HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS		QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

J. Biol. Chem., Vol. 276, Issue 37, 34579-34585, September 14, 2001

This Article Abstract Full Text (PDF) All Versions of this Article: 276/37/34579    most recent M103025200v1 Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Honda, A. Articles by Shefer, S. Search for Related Content PubMed PubMed Citation Articles by Honda, A. Articles by Shefer, S. Social Bookmarking                      What's this?

Side Chain Hydroxylations in Bile Acid Biosynthesis Catalyzed by CYP3A Are Markedly Up-regulated in Cyp27^/ Mice but Not in Cerebrotendinous Xanthomatosis^*

Akira Honda, Gerald Salen^§¶, Yasushi Matsuzaki, Ashok K. Batta^§, Guorong Xu^§¶, Eran Leitersdorf^**, G. Stephen Tint^§¶, Sandra K. Erickson, Naomi Tanaka, and Sarah Shefer^§

From the  Department of Gastroenterology, University of Tsukuba, Tsukuba-city 305-8575, Japan, the ^§ Gastrointestinal Division, Department of Medicine, and Liver Center, the University of Medicine and Dentistry of New Jersey-New Jersey Medical School, Newark, New Jersey 07103, the ^¶ Veterans Affairs Medical Center, East Orange, New Jersey 07018, the ^** Department of Medicine, Center for Research, Prevention, and Treatment of Atherosclerosis, Hadassah University Hospital, 91120 Jerusalem, Israel, and the  Department of Medicine, University of California and Veterans Affairs Medical Center, San Francisco, California 94121

Received for publication, April 5, 2001, and in revised form, July 11, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The accumulation of various 25-hydroxylated C₂₇-bile alcohols in blood and their excretion in urine are characteristic features of cerebrotendinous xanthomatosis (CTX) a recessively inherited inborn error of bile acid synthesis caused by mutations in the mitochondrial sterol 27-hydroxylase (CYP27) gene. These bile alcohols may be intermediates in the alternative cholic acid side chain cleavage pathway. The present study was undertaken to identify enzymes and reactions responsible for the formation of these bile alcohols and to explain why Cyp27^/ mice do not show CTX-related abnormalities. Microsomal activities of 5-cholestane-3,7,12-triol 25- and 26-hydroxylases, 5-cholestane-3,7,12,25-tetrol 23R-, 24S-, and 27-hydroxylases and testosterone 6-hydroxylase, a marker enzyme for CYP3A, in Cyp27^/ mice livers were markedly up-regulated (5.5-, 3.5-, 6.5-, 7.5-, 2.9-, and 5.4-fold, respectively). In contrast, these enzyme activities were not increased in CTX. The activities of 5-cholestane-3,7,12-triol 25- and 26-hydroxylases and 5-cholestane-3,7,12,25-tetrol 23R-, 24R-, 24S-, and 27-hydroxylases were strongly correlated with the activities of testosterone 6-hydroxylase in control human liver microsomes from eight unrelated donors. Troleandomycin, a specific inhibitor of CYP3A, markedly suppressed these microsomal side chain hydroxylations in both mouse and human livers in a dose-dependent manner. In addition, experiments using recombinant overexpressed human CYP3A4 confirmed that these microsomal side chain hydroxylations were catalyzed by a single enzyme, CYP3A4. The results demonstrate that microsomal 25- and 26-hydroxylations of 5-cholestane-3,7,12-triol and microsomal 23R-, 24R-, 24S-, and 27-hydroxylations of 5-cholestane-3,7,12,25-tetrol are mainly catalyzed by CYP3A in both mice and humans. Unlike Cyp27^/ mice, CYP3A activity was not up-regulated despite marked accumulation of 5-cholestane-3,7,12-triol in CTX.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

In the classic bile acid biosynthetic pathway, a series of ring modifications precede side chain cleavage to yield 5-cholestane-3,7-diol and 5-cholestane-3,7,12-triol. The side chain of the diol is hydroxylated by mitochondrial sterol 27-hydroxylase (CYP27) (1-3) and finally transformed to chenodeoxycholic acid. On the other hand, the side chain of 5-cholestane-3,7,12-triol is hydroxylated by either mitochondrial CYP27 (2-4) or microsomal 5-cholestane-3,7,12-triol 25-hydroxylase (2, 5, 6), and the formed tetrols (5-cholestane-3,7,12,27-tetrol or 5-cholestane-3,7,12,25-tetrol) are eventually converted into cholic acid. In the 25-hydroxylation side chain cleavage pathway, either C-23R, C-24R, C-24S, or C-27 position of 5-cholestane-3,7,12,25-tetrol is further hydroxylated by microsomal enzymes (7, 8), and 5-cholestane-3,7,12,24S,25-pentol is exclusively cleaved to cholic acid by cytosolic fraction (7, 9).

