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J. Biol. Chem., Vol. 276, Issue 40, 37004-37010, October 5, 2001

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From Brain to Bile

EVIDENCE THAT CONJUGATION AND -HYDROXYLATION ARE IMPORTANT FOR ELIMINATION OF 24S-HYDROXYCHOLESTEROL (CEREBROSTEROL) IN HUMANS^*

Ingemar Björkhem^§, Ulla Andersson, Ewa Ellis^¶, Gunvor Alvelius, Lars Ellegård, Ulf Diczfalusy, Jan Sjövall^**, and Curt Einarsson^¶

From the  Divisions of Clinical Chemistry and ^¶ Gastroenterology and Hepatology, Karolinska Institutet, Huddinge University Hospital, S-141 86 Huddinge, Sweden, the ^** Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177 Stockholm, Sweden, and the  Division of Clinical Nutrition, Sahlgrenska University Hospital, University of Gothenburg, SE-41345 Gothenburg, Sweden

Received for publication, April 30, 2001, and in revised form, July 19, 2001

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

The brain is the almost exclusive site of formation of 24S-hydroxycholesterol in man, and there is a continuous flux of this oxysterol across the blood-brain barrier into the circulation. The hepatic metabolism of 24S-hydroxycholesterol was studied here by three different approaches: incubation of tritium-labeled 24S-hydroxycholesterol with human primary hepatocytes, administration of tritium-labeled 24S-hydroxycholesterol to a human volunteer, and quantitation of free and conjugated 24S-hydroxycholesterol and its neutral metabolites in ileocecal fluid from patients with ileal fistulae. 24S-Hydroxycholesterol as well as 24R-hydroxycholesterol were converted into bile acids by human hepatocytes at a rate of about 40% of that of the normal intermediate in bile acid synthesis, 7-hydroxycholesterol. There was also a conversion of 24S-hydroxycholesterol into conjugate(s) of 5-cholestene-3,24S,27-triol at a rate similar to the that of conversion into bile acids. When administered to a human volunteer, labeled 24S-hydroxycholesterol was converted into bile acids at about half the rate of simultaneously administered labeled 7-hydroxycholesterol. Free, sulfated, and glucuronidated 24S-hydroxycholesterol and 5-cholestene-3,24,27-triol were identified in ileocecal fluid. The excretion of these steroids was about 3.5 mg/24 h, amounting to more than 50% of the total estimated flux of 24S-hydroxycholesterol from the brain. It is concluded that 24S-hydroxycholesterol is a less efficient precursor to bile acids and that about half of it is conjugated and eliminated in bile as such or as a conjugate of a 27-hydroxylated metabolite. The less efficient metabolism of 24S-hydroxycholesterol may explain the surprisingly high levels of this oxysterol in the circulation and is of interest in relation to the suggested role of 24S-hydroxycholesterol as a regulator of cholesterol homeostasis.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

A new mechanism was described for the elimination of cholesterol from the brain involving conversion of cholesterol into 24S-hydroxycholesterol and a flux of this oxysterol over the blood-brain barrier into the circulation (1-3). The enzyme involved in this conversion has been identified recently as a cytochrome P-450 species denoted CYP46 (4). This enzyme is almost exclusively located in the brain (4), and evidence has been presented that more than 90% of the 24S-hydroxycholesterol in the human circulation originates from this organ (3, 5). Measurements of arteriovenous concentration differences across the brain and liver have shown that the liver eliminates an amount of 24-hydroxycholesterol similar to that produced by the brain (3).

