Metabolism of 20-hydroxyvitamin D3 by mouse liver microsomes

Abstract

20-Hydroxyvitamin D3 [20(OH)D3], the major product of CYP11A1 action on vitamin D3, is biologically active and like 1,25-dihydroxyvitamin D3 [1,25(OH)2D3] can inhibit proliferation and promote differentiation of a range of cells, and has anti-inflammatory properties. However, unlike 1,25(OH)2D3, it does not cause toxic hypercalcemia at high doses and is therefore a good candidate for therapeutic use to treat hyperproliferative and autoimmune disorders. In this study we analyzed the ability of mouse liver microsomes to metabolize 20(OH)D3. The two major products were identified from authentic standards as 20,24-dihydroxyvitamin D3 [20,24(OH)2D3] and 20,25-dihydroxyvitamin D3 [20,25(OH)2D3]. The reactions for synthesis of these two products from 20(OH)D3 displayed similar Km values suggesting that they were catalyzed by the same cytochrome P450. Some minor metabolites were produced by reactions with higher Km values for 20(OH)D3. Some metabolites gave mass spectra suggesting that they were the result of hydroxylation followed by dehydrogenation. One product had an increase in the wavelength for maximum absorbance from 263 nm seen for 20(OH)D3, to 290 nm, suggesting a new double bond was interacting with the vitamin D-triene chromophore. The two major products, 20,24(OH)2D3 and 20,25(OH)2D3 have both previously been shown to have higher potency for inhibition of colony formation by melanoma cells than 20(OH)D3, thus it appears that metabolism of 20(OH)D3 by mouse liver microsomes can generate products with enhanced activity.

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

“…The most extensively characterized secosteroids produced from this pathway with respect to biological activity are 20(OH)D3 and 20,23(OH) 2 D3 [16, 22, 23, 25–28, 32–34, 46, 47]. In the present study we show that these two biologically active secosteroids can be metabolized by both rat and human CYP24A1.…”

“…Human CYP27A1 converts 20(OH)D3 to 20 S ,25-dihydroxyvitamin D3 [20,25(OH) 2 D3] and 20 S ,26-dihydroxyvitamin D3 [20,26(OH) 2 D3], whereas rat CYP24A1 produces 20 S ,24-dihydroxyvitamin D3 [20,24(OH) 2 D3] and 20,25(OH) 2 D3 [32, 33]. Other P450 isoforms found in mouse liver microsomes also produce 20,25(OH) 2 D3 and 20,26(OH) 2 D3 [34]. These resulting secosteroids are more potent than the parent 20(OH)D3 in the inhibition of melanoma colony formation [33].…”

Hydroxylation of 20-hydroxyvitamin D3 by human CYP3A4

“…The procedure used was similar to the one described previously for mouse liver microsomes (33). Human liver microsomes (1.5 mg/mL) were incubated with 20(OH)D3 solubilised in 0.45% HP-β-CD, in buffer comprising 0.25 M sucrose, 50 mM Hepes (pH 7.4), 20 mM KCl, 5 mM MgSO 4 , 0.2 mM EDTA, 500 μM NADPH, 2 mM glucose-6-phosphate and 2 U/mL glucose-6-phosphate dehydrogenase.…”

“…The kinetic parameters were obtained by fitting the Michaelis-Menten equation to experimental data using Kaleidagraph 4.0 (Synergy Software). For mass spectrometry, metabolites were purified and collected using both acetonitrile-water and methanol-water solvent systems (33,37). The purified metabolites (2 nmol) were reconstituted in 30 μL of 70% methanol + 0.1% formic acid, with 20 μL being injected into the LC/MS.…”

“…Mouse liver microsomes can act on 20(OH)D3 and like CYP24A1 in the kidney, produce the biologically active products, 20,24(OH) 2 D3 and 20,25(OH) 2 D3 (33). Mouse liver microsomes do not express CYP3A4, but do express six other family 3A isoforms (34).…”