Cerebrotendinous xanthomatosis (CTX)¹ is recessively inherited and caused by mutations in the CYP27 gene located on human chromosome 2 (10-12). Clinical features in patients are tendon and brain xanthomas, premature atherosclerosis, and nervous system dysfunction including mental retardation, dementia, cerebellar ataxia, epileptic seizures, and peripheral neuropathy (13). Biochemically, chenodeoxycholic acid production is markedly reduced (2), and large amounts of 25-hydroxylated C₂₇-bile alcohols including 5-cholestane-3,7,12,25-tetrol, 5-cholestane-3,7,12,23R,25-pentol, 5-cholestane-3,7,12,24R,25-pentol, and 5-cholestane-3,7,12,24S,25-pentol are excreted into bile and urine (14, 15). Since hydroxylations of the C-27 position of cholesterol, 5-cholestane-3,7-diol, and 5-cholestane-3,7,12-triol are all catalyzed by CYP27 (3), virtually all bile acids are synthesized from 5-cholestane-3,7,12-triol via the microsomal 25-hydroxylation side chain cleavage pathway in CTX (Fig. 1).

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Bile acid biosynthetic pathway in CTX. Reactions catalyzed by sterol 27-hydroxylase (CYP27) are indicated by the dashed lines. I, cholesterol 7-hydroxylase (CYP7A1); II, 3-hydroxy-5-C27-steroid dehydrogenase/isomerase (3-HSD); III, 7-hydroxy-4-cholesten-3-one 12-hydroxylase (CYP8B1); IV, 5-cholestane-3,7,12-triol 25-hydroxylase (CYP3A); V, 5-cholestane-3,7,12,25-tetrol 24S-hydroxylase (CYP3A).

While a disrupted CYP27 gene causes CTX in humans, CYP27 knockout mice do not show typical CTX-related pathological or biochemical abnormalities (16). Recently we measured hepatic concentrations of intermediates in bile acid biosynthesis in Cyp27^/ mice and compared them with those in CTX (17). In Cyp27^/ mice, microsomal concentrations of early intermediates in the bile acid biosynthetic pathway (7-hydroxycholesterol, 7-hydroxy-4-cholesten-3-one, 7,12-dihydroxy-4-cholesten-3-one, and 5-cholestane-3,7,12-triol), and 25-hydroxylated bile alcohols (5-cholestane-3,7,12,25-tetrol, 5-cholestane-3,7,12,23R,25-pentol, and 5-cholestane-3,7,12,24R,25-pentol) were all significantly elevated compared with those in Cyp27^+/+ mice, but the levels were much lower than those in CTX patients. Therefore, we speculated that these intermediates were more efficiently metabolized by the microsomal 25-hydroxylation side chain cleavage pathway in Cyp27^/ mice than in CTX.

Until recently, the microsomal enzymes involved in the 25-hydroxylation pathway had not been characterized. However, Furster and Wikvall (18) demonstrated in 1999 that CYP3A4 was the predominant enzyme responsible for 25-hydroxylation of 5-cholestane-3,7,12-triol in human liver microsomes. The present study was undertaken to identify the enzymes responsible for the formation of bile alcohols other than 5-cholestane-3,7,12,25-tetrol, and to explain why Cyp27^/ mice accumulate less amounts of bile acid intermediates compared with CTX. We measured several enzyme activities in the 25-hydroxylation pathway by an improved assay method based on high-resolution GC-MS (19) and compared the values with testosterone 6-hydroxylase activity, the marker activity for CYP3A. The results showed that CYP3A catalyzed not only 25-hydroxylation of 5-cholestane-3,7,12-triol but also carried out other side chain hydroxylations on 5-cholestane-3,7,12-triol and 5-cholestane-3,7,12,25-tetrol.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Chemicals-- 7-Hydroxycholesterol was obtained from Steraloids (Wilton, NH). 27-Hydroxycholesterol was synthesized from diosgenin (20) and the pure compound was obtained by preparative thin-layer chromatography (TLC). 7-Hydroxy-4-cholesten-3-one, 7,12-dihydroxy-4-cholesten-3-one, and 5-cholestane-3,7,12,25,27-pentol were gifts from Drs. T. Hoshita and K. Kihira (Pharmaceutical Institute, Hiroshima University, Hiroshima, Japan). 5-Cholestane-3,7,12-triol was prepared by electrolytic coupling of cholic acid with isovaleric acid according to Bergström and Krabisch (21). 5-Cholestane-3,7,12,25-tetrol was synthesized from cholic acid by the method of Dayal et al. (22). 5-Cholestane-3,7,12,27-tetrol was prepared by lithium aluminum hydride reduction of the methyl ester of 3,7,12-trihydroxy-5-cholestan-27-oic acid which was isolated from the bile of Alligator mississippiensis. 5-Cholestane-3,7,12,23R,25-pentol was isolated from bile and feces of patients with CTX (23). 5-Cholestane-3,7,12,24R,25-pentol and 5-cholestane-3,7,12,24S,25-pentol were prepared from 5-cholestane-3,7,12,25-tetrol by the method of Hoshita (24). [25,26,26,26,27,27,27-²H₇]Cholesterol was obtained from MSD Isotopes (Montreal, Canada). [²H₇]7-Hydroxycholesterol (25), [²H₇]27-hydroxycholesterol (26), and [²H₇]7-hydroxy-4-cholesten-3-one (27) were prepared by previously described methods. Troleandomycin and 6-hydroxytestosterone were purchased from Sigma.

