• vitamin D;
  • 25-hydroxyvitamin D;
  • vitamin D 25-hydroxylase;
  • liver microsomes;
  • cytochrome P450 enzymes


  1. Top of page
  2. Abstract
  7. Acknowledgements

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 6β-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.

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

Materials and 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 1α-hydroxyvitamin D2 [1α(OH)D2], 1α-hydroxyvitamin D3 [1α(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 1α(OH)D2 as substrate. CYP3A4 25-hydroxylase activity was four times higher with 1α(OH)D2 than with 1α(OH)D3 as substrate, was much less with vitamin D2, and was not detected with vitamin D3. 1α(OH)D2 was the substrate in subsequent experiments. In a panel of characterized human liver microsomes, 25-hydroxylase activity correlated with CYP3A4 testosterone 6β-hydroxylase activity (r = 0.93, p < 0.001) and CYP2C91 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 α-naphthoflavone, known inhibitors of CYP3A4. Activity in pooled liver microsomes was inhibited by antibodies to CYP3A2 that are known to inhibit CYP3A4 activity.

Conclusion: CYP3A4 is a vitamin D 25-hydroxylase for vitamin D2 in human hepatic microsomes and hydroxylates both 1α(OH)D2 and 1α(OH)D3.


  1. Top of page
  2. Abstract
  7. Acknowledgements

TO BECOME BIOLOGICALLY ACTIVE, vitamin D must first be converted to 25-hydroxyvitamin D [25(OH)D] in the liver(1, 2) and to 1,25-dihydroxyvitamin D [1,25(OH)2D] in the kidney.(3-5) 25(OH)D and 1,25(OH)2D undergo further hydroxylation in the kidney and elsewhere by 25(OH)D 24-hydroxylase (CYP24A1), the rate-limiting enzyme for their catalytic degradation, to form calcitroic acid.(6-8) In humans, hepatic 25-hydroxylase activity is present in both mitochondria and microsomes.(1, 2, 9-11) Recombinant CYP27A1, a mitochondrial enzyme involved in the alternative pathway of bile acid metabolism, 25-hydroxylated 1α-hydroxyvitamin D3 [1α(OH)D3], vitamin D3, and 1α-hydroxyvitamin D2 [1α(OH)D2] in the ratio 13/2/1, respectively, but not vitamin D2.(10, 11) Mutations of the CYP27A1 gene cause cerebrotendinous xanthomatosis.(12, 13) Some patients have a 50% reduction in serum 25(OH)D, which is associated with osteoporosis but not rickets or osteomalacia.(13) Whereas the genes for human CYP27A1, 25(OH)D 1α-hydroxylase (CYP27B1), and CYP24A1 have each been identified and sequenced,(10, 14, 15) an important question in vitamin D biology is the identification of human microsomal vitamin D 25-hydroxylases and their substrate specificity.

Based on these considerations and the possibility that one or more of the cytochrome P450 family of the microsomal enzymes 25-hydroxylates vitamin D, the purpose of these studies was to screen recombinant human hepatic cytochrome P450 enzymes expressed in baculovirus-infected insect cells for vitamin D 25-hydroxylase activity. Studies also were performed to correlate activity with known activities of the enzymes in human liver microsomes and to determine the effects of known inhibitors and inhibitory antibodies on activity in pooled human liver microsomes and in the recombinant enzymes.


  1. Top of page
  2. Abstract
  7. Acknowledgements


Human recombinant cytochrome P450 coexpressed in baculovirus-infected insect cells with human cytochrome P450 reductase, and in some cases cytochrome b5, individual human liver microsomes with characterized enzyme activity, pooled human liver microsomes, antibodies against CYP1A1, CYP2B6, CYP2C, CYP3A2, and a NADPH-regenerating system were purchased from BD Gentest (Woburn, MA, USA); troleandomycin, isoniazid, ketoconazole, α-naphthoflavone, dianilinoethane, vitamin D2, vitamin D3 and 1,25(OH)2D3 were from Sigma Aldrich (St Louis, MO, USA); and acetonitrile, dichloromethane, hexanes, methanol, and 2-propanol were from Fisher Scientific (Norcross, GA, USA). [3H-26,27-methyl]-1,25(OH)2D3 was purchased from Amersham Biosciences (Piscataway, NJ, USA). Cytochrome P450 b5 was coexpressed with all of the enzymes except CYP1A1, CYP1A2, CYP1B1, CYP2C18, CYP3A5, and CYP4A11. 1α(OH)D2 and 1α(OH)D3 were generously provided by Bone Care International (Madison, WI, USA) and Leo Pharmaceutical Products (Ballerup, Denmark), respectively. Characterized liver microsomes had been assayed by the vendor for enzymatic activities of coumarin 7-dehydroxylase (CYP2A6), (S)-mephenytoin N-demethylase (CYP2B6), paclitaxel 6α-hydroxylase (CYP2C8), diclofenac 4′-hydroxylase (CYP2C91), (S)-mephenytoin 4′-hydroxylase (CYP2C19), testosterone 6β-hydroxylase (CYP3A4), and lauric acid ω-hydroxylase (CYP4A11) as described.(16)

Assay of 25-hydroxylase

A 1-ml reaction mixture contained either 50 μl of hepatic microsomes (50-150 μg) or 50 μl of microsomes from baculovirus-infected insect cells and 100 μl of 0.5 M Na2HPO4 (pH 7.4), 50 μl of 60 mM EDTA, 50 μl of NADPH regenerating solution A (BD Gentest), 10 μl of regenerating solution B (BD Gentest), 2 μl of 5 mM dianilinoethane, and 2 μl of 1α(OH)D2, 1α(OH)D3, vitamin D2, or vitamin D3. The final concentration of substrate was 10 μM. Unless otherwise mentioned, the reaction was performed at 37°C for 1.5 h and terminated with the addition of 1 ml of acetonitrile. Protein was measured by the bicinchoninic acid method.(17) The reaction with 1α(OH)D2 as substrate was linear for 15 minutes. For enzyme kinetics, incubations were carried out for 10 minutes.

