH)2D3] and 20S,25-dihydroxyvitamin D3, with both producing greater inhibition of melanoma colony formation than 20(OH)D3. The aim of this study was to characterize the ability of rat and human CYP24A1 to metabolize 20(OH)D3 and 20,23(OH)2D3. Both isoforms metabolized 20(OH)D3 to the same dihydroxyvitamin D species with no secondary metabolites being observed. Hydroxylation at C24 produced both enantiomers of 20,24(OH)2D3. For rat CYP24A1 the preferred initial site of hydroxylation was at C24 whereas the human enzyme preferred C25. 20,23(OH)2D3 was initially metabolized to 20S,23,24-trihydroxyvitamin D3 and 20S,23,25-trihydroxyvitamin D3 by rat and human CYP24A1 as determined by NMR, with both isoforms showing a preference for initial hydroxylation at C25. CYP24A1 was able to further oxidize these metabolites in a series of reactions which included the cleavage of C23-C24 bond, as indicated by high resolution mass spectrometry of the products, analogous to the catabolism of 1,25(OH)2D3 via the C24-oxidation pathway. Similar catalytic efficiencies were observed for the metabolism of 20(OH)D3 and 20,23(OH)2D3 by human CYP24A1 and were lower than for the metabolism of 1,25(OH)2D3. We conclude that rat and human CYP24A1 metabolizes 20(OH)D3 producing only dihydroxyvitamin D3 species as products which retain biological activity, whereas 20,23(OH)2D3 undergoes multiple oxidations which include cleavage of the side chain.

Tieu EW ，Li W ，Chen J ，Kim TK ，Ma D ，Slominski AT ，Tuckey RC ... -  《-》

C-25 hydroxylation of 1alpha,24(R)-dihydroxyvitamin D3 is catalyzed by 25-hydroxyvitamin D3-24-hydroxylase (CYP24A1): metabolism studies with human keratinocytes and rat recombinant CYP24A1.

Astecker N ，Bobrovnikova EA ，Omdahl JL ，Gennaro L ，Vouros P ，Schuster I ，Uskokovic MR ，Ishizuka S ，Wang G ，Reddy GS ... -  《ARCHIVES OF BIOCHEMISTRY AND BIOPHYSICS》

Calcioic acid: In vivo detection and quantification of the terminal C24-oxidation product of 25-hydroxyvitamin D(3) and related intermediates in serum of mice treated with 24,25-dihydroxyvitamin D(3).

Calcitroic acid, the excretory form of vitamin D, is the terminal product of a 5-step pathway catalyzed by CYP24A1, commencing with C24-hydroxylation of 1,25-dihydroxyvitamin D3 (1,25-(OH)2D3). Catabolism of 25-hydroxyvitamin D3 (25-OH-D3) proceeds via analogous steps culminating in calcioic acid; however this C23-truncated acid has not been reported in the circulation. It has recently been shown that 24,25-dihydroxyvitamin D3 (24,25-(OH)2D3) is an important factor in optimal bone fracture healing acting via an effector molecule FAM57B2 to produce lactosylceramide. Administration of 24,25-(OH)2D3 was found to restore normal fracture repair in Cyp24a1-/- mice devoid of 24,25-(OH)2D3. We set out to study the multi-step catabolism of D3 metabolites in vivo using LC-MS/MS methods in vehicle or 24,25-(OH)2D3-treated mice. Vehicle-treated Cyp24a1+/- mice possessed normal levels of serum 24,25-(OH)2D3 (7 ng/mL) and 25-OH-D3-26,23-lactone (4 ng/mL). We also detected 24-oxo-25-OH-D3 (3 ng/mL) and 24-oxo-23,25-(OH)2D3 (0.4 ng/mL); which were not detectable in vehicle-treated Cyp24a1-/- mice. In 24,25-(OH)2D3-treated Cyp24a1+/- mice, serum 24,25-(OH)2D3 rose to 200 ng/mL while 25-OH-D3-26,23-lactone remained unchanged in comparison to vehicle-treated Cyp24a1+/- mice Concentration of serum 24-oxo-25-OH-D3 and 24-oxo-23,25-(OH)2D3 rose by 10-fold, when Cyp24a1+/- mice were treated with 24,25-(OH)2D3 Calcioic acid was increased to 0.030 ng/mL for 24,25-(OH)2D3-treated Cyp24a1+/- mice. In 24,25-(OH)2D3-treated Cyp24a1-/- mice, serum 24,25-(OH)2D3 rose further to a striking 830 ng/mL due to lack of catabolism of the 24,25-(OH)2D3 dose. Serum 1,25-(OH)2D3 levels were suppressed in 24,25-(OH)2D3-treated Cyp24a1+/- and Cyp24a1-/- mice. Circulating 1,24,25-(OH)3D3 rose from 73 pg/mL to 106 pg/mL when Cyp24a1+/- mice were treated with 24,25-(OH)2D3. While undetectable in vehicle-treated Cyp24a1-/- mice, 1,24,25-(OH)3D3 rose unexpectedly to 153 pg/mL in 24,25-(OH)2D3-treated nulls suggesting conversion of 24,25-(OH)2D3 to 1,24,25-(OH)3D3 via 1-hydroxylation. Taken together, amplification of 24,25-(OH)2D3 catabolism by exogenous doses of this metabolite have enabled detection of downstream C24-oxidation pathway products in vivo, including calcioic acid; and provides a platform for studying alternative routes of vitamin D metabolism that may occur in pathological states including hypervitaminosis D and idiopathic infantile hypercalcemia caused by mutations of CYP24A1.