In view of the fact that a hydroxyl group is introduced in the 24 position during the normal transformation of cholesterol into bile acids (6), a conversion of 24S-hydroxycholesterol into bile acids would be expected. In the major pathway for biosynthesis of bile acids, the first and rate-limiting step is the introduction of a hydroxyl group in the 7 position by the cytochrome P-450 enzyme CYP7A. When human CYP7A was expressed in Escherichia coli and in simian COS cells, there was a significant activity toward 24-hydroxycholesterol (7). There is an alternative pathway for the conversion of cholesterol into bile acids in humans, starting with the oxidation of the steroid side chain followed by a 7-hydroxylation catalyzed by the oxysterol 7-hydroxylase (CYP7B) (8). Surprisingly this enzyme has no significant activity toward 24S-hydroxycholesterol (7, 9). Very recently, a new species of cytochrome P-450 denoted CYP39 was identified and found to have a high 7-hydroxylase activity toward 24S-hydroxycholesterol (10).

Because both CYP7A and CYP39 are present in human liver and because both are able to 7-hydroxylate 24-hydroxycholesterol, one would expect an efficient conversion of 24-hydroxycholesterol into bile acids. In a recent pilot study, we incubated a racemic mixture of tritium-labeled 24R- and 24S-hydroxycholesterol with human primary hepatocytes. The conversion of this racemic mixture into the primary bile acids cholic acid and chenodeoxycholic acid was less than 10% of that of 7-hydroxycholesterol. However, because only the 24S isomer occurs naturally and one isomer may inhibit the metabolism of the other, no firm conclusions could be drawn, from that study.

To our knowledge, there is only one previous report concerned with the metabolism of 24-hydroxycholesterol in the mammalian liver (11). In that report tritium-labeled 24R- and 24S-hydroxycholesterol were administered to mice. Because the tritium was located in positions 3 and 24 and would be expected to be lost in a conversion of the oxysterol into bile acids, no conclusion could be drawn regarding a possible metabolism into bile acids. More polar metabolites were formed from the two oxysterols, but their identities were never established.

In the present work we have prepared 24R and 24S isomers of tritium-labeled 24-hydroxycholesterol and incubated them separately with human primary hepatocytes. In addition we have injected tritium-labeled 24S-hydroxycholesterol together with ¹⁴C-labeled 7-hydroxycholesterol in two human volunteers and studied the relative conversion of the two oxysterols into bile acids. Furthermore, we have identified and quantitated conjugates and a major hydroxylated metabolite of 24S-hydroxycholesterol in ileocecal contents from patients with ileocecal fistulae. The results show that 24S-hydroxycholesterol is less efficient than 7-hydroxycholesterol as a precursor to bile acids in humans and that a considerable part is excreted as conjugates in bile with or without an additional hydroxyl group.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Materials and Methods-- Unlabeled and ²H₃-labeled 24R- and 24S-hydroxycholesterol were obtained as a racemic mixture using a previously described synthetic procedure (12). The two isomers were separated by HPLC¹ using reversed-phase C₁₈ columns (YMC-Pack ODS-A, 5 µm) using methanol/water (85:15) as solvent at a flow rate of 1.5 ml/min. 24S-Hydroxycholesterol was also isolated from a lipid extract of a homogenate of pig brain and subsequent preparative thin-layer chromatography with ethyl acetate/toluene (7:3 (v/v)) as solvent.

Tritium-labeled 24R-hydroxycholesterol was prepared from racemic 24-hydroxycholesterol subjected to Wilzbach tritiation (custom synthesis performed by PerkinElmer Life Sciences). The crude material after the tritiation was subjected to alkaline hydrolysis and preparative thin-layer chromatography as above. The 24R isomer was isolated by preparative HPLC as described above. The material used for incubation had a specific radioactivity of 0.23 × 10⁶ cpm/µg. Tritium-labeled 24S-hydroxycholesterol was prepared biosynthetically from 1,2-³H-labeled cholesterol (PerkinElmer Life Sciences) as described below. The material incubated had a specific radioactivity of 0.5 × 10⁶ cpm/µg.