Extraction of samples

The acetonitrile mixture was mixed with a vortex, and 1000 cpm [3H]-1,25(OH)2D3 or [3H]-25(OH)D3, depending on the substrate, was added for recovery and centrifuged at 4°C and 2000g for 10 minutes.(18) The supernatant was decanted to another 13 × 100-mm glass tube containing 1 ml of 0.4 M K2HPO4 (pH 10.4, pH adjusted with KOH) and mixed. The solution containing 1,25(OH)2D3 was mixed and transferred to a C18OH Cartridge (DiaSorin, Stillwater, MN, USA) that had been conditioned twice with 1.5 ml methanol. The cartridge was washed with 5 ml solvent A (methanol:water, 70:30), 5 ml solvent B (hexanes: dichloromethane, 88:12), and 3 ml solvent C (hexanes:2-propanol, 99:1), and eluted with 5 ml solvent D (hexanes:2-propanol, 95:5).(18) The cartridges were washed with 1 ml 2-propanol and conditioned with methanol as above for further use. The eluted extracts were evaporated, and the residue was dissolved in 200 μl dichloromethane:hexanes:2-propanol 50:50:2 and subjected to HPLC. When vitamin D2 or vitamin D3 was the substrate, the C18 cartridge was washed twice with 2 ml 2-propanol and regenerated twice with 2 ml methanol. The supernatant, mixed with 0.5 M K2HPO4 (pH 10.4), was transferred as described above onto the regenerated C18 cartridges. The columns were washed with 5 ml solvent A (methanol:water, 70:30) and eluted with 3.5 ml acetonitrile. The solution was evaporated under N2, dissolved in 200 μl hexanes:methylene chloride:2-propanol (50:50:2.5), and subjected to HPLC.

Isolation and measurement of 1α,25(OH)D2 and 1α,25(OH)D3

Extracts in 200 μl solution were loaded onto a Zorbax Sil 4.6 × 250 mm column, and the metabolites were separated by HPLC with hexanes:2-propanol (85:15). 1α,25(OH)2D2 and 1α,25(OH)2D3 were quantified by measuring the area of the separated peak that eluted at 8.3 and 8.8 minutes, respectively. Recovery was assessed with [3H]-1,25(OH)2D3. Results were expressed as nmol/nmol/1.5 h for human recombinant cytochrome P450 enzymes expressed in baculovirus-infected insect cells and nmol/mg protein/1.5 h for hepatic microsomes. All measurements were carried out in triplicate. For structural confirmation, both sterols were quantified by radioimmunoassay.(19)

Isolation and measurement of 25(OH)D2 and 25(OH)D3

The HPLC was performed as described above. 25(OH)D2 or 25(OH)D3 were separated with the mobile phase hexanes:2-propanol (24:1). Sterols eluted at 7.9 and 9.2 minutes, respectively, and were quantified by radioimmunoassay as previously described.(20) Results are given as nmol/nmol/1.5 h or nmol/mg protein/1.5 h.

Mass spectrometry of 1,25(OH)2D2 and 1,25(OH)2D3

The structural characterization of these two metabolites was by liquid chromatography-mass spectrometry (LC-MS) and gas chromatography-MS (GC-MS). The LC-MS (API QStar Pulsar; PE Sciex Instruments, Boston, MA, USA) provided molecular ions consistent with these hydroxylated species. The spray voltage was 800 V and the collision energy was 25 V. N2 was the collision gas.(21) Structural confirmation of 1,25(OH)2D2 and 1,25(OH)2D3 was by GC-MS (6890 GC-5973N MS with Chemstation and Autosampler; Agilent, Wilmington, DE, USA). A fraction containing a metabolite from the HPLC separation or a reference standard in ethanol was evaporated to dryness in a tapered microvial insert (200 μl; spring fitted) with a stream of nitrogen. N,O-bis(trimethylsilyl)trifluoroacetamide (25 μl; Supelco, Bellefonte, PA, USA) was added, and the microvial was sealed in a 1.5-ml Autosampler vial with a Teflon-lined cap. After heating ∼24 h at 42°C, 1-2 μl were injected in the pulsed splitless mode onto a 5% phenylmethylpolysiloxane GC column (30 m × 0.25 mm; film 0.25 μm) with the helium linear velocity at 55 cm/s. The oven was held at 200°C for 1.5 minutes, followed by a 20°C/minute ramp to a hold at 280°C. Under these GC conditions, the tristrimethylsilyl derivative of metabolite or standard of 1,25(OH)2D2 eluted at 17.6 minutes, and the corresponding derivatives of 1,25(OH)2D3 eluted at 16.2 minutes after injection. MS ionization was by electron impact at 70 eV, acquiring m/z 50-700.

Enzyme antibody inhibition

For these studies, immune and nonimmune sera were added for determination of the inhibitory effects in pooled human microsomes and recombinant cytochrome P450 enzymes. Results are presented as percent control with nonimmune serum.


Linear regression analysis was used to correlate hepatic microsomal vitamin D 25-hydroxylase activity with known activities of cytochrome P450 enzymes in characterized liver microsomes. ANOVA was used to assess the response of pooled human liver microsomes to antibodies to cytochrome P450 proteins.


