Original Article

Regulation of 25- and 27-hydroxylation side chain cleavage pathways for cholic acid biosynthesis in humans, rabbits, and mice: assay of enzyme activities by high-resolution gas chromatography;–mass spectrometry

Correspondence to: Gerald Salen

	ABSTRACT

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In classic cholic acid biosynthesis, a series of ring modifications of cholesterol precede side chain cleavage and yield 5ß-cholestane-3, 7, 12-triol. Side chain reactions of the triol then proceed either by the mitochondrial 27-hydroxylation pathway or by the microsomal 25-hydroxylation pathway. We have developed specific and precise assay methods to measure the activities of key enzymes in both pathways, 5ß-cholestane-3, 7, 12-triol 25- and 27-hydroxylases and 5ß-cholestane-3, 7, 12, 25-tetrol 23R-, 24R-, 24S- and 27-hydroxylases. The extracts from either the mitochondrial or microsomal incubation mixtures were purified by means of a disposable silica cartridge column, derivatized into trimethylsilyl ethers, and quantified by gas chromatography;–mass spectrometry with selected-ion monitoring in a high resolution mode. Compared with the addition of substrates in acetone, those in 2-hydroxypropyl-ß-cyclodextrin increased mitochondrial triol 27-hydroxylase activity 132% but decreased activities of the enzymes in microsomal 25-hydroxylation pathway (triol 25-hydroxylase and 5ß-cholestane-3, 7, 12, 25-tetrol 23R-, 24R-, 24S- and 27-hydroxylases) 13;–60% in human liver. The enzyme activities in both pathways were generally 2- to 4-times higher in mouse and rabbit livers compared with human liver. In all species, microsomal triol 25-hydroxylase activities were 4- to 11-times larger than mitochondrial triol 27-hydroxylase activities but the activities of tetrol 24S-hydroxylase were similar to triol 27-hydroxylase activities in our assay conditions. The regulation of both pathways in rabbit liver was studied after bile acid synthesis was perturbed. Cholesterol feeding up-regulated enzyme activities involved in both 25- (64;–142%) and 27- (77%) hydroxylation pathways, while bile drainage up-regulated only the enzymes in the 25-hydroxylation pathway (178;–371%).

Using these new assays, we demonstrated that the 25- and 27-hydroxylation pathways for cholic acid biosynthesis are more active in mouse and rabbit than human livers and are separately regulated in rabbit liver.—Honda, A., G. Salen, S. Shefer, Y. Matsuzaki, G. Xu, A. K. Batta, G. S. Tint, and N. Tanaka. Regulation of 25- and 27-hydroxylation side chain cleavage pathways for cholic acid biosynthesis in humans, rabbits, and mice: assay of enzyme activities by high-resolution gas chromatography;–mass spectrometry. J. Lipid Res. 2000. 41: 442;–451.

Supplementary key words: bile acids, 5ß-cholestane-3, 7, 12-triol 27-hydroxylase, 5ß-cholestane-3, 7, 12-triol 25-hydroxylase, 5ß-cholestane-3, 7, 12, 25-tetrol 23R-hydroxylase, 5ß-cholestane-3, 7, 12, 25-tetrol 24R-hydroxylase, 5ß-cholestane-3, 7, 12, 25-tetrol 24S-hydroxylase, 5ß-cholestane-3, 7, 12, 25-tetrol 27-hydroxylase

	INTRODUCTION

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In classic cholic acid biosynthesis, cholesterol is first converted to 7-hydroxycholesterol by hepatic microsomal cholesterol 7-hydroxylase, the rate-limiting enzyme in the pathway (1). Hepatic microsomal and cytosolic enzymes further metabolize 7-hydroxycholesterol to 5ß-cholestane-3, 7, 12-triol by a series of ring modifications. The side chain of 5ß-cholestane-3, 7, 12-triol is then hydroxylated by either mitochondrial 27-hydroxylase (2) (3) or microsomal 25-hydroxylase (4) (5) ( Fig 1). In the 27-hydroxylation pathway, 5ß-cholestane-3, 7, 12, 27-tetrol is oxidized to 3, 7, 12-trihydroxy-5ß-cholestanoic acid by the same mitochondrial 27-hydroxylase (6) or cytosolic alcohol and aldehyde dehydrogenases (7) (8) and finally converted into cholic acid by cleavage of the terminal three carbons as propionic acid. In the 25-hydroxylation pathway, either C-23R, C-24R, C-24S, or C-27 position of 5ß-cholestane-3, 7, 12, 25-tetrol is hydroxylated by microsomal enzymes (9) (10), and the cytosolic fraction exclusively cleaves 5ß-cholestane-3, 7, 12, 24S, 25-pentol to cholic acid by releasing the terminal three carbons as acetone (9) (11).

View larger version (26K): [in this window] [in a new window]   Figure 1. Cholic acid biosynthesis from cholesterol. In classic cholic acid biosynthetic pathway, a series of ring modifications of cholesterol precedes side chain oxidation to yield 5ß-cholestane-3, 7, 12-triol. Side chain cleavage of the triol proceeds either by the mitochondrial 27-hydroxylation pathway or by the microsomal 25-hydroxylation pathway.

Although there is no direct evidence, it has been presumed that the mitochondrial 27-hydroxylation pathway is the major route for side chain cleavage in cholic acid biosynthesis in normal human (12) and rat (13). However, little is known about the regulation of the alternative microsomal 25-hydroxylation side chain cleavage pathway when bile acid synthesis is perturbed. A rare, recessive lipid storage disease, cerebrotendinous xanthomatosis (CTX), has genetically defective mitochondrial sterol 27-hydroxylase (CYP27) (14). The patients show markedly reduced production of chenodeoxycholic acid whereas cholic acid synthesis is almost normal (15). In addition, large amounts of 25-hydroxylated C₂₇-bile alcohols are excreted in bile and urine of CTX subjects (16) (17). These data suggest that in CTX virtually all cholic acid is synthesized from 5ß-cholestane-3, 7, 12-triol via the microsomal 25-hydroxylation pathway. However, a recent report by Rosen et al. (18) showed that mice with disrupted sterol 27-hydroxylase gene (knockout model) had markedly reduced cholic acid and chenodeoxycholic acid production without accumulation of bile alcohols. They speculated that the mice could not compensate for the lack of the sterol 27-hydroxylase through the alternative 25-hydroxylation pathway because microsomal 25-hydroxylase activity was not active in mice.

