Metabolism of vitamin D2 to 17,20,24-trihydroxyvitamin D2 by cytochrome P450scc (CYP11A1)

Minh N. Nguyen, Andrzej Slominski, Wei Li, Yun Rui Ng, Robert C. Tuckey

Research output: Contribution to journalArticle

37 Citations (Scopus)

Abstract

As well as catalyzing the conversion of cholesterol to pregnenolone for steroid synthesis, cytochrome P450scc (P450scc) can also metabolize vitamins D2 (D2) and D3 (D3). Two products of D2 metabolism by P450scc, 20-hydroxyvitamin D2 and 17,20-dihydroxyvitamin D2, have been identified and shown to exert biological activity on cultured keratinocytes. The aim of this study was to fully characterize the metabolism of D2 by P450scc, including identifying additional products and determining the kinetics of D2 metabolism. Two new products were isolated by reverse-phase high-performance liquid chromatography: a dihydroxy metabolite with a hydroxyl group at C20 plus another unidentified position, and a trihydroxy metabolite identified by NMR as 17,20,24-trihy- droxyvitamin D2. Kinetics of D2 metabolism was determined with substrate solubilized by 2-hydroxypropyl-β-cyclodextrin or incorporated into phospholipid vesicles. In 2-hydroxypropyl-β-cyclodextrin, D2 was hydroxylated at C20 with a k cat/K m 5-fold lower than that for cholesterol metabolism. 20-Hydroxyvitamin D2 was hydroxylated with a similar k cat/K m to D2, whereas 17,20-dihy- droxyvitamin D2 was hydroxylated with a lower k cat/K m than that for D2 in 2-hydroxypropyl-β-cyclodextrin. In vesicles, D2 displayed a high K m relative to that for cholesterol, but hydroxylation resulted in products that could be further hydroxylated with relatively low K m values. We conclude that P450scc catalyzes three sequential hydroxylations of D2 producing 20-hydroxyvitamin D2,17,20-dihy- droxyvitamin D2, and 17,20,24-trihydroxyvitamin D2, which dissociate from the active site of P450scc and accumulate in the reaction mixture. D2 metabolism occurs with lower efficiency (k cat/K m) than that observed for both cholesterol and D3 metabolism by P450scc.

Original languageEnglish (US)
Pages (from-to)761-767
Number of pages7
JournalDrug Metabolism and Disposition
Volume37
Issue number4
DOIs
StatePublished - Apr 1 2009

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Cholesterol Side-Chain Cleavage Enzyme
Ergocalciferols
Cyclodextrins
Cats
Cholesterol
Hydroxylation
Pregnenolone
Reverse-Phase Chromatography
17,20,24-trihydroxyvitamin D2
Keratinocytes
Hydroxyl Radical
Catalytic Domain
Phospholipids
Steroids
High Pressure Liquid Chromatography

All Science Journal Classification (ASJC) codes

  • Pharmacology
  • Pharmaceutical Science

Cite this

Metabolism of vitamin D2 to 17,20,24-trihydroxyvitamin D2 by cytochrome P450scc (CYP11A1). / Nguyen, Minh N.; Slominski, Andrzej; Li, Wei; Ng, Yun Rui; Tuckey, Robert C.

In: Drug Metabolism and Disposition, Vol. 37, No. 4, 01.04.2009, p. 761-767.

Research output: Contribution to journalArticle

Nguyen, Minh N. ; Slominski, Andrzej ; Li, Wei ; Ng, Yun Rui ; Tuckey, Robert C. / Metabolism of vitamin D2 to 17,20,24-trihydroxyvitamin D2 by cytochrome P450scc (CYP11A1). In: Drug Metabolism and Disposition. 2009 ; Vol. 37, No. 4. pp. 761-767.
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T1 - Metabolism of vitamin D2 to 17,20,24-trihydroxyvitamin D2 by cytochrome P450scc (CYP11A1)

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N2 - As well as catalyzing the conversion of cholesterol to pregnenolone for steroid synthesis, cytochrome P450scc (P450scc) can also metabolize vitamins D2 (D2) and D3 (D3). Two products of D2 metabolism by P450scc, 20-hydroxyvitamin D2 and 17,20-dihydroxyvitamin D2, have been identified and shown to exert biological activity on cultured keratinocytes. The aim of this study was to fully characterize the metabolism of D2 by P450scc, including identifying additional products and determining the kinetics of D2 metabolism. Two new products were isolated by reverse-phase high-performance liquid chromatography: a dihydroxy metabolite with a hydroxyl group at C20 plus another unidentified position, and a trihydroxy metabolite identified by NMR as 17,20,24-trihy- droxyvitamin D2. Kinetics of D2 metabolism was determined with substrate solubilized by 2-hydroxypropyl-β-cyclodextrin or incorporated into phospholipid vesicles. In 2-hydroxypropyl-β-cyclodextrin, D2 was hydroxylated at C20 with a k cat/K m 5-fold lower than that for cholesterol metabolism. 20-Hydroxyvitamin D2 was hydroxylated with a similar k cat/K m to D2, whereas 17,20-dihy- droxyvitamin D2 was hydroxylated with a lower k cat/K m than that for D2 in 2-hydroxypropyl-β-cyclodextrin. In vesicles, D2 displayed a high K m relative to that for cholesterol, but hydroxylation resulted in products that could be further hydroxylated with relatively low K m values. We conclude that P450scc catalyzes three sequential hydroxylations of D2 producing 20-hydroxyvitamin D2,17,20-dihy- droxyvitamin D2, and 17,20,24-trihydroxyvitamin D2, which dissociate from the active site of P450scc and accumulate in the reaction mixture. D2 metabolism occurs with lower efficiency (k cat/K m) than that observed for both cholesterol and D3 metabolism by P450scc.

AB - As well as catalyzing the conversion of cholesterol to pregnenolone for steroid synthesis, cytochrome P450scc (P450scc) can also metabolize vitamins D2 (D2) and D3 (D3). Two products of D2 metabolism by P450scc, 20-hydroxyvitamin D2 and 17,20-dihydroxyvitamin D2, have been identified and shown to exert biological activity on cultured keratinocytes. The aim of this study was to fully characterize the metabolism of D2 by P450scc, including identifying additional products and determining the kinetics of D2 metabolism. Two new products were isolated by reverse-phase high-performance liquid chromatography: a dihydroxy metabolite with a hydroxyl group at C20 plus another unidentified position, and a trihydroxy metabolite identified by NMR as 17,20,24-trihy- droxyvitamin D2. Kinetics of D2 metabolism was determined with substrate solubilized by 2-hydroxypropyl-β-cyclodextrin or incorporated into phospholipid vesicles. In 2-hydroxypropyl-β-cyclodextrin, D2 was hydroxylated at C20 with a k cat/K m 5-fold lower than that for cholesterol metabolism. 20-Hydroxyvitamin D2 was hydroxylated with a similar k cat/K m to D2, whereas 17,20-dihy- droxyvitamin D2 was hydroxylated with a lower k cat/K m than that for D2 in 2-hydroxypropyl-β-cyclodextrin. In vesicles, D2 displayed a high K m relative to that for cholesterol, but hydroxylation resulted in products that could be further hydroxylated with relatively low K m values. We conclude that P450scc catalyzes three sequential hydroxylations of D2 producing 20-hydroxyvitamin D2,17,20-dihy- droxyvitamin D2, and 17,20,24-trihydroxyvitamin D2, which dissociate from the active site of P450scc and accumulate in the reaction mixture. D2 metabolism occurs with lower efficiency (k cat/K m) than that observed for both cholesterol and D3 metabolism by P450scc.

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