  1. Top of page
  2. Abstract
  7. Acknowledgements

Measurement of enzyme activity for 25-hydroxylation was carried out for 16 major hepatic cytochrome P450s expressed in baculovirus-infected insect cells with 1α(OH)D2 and 1α(OH)D3 as substrates. Eleven of them showed varying degrees of activity, but CYP3A4 had the greatest activity (Fig. 1). The Km for 1α(OH)D2 was 3.98 ± 0.70 μM and Vmax was 1.33 ± 0.08 nmol/nmol/minute. The Km for 1α(OH)D3 was 9.7 ± 2.2 μM and Vmax was 144.9 ± 9.5 pmol/nmol/minute. With 1α(OH)D2 as substrate, 25-hydroxylase activity of CYP3A4 was 4-fold that of 1α(OH)D3 and 7-fold that of any of the other cytochrome P450s. With vitamin D2 as substrate, activity was 0.17 ± 0.06, 0.08 ± 0.03, 0.07 ± 0.02, 0.81 ± 0.17, 0.10 ± 0.01, 0.36 ± 0.15, and 1.58 ± 0.25 nmol/nmol/1.5 h, respectively, for CYP1A1, CYP1A2, CYP2B6, CYP2C8, CYP2C91, CYP2C18, and CYP3A4, and was either 0.05 nmol/nmol/1.5 h or less or not detectable for the other cytochrome P450 enzymes. With vitamin D3 as substrate, none of the recombinant enzymes showed any 25-hydroxylase activity (data not shown). Reported abundances of individual cytochrome P450 proteins in human liver range from 2% to 29% of total cytochrome P450 protein.(22-24) 25-Hydroxylase activity of CYP3A4 with vitamin D2 and vitamin D3 as substrates was compared with that of 1α(OH)D2 and 1α(OH)D3 (Table 1). Activity with 1α(OH)D2 was almost 4-fold that with 1α(OH)D3 and was 7-fold that with vitamin D2. No activity was found with vitamin D3 as substrate when incubations were carried out for as long as 24 h or when pooled human liver microsomes were used (data not shown). In subsequent experiments, 1α(OH)D2 was used as substrate.

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Figure FIG. 1.. Vitamin D 25-hydroxylase activity in recombinant human cytochrome P450 enzymes. Incubations were carried out with enzymes for 1.5 h. Substrate concentration of 1α(OH)D2 and 1α(OH)D3 was 10 μM. At the end of incubation, samples were extracted, separated by HPLC, and analyzed by measuring the peaks of 1,25(OH)2D2 or 1,25(OH)2D3 in the eluates. Results are mean ± SE of three determinations.

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Table Table 1.. 25-Hydroxylation by Human Recombinant CYP3A4 With Different Substrates: Substrate Concentration was 10 μM
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Structural identity of the extracted and HPLC-fractionated 1,25(OH)2D3, 1,25(OH)2D2, and 25(OH)D2 were supported by results of studies with radioimmunoassay, where it was demonstrated that unknowns in the eluates diluted in parallel with standards (Fig. 2). Structure of the metabolites 1,25(OH)2D2 and 1,25(OH)2D3 was confirmed by comparison of their respective LC and GC retention times, molecular ions (M+; LC-MS and GC-MS), and electron impact fragmentation patterns (Fig. 3) with those of the respective reference standards. Electron impact MS spectra of tristrimethylsilyl 1,25(OH)2D3(3, 4, 11) matched that of our study. As noted in Fig. 3, each base peak ion (m/z 131) resulted from the α-cleavage fragmentation process (C24-C25 bond scission) characteristic of 25-hydroxyl isomers.(4) The corresponding α-cleavage product of the 26,27-hydroxyl isomer yielded m/z 103 ions (C25-C26 bond scission).(4) Molecular ions (M+) were detectable for all four electron impact-MS samples: m/z 644 for 1,25(OH)2D2 and m/z 632 for 1,25(OH)2D3. However, the M+ ion (1% relative abundance) for the reference standard of 1,25(OH)2D3 was not assigned a printed mass number (Fig. 3D) because of space constraints from m/z 617.

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Figure FIG. 2.. Radioimmunoassay of reference standards and metabolically formed 1,25(OH)2D3, 1,25(OH)2D2, and 25(OH)D2. Assay of standards (○) was carried out in duplicate and assay of metabolically formed metabolites (•) in triplicate. Note that metabolically formed and standards of 1,25(OH)2D3, 1,25(OH)2D2, and 25(OH)D2 diluted in parallel.

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Figure FIG. 3.. (A) Electron impact mass spectra of metabolically formed 1,25(OH)2D2. (B) Reference standard of 1,25(OH)2D2. (C) Metabolically formed 1,25(OH)2D3.(D) Reference standard of 1,25(OH)2D3. All samples were analyzed as tristrimethylsilyl derivatives. The m/z 632 molecular ion (M+) of metabolite 1,25(OH)2D3 was present at 1% relative abundance.

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If two different substrates are metabolized by a single enzyme, enzymatic activity of the two substrates should correlate when their activities are measured in samples from different livers. In a panel of 12 characterized human liver microsomes in which enzyme activities had been predetermined,(24) CYP3A4 activity correlated significantly with testosterone 6β-hydroxylase activity, an enzyme activity characteristic of CYP3A4 (Fig. 4). Vitamin D 25-hydroxylase activity varied 13-fold in these different liver samples. Activity correlated less significantly with diclofenac 4′-hydroxylase activity (CYP2C91) but not with enzymatic activities of coumarin 7-hydroxylase (CYP2A6), paclitaxel 6α-hydroxylase (CYP2C8), (S)-mephenytoin 4′-hydroxylase (CYP2C19), or lauric acid ω-hydroxylase (CYP4A11; Table 2). In these different liver microsomes, microsomal CYP3A4 testosterone 6β-hydroxylase activity varied 28-fold and CYP2C9 diclofenac 4′-hydroxylase activity varied 8-fold (data not shown). 25-hydroxylation activity by pooled human liver microsomes with 1α(OH)D2 as substrate for 11 experiments was 0.54 ± 0.04 nmol/mg protein/1.5 h.

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Figure FIG. 4.. Correlation between vitamin D 25-hydroxylase activity and CYP3A4 testosterone. 6β-Hydroxylase activity in a panel of 12 characterized human liver microsomes. Incubations were carried out with microsomes for 1.5 h. Substrate concentration of 1α(OH)D3 was 10 μM. At the end of incubation, samples were extracted, separated by HPLC, and analyzed by measuring the peaks of 1,25(OH)2D2 in the eluates. Results are mean ± SE of three determinations.