Human Liver Microsomes and Mitochondria-- The CTX patient is a 45-year-old male with dementia, spinal cord paresis, and cerebellar ataxia. Xanthomas were located in both Achilles tendons. Serum cholestanol concentration was 6.4 mg/dl (normal level; 0.2 ± 0.2 mg/dl). The results of mutation analysis of this patient (Patient 1100-3) has been described in a recent report (28). A liver biopsy was obtained for diagnostic histology and the extra tissue was available for biochemical analyses. Control liver specimens were from 10 healthy adults who died unexpectedly and whose livers became available because no suitable recipient for liver transplantation could be found (University of Minnesota Hospital NIH contract 1-DK-62274). All liver specimens were immediately frozen and stored at 70 °C until used. Microsomal and mitochondrial fractions were prepared by differential ultracentrifugation as described previously (17). The research protocol was approved by the Human Studies Committees at the University of Medicine and Dentistry of New Jersey-New Jersey Medical School (Newark, NJ) and Veterans Affairs Medical Center (East Orange, NJ).

Mice Liver Microsomes and Mitochondria-- Cyp27^/ mice were produced by Rosen et al. (16) at the Hadassah-Hebrew University animal facility in a specific pathogen-free unit. Six male and four female Cyp27^/ mice, and three male and four female Cyp27^+/+ mice, were fed a normal chow diet. The animals, 3 months of age, were sacrificed at noon and livers were immediately frozen and stored at 70 °C until used. Microsomes and mitochondria were prepared as described previously (17). The animal protocol was approved by Subcommittee on Animal Studies at Veterans Affairs Medical Center (East Orange, NJ) and Institutional Animal Care and Use Committee at University of Medicine and Dentistry of New Jersey-New Jersey Medical School.

Recombinant Overexpressed Human CYP3A4-- Microsomes (Baculosomes) prepared from insect cells that were infected with a baculovirus containing the cDNA for human CYP3A4 and rabbit cytochrome P450 reductase were purchased from PanVera Corp. (Madison, WI).

Assays of Cholesterol 7-Hydroxylase and 27-Hydroxylase Activities-- The activities of microsomal cholesterol 7-hydroxylase and mitochondrial 27-hydroxylase were measured by the stable-isotope dilution mass spectrometry method using [²H₇]7-hydroxycholesterol and [²H₇]27-hydroxycholesterol as internal recovery standards (29).

Assay of 3-Hydroxy-⁵-C₂₇-steroid Dehydrogenase/Isomerase Activity-- The assay was performed according to the method described by Björkhem (30) with some modifications. The microsomal fraction was incubated for 20 min at 37 °C with 7-hydroxycholesterol (1 µg dissolved in 5 µl of isopropyl alcohol), NAD (1 mM), and 100 mM potassium phosphate buffer (pH 7.4) containing 0.1 mM EDTA in a total volume of 0.3 ml. The reaction was stopped by adding 0.3 ml of methanol and 3 ml of petroleum ether. Formed 7-hydroxy-4-cholesten-3-one was extracted and quantified by HPLC (31).

