CYP3A4 is a human microsomal vitamin D 25-hydroxylase.
The human hepatic microsomal vitamin D 25-hydroxylase protein and gene have not been identified with certainty. Sixteen hepatic recombinant microsomal enzymes were screened for 25-hydroxylase activity; 11 had some 25-hydroxylase activity, but CYP3A4 had the highest activity. In characterized liver microsomes, 25-hydroxylase activity correlated significantly with CYP3A4 testosterone 6beta-hydroxylase activity. Activity in pooled liver microsomes was inhibited by known inhibitors of CYP3A4 and by an antibody to CYP3A2. Thus, CYP3A4 is a hepatic microsomal vitamin D 25-hydroxylase.


BACKGROUND
Studies were performed to identify human microsomal vitamin D-25 hydroxylase.


METHODS
Sixteen major hepatic microsomal recombinant enzymes derived from cytochrome P450 cDNAs expressed in baculovirus-infected insect cells were screened for 25-hydroxylase activity with 1alpha-hydroxyvitamin D2 [1alpha(OH)D2], 1alpha-hydroxyvitamin D3 [1alpha(OH)D3], vitamin D2, and vitamin D3 as substrates. Activity was correlated with known biological activities of enzymes in a panel of 12 characterized human liver microsomes. The effects of known inhibitors and specific antibodies on activity also were determined.


RESULTS
CYP3A4, the most abundant cytochrome P450 enzyme in human liver and intestine, had 7-fold greater activity than that of any of the other enzymes with 1alpha(OH)D2 as substrate. CYP3A4 25-hydroxylase activity was four times higher with 1alpha(OH)D2 than with 1alpha(OH)D3 as substrate, was much less with vitamin D2, and was not detected with vitamin D3. 1alpha(OH)D2 was the substrate in subsequent experiments. In a panel of characterized human liver microsomes, 25-hydroxylase activity correlated with CYP3A4 testosterone 6beta-hydroxylase activity (r = 0.93, p < 0.001) and CYP2C9*1 diclofenac 4'-hydroxylase activity (r = 0.65, p < 0.05), but not with activity of any of the other enzymes. Activity in recombinant CYP3A4 and pooled liver microsomes was dose-dependently inhibited by ketoconazole, troleandomycin, isoniazid, and alpha-naphthoflavone, known inhibitors of CYP3A4. Activity in pooled liver microsomes was inhibited by antibodies to CYP3A2 that are known to inhibit CYP3A4 activity.


CONCLUSIONS
CYP3A4 is a vitamin D 25-hydroxylase for vitamin D2 in human hepatic microsomes and hydroxylates both 1alpha(OH)D2 and 1alpha(OH)D3.
Department of Medicine, Medical University of South Carolina, Charleston, South Carolina 29425, USA.
Ram P
RP
Gupta
Bruce W
BW
Hollis
Shailendra B
SB
Patel
Kennerly S
KS
Patrick
Norman H
NH
Bell
D053493:Cholestanetriol 26-Monooxygenase; D003001:Cloning, Molecular; D051544:Cytochrome P-450 CYP3A; D065607:Cytochrome P-450 Enzyme Inhibitors; D003577:Cytochrome P-450 Enzyme System / Q000737:chemistry* / Q000235:genetics; D004791:Enzyme Inhibitors / Q000494:pharmacology; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D006801:Humans; D008862:Microsomes, Liver / Q000201:enzymology*; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000737:chemistry / Q000235:genetics; D013250:Steroid Hydroxylases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000235:genetics; D013379:Substrate Specificity / Q000187:drug effects / Q000235:genetics; D014807:Vitamin D / Q000737:chemistry*
D065607:Cytochrome P-450 Enzyme Inhibitors; D004791:Enzyme Inhibitors; D011994:Recombinant Proteins; D014807:Vitamin D; D003577:Cytochrome P-450 Enzyme System; D013250:Steroid Hydroxylases; C104464:CYP3A protein, human; D051544:Cytochrome P-450 CYP3A; C510163:CYP3A4 protein, human; C506831:CYP27A1 protein, human; D053493:Cholestanetriol 26-Monooxygenase