We now report new precise assays for several key enzyme activities in the microsomal 25- and mitochondrial 27-hydroxylation pathways based on high-resolution gas chromatography;–mass spectrometry (GC;–MS). Two advantages are that the method does not require radio-labeled substrates and bile alcohol reaction products can be easily purified from the crude lipid extract of the incubation mixture by disposable silica cartridge column. By using the new assay methods, the enzyme activities were compared among human, mouse, and rabbit. In addition, the effects of cholesterol feeding and depletion of the bile acid pool on the enzyme activities were studied in rabbit liver. The results did not support the idea that the microsomal 25-hydroxylation pathway in mice was much less active than in human. Further, rabbits fed cholesterol up-regulated both 25- and 27-hydroxylation cleavage pathways, while bile drainage eliminated the bile acid pool and up-regulated only the 25-hydroxylation pathway.

	MATERIALS AND METHODS

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Chemicals
Sitosterol (24-ethyl-5-cholesten-3ß-ol) was purchased from Sigma Chemical Co. (St. Louis, MO) and purified by recrystallization. 5ß-Cholestane-3, 7, 12-triol was prepared by electrolytic coupling of cholic acid with isovaleric acid according to Bergström and Krabisch (19). 5ß-Cholestane-3, 7, 12, 25-tetrol was synthesized from cholic acid by the method of Dayal et al. (20). 5ß-Cholestane-3, 7, 12, 27-tetrol was prepared by lithium aluminum hydride reduction of the methyl ester of 3,7,12-trihydroxy-5ß-cholestan-27-oic acid which was isolated from the bile of Alligator mississippiensis. 5ß-Cholestane-3, 7, 12, 23R, 25-pentol was isolated from bile and feces of patients with CTX (21). 5ß-Cholestane-3, 7, 12, 24R, 25-pentol and 5ß-cholestane-3, 7, 12, 24S, 25-pentol were prepared from 5ß-cholestane-3, 7, 12, 25-tetrol by the method of Hoshita (22). 5ß-Cholestane-3, 7, 12, 25, 27-pentol was a gift from Dr. T. Hoshita (Pharmaceutical Institute, Hiroshima University, Hiroshima, Japan). 2-Hydroxypropyl-ß-cyclodextrin (ß-CD) and Tween-80 were purchased from Pharmatec Inc. (Alachua, FL) and Fisher Scientific Co. (Springfield, NJ), respectively.

Preparation of liver specimens
Human liver specimens became available from five healthy people who died unexpectedly when no suitable recipient for liver transplantation could be found (University of Minnesota Hospital NIH contract No. 1-DK-62274). Five mice, 3 months of age, were fed a normal chow diet and were killed and livers were removed at the end of the 12-h dark phase of the cycle. In rabbit experiments, 14 New Zealand white strain rabbits weighing 2.5;–3.2 kg were purchased from Hazleton Laboratories (Denver, PA). The control rabbits (n = 5) were fed normal rabbit chow for 10 days. Some rabbits (n = 5) were fed rabbit chow containing 2% cholesterol in the diet (about 3,000 mg/day) for 10 days. For other rabbits (n = 4), bile fistulas were constructed under anesthesia (ketamine 35;–40 mg/kg body weight combined with xylazine 3;–4 mg/kg body weight intramuscularly). During the period of bile drainage (for 7 days), 0.9% NaCl was administered intravenously, and chow and water were provided ad libitum. The rabbits were killed at the end of the 12-h dark phase of the cycle, and livers were collected. The animal protocols were approved by Subcommittee on Animal Studies at the VA Medical Center (East Orange, NJ) and Institutional Animal Care and Use Committee at the University of Medicine and Dentistry of New Jersey-New Jersey Medical School (Newark, NJ). All liver tissues were stored at -70°C until used.

Preparation of liver microsomes and mitochondria
The liver specimen was weighed, minced, and homogenized with a loose-fitting Teflon pestle in 4 volumes of 3 mM Tris-HCl buffer (pH 7.4) containing 0.25 M sucrose, and 0.1 mM EDTA. The homogenate was centrifuged at 700 g for 10 min, and the supernatant was centrifuged at 7,000 g for 20 min. The 7,000 g pellet (mitochondrial fraction) was washed twice with homogenizing medium and suspended in 100 mM potassium phosphate buffer (pH 7.4) containing 1 mM EDTA, 5 mM DTT, 50 mM KCl, and 20% glycerol (v/v) (storage buffer). The 7,000 g supernatant was then centrifuged at 105,000 g for 90 min and the pellet (microsomal fraction) was suspended in storage buffer. Protein concentrations of the mitochondrial and microsomal fractions were determined by the method of Lowry et al. (23).

