Mutational analysis of CYP27A1: assessment of 27-hydroxylation of cholesterol and 25-hydroxylation of vitamin D.

The CYP27A1 gene encodes a mitochondrial enzyme that modulates the acidic biosynthetic pathway for bile acids beginning with the 27-hydroxylation of cholesterol. CYP27A1 also 25-hydroxylates vitamin D(3). Gene mutations cause cerebrotendinous xanthomatosis (CTX), an autosomal recessive disorder, and may cause 25-hydroxyvitamin D deficiency and early-onset osteoporosis and fractures in affected patients. To examine the effects of mutations of CYP27A1 on vitamin D and cholesterol hydroxylating activity, recombinant CYP27A1 and mutant complementary DNAs produced by site-directed mutagenesis were stably expressed in either Escherichia coli or COS-1 cells. Activities of wild-type and mutant enzymes were determined with cholesterol, vitamin D(3), and 1alpha-hydroxyvitamin D(3) (1alphaOHD(3)) as substrates. Of the 15 mutants tested, 11 expressed protein and 4 expressed little or no protein. Functional heme activity, estimated by reduced CO difference spectra at 450 nm, was absent in 12 mutants. When expressed in E. coli, 3 mutants, K226R, D321G, and P408S, each known to cause clinically CTX, showed modest decreases in reduced CO spectra peak and either no change or decreases of less than 50% in hydroxylation of cholesterol, vitamin D(3), and 1alphaOHD(3) compared with wild type. When expressed transiently in COS-1 cells, each of these mutants showed 25-hydroxylation activity for 1alphaOHD(3) as well as wild type. Thus, 3 mutants, K226R, D321G, and P408S, known to occur clinically with nonfunctioning mutants, hydroxylated cholesterol, vitamin D(3), and 1alphaOHD(3). How they contribute to the pathogenesis of CTX despite being biologically active in vitro remains to be determined.

CYP3A4 is a vitamin D-24- and 25-hydroxylase: analysis of structure function by site-directed mutagenesis.

Studies were performed to identify the microsomal enzyme that 24-hydroxylates vitamin D, whether 25-hydroxylation occurs, and structure function of the enzyme. Sixteen hepatic recombinant microsomal cytochrome P450 enzymes expressed in baculovirus-infected insect cells were screened for 24-hydroxylase activity. CYP3A4, a vitamin D-25-hydroxylase, and CYP1A1 had the highest 24-hydroxylase activity with 1 alpha-hydroxyvitamin D(2) (1 alpha OHD(2)) as substrate. The ratio of rates of 24-hydroxylation of 1 alpha-hydroxyvitamin D(3) (1 alpha OHD(3)), 1 alpha OHD(2), and vitamin D(2) by CYP3A4 was 3.6/2.8/1.0. Structures of 24-hydroxyvitamin D(2), 1,24(S)-dihydroxyvitamin D(2), and 1,24-dihydroxyvitamin D(3) were confirmed by HPLC and gas chromatography retention time and mass spectroscopy. In characterized human liver microsomes, 24-hydroxylation of 1 alpha OHD(2) by CYP3A4 correlated significantly with 6 beta-hydroxylation of testosterone, a marker of CYP3A4 activity. 24-Hydroxylase activity in recombinant CYP3A4 and pooled human liver microsomes showed dose-dependent inhibition by ketoconazole, troleandomycin, alpha-naphthoflavone, and isoniazid, known inhibitors of CYP3A4. Rates of 24- and 25-hydroxylation of 1 alpha OHD(2) and 1 alpha OHD(3) were determined in recombinant wild-type CYP3A4 and site-directed mutants and naturally occurring variants expressed in Escherichia coli. Substitution of residues showed the most prominent alterations of function at residues 119, 120, 301, 305, and 479. Thus, CYP3A4 is both a 24- and 25-hydroxylase for vitamin D(2), 1 alpha OHD(2), and 1 alpha OHD(3).

Significant differential effects of alendronate, estrogen, or combination therapy on the rate of bone loss after discontinuation of treatment of postmenopausal osteoporosis. A randomized, double-blind, placebo-controlled trial.