Assay of 7-Hydroxy-4-cholesten-3-one 12-Hydroxylase Activity-- The activity of 12-hydroxylase was assayed as described by Noshiro et al. (32) with minor modifications. Microsomes were incubated for 10 min at 37 °C with 7-hydroxy-4-cholesten-3-one (6 µg dissolved in 5 µl of isopropyl alcohol), NADPH (1 mM), and 100 mM potassium phosphate buffer (pH 7.4) containing 0.1 mM EDTA in a total volume of 0.3 ml. The incubation was stopped by addition of 5 ml of benzene. Formed 7,12-dihydroxy-4-cholesten-3-one was quantified by HPLC (32).

Assay of 5-Cholestane-3,7,12-triol 25-Hydroxylase Activity-- Microsomal 5-cholestane-3,7,12-triol 25-hydroxylase activity was measured by our method described previously (19). Microsomes were incubated for 20 min at 37 °C with 5-cholestane-3,7,12-triol (25 nmol dissolved in 10 µl of acetone), NADPH (1.2 mM), glucose 6-phosphate (3.6 mM), 2 units of glucose-6-phosphate dehydrogenase, and 100 mM potassium phosphate buffer (pH 7.4) containing 0.1 mM EDTA in a total volume of 0.5 ml. The reaction was stopped by adding 2 ml of ethyl acetate. After addition of 1 µg of 5-cholestane-3,7,12,27-tetrol as an internal recovery standard, tetrols were extracted twice with 2 ml of ethyl acetate, purified by a Bond Elut SI cartridge, derivatized to trimethylsilyl ether, and quantified by high-resolution GC-MS with selected ion monitoring.

Assays of 5-Cholestane-3,7,12-triol 26- and 27-Hydroxylase Activities-- The activities of microsomal 5-cholestane-3,7,12-triol 26-hydroxylase and mitochondrial 5-cholestane-3,7,12-triol 27-hydroxylase were determined by previously described methods (19). The microsomes (for 26-hydroxylase) or mitochondria (for 27-hydroxylase) were incubated for 20 min at 37 °C with 5-cholestane-3,7,12-triol (50 nmol dissolved in 12 µl of a 33% aqueous solution of -cyclodextrin), NADPH (1.2 mM), isocitrate (5 mM), and 0.2 unit of isocitrate dehydrogenase, and 100 mM potassium phosphate buffer (pH 7.4) containing 0.1 mM EDTA in a total volume of 0.5 ml. The reaction was stopped with 2 ml of ethyl acetate. After addition of 1 µg of 5-cholestane-3,7,12,25-tetrol as an internal recovery standard, 5-cholestane tetrols were extracted twice with 2 ml of ethyl acetate, purified by a Bond Elut SI cartridge, derivatized to trimethylsilyl ether, and quantified by high-resolution GC-MS with selected ion monitoring.

Assay of 5-Cholestane-3,7,12,25-tetrol 23R-, 24R-, 24S-, and 27-Hydroxylase Activities-- As reported previously (19), microsomes were incubated for 20 min at 37 °C with 5-cholestane-3,7,12,25-tetrol (100 nmol dissolved in 10 µl of 0.75% (w/v) Tween 80 solution), NADPH (1.2 mM), glucose 6-phosphate (3.6 mM), 2 units of glucose-6-phosphate dehydrogenase, and 100 mM potassium phosphate buffer (pH 7.4) containing 0.1 mM EDTA in a total volume of 0.5 ml. The reaction was stopped by adding 2 ml of ethyl acetate. After addition of 1 µg of 5-cholestane-3,7,12,27-tetrol as an internal recovery standard, bile alcohols were extracted twice with 2 ml of ethyl acetate, purified by a Bond Elut SI cartridge, derivatized to trimethylsilyl ether and quantified by high-resolution GC-MS with selected ion monitoring.

Assay of Testosterone 6-Hydroxylase Activity-- The activity of testosterone 6-hydroxylase was measured according to the method described by Hayashi et al. (33) with minor modifications. Microsomes were incubated for 30 min at 37 °C with testosterone (300 nmol dissolved in 5 µl of methanol), NADPH (1.2 mM), glucose 6-phosphate (3.6 mM), 2 units of glucose-6-phosphate dehydrogenase, and 100 mM potassium phosphate buffer (pH 7.4) containing 0.1 mM EDTA in a total volume of 0.5 ml. The incubation was stopped by addition of 3 ml of benzene. Formed 6-hydroxytestosterone was quantified by HPLC (33).

