Pathways and products for the metabolism of vitamin D3 by cytochrome P450scc

Abstract

Cytochrome P450scc (CYP11A1) catalyzes the first enzymatic step in steroid synthesis: the cleavage of the side chain of cholesterol to produce pregnenolone [1]. This three-step reaction occurs in mitochondria and involves sequential hydroxylation at C22 and C20, and subsequent oxidative cleavage of the bond between C20 and C22. It has now been established that cytochrome P450scc can also act on both vitamins D2 and D3, as well as their precursors ergosterol and 7-dehydrocholesterol [2][3][4][5][6] Cytochrome P450scc (CYP11A1) can hydroxylate vitamin D3 to produce 20-hydroxyvitamin D3 and other poorly characterized hydroxylated products. The present study aimed to identify all the products of vitamin D3 metabolism by P450scc, as well as the pathways leading to their formation. Besides 20-hydroxyvitamin D3, other major metabolites of vitamin D3 were a dihydroxyvitamin D3 and a trihydroxyvitamin D3 product. The dihydroxyvitamin D3 was clearly identified as 20,23-dihydroxyvitamin D3 by NMR, in contrast to previous reports that postulated hydroxyl groups in positions 20 and 22. NMR of the trihydroxy product identified it as 17a,20,23-trihydroxyvitamin D3. This product could be directly produced by P450scc acting on 20,23-dihydroxyvitamin D3, confirming that hydroxyl groups are present at positions 20 and 23. Three minor products of D3 metabolism by P450scc were identified by MS and by examining their subsequent metabolism by P450scc. These products were 23-hydroxyvitamin D3, 17a-hydroxyvitamin D3 and 17a,20-dihydroxyvitamin D3 and arise from the three P450scc-catalysed hydroxylations occurring in a different order. We conclude that the major pathway of vitamin D3 metabolism by P450scc is: vitamin D3 fi 20-hydroxyvitamin D3 fi 20,23-dihydroxyvitamin D3 fi 17a,20,23-trihydroxyvitamin D3. The major products dissociate from the P450scc active site and accumulate at a concentration well above the P450scc concentration. Our new identification of the major dihydroxyvitamin D3 product as 20,23-dihydroxyvitamin D3, rather than 20,22-dihydroxyvitamin D3, explains why there is no cleavage of the vitamin D3 side chain, unlike the metabolism of cholesterol by P450scc.Abbreviations cyclodextrin, 2-hydroxypropyl-b-cyclodextrin; HMBC, heteronuclear multiple bond correlation; HSQC, heteronuclear single quantum correlation; RT, retention time.

The Vitamin D Receptor Regulates Rheumatoid Arthritis Synovial Fibroblast Invasion and Morphology

“…Although the vitamin D receptor (VDR) (also an NR) itself was not differentially expressed, there was an overrepresentation of VDR target genes among the genes with increased expression in synovial tissues from arthritisprotected rats, suggesting increased VDR activity. Furthermore, genes implicated in the hydroxylation and synthesis of alternative vitamin D active metabolites such as Cyp27a1, Cyp2j3 and Cyp11a1 (6,7) were also expressed in increased levels in the tissues of the protected rats, suggesting that the increased expression of target genes could be related at least in part to increased local production of VDR agonists (5).…”

Vitamin D supplementation during pregnancy: safety considerations in the design and interpretation of clinical trials

“…Although vitamin D3 itself is believed to be biologically inactive, there are a variety of non-25-hydroxylated metabolites that have been derived from the D3 parent molecule. 84 For example, secosteroids resulting from a 20-hydroxylation reaction catalyzed by cytochrome P450 side chain cleavage enzyme 85 are biologically active in vitro (for example, promote keratinocyte differentiation). 86 Theoretical implications in the setting of pregnancy are of interest because P450 side chain cleavage enzyme is expressed at high levels in the placental tissue.…”

Kinetics of vitamin D3 metabolism by cytochrome P450scc (CYP11A1) in phospholipid vesicles and cyclodextrin

“…20(OH)D3 and 20,23(OH) 2 D3 were prepared enzymatically from 50 ml incubations of P450scc with D3 and purified by preparative TLC as described before (Slominski et al, 2005b). The purity of these samples was checked prior to use by HPLC (Tuckey et al, 2008). The concentration of hydroxyvitamin D3 was determined using an extinction coefficient of 18,000 M −1 cm −1 at 263 nm (Hiwatashi et al, 1982).…”

“…The reaction involves removal of the side chain of cholesterol via production of enzyme-bound intermediates, 22R-hydroxycholesterol and 20α,22R-dihydroxycholesterol (Hume et al, 1984;Lambeth et al, 1982;Tuckey, 1990Tuckey, , 2005. Cytochrome P450scc can also metabolize vitamin D3 (D3) and vitamin D2 (D2), as well as their precursors 7-dehydrocholesterol and ergosterol (Guryev et al, 2003;Slominski et al, 2004Slominski et al, , 2005aSlominski et al, ,b, 2006Tuckey et al, 2008). Our most recent study shows that P450scc can hydroxylate D3 at carbons 17, 20 and 23 (Tuckey et al, 2008).…”

“…Cytochrome P450scc can also metabolize vitamin D3 (D3) and vitamin D2 (D2), as well as their precursors 7-dehydrocholesterol and ergosterol (Guryev et al, 2003;Slominski et al, 2004Slominski et al, , 2005aSlominski et al, ,b, 2006Tuckey et al, 2008). Our most recent study shows that P450scc can hydroxylate D3 at carbons 17, 20 and 23 (Tuckey et al, 2008). The major pathway of metabolism involves initial hydroxylation at C20 followed by C23 and C17, D3),20,23(OH) 2 D3) and 17,20,20,23(OH) 3 D3) as the major products.…”