Assay of mitochondrial 5ß-cholestane-3, 7, 12-triol 27-hydroxylase activity
The composition of the standard reaction mixture (final volume 0.5 ml) was 100 mM potassium phosphate buffer (pH 7.4) containing 0.1 mM EDTA, 50;–400 µg of mitochondrial protein, and 50 nmol of 5ß-cholestane-3, 7, 12-triol (final concentration 100 µM) dissolved in 12 µl of a 33% aqueous solution of ß-CD. The reaction was initiated by adding a mixture of NADPH (final concentration 1.2 mM), isocitrate (final concentration 5 mM) and 0.2 unit of isocitrate dehydrogenase, and continued for 20 min at 37°C. The reaction was stopped with 2 ml of ethyl acetate. After addition of 1 µg of 5ß-cholestane-3, 7, 12, 25-tetrol as an internal recovery standard, bile alcohols were extracted twice with 2 ml of ethyl acetate and the extracts were evaporated to dryness under nitrogen. The residue was dissolved in 100 µl of chloroform;–acetone;–methanol 35:25:2 (v/v/v), and applied to a Bond Elut SI (unbonded silica, 500 mg) cartridge (Varian, Harbor City, CA) which was prewashed with 3 ml of the same solvent. After washing out sterols with 5 ml of the same solvent, bile alcohols were eluted with 3 ml of chloroform;–acetone;–methanol 35:25:20 (v/v/v). The eluate was evaporated to dryness and bile alcohols were converted into trimethylsilyl (TMS) ether derivatives by addition of 100 µl of TMSI-H (GL Sciences Inc., Tokyo, Japan) and incubation at 55°C for 15 min. High-resolution gas chromatography;–mass spectrometry (GC-MS) with selected-ion monitoring (SIM) was performed using a JMS-SX102 instrument equipped with a data-processing system JMA DA-6000 (JEOL, Tokyo, Japan). The accelerating voltage was 10 kV, the ionization energy 70 eV, the trap current 300 µA, and the mass spectral resolution about 10,000. An Ultra Performance capillary column (25 m x 0.32 mm ID) coated with methylsilicone (Hewlett-Packard, Palo Alto, CA) was used with a flow-rate of helium carrier gas of 1.0 ml/min. The column oven was programmed to change from 100 to 265°C at 30°C/min, after a 1-min delay from the start time.

Assay of 5ß-cholestane-3, 7, 12-triol 25-hydroxylase activity
The reaction mixture (final volume 0.5 ml) consisted of 100 mM potassium phosphate buffer (pH 7.4) containing 0.1 mM EDTA, 50;–400 µg of microsomal protein, and 25 nmol of 5ß-cholestane-3, 7, 12-triol (final concentration 50 µM) dissolved in 10 µl of acetone. The incubation was started by the addition of a mixture of NADPH (final concentration 1.2 mM), glucose-6-phosphate (final concentration 3.6 mM), and 2 units of glucose-6-phosphate dehydrogenase, and continued for 20 min at 37°C. The reaction was stopped by adding 2 ml of ethyl acetate. After addition of 1 µg of 5ß-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 TMS ether, and quantified by the same procedure as the triol 27-hydroxylase assay described above.

Assay of 5ß-cholestane-3, 7, 12, 25-tetrol 23R, 24R, 24S and 27-hydroxylase activities
The reaction mixture (final volume 0.5 ml) consisted of 100 mM potassium phosphate buffer (pH 7.4) containing 0.1 mM EDTA, 100;–400 µg of microsomal protein, and 100 nmol of 5ß-cholestane-3, 7, 12, 25-tetrol (final concentration 200 µM) dissolved in 10 µl of 0.75% (w/v) Tween-80 solution. The incubation was started by the addition of a mixture of NADPH (final concentration 1.2 mM), glucose-6-phosphate (final concentration 3.6 mM), and 2 units of glucose-6-phosphate dehydrogenase, and continued for 20 min at 37°C. The reaction was stopped by adding 2 ml of ethyl acetate. After addition of 1 µg of 5ß-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, and derivatized to TMS ether by the same procedure as triol 27- and 25-hydroxylase assays described above. The column oven was programmed to change from 100 to 265°C at 30°C/min, after a 1-min delay from the start time.

Statistics
Data are reported here as the mean ± SEM. The statistical significance of differences between the results in the different groups was evaluated by the Student's two-tailed t-test and significance was accepted at the level of P < 0.05.

	RESULTS

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The recoveries of 5ß-cholestane-3, 7, 12, 25-tetrol, 5ß-cholestane-3, 7, 12, 27-tetrol, 5ß-cholestane-3, 7, 12, 24R, 25-pentol, and 5ß-cholestane-3, 7, 12, 24S, 25-pentol from the Bond Elut SI cartridge were checked by adding a mixture of 100 ng each of the bile alcohol, 5ß-cholestane-3, 7, 12 triol and cholesterol to the column. The bile alcohols and sterols in the eluate were quantified by GC/SIM using sitosterol as an internal standard. The recoveries of 5ß-cholestane-3, 7, 12, 25-tetrol, 5ß-cholestane-3, 7, 12, 27-tetrol, 5ß-cholestane-3, 7, 12, 24R, 25-pentol, and 5ß-cholestane-3, 7, 12, 24S, 25-pentol were 104.2 ± 3.5% (n = 4), 98.8 ± 3.8% (n = 4), 99.0 ± 3.3% (n = 4), and 100.2 ± 2.5% (n = 4), respectively, while more than 99.9% of 5ß-cholestane-3, 7, 12 triol and cholesterol (n = 4) were eliminated by the purification procedure.