Statistics-- Data are reported here as the mean ± S.E. The statistical significance of differences between the results in the different groups was evaluated by the Student's two-tailed t test. Correlation was tested by calculating Pearson's correlation coefficient, r. In all statistical tests, significance was accepted at the level of p < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Table I shows hepatic mitochondrial 27-hydroxylase activities toward cholesterol and 5-cholestane-3,7,12-triol. The activities of 27-hydroxylation of both cholesterol and 5-cholestane-3,7,12-triol were virtually absent in Cyp27^/ mice while very low but significant activities were detected in the CTX patient.

View this table: [in this window] [in a new window]   Table I Hepatic mitochondrial 27-hydroxylase activities toward cholesterol and 5-cholestane-3,7,12-triol in female Cyp27/ mice and a CTX patient

The activities of enzymes involved in cholic acid biosynthesis from cholesterol via 25-hydroxylation side chain cleavage pathway are summarized in Fig. 2. Cholesterol 7-hydroxylase (CYP7A1) activities were up-regulated in both CTX patient and Cyp27^/ mice compared with the controls. In contrast, microsomal 5-cholestane-3,7,12-triol 25-hydroxylase and 5-cholestane-3,7,12,25-tetrol 24S-hydroxylase activities were not stimulated in the CTX patient, whereas both enzyme activities in Cyp27^/ mice were markedly up-regulated 5.5- and 7.5-fold, respectively, compared with those in Cyp27^+/+ mice.

View larger version (20K): [in this window] [in a new window]   Fig. 2.   Hepatic microsomal enzyme activities involved in cholic acid biosynthetic pathway in CTX and female Cyp27/ mice. 7-OHase, cholesterol 7-hydroxylase (CYP7A1); 3-HSD, 3-hydroxy-5-C27-steroid dehydrogenase/isomerase; 12-OHase, 7-hydroxy-4-cholesten-3-one 12-hydroxylase (CYP8B1); 25-OHase, 5-cholestane-3,7,12-triol 25-hydroxylase (CYP3A); 24S-OHase, 5-cholestane-3,7,12,25-tetrol 24S-hydroxylase (CYP3A). An asterisk denotes that the value for the CTX patient is above the 95% confidence interval for control mean.

Fig. 3 shows data comparing up-regulation of cholesterol 7-hydroxylase and 5-cholestane-3,7,12-triol 25-hydroxylase in male versus female Cyp27^/ mice. Cholesterol 7-hydroxylase activity was elevated 6.5-fold in male Cyp27^/ mice while the elevation was only 2-fold in female Cyp27^/ mice. In contrast, up-regulation of 25-hydroxylase in female Cyp27^/ mice (5.5-fold) was similar to that in male Cyp27^/ mice (5.3-fold).

View larger version (27K): [in this window] [in a new window]   Fig. 3.   Hepatic microsomal cholesterol 7-hydroxylase and 5-cholestane-3,7,12-triol 25-hydroxylase activities in male and female Cyp27/ mice.

Activities of side chain hydroxylations of 5-cholestane-3,7,12-triol and 5-cholestane-3,7,12,25-tetrol in CTX patient and control humans are shown in Table II. Significant activities for the 26-hydroxylation of 5-cholestane-3,7,12-triol and 23R-, 24R-, and 27-hydroxylations of 5-cholestane-3,7,12,25-tetrol were detected in microsomes from the CTX patient, but these activities were within the 95% confidence intervals for control mean activities.

View this table: [in this window] [in a new window]   Table II Hepatic microsomal hydroxylase activities toward 5-cholestane-3,7,12-triol and 5-cholestane-3,7,12,25-tetrol in a CTX patient

Table III shows microsomal side chain hydroxylations of 5-cholestane-3,7,12-triol and 5-cholestane-3,7,12,25-tetrol in female Cyp27^/ mice. Not only 25-hydroxylation of 5-cholestane-3,7,12-triol and 24S-hydroxylation of 5-cholestane-3,7,12,25-tetrol but also 26-hydroxylation of 5-cholestane-3,7,12-triol and 23R- and 27-hydroxylations of 5-cholestane-3,7,12,25-tetrol were markedly up-regulated compared with those in Cyp27^+/+ mice. However, 24R-hydroxylation of 5-cholestane-3,7,12,25-tetrol in Cyp27^/ mice was not elevated.