BACKGROUND: Combination therapy with alendronate and estrogen for 2 years increases bone mineral density at the spine and hip more than does therapy with either agent alone. Changes in bone mineral density after discontinuation of therapy have not been compared directly. OBJECTIVE: To determine the rate of bone loss when therapy with alendronate, estrogen, or both agents is discontinued. DESIGN: Double-blind, placebo-controlled discontinuation trial. SETTING: 18 U.S. centers. PATIENTS: 244 postmenopausal, hysterectomized women 44 to 77 years of age. INTERVENTION: 2 years of therapy with alendronate, 10 mg/d (n = 92); conjugated estrogen, 0.625 mg/d (n = 143); alendronate and conjugated estrogen (n = 140); or placebo (n = 50). At year 3, women were allocated into five groups: Twenty-eight women continued to take placebo and 44 women continued to take combination therapy, but 50 women taking alendronate, 81 taking conjugated estrogen, and 41 taking combination therapy were switched to placebo. MEASUREMENTS: Bone mineral density and biochemical markers of bone turnover. RESULTS: Women taking alendronate or combination therapy who were switched to placebo for year 3 of the study maintained bone mass. Bone mineral density in these women was 4.1% (CI, 2.6% to 5.7%) and 6.6% (CI, 5.0% to 8.2%) higher, respectively, at the spine (P < 0.001 for both treatment comparisons) and 3.5% (CI, 2.3% to 4.6%) and 3.0% (CI, 1.8% to 4.2%) higher, respectively, at the trochanter (P < 0.001 for both treatment comparisons) than that in women previously taking estrogen who were switched to placebo. In contrast, women who were taking estrogen and were switched to placebo during year 3 experienced a 4.5% decrease at the spine (95% CI, -5.0% to -4.0%) and a 2.4% decrease at the trochanter (CI, -2.7% to -2.1%) (P < 0.001 for both changes). Compared with women who took placebo for 3 years, women who took estrogen for 2 years and were then switched to placebo had a bone mineral density that was 2.9% higher (CI, 1.2% to 4.6%) at the spine (P < 0.05) and 2.9% higher (CI, 1.6% to 4.2%) at the trochanter (P < 0.001). Changes in biochemical markers during year 3 did not differ among the groups that discontinued active treatment. CONCLUSIONS: Accelerated bone loss is seen after withdrawal of estrogen therapy but not after withdrawal of alendronate or combination therapy. The differential effects after withdrawal of therapy should be considered in the management of postmenopausal osteoporosis.

CYP3A4 is a human microsomal vitamin D 25-hydroxylase.

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

Alendronate increases bone mass and reduces bone markers in postmenopausal African-American women.

Previous studies indicated that aminobisphosphonate alendronate sodium, a potent inhibitor of bone resorption, increases bone mineral density (BMD) at the hip and spine, reduces markers of bone turnover, and reduces the risk of fractures in Caucasian postmenopausal women. The purpose of the present study was to investigate whether alendronate increases BMD and reduces markers of bone turnover in African-American postmenopausal women. In a multicenter, randomized, double-blind, placebo-controlled study, 65 African-American women, aged 45 to 88 yr, were randomly assigned to either placebo (n = 33) or alendronate 10 mg daily (n = 32) for 2 yr. Mean BMD T scores of the lumbar spine at baseline were -3.18 in the placebo-treated group and -3.09 in the alendronate-treated group. All women took 500 mg elemental calcium daily in the form of calcium carbonate and 500 IU vitamin D. Alendronate significantly increased BMD and reduced markers of bone formation and resorption, compared with placebo. At 2 yr, mean changes +/- SE in BMD were 6.5% +/- 0.7% for the lumbar spine (P < 0.001), 4.5% +/- 1.0% for the femoral neck (P < 0.001), 6.4% +/- 0.6% for the femoral trochanter (P < 0.001), 4.1% +/- 0.7% for the total hip (P < 0.001), 0.7% +/- 0.5% for the one third forearm (NS), and 2.0% +/- 0.4% for the total body (P < 0.001) in women treated with alendronate, compared with 0.9% +/- 0.6% (NS), 0.5% +/- 1.1% (NS), -0.2 +/- 0.8 (NS), -1.1 +/- 0.7% (NS), -0.8% +/- 0.6% (NS), and -1.2% +/- 0.6% (P < 0.05) for the lumbar spine, femoral neck, trochanter, total hip, one third forearm, and total body, respectively, in women treated with placebo. At 2 yr, mean serum bone-specific alkaline phosphatase had declined by 46.3% with alendronate (P < 0.001) and 13.6% with placebo (P < 0.01), and mean urinary N-telopeptide of type I collagen/creatinine ratio had declined by 70.5% with alendronate (P < 0.001) and 6.7% with placebo (NS). The incidence of adverse experiences was not different between the two groups. We conclude that in postmenopausal African-American women with osteoporosis, alendronate, 10 mg daily for 2 yr, increases BMD at the lumbar spine, hip, and total body and reduces markers of bone remodeling and is well tolerated.

25-Hydroxylation of vitamin D3: relation to circulating vitamin D3 under various input conditions.