TMS ether derivatives of bile alcohols were well separated by capillary gas chromatography. Retention times were: 5ß-cholestane-3, 7, 12, 25-tetrol (13.7 min), 5ß-cholestane-3, 7, 12, 27-tetrol (14.5 min), 5ß-cholestane-3, 7, 12, 23R, 25-pentol (16.4 min), 5ß-cholestane-3, 7, 12, 24R, 25-pentol (17.0 min), 5ß-cholestane-3, 7, 12, 24S, 25-pentol (17.2 min), and 5ß-cholestane-3, 7, 12, 25, 27-pentol (18.4 min). The major fragments of bile alcohols generated by GC-MS in the electron ionization (EI) mode are described previously (24). The calibration curves for the bile alcohols were established by focusing the multiple ion detector on m/z 253.1956 for 5ß-cholestane-3, 7, 12, 25-tetrol and 5ß-cholestane-3, 7, 12, 27-tetrol, m/z 307.2425 for 5ß-cholestane-3, 7, 12, 23R, 25-pentol, m/z 321.2582 for 5ß-cholestane-3, 7, 12, 24R, 25-pentol and 5ß-cholestane-3, 7, 12, 24S, 25-pentol, and m/z 349.2895 for 5ß-cholestane-3, 7, 12, 25, 27-pentol ( Fig 2). Good linearities were obtained at least up to 100 ng for 5ß-cholestane-3, 7, 12, 27-tetrol and up to 1000 ng for the other bile alcohols. Fig 3 and Fig 4 show representative chromatograms obtained by the analysis of standard incubation mixture from a control human liver at zero time (before) and at 20 min (after). The peaks of 5ß-cholestane-3, 7, 12, 25-tetrol (Fig 3A) and 5ß-cholestane-3, 7, 12, 27-tetrol (Fig 3B) at 20 min incubation corresponded to about 3.9 ng (9.0 pmol) and 220 pg (0.5 pmol), respectively. 5ß-Cholestane-3, 7, 12, 23R, 25-pentol, 5ß-cholestane-3, 7, 12, 24R, 25-pentol, 5ß-cholestane-3, 7, 12, 24S, 25-pentol, and 5ß-cholestane-3, 7, 12, 25, 27-pentol peaks shown after incubation (Fig 4) corresponded to about 9.0 ng (20 pmol), 448 pg (0.96 pmol), 536 pg (1.2 pmol), and 240 pg (0.53 pmol), respectively.

View larger version (16K): [in this window] [in a new window]   Figure 2. Calibration curves for formation of (A) 5ß-cholestane-3, 7, 12, 25-tetrol, (B) 5ß-cholestane-3, 7, 12, 27-tetrol, and (C) 5ß-cholestanepentols. Varying amounts of the bile alcohols were mixed with 1 µg of 5ß-cholestane-3, 7, 12, 25-tetrol (for B) or 5ß-cholestane-3, 7, 12, 27-tetrol (for A and C) as an internal recovery standard, converted into TMS derivatives and determined by high-resolution GC;–SIM. The linearities were checked by simple linear regression, and the lines satisfied the relationships x = 663.44y + 22.03 (n = 4; r = 0.998; P < 0.005) for 5ß-cholestane-3, 7, 12, 25-tetrol; x = 1507.46y + 4.04 (n = 4; r = 0.996; P < 0.005) for 5ß-cholestane-3, 7, 12, 27-tetrol, x = 18289.32y - 4.22 (n = 4; r = 1.000; P < 0.0001) for 5ß-cholestane-3, 7, 12, 23R, 25-pentol; x = 16503.95y - 3.89 (n = 4; r = 1.000; P < 0.0001) for 5ß-cholestane-3, 7, 12, 24, 25-pentol; and x = 10314.59y - 4.49 (n = 4; r = 1.000; P < 0.0001) for 5ß-cholestane-3, 7, 12, 25, 27-pentol.

View larger version (18K): [in this window] [in a new window]   Figure 3. Representative high-resolution GC;–SIM chromatograms of standard assay mixture for (A) microsomal 5ß-cholestane-3, 7, 12-triol 25-hydroxylase and (B) mitochondrial 5ß-cholestane-3, 7, 12-triol 27-hydroxylase in control human liver. Before, without incubation (0 time); After, after 20 min incubation; IS, internal standard.

View larger version (28K): [in this window] [in a new window]   Figure 4. Representative high-resolution GC;–SIM chromatograms of standard assay mixture for microsomal 5ß-cholestane-3, 7, 12, 25-tetrol 23R, 24R, 24S, and 27-hydroxylases in control human liver. Before, without incubation (0 time); After, after 20 min incubation; IS, internal standard.

Optimal conditions for the determination of the enzyme activities were established by using a control human liver. The enzymatic reactions were linear with up to 400 µg of either microsomal or mitochondrial proteins ( Fig 5A and Fig 6A) and with time up to 20 min (Fig 5B and Fig 6B). The effects of 5ß-cholestane-3, 7, 12-triol concentration on the activities of microsomal triol 25-hydroxylase and mitochondrial triol 27-hydroxylase are shown in Fig 5C. The highest 25-hydroxylase activity was observed when 50 µM of the substrate was added to the incubation mixture while optimal substrate concentration for 27-hydroxylase was found at 100 µM. Fig 6C shows the effects of 5ß-cholestane-3, 7, 12, 25-tetrol concentration on the activities of microsomal tetrol 23R, 24R, 24S, and 27-hydroxylases. Increasing enzyme activities were observed up to 200 µM of substrate for the tetrol hydroxylases.

View larger version (19K): [in this window] [in a new window]   Figure 5. Determination of optimal conditions for assay of microsomal 5ß-cholestane-3, 7, 12-triol 25-hydroxylase and mitochondrial 5ß-cholestane-3, 7, 12-triol 27-hydroxylase in human liver. Microsomal and mitochondrial fractions from control human liver were prepared and assayed as described under Materials and Methods with the following changes: (A) amount of microsomal or mitochondrial protein was varied; (B) time of incubation was varied; (C) concentration of 5ß-cholestane-3, 7, 12-triol was varied.

View larger version (23K): [in this window] [in a new window]   Figure 6. Determination of optimal conditions for assay of microsomal 5ß-cholestane-3, 7, 12, 25-tetrol 23R, 24R, 24S, and 27-hydroxylases in human liver. Microsomal fraction from control human liver was prepared and assayed as described under Materials and Methods with the following changes: (A) amount of microsomal protein was varied; (B) time of incubation was varied; (C) concentration of 5ß-cholestane-3, 7, 12, 25-tetrol was varied.

Table 1 shows the effects of acetone, ß-CD, and Tween-80 as substrate vehicles on enzyme activities in human liver. Compared with the addition of substrates dissolved in acetone, those in ß-CD increased mitochondrial 5ß-cholestane-3, 7, 12-triol 27-hydroxylase activity 132% but decreased activities of the enzymes in microsomal 25-hydroxylation pathway 13;–60%. Tween-80 raised 5ß-cholestane-3, 7, 12-triol 27-hydroxylase and 5ß-cholestane-3, 7, 12, 25-tetrol 23R-hydroxylase activities 55% and 96%, respectively, but reduced 5ß-cholestane-3, 7, 12-triol 25-hydroxylase activity 47%.