View this table: [in this window] [in a new window]   Table III Hepatic microsomal hydroxylase activities toward 5-cholestane-3,7,12-triol and 5-cholestane-3,7,12,25-tetrol in female Cyp27/ mice

Fig. 4 shows testosterone 6-hydroxylase activities in the CTX patient and female Cyp27^/ mice. The activity in CTX was not significantly different from those in controls while a 5.4-fold up-regulation was observed in Cyp27^/ mice.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Hepatic microsomal activities of testosterone 6-hydroxylase, a marker enzyme for CYP3A, in CTX and female Cyp27/ mice. Ninety-five percent confidence interval for mean of human controls is 73-419 pmol/min/mg of protein.

Correlations between the activity of microsomal testosterone 6-hydroxylase (CYP3A) and other hydroxylase activities from eight control human subjects are summarized in Table IV. Microsomal activities of 5-cholestane-3,7,12-triol 26-hydroxylase and 5-cholestane-3,7,12,25-tetrol 23R-, 24R-, 24S-, and 27-hydroxylases were all significantly correlated with testosterone 6-hydroxylase activity. However, microsomal cholesterol 7-hydroxylase, 12-hydroxylase, and mitochondrial 5-cholestane-3,7,12-triol 27-hydroxylase activities were not correlated with testosterone 6-hydroxylase activity.

View this table: [in this window] [in a new window]   Table IV Correlations between the activities of testosterone 6-hydroxylase, a marker enzyme for CYP3A, and other hydroxylase activities in control human liver (n = 8)

Recombinant overexpressed human CYP3A4 (9.2 pmol of P450) was incubated at 37 °C for 5 h with 50 µM 5-cholestane-3,7,12-triol, and the bile alcohol fraction was derivatized as trimethylsilyl ethers and analyzed by GC-MS. Fig. 5A represents the total ion chromatogram of the sample. Mass spectrometric analysis confirmed the formations of 5-cholestane-3,7,12,25-tetrol (III), 5-cholestane-3,7,12,26-tetrol (IV), 5-cholestane-3,7,12,23R,25-pentol (V), 5-cholestane-3,7,12,24R,25-pentol (VI), and 5-cholestane-3,7,12,24S,25-pentol (VII). Reference compounds for 5-cholestane-3,7,12,23-tetrol (I) and 5-cholestane-3,7,12,24-tetrol (II) were not available, but they were identified from previous GC-MS data (34). In addition, several unknown 25-, 26-, or 27-hydroxylated bile alcohol peaks (a-e) were detected in the incubation mixture. These bile alcohol peaks were not observed when boiled (for 5 min) CYP3A4 was used for the assay (Fig. 5B). The activities of 25- and 26-hydroxylations of 5-cholestane-3,7,12-triol and 23R-, 24R-, 24S-, and 27-hydroxylations of 5-cholestane-3,7,12,25-tetrol by the recombinant CYP3A4 were 4894, 318, 1820, 166, 245, and 46 pmol/min/nmol P450, respectively. The ratio of these six hydroxylation activities in CYP3A4 was similar to that in human liver microsomes and a strong correlation (r = 0.939, p < 0.01, n = 6) existed between the six enzyme activities by CYP3A4 and those in human liver microsomes (mean activities from 10 control humans).

View larger version (19K): [in this window] [in a new window]   Fig. 5.   Total ion chromatogram obtained in a GC-MS analysis of bile alcohol fraction from incubation mixture of recombinant overexpressed human CYP3A4 (A) or boiled CYP3A4 with 5-cholestane-3,7,12-triol (B). I, 5-cholestane-3,7,12,23-tetrol; II, 5-cholestane-3,7,12,24-tetrol; III, 5-cholestane-3,7,12,25-tetrol; IV, 5-cholestane-3,7,12,26-tetrol; V, 5-cholestane-3,7,12,23R,25-pentol; VI, 5-cholestane-3,7,12,24R,25-pentol; VII, 5-cholestane-3,7,12,24S,25-pentol; a-e, unidentified 25-, 26-, or 27-hydroxylated bile alcohols.

IC₅₀ values for troleandomycin, a specific inhibitor of CYP3A, determined by using recombinant human CYP3A4 and pooled human liver microsomes are shown in Table V. Troleandomycin markedly inhibited 6-hydroxylation of testosterone, 25- and 26-hydroxylations of 5-cholestane-3,7,12-triol and 23R-, 24R-, 24S-, and 27-hydroxylations of 5-cholestane-3,7,12,25-tetrol in both recombinant CYP3A4 and microsomes, but IC₅₀ values for microsomes were somewhat higher than those for recombinant CYP3A4.