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Table Table 2.. Correlation Between Vitamin D-25-hydroxylase Activity and Other Specific Cytochrome P450 Enzyme Activities in 12 Characterized Human Liver Microsomes
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We used inhibition of some of the cytochrome P450 enzyme activities to identify which of them was responsible for the 25-hydroxyase activity. 25-Hydroxylase activity was inhibited in a dose-response fashion by ketoconazole, α-naphthoflavone, troleandomycin, and isoniazid, known inhibitors of CYP3A enzyme activity in human recombinant CYP3A4 and pooled liver microsomes (Fig. 5).(25-28) Inhibition in a dose-dependent fashion by α-naphthoflavone, also an inhibitor of CYP1A1, and by sulfaphenazole, an inhibitor of CYP2C91 (Fig. 6), was observed.(26, 29, 30) 25-Hydroxylase activity in pooled human microsomes was inhibited 23% by an anti-CYP1A1 antibody, 52% by an anti-CYP2C antibody, and 71% by an anti-CYP3A2 antibody that inhibits CYP3A4,(28) and was not altered by an anti-CYP2A6 antibody (Fig. 7).

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Figure FIG. 5.. Effects of inhibitors of CYP3A4 on vitamin D 25-hydroxylase activity in pooled human liver microsomes (HLM) and recombinant CYP3A4. Incubations were carried out with microsomes or enzymes for 1.5 h. Substrate concentration of 1α(OH)D2 was 10 μM. At the end of incubation, samples were extracted, separated by HPLC, and analyzed by measuring the peaks of 1,25(OH)2D2 in the eluates. Results are mean ± SE of three determinations.

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Figure FIG. 6.. Effects of inhibitors of CYP1A1 and CYP2C9*1 on vitamin D 25-hydroxylase activity in pooled human liver microsomes (HLM) and recombinant enzymes. Incubations were carried out with microsomes or enzymes for 1.5 h. Substrate concentration of 1α(OH)D2 was 10 μM. At the end of incubation, samples were extracted, separated by HPLC, and analyzed by measuring the peaks of 1,25(OH)2D2 in the eluates. Results are mean ± SE of three determinations.

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Figure FIG. 7.. Effects of antibodies against cytochrome P450 enzymes on vitamin D 25-hydroxylase activity in pooled human hepatic microsomes. Incubations were carried out with microsomes for 1.5 h. Substrate concentration of 1α(OH)D2 was 10 μM. At the end of incubation, samples were extracted, separated by HPLC, and analyzed by measuring the peak of 1,25(OH)2D2 in the eluates. Results are mean ± SE of three determinations. *p < 0.05, **p < 0.01. Antibodies used were goat polyclonal anti-rat CYP1A1 for human CYP1A1, monoclonal anti-human CYP2A6 for human CYP2A6, goat polyclonal anti-rat CYP2C13 for human CYP3C9, and rabbit anti-rat CYP3A2 for human CYP3A4. Controls for each volume of antiserum contained the same volume of nonimmune goat or rabbit serum.

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  1. Top of page
  2. Abstract
  7. Acknowledgements

CYP3A4 is the most abundant cytochrome P450 enzyme, and together with CYP3A5 and CYPA7, represents almost 30% of cytochrome P450 content in the human liver, whereas CYP4A11 and CYP2C9 each account for about 14%. CYP1A1 is expressed at low levels in liver and is induced by smoking.(31) CYP3A5 is polymorphically expressed in one-third of the livers of whites and one-half of the livers of blacks.(32) CYP3A7 accounts for 1.5% of CYP3A4 and CYP3A7 mRNA transcripts together in adult liver tissue.(33) However, the relative quantitative amounts of CYP3A5 and CYP3A7 proteins compared with CYP3A4 protein in human liver have not been determined. The 25-hydroxylase activities of CYP3A5 and CYP3A7 were extremely low compared with the activity of CYP3A4.

The present studies provide evidence that CYP3A4 is a hepatic microsomal vitamin D 25-hydroxylase. First, CYP3A4 showed the highest vitamin D 25-hydroxylase activity of the 16 cytochrome P450 enzymes examined. However, like CYP27A1, CYP3A4 hydroxylated both 1α(OH)D2 and 1α(OH)D3, but unlike CYP27A1, CYP3A4 hydroxylated vitamin D2 and not vitamin D3.(11) Second, correlation of 25-hydroxylase activity with CYP3A4 testosterone 6β-hydroxylase activity in characterized liver microsomes(25) was highly significant; that is, those liver microsomes that had the highest testosterone 6β-hydroxylase activity also had the highest vitamin D 25-hydroxylase activity. Third, 25-hydroxylase activity in hepatic microsomes and recombinant CYP3A4 was inhibited by ketoconazole, α-naphthoflavone, and troleandomycin, known inhibitors of CYP3A enzymes.(26) Furthermore, activity in hepatic microsomes was inhibited by a polyclonal antibody to CYP3A2 that inhibits CYP3A4.(26) Activity also was inhibited by isoniazid, a drug that inhibits CYP3A4.(30) In addition, previous studies demonstrated that CYP3A4 mRNA in cultured human hepatocytes was increased 15-fold by 1,25(OH)2D3 at a low normal concentration of 10 nM and much less at a high normal concentration of 100 nM,(34) and 9-fold by growth hormone.(35) CYP3A4 gene expression was also induced by 1,25(OH)2D3 in the human fetal small intestine.(36) Induction of the enzyme by 1,25(OH)2D3 could provide a strong, positive feedback mechanism to ensure an adequate supply of substrate for renal 25(OH)D 1α-hydroxylase at low concentrations of 1,25(OH)2D. Whether the effects of growth hormone on CYP3A4 are physiological is not clear because studies in growth hormone-deficient subjects showed that CYP3A4 activity, estimated with the erythromycin breath test, was increased when growth hormone was given by continuous infusion but not by the pulsatile administration that occurs physiologically.(37) CYP2B6 and CYP2C91 also were induced by 1,25(OH)2D3, but only 3.5- and 2.6-fold, respectively.(34) Because CYP2B6 has no 25-hydroxylase activity, it has no apparent role in vitamin D metabolism. On the other hand, CYP2C91 may play a modest role because the recombinant enzyme has some 25-hydroxylase activity. CYP2C91 diclofenac 4′-hydroxylase activity correlated significantly with 25-hydroxylase activity in characterized liver microsomes, and activity in pooled liver microsomes was inhibited by an antibody to CYP2C and by sulfaphenazole, an inhibitor of CYP2C9.(26, 30) Similarly, 25-hydroxylase activity in pooled liver microsomes was inhibited by an antibody to CYP1A1.