View this table: [in this window] [in a new window]   Table 1. Effects of acetone, ß'''o< T'''°'' o =ß'''o'''x<<+-'''o =ß'''o'''x<<+-<-='''o o µ

Table 2 compares the enzyme activities in human, mouse, and rabbit livers. In mice and rabbits, enzyme activities involved in both mitochondrial 27-hydroxylation and microsomal 25-hydroxylation pathways were generally 2- to 4-times higher than those in humans. In all species, microsomal 5ß-cholestane-3, 7, 12-triol 25-hydroxylase activities were 4- to 11-times greater than mitochondrial 5ß-cholestane-3, 7, 12-triol 27-hydroxylase activities. In human and rabbit, the activities of microsomal 5ß-cholestane-3, 7, 12, 25-tetrol 24S-hydroxylase were not significantly different from mitochondrial triol 27-hydroxylase. However, tetrol 24S-hydroxylase activity was significantly lower than triol 27-hydroxylase activity in mouse (P < 0.05).

View this table: [in this window] [in a new window]   Table 2. Activities of 5ß'''o'''x<<+-'''o =ß'''o'''x<<+-<-='''o o µ< µo< ßß

The effects of cholesterol feeding and bile fistula on the hepatic enzyme activities were studied in rabbits. As shown in Table 3, cholesterol feeding significantly up-regulated 5ß-cholestane-3, 7, 12-triol 25- (69%) and 27-hydroxylase activities (77%). In contrast, bile fistula significantly up-regulated microsomal 25-hydroxylase activity (178%) but significant up-regulation was not observed in mitochondrial 27-hydroxylase activity. Microsomal 5ß-cholestane-3, 7, 12, 25-tetrol 23R, 24R, 24S and 27-hydroxylase activities were coordinately up-regulated with microsomal 25-hydroxylase activity by cholesterol (64;–142%) and bile fistula treatments (188;–371%).

View this table: [in this window] [in a new window]   Table 3. Effects of cholesterol feeding and bile fistula on 5ß'''o'''x<<+-'''o =ß'''o'''x<< +-<-='''o o ßß

	DISCUSSION

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The activities of 5ß-cholestane-3, 7, 12-triol 27-hydroxylase (2) (3), 25-hydroxylase (11) (25), and 5ß-cholestane-3, 7, 12, 25-tetrol 23R, 24R, 24S and 27-hydroxylases (10) have generally been assayed by isotope incorporation methods using radioactive substrates. Formed bile alcohol products in the incubation mixtures were separated by thin-layer chromatography (TLC) and radioactivities were measured. The methods are simple but the separation of each radioactive bile alcohol by TLC was not always precise because hydroxy groups can be introduced in a number of different positions in the side chain of 5ß-cholestane-3, 7, 12-triol, such that a mixture of C-23R, C-24R, C-24S, C-25, C-26 and C-27 tetrols can be formed (1). To overcome the above disadvantage, Oftebro et al. (26) developed a GC-SIM method for assaying 5ß-cholestane-3, 7, 12-triol 25- and 27-hydroxylase activities. Although the method detected the masses of 5ß-cholestane-3, 7, 12, 25-tetrol and 5ß-cholestane-3, 7, 12, 27-tetrol, which are usually more reliable than radioactivities, their separation by low-resolution GC-SIM was not sufficient for accurate quantification. Theoretically, the present high-resolution GC-SIM method is more specific and reliable than an ordinary low-resolution GC-SIM. In addition, capillary GC column conditions completely resolved 5ß-cholestane-3, 7, 12, 25- and 27-tetrols, and the separation of 5ß-cholestanepentols was also complete enough for quantification.

As convenient internal recovery standards, we used 1 µg of 5ß-cholestane-3, 7, 12, 25-tetrol for quantification of mitochondrial 5ß-cholestane-3, 7, 12, 27-tetrol and 1 µg of 5ß-cholestane-3, 7, 12, 27-tetrol for microsomal 5ß-cholestane-3, 7, 12, 25-tetrol and 5ß-cholestanepentols. The purification of the bile alcohols from the lipid extract was facilitated by a disposable silica cartridge column. The 5ß-cholestanetetrols and 5ß-cholestanepentols were almost completely recovered while more than 99.9% of 5ß-cholestane-3, 7, 12 triol and cholesterol were eliminated. Isolated microsomal and mitochondrial fractions contained small amounts of endogenous 5ß-cholestane-3, 7, 12, 27-tetrol and 5ß-cholestane-3, 7, 12, 25-tetrol, respectively. However, these quantities were less than 1% of the added internal standards.

ß-CD is a cyclic oligosaccharide of seven glucopyranose units and has been used to deliver hydrophobic drugs to target sites both in vitro and in vivo (27) (28). It was reported that the addition of cholesterol dissolved in ß-CD markedly raised cholesterol 27-hydroxylase activity in rat liver mitochondria compared with that in acetone (29). Our results showed that ß-CD also increased mitochondrial 5ß-cholestane-3, 7, 12 triol 27-hydroxylase activity, but diminished microsomal 5ß-cholestane-3, 7, 12 triol 25-hydroxylase and 5ß-cholestane-3, 7, 12, 25-tetrol 23R, 24R, 24S and 27-hydroxylase activities in human liver (Table 1).