Fig. 6 illustrates the effects of adding increasing amounts of troleandomycin on side chain hydroxylation enzyme activities in Cyp27^/ mice liver. When troleandomycin was added to the incubation mixtures, microsomal 25- and 26-hydroxylations of 5-cholestane-3,7,12-triol and 23R-, 24R-, 24S-, and 27-hydroxylations of 5-cholestane-3,7,12,25-tetrol were all inhibited in a dose-dependent manner. In contrast, mitochondrial 27-hydroxylation of 5-cholestane-3,7,12-triol was not suppressed by troleandomycin.

View larger version (25K): [in this window] [in a new window]   Fig. 6.   Effects of troleandomycin on 25-, 26-, and 27-hydroxylations of 5-cholestane-3,7,12-triol and 23R-, 24R-, 24S-, and 27-hydroxylations of 5-cholestane-3,7,12,25-tetrol in mice liver. 5-Cholestane-3,7,12-triol 27-hydroxylase activity was measured by using the mitochondrial fraction from a female CYP27+/+ mouse. The other enzyme activities were determined using the microsomal fraction from a female CYP27/ mouse. The final concentrations of 5-cholestane-3,7,12-triol and 5-cholestane-3,7,12,25-tetrol in incubation mixtures were 25 µM. Data points represent the mean of duplicate determinations. The absolute activities for 5-cholestane-3,7,12-triol 25-, 26-, and 27-hydroxylases without addition of troleandomycin were 533, 13, and 12 pmol/min/mg of protein, respectively. 5-Cholestane-3,7,12,25-tetrol 23R-, 24R-, 24S-, and 27-hydroxylase activities without addition of troleandomycin were 321, 169, 34, and 5.0 pmol/min/mg of protein, respectively.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Our previous studies showed that several side chain hydroxylations of 5-cholestane-3,7,12-triol and 5-cholestane-3,7,12,25-tetrol were coordinately up-regulated by phenobarbital administration in rats (8) and cholesterol or bile fistula treatment in rabbits (19). In addition, we have partially purified 5-cholestane-3,7,12,25-tetrol 24S-hydroxylase in rat liver microsomes by polyethylene glycol precipitation, octylamine-agarose chromatography, hydroxylapatite chromatography, and diethylaminoethyl-Sepharose chromatography. However, the 24S-hydroxylase fraction still contained substantial 5-cholestane-3,7,12-triol 25-hydroxylase, and 5-cholestane-3,7,12,25-tetrol 23R-, 24R-, and 27-hydroxylase activities.² It was reported that mitochondrial sterol 27-hydroxylase (CYP27) catalyzed not only 27-hydroxylation of cholesterol and 5-cholestane-3,7,12-triol but also 24- and 25-hydroxylations of cholesterol (35) and 25-hydroxylation of vitamin D (36). Therefore, we speculated that the microsomal 25-hydroxylation of 5-cholestane-3,7,12-triol and some other hydroxylations of 5-cholestane-3,7,12,25-tetrol were catalyzed by a common enzyme.

Recently, Furster and Wikvall (18) demonstrated that human microsomal 25-hydroxylation of 5-cholestane-3,7,12-triol was catalyzed mainly by CYP3A4. This hydroxylation was also catalyzed by recombinant overexpressed human CYP2B6, CYP2C19, and CYP3A5. However, the most abundantly expressed form of P450 in human liver is CYP3A4 (as much as 60% of all hepatic P450) (37), and the rates of 25-hydroxylation by CYP2B6 and CYP2C19 were only 5 and 8% as active as that by CYP3A4, respectively (18). Recombinant CYP3A5 showed 23% of the activity by Cyp3A4 (18) but hepatic CYP3A5 is polymorphically expressed in only 10 to 30% of humans (38). We did not measure side chain hydroxylase activities by other recombinant expressed human P-450 enzymes, but the following evidence lends support to this hypothesis that not only 25-hydroxylation of 5-cholestane-3,7,12-triol but also many other microsomal side chain hydroxylations are catalyzed by CYP3A (3A4 and 3A5) in humans. 1) Strong positive correlations existed between testosterone 6-hydroxylase activity, a marker activity for CYP3A, and the six microsomal side chain hydroxylase activities in human liver microsomes from eight unrelated donors (Table IV); 2) these seven microsomal enzyme activities were markedly suppressed by troleandomycin, a specific inhibitor of CYP3A (39) (Table V); 3) the six microsomal side chain hydroxylase activities were detected in recombinant human CYP3A4 (Fig. 5); and 4) the ratio of these six hydroxylase activities in human liver microsomes was similar to that of recombinant CYP3A4 with a strong correlation (r = 0.939). In the IC₅₀ experiments, troleandomycin markedly inhibited all microsomal hydroxylations we tested, but IC₅₀ values for human pooled microsomes were somewhat higher than those for recombinant CYP3A4. The difference appears to be caused by the existence of microsomal CYP3A5 which is polymorphically expressed in some human subjects. Although troleandomycin is a specific inhibitor of CYP3A4 and 3A5, an IC₅₀ value for CYP3A5 is larger than that for CYP3A4 (39). Actually, the microsomes showed higher IC₅₀ value than recombinant CYP3A4 when they were determined by assaying testosterone 6-hydroxylase that is a marker enzyme for CYP3A4 and 3A5.