CYP3A4 is the most abundant constitutively expressed cytochrome P450 enzyme in liver and small intestine and also is present in esophagus, colon, kidney, and leukocytes.(38-41) Hepatic expression of CYP3A4 is known to vary by as much as 40-fold,(22, 38) and testosterone 6β-hydroxylase activity in hepatic microsomes by as much as 31-fold,(42) a finding very similar to the 28-fold variation in this study.

We found that pooled liver microsomes, like recombinant CYP3A4 and the other recombinant enzymes, did not have any 25-hydroxylase activity when vitamin D3 was used as substrate. In a previous study, activity was found only in mitochondria and not in microsomes from human liver, even when five times the standard amount of incubation volume, microsomal protein, cofactors, and substrate were used.(2) In that report, the finding of 25-hydroxylase activity in microsomes as well as mitochondria in an earlier study(1) was attributed to contamination with mitochondria.

Vitamin D3 and its metabolites are the predominant forms of vitamin D in humans.(43) CYP27A1, a mitochondrial enzyme, has specificity for vitamin D3 as substrate.(11) The specificity of CYP3A4 for vitamin D2 and its analog raises questions about the relative physiologic importance of the two enzymes regarding vitamin D metabolism. As noted, patients with mutations of CYP27 develop cerebrotendinous xanthomatosis(12, 13) but not rickets or osteomalacia, whereas some of them were reported to have reductions of about 50% in serum 25(OH)D and to have early-onset, severe osteoporosis.(13) However, whether CYP27A1 plays an important role in the 25-hydroxylation of vitamin D3 is not established.(44) This study shows that 1α(OH)D2 and 1α(OH)D3, drugs that are used clinically and are substrates for microsomal CYP3A4 as well as mitochondrial CYP27A1, are converted to 1,25(OH)2D2 and 1,25(OH)2D3, respectively, to become biologically active.(45)

CYP2R1, a microsomal enzyme recently cloned, was found to 25-hydroxylate vitamin D2 and D3 equally and to be abundantly expressed and was proposed as a putative microsomal vitamin D 25-hydroxylase.(46) This may be so, but there needs to be reconciliation with the present and previous observation(2) that vitamin D3 is not hydroxylated by human liver microsomes. The possible role of this enzyme in vitamin D metabolism remains to be determined.

CYP3A4 regulates the metabolism of more than one-half of all commonly used drugs. Exogenous substrates for the enzyme include erythromycin, midazolam, cyclosporin, lovastatin, and nifedipine.(47, 48) Endogenous substrates include testosterone, 17β-estradiol, progesterone, hydrocortisone, and lithocholic acid.(47) Vitamin D2, an exogenous substrate, can now be included in this group. The large number of inducers and inhibitors of CYP3A4(47, 49) provide the basis for a wide range of potential drug interactions with vitamin D2, 1α(OH)D2, and 1α(OH)D3.

Induction of CYP2 and CYP3 genes by xenobiotics is mediated by the glucocorticoid receptor and two nuclear orphan receptors, the pregnane X receptor (hPXR) and the constitutive androstane receptor (CAR).(45-55) The effects of 1,25(OH)2D3 are mediated by the vitamin D receptor (VDR) and the VDR-RXR complex binds PXR-responsive elements of the CYP3A4 promoter to initiate transcription.(34)

A number of drugs alter vitamin D metabolism. Previous studies showed that serum 25(OH)D was significantly reduced in normal men treated with isoniazid.(56) The reduction in serum 25(OH)D also was associated with decreases in serum 1,25(OH)2D and serum calcium and increases in serum intact parathyroid hormone, indicating that isoniazid also inhibited CYP27B1 activity. A 70% reduction in serum 25(OH)D also occurred in normal men treated with rifampin,(57) a profound inducer of CYP3A4.(51, 53) Treatment of patients with tuberculosis for 9 months with isoniazid and rifampin resulted in wide individual variation but no significant change in mean serum 25(OH)D.(58) Diltiazem inhibits CYP3A4 activity by forming a metabolite intermediate complex with the enzyme.(59) Metabolite intermediate complexes inhibit activity by removing active cytochrome P450 enzymes from the enzyme pool. In addition to inhibiting CYP3A4, ketoconazole also inhibits CYP2B1 and has been used to treat hypercalcemia caused by elevated circulating 1,25(OH)2D in patients with sarcoidosis.(60) Phenobarbital, an inducer of CYP3A4(55) through CAR, is known to cause vitamin D deficiency and rickets in patients when given long term together with other anticonvulsants.(61, 62) It could act either by enhancing 25-hydroxylase activity, transiently increasing serum 25(OH)D and causing depletion of vitamin D as occurred in Japanese children,(62) by enhancing conversion by phenobarbital to more polar biologically inactive metabolites,(63) or both. How these drugs alter vitamin D metabolism and what 25-hydroxylases are involved is yet to be determined.