Tritium-labeled 7-hydroxycholesterol (with a 7-³H label) was prepared as described previously (13) and had a specific radioactivity of 0.5 × 10⁶ cpm/µg. ¹⁴C-Labeled 7-hydroxycholesterol was prepared as described previously (13) and had a specific radioactivity of 14 × 10³ cpm/µg. Helix pomatia intestinal juice was purchased from Sigma-Aldrich. Sep-Pak tC₁₈ cartridges were obtained from Waters Corp. (Milford, MA).

Human liver tissue (from four separate donors) was obtained from the Department of Transplantation at Huddinge University Hospital. In two of the three experiments shown in Table I, the patients had familial amyloidosis and polyneuropathy. In the other experiment shown in Table I, the patient had metastases from a colon cancer in the liver. Only the parts of the liver visually free from metastases were used. In one additional experiment described in the text only, the material was obtained in connection with reduction hepatectomy performed prior to a pediatric liver transplantation of size-mismatched donor liver. The preparation obtained from the latter liver was considerably less active than those from the other livers (cf. "Results").

Ileocecal content was obtained from five subjects with a well functioning conventional ileostomy after proctocolectomy because of ulcerative colitis (n = 4) or Mb Crohn (n = 1). None had any further intestinal resection in excess of the necessary 5-10 cm of distal ileum used to create the stoma. The patients were all stable in weight and without nutritional problems. At the time of collection the patients had no signs of anemia, inflammation, diabetes, or hepatic, renal, or thyroid disease as judged by history, hospital records, and standard laboratory tests. Ileostomy bags were changed every second hour during the daytime and directly after awakening. In one of the cases (Mb Crohn) the ileocecal content collected during a 24-h period was analyzed directly. In the other four cases (ulcerative colitis) the ileocecal content was immediately frozen on solid carbon ice in Dewar vessels, which the patients kept at home. Each morning the bags were delivered to the metabolic ward, weighed, and stored at 20 °C before freeze-drying to a constant weight. The dry frozen ileostomy content was meticulously ground and mixed before analysis. Aliquots of the freeze-dried powder were used for the analysis. Details of the procedure for collection and treatment of the ileocecal content have been published previously (14).

Preparation of Tritium-labeled 24S-Hydroxycholesterol-- Human embryonic 293 cells (ATCC CRK 1573) at a confluence of ~60% were transfected with cDNA of CYP46 inserted in a pcDNA 3.1 vector using Tfx^TM20 (Promega). The cDNA was supplied generously by Dr. D. Russell (Southwestern School of Medicine, Texas University, Dallas, Texas) (4). The cells were cultured in minimum Eagle's medium (Sigma M-4655) (10 ml) containing 10% horse serum (Life Technologies, Inc.), 1% minimum Eagle's medium nonessential amino acid mixture (Sigma M-7145), 1% sodium pyruvate (Sigma S-8636), and 1% penicillin-streptomycin (Life Technologies, Inc., 15140-114). The cells were selected with geneticin G418 (Sigma G-9516) at 37 °C in an atmosphere of 5% CO₂. The cells were maintained in a 100-mm² tissue culture dish (FALCON 353003) at 37 °C in an atmosphere of 5% CO₂.

Cells were cultured as described above in the absence of geneticin to a confluence of 70%. Cholesterol was removed from the cells by treatment with 2-hydroxypropyl- -cyclodextrin (Sigma C-0926) and 20 mg/ml for 1 h. In subsequent stages/cultures the horse serum was replaced with delipidated calf serum (Sigma C-1696). The cells were then incubated for 48 h with 0.33 mCi of 1,2-³H-labeled cholesterol (PerkinElmer Life Sciences NET 139) dissolved in 40 µl of ethanol. The medium was then changed, and incubation was continued for another 48 h. The medium was removed and extracted according to the Folch procedure (chloroform/methanol 2:1 (v/v), 5 parts, and aqueous medium, 1 part), and the labeled 24-hydroxycholesterol was isolated by thin-layer chromatography using the same solvent system as above. The material (dissolved in toluene) was then purified further by application to an Isolute silica cartridge column (International Sorbent Technology, Mid Glamorgan, UK) previously equilibrated with hexane. 24S-Hydroxycholesterol was eluted from the column with 50% 2-propanol in hexane. In some cases the material needed further purification by preparative HPLC as described above. The overall yield of tritium-labeled 24S-hydroxycholesterol from the tritium-labeled substrate varied between 5 and 10% in different incubations.