BACKGROUND: Neither the efficiency of the 25-hydroxylation of vitamin D nor the steady state relation between vitamin D(3) and 25-hydroxyvitamin D [25(OH)D] has been studied in humans. OBJECTIVE: We aimed to examine the relation between serum vitamin D(3) and 25(OH)D in normal subjects after either oral administration of vitamin D(3) or ultraviolet-B radiation across a broad range of inputs. DESIGN: Values for serum vitamin D(3) and (OH)D(3) were aggregated from 6 studies--1 acute and 5 near-steady state--at various vitamin D(3) inputs. In 3 of the steady state studies, vitamin D(3) had been administered for 18-26 wk in doses of 0 to 11000 IU/d; in 2 studies, subjects had received solar or ultraviolet-B irradiation. RESULTS: In the acute study, subjects receiving a single 100000-IU dose of vitamin D(3) had a rise in serum cholecalciferol to a mean of 521 nmol/L at 1 d and then a fall to near-baseline values by 7-14 d. Serum 25(OH)D peaked at 103 nmol/L on day 7 and fell slowly to baseline by day 112. In the 5 steady state studies, the relation of serum 25(OH)D to serum vitamin D(3) was biphasic and was well described by a combined exponential and linear function: Y = 0.433X + 87.81[1-exp (-0.468X)], with R(2) = 0.448. CONCLUSIONS: At physiologic inputs, there is rapid conversion of precursor to product at low vitamin D(3) concentrations and a much slower rate of conversion at higher concentrations. These data suggest that, at typical vitamin D(3) inputs and serum concentrations, there is very little native cholecalciferol in the body, and 25(OH)D constitutes the bulk of vitamin D reserves. However, at supraphysiologic inputs, large quantities of vitamin D(3) are stored as the native compound, presumably in body fat, and are slowly released to be converted to 25(OH)D.

Characterization of recombinant CYP2C11: a vitamin D 25-hydroxylase and 24-hydroxylase.

Studies were performed to further characterize the male-specific hepatic recombinant microsomal vitamin D 25-hydroxlase CYP2C11, expressed in baculovirus-infected insect cells, and determine whether it is also a vitamin D 24-hydroxylase. 25- and 24-hydroxylase activities were compared with those of 10 other recombinant hepatic microsomal cytochrome P-450 enzymes expressed in baculovirus-infected insect cells. Each of them 25-hydroxylated vitamin D2, vitamin D3, 1alpha-hydroxyvitamin D2 (1alphaOHD2), and 1alpha-hydroxyvitamin D3 (1alphaOHD3). CYP2C11 had the greatest activity with these substrates, except vitamin D3, which had the same activity as four of the other enzymes. The descending order of 25-hydroxylation by CYP2C11 was 1alphaOHD3 > 1alphaOHD2 > vitamin D2 > vitamin D3. Each of the recombinant cytochrome P-450 enzymes 24-hydroxylated 1alphaOHD2. CYP2C11 had the greatest activity. 24-Hydroxylation of 1alphaOHD3 was very low, and there was none with vitamin D3. Only CYP2C11 24-hydroxylated vitamin D2. Structures of vitamin D metabolites, including 24-hydroxyvitamin D2, 1,24(S)-dihydroxyvitamin D2, and 1,24-dihydroxyvitamin D3, were confirmed by HPLC and gas chromatography retention times and characteristic mass spectrometric fragmentation patterns. In male rats, hypophysectomy significantly reduced body weight, liver weight, hepatic CYP2C11 mRNA expression, and 24- and 25-hydroxylation of 1alphaOHD2. Expression of CYP2J3 and CYP2R1 mRNA did not change. In male rat hepatocytes, CYP2C11 mRNA expression and 24- and 25-hydroxylation were significantly reduced after culture for 24 h compared with uncultured cells. Expression of CYP2J3 and CYP2R1 either increased or did not change. It is concluded that CYP2C11 is a male-specific hepatic microsomal vitamin D 25-hydroxylase that hydroxylates vitamin D2, vitamin D3, 1alphaOHD2, and 1alphaOHD3. CYP2C11 is also a vitamin D 24-hydroxylase.

Genetic evidence that the human CYP2R1 enzyme is a key vitamin D 25-hydroxylase.

The synthesis of bioactive vitamin D requires hydroxylation at the 1 alpha and 25 positions by cytochrome P450 enzymes in the kidney and liver, respectively. The mitochondrial enzyme CYP27B1 catalyzes 1 alpha-hydroxylation in the kidney but the identity of the hepatic 25-hydroxylase has remained unclear for >30 years. We previously identified the microsomal CYP2R1 protein as a potential candidate for the liver vitamin D 25-hydroxylase based on the enzyme's biochemical properties, conservation, and expression pattern. Here, we report a molecular analysis of a patient with low circulating levels of 25-hydroxyvitamin D and classic symptoms of vitamin D deficiency. This individual was found to be homozygous for a transition mutation in exon 2 of the CYP2R1 gene on chromosome 11p15.2. The inherited mutation caused the substitution of a proline for an evolutionarily conserved leucine at amino acid 99 in the CYP2R1 protein and eliminated vitamin D 25-hydroxylase enzyme activity. These data identify CYP2R1 as a biologically relevant vitamin D 25-hydroxylase and reveal the molecular basis of a human genetic disease, selective 25-hydroxyvitamin D deficiency.