In a previous investigation, none of the recombinant enzymes used in the present study showed any microsomal 25-hydroxylase activity when expressed in a B-lymphoblastoid cell line with 1α(OH)D3 used as a substrate.(64) We found that CYP3A4 expressed in the B-lymphoblastoid cell line had no 25-hydroxylase activity with 1α(OH)D2 as substrate (RP Gupta and NH Bell, unpublished observations, 2003). Despite the fact that these cell lines are widely used to investigate drug metabolism by CYP3A4 and other cytochrome P450 enzymes, we have no explanation for the discordance in vitamin D 25-hydroxylase activity expressed in B-lymphoblastoid and baculovirus-infected insect cells.

In conclusion, our studies indicate that CYP3A4 is a hepatic microsomal vitamin D 25-hydroxylase with specificity for vitamin D2 and not vitamin D3. Like CYP27A1, it hydroxylates both 1α(OH)D2 and 1α(OH)D3. Because CYP3A4 metabolizes over one-half of commonly used drugs and is inhibited and induced by a number of drugs, there is great potential for drug-induced alteration in the metabolism of vitamin D2, 1α(OH)D2, and 1α(OH)D3.


  1. Top of page
  2. Abstract
  7. Acknowledgements

These studies were supported by National Institutes of Health Grant DK56603-01A1.