In this report, we systematically studied the regulations of 5ß-cholestane-3, 7, 12-triol 25- and 27-hydroxylase and 5ß-cholestane-3, 7, 12, 25-tetrol 23R, 24R, 24S and 27-hydroxylase activities in rabbit liver. Hydroxylations of C-27 position of cholesterol and 5ß-cholestane-3, 7, 12-triol are believed to be catalyzed by the same mitochondrial enzyme, sterol 27-hydroxylase (CYP27) (3). It was reported that cholic acid or cholestyramine feeding did not affect 27-hydroxylation of 5ß-cholestane-3, 7, 12-triol in rabbits (30). Fig 7 shows the effects of cholesterol feeding and bile drainage on cholesterol 27-hydroxylase and 7-hydroxylase activities in rabbit liver, and which were reported previously by Xu et al. (31) (32) (33) (34). The results clearly demonstrated that bile drainage had no effect while cholesterol feeding stimulated 27-hydroxylation of cholesterol in rabbits. The present results again showed that 5ß-cholestane-3, 7, 12-triol 27-hydroxylase activities were not increased by bile drainage but were significantly up-regulated (+77%) by cholesterol feeding (Table 3). The results and the effect of ß-CD on both 27-hydroxylations support the contention that there is a single 27-hydroxylase enzyme (sterol 27-hydroxylase) that catalyzes C-27 hydroxylation of both cholesterol and 5ß-cholestane-3, 7, 12-triol.

View larger version (14K): [in this window] [in a new window]   Figure 7. Effects of cholesterol feeding and bile fistula on cholesterol 7-hydroxylase and cholesterol 27-hydroxylase activities in rabbit liver. Data are shown as mean and SEM. Control, chow fed for 10 days (n = 5). CHOL, 2% cholesterol in chow fed for 10 days (n = 5). BF, chow-fed rabbits with bile fistula for 7 days (n = 5). * P < 0.001, ** P < 0.05. These results were reported previously by Xu et al. (31) (32) (33) (34).

In contrast to C-27-hydroxylation, 5ß-cholestane-3, 7, 12-triol 25-hydroxylase seems to be a different enzyme from cholesterol 25-hydroxylase. Cholesterol 25-hydroxylase cDNAs were recently cloned by Lund et al. (35). Unlike most other sterol hydroxylases, cholesterol 25-hydroxylase was not a cytochrome P450, while 5ß-cholestane-3, 7, 12-triol 25-hydroxylase is believed to be cytochrome P450 (CYP3A4) (36). To the best of our knowledge, there is no report about enzyme activities in the side chain 25-hydroxylation pathway in rabbits, and data on side chain hydroxylation activities in other mammals are also limited. In rats, 25-hydroxylation of 5ß-cholestane-3, 7, 12-triol was not affected by cholestyramine, bile fistula, or starvation (37). In addition, Ahlberg et al. (25) reported that the enzyme activity was normal in hyperlipidemic humans and was unaffected by chenodeoxycholic acid treatment in patients with cholesterol gallstones (38). Unlike these previous reports, our results showed that 25-hydroxylase activity was significantly up-regulated by bile drainage (bile acid depletion) and cholesterol feeding in rabbits. It is not clear whether the inconsistency is due to different species or methods used. Regulation of 5ß-cholestane-3, 7, 12, 25-tetrol 23R, 24R, 24S and 27-hydroxylase activities were studied in rats by Cheng et al. (10). Phenobarbital administration stimulated the formation of all four pentols studied while fasting had no significant effect on these hydroxylations. The present data showed that 23R, 24R, 24S, and 27-hydroxylations of 5ß-cholestane-3, 7, 12, 25-tetrol were also coordinately up-regulated with 25-hydroxylase by both bile acid depletion and cholesterol feeding in rabbits.

It is well established that cholesterol 7-hydroxylase activity is up-regulated by bile drainage (32) (33) while the activity is down-regulated by expansion of the bile acid pool during cholesterol feeding in rabbits (32) (34) (Fig 7). Therefore, in rabbits, depletion of the bile acid pool by bile fistula drainage up-regulates the microsomal cholesterol 7-hydroxylase and 25-hydroxylation pathways (5ß-cholestane-3, 7, 12-triol 25-hydroxylase and 5ß-cholestane-3, 7, 12, 25-tetrol 23R, 24R, 24S and 27-hydroxylases) but does not stimulate mitochondrial sterol 27-hydroxylase. In distinction, cholesterol feeding down-regulates cholesterol 7-hydroxylase but up-regulates the sterol 27-hydroxylase and 25-hydroxylation pathways. Thus, the side chain microsomal 25-hydroxylation pathway is separately regulated from cholesterol 7-hydroxylase and sterol 27-hydroxylase. In rabbits with bile fistulas, 98% of the newly synthesized bile acids is cholic acid (33). Therefore, the regulation of the 25-hydroxylation pathway may be more important in rabbits than in other mammals.

Defective mitochondrial sterol 27-hydroxylase causes CTX in human (14). However, sterol 27-hydroxylase knockout mice did not show CTX-related pathological or biochemical abnormalities including the increased formation of 25-hydroxylated C₂₇-bile alcohols and cholestanol, but the production of both cholic and chenodeoxycholic acids was markedly reduced (18). CTX patients have almost normal cholic acid production (15) and it appears to be synthesized from 5ß-cholestane-3, 7, 12-triol via the 25-hydroxylation pathway (9) (11). Therefore, it was suspected that compared with humans, the 25-hydroxylation pathway was not active in mice (18). However, these data showed that both 5ß-cholestane-3, 7, 12-triol 25-hydroxylase and 5ß-cholestane-3, 7, 12, 25-tetrol 24S-hydroxylase activities were much higher in mouse than in human livers (Table 2). Thus, the results do not support the idea that the 25-hydroxylation pathway in mice was not as active as that in human.

In summary, we have developed improved precise assays for the activities of hepatic 5ß-cholestane-3, 7, 12-triol 25- and 27-hydroxylases and 5ß-cholestane-3, 7, 12, 25-tetrol 23R, 24R, 24S and 27-hydroxylases by high-resolution GC-SIM. The enzyme activities in mice and rabbits were generally higher than those in humans. When bile acid synthesis was perturbed by cholesterol feeding or bile drainage, cholesterol 7-hydroxylase, sterol 27-hydroxylase, and the 25-hydroxylation pathway (5ß-cholestane-3, 7, 12-triol 25-hydroxylase and 5ß-cholestane-3, 7, 12, 25-tetrol 23R, 24R, 24S and 27-hydroxylases) were independently regulated in rabbits..