In mice, CYP3A11 is the major CYP3A enzyme in the liver and it has been reported that the purified CYP3A11, called P450_UT as a trivial name, possesses high activity for testosterone 6-hydroxylation (40). In addition, the sizes of exons and amino acid sequences of CYP3A11 are highly homologous to those of human CYP3A4 (41). In our experiments, side chain hydroxylations in mouse microsomes were markedly suppressed by troleandomycin (Fig. 6) and the IC₅₀ values were similar to those for human microsomes. Therefore, CYP3A11 seems to be the predominant enzyme responsible for side chain hydroxylations of 5-cholestane-3,7,12-triol and 5-cholestane-3,7,12,25-tetrol in mice liver microsomes.

Accumulation of 25-hydroxylated C₂₇-bile alcohols is a characteristic feature of CTX. In the bile and plasma, the major bile alcohol is the glucuronic acid conjugate of 5-cholestane-3,7,12,25-tetrol, whereas in urine, the glucuronic acid conjugates of 5-cholestane-3,7,12,23R,25-pentol, 5-cholestane-3,7,12,24R,25-pentol, and 5-cholestane-3,7,12,24S,25-pentol predominate (14, 15, 42). The virtual absence of 5-cholestane-3,7,12,25-tetrol and the large quantities of pentahydroxy bile alcohols in urine suggested that the urinary pentahydroxy bile alcohols, at least in part, might be formed by the renal hydroxylation of 5-cholestane-3,7,12,25-tetrol which was produced in the liver (42). In contrast to human liver, CYP3A5 is consistently present while CYP3A4 is expressed in 40 to 90% of subjects in human kidney (43, 44). Thus, CYP3A5 may be the predominant enzyme responsible for the formation of pentahydroxy bile alcohols in the kidney of CTX patients. It should be mentioned that as reported previously (18), the incubation of human CYP3A4 with 5-cholestane-3,7,12-triol produced significant amounts of 5-cholestane-3,7,12,23-tetrol and 5-cholestane-3,7,12,24-tetrol in addition to a large amount of 5-cholestane-3,7,12,25-tetrol (Fig. 5A). Therefore, some of the pentahydroxy bile alcohols produced in CTX may be synthesized by 25-hydroxylation of 5-cholestane-3,7,12,23-tetrol and 5-cholestane-3,7,12,24-tetrol.

In Cyp27^/ mice, hepatic concentrations of early intermediates in bile acid biosynthesis and 25-hydroxylated bile alcohols were significantly elevated compared with those in Cyp27^+/+ mice (17), but the levels were much lower than found in the CTX patient. In particular, female Cyp27^/ mice showed less accumulation of the early bile acid intermediates than male Cyp27^/ mice. In comparison with the CTX patient and male Cyp27^/ mice, female Cyp27^/ mice had limited up-regulation of cholesterol 7-hydroxylase activity (2-fold versus 6.5-fold in male Cyp27^/ mice and 22-fold in CTX). In contrast, CYP3A was markedly stimulated in both male and female Cyp27^/ mice but not up-regulated in CTX. Thus, the low accumulation of the intermediates in female mice appears to be due to the limited up-regulation of cholesterol 7-hydroxylase with markedly stimulated CYP3A. However, the reason for the limited up-regulation of cholesterol 7-hydroxylase in female Cyp27^/ mice remains unknown. Markedly reduced pool size of bile acids including cholic acid is another feature of Cyp27^/ mice (16, 45), which is different from CTX patients where the pool size of cholic acid was normal while that of chenodeoxycholic acid was markedly reduced (2). Despite coordinate up-regulation of cholesterol 7-hydroxylase and CYP3A, Cyp27^/