Kaufmann M ，Martineau C ，Arabian A ，Traynor M ，St-Arnaud R ，Jones G ... -  《-》

Hydroxylation of 20-hydroxyvitamin D3 by human CYP3A4.

20S-Hydroxyvitamin D3 [20(OH)D3] is the biologically active major product of the action of CYP11A1 on vitamin D3 and is present in human plasma. 20(OH)D3 displays similar therapeutic properties to 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], but without causing hypercalcaemia and therefore has potential for development as a therapeutic drug. CYP24A1, the kidney mitochondrial P450 involved in inactivation of 1,25(OH)2D3, can hydroxylate 20(OH)D3 at C24 and C25, with the products displaying more potent inhibition of melanoma cell proliferation than 20(OH)D3. CYP3A4 is the major drug-metabolising P450 in liver endoplasmic reticulum and can metabolise other active forms of vitamin D, so we examined its ability to metabolise 20(OH)D3. We found that CYP3A4 metabolises 20(OH)D3 to three major products, 20,24R-dihydroxyvitamin D3 [20,24R(OH)2D3], 20,24S-dihydroxyvitamin D3 [20,24S(OH)2D3] and 20,25-dihydroxyvitamin D3 [20,25(OH)2D3]. 20,24R(OH)2D3 and 20,24S(OH)2D3, but not 20,25(OH)2D3, were further metabolised to trihydroxyvitamin D3 products by CYP3A4 but with low catalytic efficiency. The same three primary products, 20,24R(OH)2D3, 20,24S(OH)2D3 and 20,25(OH)2D3, were observed for the metabolism of 20(OH)D3 by human liver microsomes, in which CYP3A4 is a major CYP isoform present. Addition of CYP3A family-specific inhibitors, troleandomycin and azamulin, almost completely inhibited production of 20,24R(OH)2D3, 20,24S(OH)2D3 and 20,25(OH)2D3 by human liver microsomes, further supporting that CYP3A4 plays the major role in 20(OH)D3 metabolism by microsomes. Since both 20,24R(OH)2D3 and 20,25(OH)2D3 have previously been shown to display enhanced biological activity in inhibiting melanoma cell proliferation, our results show that CYP3A4 further activates, rather than inactivates, 20(OH)D3.

Cheng CY ，Slominski AT ，Tuckey RC 《-》

A High-Calcium and Phosphate Rescue Diet and VDR-Expressing Transgenes Normalize Serum Vitamin D Metabolite Profiles and Renal Cyp27b1 and Cyp24a1 Expression in VDR Null Mice.

Kaufmann M ，Lee SM ，Pike JW ，Jones G ... -  《-》