  1. Top of page
  2. Abstract
  7. Acknowledgements
  • 1
    Björkhem I, Holmberg I 1978 Assay and properties of a mitochondrial 25-hydroxylase active on vitamin D. J Biol Chem 253:842849.
  • 2
    Saarem K, Bergseth S, Oftebro H, Pedersen JI 1984 Subcellular localization of vitamin D3 25-hydroxylase in human liver. J Biol Chem 259:1093610960.
  • 3
    Lawson DEM, Fraser DR, Kocdicek E, Morris HR, Williams DH 1971 Identification of 1,25-dihydroxycholecalciferol, a new kidney hormone controlling calcium metabolism. Nature 230:228230.
  • 4
    Holick MF, Schnoes HK, DeLuca HF 1971 Identification of 1,25-dihydroxycholecal-ciferol, a form of vitamin D3 metabolically active in the intestine. Proc Natl Acad Sci USA 68:803804.
  • 5
    Norman AW, Myrtle JF, Midgett RJ, Nowicki HG, Williams V, Popjak G 1971 1,25-Dihydroxycholecalciferol: Identification of the proposed active form of vitamin D3 in the intestine. Science 173:5154.
  • 6
    Holick MR, Schnoes HK, DeLuca HF, Gray RW, Boyle IT, Suda T 1972 Isolation and identification of 24,25-dihydroxycholecalciferol, a metabolite of vitamin D3 made in kidney. Biochemistry 11:45214255.
  • 7
    Sakaki T, Sawada N, Nonaka Y, Ohyama Y, Inouye K 1999 Metabolic studies using recombinant Escherichia coli cells producing rat mitochondrial CYP24: CYP24 can convert 1α,25-dihydroxyvitamin D3 to calcitroic acid. Eur J Biochem 262:4348.
  • 8
    Zimmerman DR, Reinhardt TA, Kremer R, Beitz DC, Reddy GS, Horst RL 2001 Calcitroic acid is a major catabolic metabolite in the metabolism of 1α,25-dihydroxyvitamin D2. Arch Biochem Biophys 392:1422.
  • 9
    Kamachi S, Sugimoto K, Yamasakis T, Hirose N, Ide H, Ohyama Y 2001 Metabolic activation of 1α-hydroxyvitamin D3 in human liver microsomes. Xenobiotica 31:701712.
  • 10
    Cali JJ, Russell DW 1991 Characterization of human sterol 27-hydroxylase. A mitochondrial cytochrome P-450 that catalyzes multiple oxidation reaction in bile acid biosynthesis. J Biol Chem 266:77747778.
  • 11
    Guo Y-D, Strugnell S, Back DW, Jones G 1993 Transfected human liver cytochrome P-450 hydroxylates vitamin D analogs at different side-chain positions. Proc Natl Acad Sci USA 90:86688672.
  • 12
    Cali JJ, Hsieh CL, Francke U, Russell DW 1991 Mutations in the bile acid biosynthetic enzyme sterol 27-hydroxylase underlie cerebrotendinous xanthomatosis. J Biol Chem 266:77797783.
  • 13
    Berginer VM, Shany S, Aklalay D, Berginer J, Dekel S, Salen G, Ting GS, Gazit D 1993 Osteoporosis and increased bone fractures in cerebrotendinous xanthomatosis. Metabolism 42:6974.
  • 14
    Chen KS, Prahl JM, DeLuca HF 1993 Isolation and expression of human 1,25-dihydroxyvitamin D3 24-hydroxylase cDNA. Proc Natl Acad Sci USA 90:45434547.
  • 15
    Fu GK, Lin D, Zhang MY, Bikle DD, Shackleton CH, Miller WL, Portale AA 1997 Cloning of human 25-hydroxyvitamin D-1 alpha-hydroxylase and mutations causing vita-min D-dependent rickets type 1. Mol Endocrinol 11:19611970.
  • 16
    Anonymous 2002 Human liver microsome assays. Available online at Accessed on May 23, 2002.
  • 17
    Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzaon MD, Fujimo-to IK, Goeke NM, Olson BJ, Klenk DC 1985 Measurement of protein using bicinchoninic acid. Anal Biochem 150:7685.
  • 18
    Hollis BW 1997 Detection of vitamin D and its major metabolites. In: FeldmanDD, GlorieuxFH, PikeJW (eds.) Vitamin D. Academic Press, San Diego, CA, USA, pp. 587606.
  • 19
    Hollis BW, Kamerud JQ, Kurkowski A, Beaulieu J, Napoli JL 1996 Quantitation of circulating 1,25-dihydroxyvitamin D by radioimmunoassay with an 125I-labeled tracer. Clin Chem 42:586592.
  • 20
    Hollis BW, Kamerud JQ, Selvaag SR, Lorenz JD, Napoli JL 1993 Determination of vitamin D status with an 125I-labeled tracer. Clin Chem 39:529533.
  • 21
    Jones G 1986 A new pathway of 25-hydroxyvitamin D3 metabolism. Methods Enzymol 123:141154.
  • 22
    Shimada T, Yamazaki H, Mimura M, Inui Y, Guengerich P 1994 Interindividual variations in human liver cytochrome P-450 enzymes involved in the oxidation of drugs, carcinogens and toxic chemicals; studies with liver microsomes of 30 Japanese and 30 Caucasians. J Pharmacol Exp Ther 270:414423.
  • 23
    Lasker JM, Wester MR, Aramsombatdee E, Raucy JL 1998 Characterization of CYP2C19 and CYP2C9 from human liver: Respective roles in microsomal tolbutamide, S-mephenytoin, and omeprazole hydroxylations. Arch Biochem Biophys 353:1628.
  • 24
    Lasker JM, Chen WB, Wolf I, Bloswick BP, Wilson PD, Powell PK 2000 Formation of 20-hydroxyeicosatetraenoic acid, a vasoactive and natriuretic eicosanoid, in human kidney. J Biol Chem 275:41184126.
  • 25
    Roy P, Yu LJ, Crespi CL, Waxman DJ 1999 Development of a substrate-activity based approach to identify the major human liver P-450 catalysts of cyclophosphamide and ifosfamide activation based on cDNA-expressed activities and liver microsomal P-450 profiles. Drug Metab Dispos 27:655666.
  • 26
    Ono S, Hatanaka T, Hotta H, Satoh T, Gonzalez FJ, Tsutsui M 1996 Specificity of substrate and inhibitor probes for cytochrome P450s: Evaluation of in vitro metabolism using cDNA-expressed human P450s and human liver microsomes. Xenobiotica 26:681693.
  • 27
    Desta Z, Soukhova NV, Flockhart DA 2001 Inhibition of cytochrome P450 (CYP450) isoforms by isoniazid: Potent inhibition of CYP2C19 and CYP3A. Antimicrob Agents Chemother 45:382392.
  • 28
    Ghosal A, Satoh H, Thomas PE, Bush E, Moore D 1996 Inhibition and kinetics of cytochrome P4503A activity in microsomes from rat, human, and cDNA-expressed human cytochrome P450. Drug Metab Dispos 24:940947.
  • 29
    Ueng Y-F, Kuwabara T, Chun Y-J, Guengerich FP 1997 Cooperativity in oxidations catalyzed by cytochrome P450 3A4. Biochemistry 36:370381.
  • 30
    Giancarlo GM, Venkatakrishnan K, Granda BW, von Moltke LL, Greenblatt DJ 2001 Relative contributions of CYP2C9 and 2C19 to phenytoin 4-hydroxylation in vitro: Inhibition by sulfaphenazole, omeprazole, and ticlopidine. Eur J Clin Pharmacol 57:3136.
  • 31
    Schweikl H, Taylor JA, Kitareewan S, Linko P, Nagorney D, Goldstein JA 1993 Expression of CYP1A1 and CYP1A2 genes in human liver. Pharmacogenetics 3:239249.
  • 32
    Khuel P, Zhang P, Lin J, Lamba J, Assem M, Schuetz J, Watkins PB, Daly A, Wrighton SA, Hall SD, Maurel P, Relling M, Brimer C, Yasuda K, Venkataramanan R, Strom S, Thummel K, Boguski MS, Schuetz E 2001 Sequence diversity in CYP3A promoters and characterization of the genetic basis of polymorphic CYP3A5 expression. Nat Genet 27:383391.
  • 33