	ACKNOWLEDGMENTS

The authors thank Bibiana Pcolinsky and Eva Paroulek for excellent technical assistance and Barbara Rouse for preparing the manuscript. This work was supported in part by grants from the Department of Veterans Affairs Research Service, by grants HD-31932, HL-17818 and DK-26756 from the National Institutes of Health, and by a grant (B)10470130 from the Ministry of Education, Science and Culture of Japan.

Manuscript received August 31, 1999; and in revised form November 30, 1999

Abbreviations: ß-CD, 2-hydroxypropyl-ß-cyclodextrin; CTX, cerebrotendinous xanthomatosis; DTT, dithiothreitol; GC, gas chromatography; MS, mass spectrometry; SIM, selected-ion monitoring; TLC, thin-layer chromatography; TMS, trimethylsilyl

	REFERENCES

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

1. Vlahcevic, Z. R., Pandak, W. M., Heuman, D. M., Hylemon, P. B. 1992. Function and regulation of hydroxylases involved in the bile acid biosynthesis pathways. Semin. Liver Dis. 12:403-419[Medline].

2. Taniguchi, S., Hoshita, N., Okuda, K. 1973. Enzymatic characteristics of CO-sensitive 26-hydroxylase system for 5ß-cholestane-3, 7, 12-triol in rat-liver mitochondria and its intramitochondrial localization. Eur. J. Biochem. 40:607-617[Medline].

3. Okuda, K., Masumoto, O., Ohyama, Y. 1988. Purification and characterization of 5ß-cholestane-3, 7, 12-triol 27-hydroxylase from female rat liver mitochondria. J. Biol. Chem. 263:18138-18142[Abstract/Free Full Text].

4. Björkhem, I., Danielsson, H., Wikvall, K. 1976. Side chain hydroxylations in biosynthesis of cholic acid. J. Biol. Chem. 251:3495-3499[Abstract/Free Full Text].

5. Andersson, S., Holmberg, I., Wikvall, K. 1983. 25-Hydroxylation of C₂₇-steroids and vitamin D₃ by a constitutive cytochrome P-450 from rat liver microsomes. J. Biol. Chem. 258:6777-6781[Abstract/Free Full Text].

6. Cali, J. J., Russell, D. W. 1991. Characterization of human sterol 27-hydroxylase. J. Biol. Chem. 266:7774-7778[Abstract/Free Full Text].

7. Okuda, K., Takigawa, N. 1968. Separation of 5ß-cholestane-3,7,12,26-tetrol oxidoreductase, ethanol-NAD oxidoreductase, 5ß-cholestane-3,7,12-triol-26-al oxidoreductase, and acetaldehyde-NAD oxidoreductase from the soluble fraction of rat liver by gel filtration. Biochem. Biophys. Res. Commun. 33:788-793[Medline].

8. Okuda, A., Okuda, K. 1983. Physiological function and kinetic mechanism of human liver alcohol dehydrogenase as 5ß-cholestane-3,7,12,26-tetrol dehydrogenase. J. Biol. Chem. 258:2899-2905[Abstract/Free Full Text].

9. Shefer, S., Cheng, F. W., Dayal, B., Hauser, S., Tint, G. S., Salen, G., Mosbach, E. H. 1976. A 25-hydroxylation pathway of cholic acid biosynthesis in man and rat. J. Clin. Invest. 57:897-903.

10. Cheng, F. W., Shefer, S., Dayal, B., Tint, G. S., Setoguchi, T., Salen, G., Mosbach, E. H. 1977. Cholic acid biosynthesis: conversion of 5ß-cholestane-3, 7, 12, 25-tetrol into 5ß-cholestane-3, 7, 12, 24ß, 25-pentol by human and rat liver microsomes. J. Lipid Res. 18:6-13[Abstract].

11. Salen, G., Shefer, S., Cheng, F. W., Dayal, B., Batta, A. K., Tint, G. S. 1979. Cholic acid biosynthesis. J. Clin. Invest. 63:38-44.

12. Duane, W. C., Pooler, P. A., Hamilton, J. N. 1988. Bile acid synthesis in man. J. Clin. Invest. 82:82-85.

13. Duane, W. C., Björkhem, I., Hamilton, J. N., Mueller, S. M. 1988. Quantitative importance of the 25-hydroxylation pathway for bile acid biosynthesis in the rat. Hepatology. 8:613-618[Medline].

14. Cali, J. J., Hsieh, C-L., Francke, U., Russell, D. W. 1991. Mutations in the bile acid biosynthetic enzyme sterol 27-hydroxylase underlie cerebrotendinous xanthomatosis. J. Biol. Chem. 266:7779-7783[Abstract/Free Full Text].

15. Salen, G., Shefer, S., Tint, G. S., Nicolau, G., Dayal, B., Batta, A. K. 1985. Biosynthesis of bile acids in cerebrotendinous xanthomatosis. J. Clin. Invest. 76:744-751.

16. Hoshita, T., Yasuhara, M., Une, M., Kibe, A., Itoga, E., Kito, S., Kuramoto, T. 1980. Occurrence of bile alcohol glucuronides in bile of patients with cerebrotendinous xanthomatosis. J. Lipid Res. 21:1015-1021[Abstract].

17. Batta, A. K., Shefer, S., Batta, M., Salen, G. 1985. Effect of chenodeoxycholic acid on biliary and urinary bile acids and bile alcohols in cerebrotendinous xanthomatosis; monitoring by high performance liquid chromatography. J. Lipid Res. 26:690-698[Abstract].

18. Rosen, H., Reshef, A., Maeda, N., Lippoldt, A., Shpizen, S., Triger, L., Eggertsen, G., Björkhem, I., Leitersdorf, E. 1998. Markedly reduced bile acid synthesis but maintained levels of cholesterol and vitamin D metabolites in mice with disrupted sterol 27-hydroxylase gene. J. Biol. Chem. 273:14805-14812[Abstract/Free Full Text].

19. Bergström, S., Krabisch, L. 1957. Preparation of some hydroxy coprostanes, 3a,7- and 3a,12-dihydroxy coprostane. Acta Chem. Scand. 11:1067.

20. Dayal, B., Shefer, S., Tint, G. S., Salen, G., Mosbach, E. H. 1976. Synthesis of 5ß-cholestane-3, 7, 12, 25-tetrol and 5ß-Cholestane-3, 7, 12, 24, 25-pentol. J. Lipid Res. 17:74-77[Abstract].

21. Shefer, S., Dayal, B., Tint, G. S., Salen, G., Mosbach, E. H. 1975. Identification of pentahydroxy bile alcohols in cerebrotendinous xanthomatosis: characterization of 5ß-cholestane-3, 7, 12, 24, 25-pentol and 5ß-cholestane-3, 7, 12, 23, 25-pentol. J. Lipid Res. 16:280-286[Abstract].

22. Hoshita, T. 1962. Stero-bile acids and bile sterols. XLVII. Syntheses of 3, 7, 12, 25, 26- and 3, 7, 12, 24, 25-pentahydroxycoprostanes. J. Biochem. 52:176-179[Free Full Text].

23. Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J. 1951. Protein measurement with the Folin phenol reagent. J. Biol. Chem. 193:265-275[Free Full Text].

24. Tint, G. S., Dayal, B., Batta, A. K., Shefer, S., Cheng, F. W., Salen, G., Mosbach, E. H. 1978. Gas-liquid chromatography–mass spectrometry of trimethylsilyl ethers of bile alcohols. J. Lipid Res. 19:956-966[Abstract].

25. Ahlberg, J., Angelin, B., Björkhem, I., Einarsson, S. K., Leijd, B. 1979. Hepatic cholesterol metabolism in normo- and hyperlipidemic patients with cholesterol gallstones. J. Lipid Res. 20:107-115[Abstract].

26. Oftebro, H., Björkhem, I., Skrede, S., Schreiner, A., Pedersen, J. I. 1980. Cerebrotendinous xanthomatosis. J. Clin. Invest. 65:1418-1430.

27. Pitha, J., Irie, T., Sklar, P. B., Nye, J. S. 1988. Drug solubilizers to aid pharmacologists: amorphous cyclodextrin derivatives. Life Sci. 43:493-502[Medline].

28. Yaksh, T. L., Jang, J., Nishiuchi, Y., Braun, K. P., Ro, S., Goodman, M. 1991. The utility of 2-hydroxypropyl-ß-cyclodextrin as a vehicle for the intracerebral and intrathecal administration of drugs. Life Sci. 48:623-633[Medline].

29. Petrack, B., Latario, B. J. 1993. Synthesis of 27-hydroxycholesterol in rat liver mitochondria: HPLC assay and marked activation by exogenous cholesterol. J. Lipid Res. 34:643-649[Abstract].

30. Araya, Z., Sjoberg, H., Wikvall, K. 1995. Different effects on the expression of CYP7 and CYP27 in rabbit liver by cholic acid and cholestyramine. Biochem. Biophys. Res. Commun. 216:868-873[Medline].

31. Xu, G., Salen, G., Shefer, S., Tint, G. S., Kren, B. T., Nguyen, L. B., Steer, C. J., Chen, T. S., Salen, L., Greenblatt, D. 1997. Increased bile acid pool inhibits cholesterol 7-hydroxylase in cholesterol-fed rabbits. Gastroenterology. 113:1958-1965[Medline].

32. Xu, G., Salen, G., Shefer, S., Tint, G. S., Nguyen, L. B., Parker, T. T., Chen, T. S., Roberts, J., Kong, X., Greenblatt, D. 1998. Regulation of classic and alternative bile acid synthesis in hypercholesterolemic rabbits: effects of cholesterol feeding and bile acid depletion. J. Lipid Res. 39:1608-1615[Abstract/Free Full Text].

33. Xu, G., Salen, G., Batta, A. K., Shefer, S., Nguyen, L. B., Niemann, W., Chen, T. S., Arora-Mirchandani, R., Ness, G. C., Tint, G. S. 1992. Glycocholic acid and glycodeoxycholic acid but not glycoursocholic acid inhibit bile acid synthesis in the rabbit. Gastroenterology. 102:1717-1723[Medline].

34. Xu, G., Salen, G., Shefer, S., Ness, G. C., Nguyen, L. B., Parker, T. S., Chen, T. S., Zhao, Z., Donnelly, T. M., Tint, G. S. 1995. Unexpected inhibition of cholesterol 7-hydroxylase by cholesterol in New Zealand white and Watanabe heritable hyperlipidemic rabbits. J. Clin. Invest. 95:1497-1504.

35. Lund, E. G., Kerr, T. A., Sakai, J., Li, W. P., Russell, D. W. 1998. cDNA cloning of mouse and human cholesterol 25-hydroxylases, polytopic membrane proteins that synthesize a potent oxysterol regulator of lipid metabolism. J. Biol. Chem. 273:34316-34327[Abstract/Free Full Text].

36. Furster, C., Wikvall, K. 1999. Identification of CYP3A4 as the major enzyme responsible for 25-hydroxylation of 5ß-cholestane-3,7,12-triol in human liver microsomes. Biochim. Biophys. Acta. 1437:46-52[Medline].

37. Johansson, G. 1970. Effect of cholestyramine and diet on hydroxylations in the biosynthesis and metabolism of bile acids. Eur. J. Biochem. 17:292-295[Medline].

38. Ahlberg, J., Angelin, B., Björkhem, I., Einarsson, K., J-Å. Gustafsson,, Rafter, J. 1980. Effects of treatment with chenodeoxycholic acid on liver microsomal metabolism of steroids in man. J. Lab. Clin. Med. 95:188-194[Medline].