    Burk O, Tegude H, Kock I, Hustert E, Wolbold R, Glaeser H, Klein K, Fromm MF, Nuessler AK, Neuhaus P, Zanger UM, Eichelbaum M, Wolnowski L 2002 Molecular mechanisms of polymorphic CYP3A7 expression in adult human liver and intestine. J Biol Chem 277:2428024288.
  • 34
    Drocourt L, Ourlin J-C, Pascussi J-M, Maurel P, Vilarem M-J 2002 Expression of CYP3A4, CYP2B6 and CYP2C9 is regulated by the vitamin D receptor pathway in primary human hepatocytes. J Biol Chem 277:2512525132.
  • 35
    Liddle C, Goodwin BJ, George J, Tapner M, Farrell GC 1998 Separate and interactive regulation of cytochrome P450 3A4 by triiodothyronine, dexamethasone, and growth hormone in cultured hepatocytes. J Clin Endocrinol Metab 83:24112416.
  • 36
    Theodoropoulos C, Demers C, Delvin E, Menard D, Gascon-Barre M 2003 Calcitriol regulates the expression of the genes encoding the three key vitamin D3 hydroxylases and the drug-metabolizing enzyme CYP3A4 in the human fetal intestine. Clin Endocrinol (Oxf) 58:489499.
  • 37
    Jaffe CA, Turgeon DK, Lown K, Demott-Friberg R, Watkins PB 2002 Growth hormone secretion pattern is an independent regulator of growth hormone actions in humans. Am J Physiol Endocrinol Metab 283:E1008E1015.
  • 38
    Hashimoto H, Toide K, Kitamura R, Fujita M, Tagawa S, Itoh S, Kamataki T 1993 Gene structure of CYP3A4, an adult-specific form of cytochrome P450 human livers, and its transcriptional control. Eur J Biochem 218:585595.
  • 39
    Schuetz JD, Beach DL, Guzelian PS 1994 Selective expression of cytochrome P450 CYP3A mRNAs in embryonic and adult human liver. Pharmacogenetics 4:1120.
  • 40
    Zhang CY, Dunbar D, Ostrowska A, Zeisloft S, Yang J, Kaminsky LS 1999 Characterization of human small intestinal cytochromes P-450. Drug Metab Dispos 27:804809.
  • 41
    de Wildt SN, Kearns GL, Leeder JS, van den Anker JN 1999 Cytochrome P450 3A: Ontogeny and drug disposition. Clin Pharmacokinet 37:485506.
  • 42
    Westlind A, Löfberg L, Tindberg N, Andersson TB, Ingel-Sundberg M 1999 Interindividual differences in hepatic expression of CYP3A4: Relationship to genetic polymorphism in the 5′-upstream regulatory region. Biochem Biophys Res Commun 259:201205.
  • 43
    Hollis BW, Pittard WB, Reinhardt TA 1986 Relationships among vitamin D, 25-hydroxyvitamin D-binding protein concentrations in the plasma and milk of humans. J Clin Endocrinol Metab 62:4144.
  • 44
    Jones G, Strugnell SA, DeLuca HF 1998 Current understanding of the molecular actions of vitamin D. Physiol Rev 78:11931231.
  • 45
    Brown AJ, Dusso AS, Slatopolsky E 2002 Vitamin D analogues for secondary hyperparathyroidism. Nephrol Dial Transplant 10(Suppl):1019.
  • 46
    Cheng JB, Motola DL, Mangelsdorf DJ, Russell DW 2003 Deorphanization of cytochrome P4502R1: A microsomal vitamin D-25-hydroxylase. J Biol Chem 278:3808438093.
  • 47
    Gibson GG, Plant NJ, Swales KE, Ayrton A, El-Sankary W 2002 Topics in xenobiochemistry. Receptor-dependent transcriptional activation of cytochrome P4503A genes: Induction mechanisms, species differences and interindividual variation in man. Xenobiotica 32:165206.
  • 48
    Venkatakrishnan K, von Moltke LL, Greenblatt DJ 2001 Human drug metabolism and the cytochromes P450: Application and relevance of in vitro methods. J Clin Pharmacol 41:11491170.
  • 49
    Dresser GK, Spence JD, Bailey DG 2000 Pharmacokinetic-pharmacodynamic consequences and clinical relevance of cytochrome P450 3A4 inhibition. Clin Pharmacokinet 38:4157.
  • 50
    Sankary W, Plant NJ, Gibson GJ, Moore DJ 2000 Regulation of the CYP3A4 gene by hydrocortisone and xenobiotics: Role of the glucocorticoid and pregnane X receptors. Drug Metab Dispos 28:493496.
  • 51
    Lehmann JM, McKee DD, Watson MA, Willson TM, Moore JT, Kliewer SA 1998 The human orphan nuclear receptor PXR is activated by compounds that regulate CYP3A4 gene expression and cause drug interactions. J Clin Invest 102:10161023.
  • 52
    Bertilsson G, Heidrich J, Svensson K, Asman M, Jendeberg L, Sydow-Backman M, Ohlsson R, Postlind H, Blomquist P, Berkenstam A 1998 Identification of a human nuclear receptor defines a new signaling pathway for CYP3A induction. Proc Natl Acad Sci USA 95:1220812213.
  • 53
    Goodwin B, Hodgson E, Liddle C 1999 The orphan human pregnane X receptor mediates the transcriptional activation of CYP3A4 by rifampicin through a distal enhancer module. Mol Pharmacol 56:13291339.
  • 54
    Moore LB, Parks DJ, Jones SA, Bledso RK, Consler TG, Stimmel JB, Goodwin B, Liddle C, Blanchard SG, Willson TM, Collins TL, Kliewer SA 2000 Orphan nuclear receptors constitutive receptor and pregnane X receptor share xenobiotic and steroid ligands. J Biol Chem 275:1512215127.
  • 55
    Pascussi J-M, Gerbal-Chaloin S, Fabre J-M, Maurel P, Vilarem M-J 2000 Dexamethasone enhances constitutive androstane receptor expression in human hepatocytes: Consequences on cytochrome P450 gene regulation. Mol Pharmacol 58:14411450.
  • 56
    Brodie MJ, Boobis AR, Hillyard CJ, Abeyasekera G, MacIntyre I, Part BK 1981 Effect of isoniazid on vitamin D metabolism and hepatic monooxygenase activity. Clin Pharmacol Ther 30:362367.
  • 57
    Brodie MJ, Boobis AR, Dollery CT, Hillyard CJ, Brown DJ, MacIntyre I, Part BK 1980 Rifampicin and vitamin D metabolism. Clin Pharmacol Ther 27:810814.
  • 58
    Williams SE, Wardman AG, Taylor GA, Peacock M, Cooke NJ 1985 Long term study of the effect of rifampin and isoniazid on vitamin D metabolism. Tuberculosis (Edinb) 66:4954.
  • 59
    Jones DR, Gorski JC, Hamman MA, Mayhew BS, Rider S, Hall SD 1999 Diltiazem inhibition of cytochrome P-450 3A activity is due to metabolite intermediate complex formation. J Pharmacol Exp Ther 290:11161125.
  • 60
    Adams JS, Sharma OP, Diz MM, Endres DB 1990 Ketoconazole decreases the serum 1,25-dihydroxyvitamin D and calcium concentration in sarcoidosis-associated hypercalcemia. J Clin Endocrinol Metab 13:407413.
  • 61
    Hahn TJ, Hendin BA, Scharp CR, Haddad JG 1972 Effect of chronic anticonvulsant therapy on serum 25-hydroxycholecalciferol levels in adults. N Engl J Med 287:900904.
  • 62
    Sumi K, Sugita T, Shimotsuji T, Seino Y, Mimaki T, Yabuuchi H 1978 Effect of anticonvulsant therapy on serum 25-hydroxyvitamin D level. Tohoku J Exp Med 125:265269.
  • 63
    Hahn TJ, Birge SJ, Scharp CR, Avioli LV 1972 Phenobarbital-induced alterations in vitamin D metabolism. J Clin Invest 51:748751.
  • 64
    Kamachi S, Sugimoto K, Yamasaki T, Hirose N, Ide H, Ohyama Y 2001 Metabolic activation of 1α-hydroxyvitamin D3 in human liver microsomes. Xenobiotica 10:701712.