Preparation of Human Hepatocytes, Incubations, and Analyses-- Hepatocytes were prepared from the human liver material according to a two-step perfusion technique with EGTA and collagenase (Type XI, Sigma) solutions as described previously (15). The hepatocytes were cultured in 6-cm dishes that had been precoated with 200 µl of EHS Matrigel containing 3 ml of culture medium (William E medium supplemented with glutamine (292 µg/ml), Na₂SeO₃ (173 µg/ml), insulin (2 milli international units/ml), penicillin G sodium (100 units/ml), streptomycin sulfate (100 µg/ml), and gentamycin (85 µg/ml)). The hepatocytes were cultured for 4 days without additions. On the fourth day the hepatocytes were incubated with 4 µg of tritium-labeled 7-hydroxycholesterol, 24R-hydroxycholesterol, or 24S-hydroxycholesterol dissolved in 10 µl of ethanol. After incubation for 48 h, the medium (3 ml) was removed and hydrolyzed at 120 °C for 48 h with 6 g of KOH dissolved in 12 ml of 50% ethanol. After dilution with saline and 50% ethanol, an alkaline extraction was carried out with diethyl ether to isolate neutral steroids. The aqueous phase was then acidified with 6 M HCl, and the bile acids were extracted with diethyl ether. The diethyl ether was washed with water until neutral. After evaporation of the ether, an aliquot of the material was assayed for radioactivity with use of a scintillation counter. Another aliquot of the material was analyzed for bile acids by radio HPLC using the same reversed-phase C₁₈ column as described above and a mixture of methanol and 15 mM KH₂PO₄ buffer, pH 5.4 (3:1 (v/v)), as solvent at a flow rate of 1.5 ml/min. The cells were extracted according to Folch, and aliquots of the extract were analyzed for radioactivity. Aliquots were also analyzed for the presence of neutral radioactive steroids by radio HPLC with the use of the same reversed-phase C₁₈ column as described above and metanol/water (85:15) as solvent.

In one of the experiments with hepatocytes (Table I), part of the medium was subjected to alkaline hydrolysis, acid solvolysis, and treatment with H. pomatia juice as described below for ileocecal content (Fig. 1) and analyzed by radio HPLC with the use of methanol/water (85:15) as solvent (Fig. 3).

Conversion of Labeled Oxysterols into Bile Acids in a Volunteer-- A mixture of [4-¹⁴C]7-hydroxycholesterol (~0.15 mg, 2.0 × 10⁶ cpm) and [1,2-³H]24S-hydroxycholesterol (~20 µg, 4 × 10⁶ cpm) in ethanol (2 ml) was passed through a Millex 0.22-µm filter (Millipore Corp., Bedford, MA) and then added to 1 ml of 20% (w/v) aqueous solution of human serum albumin and 7 ml of saline. The mixture was injected intravenously in a fasting healthy male volunteer (age 59 years, body mass index 25) and a fasting healthy female volunteer (age 64 years, body mass index 24). Bile was collected 24 h and, in the male subject, also 48 h later by duodenal intubation. At each occasion, the yield of bile obtained was between 10 and 50 ml. Aliquots of the bile were hydrolyzed in alkali, extracted, methylated, and subjected to preparative thin-layer chromatography as described previously (16). The chromatographic zones corresponding to cholic acid and chenodeoxycholic acid were visualized by exposure to iodine vapor. The radioactivity eluted from the two different zones was assayed by scintillation counting, differentiating between ³H and ¹⁴C. Under the conditions used, the counting efficiency for ³H was ~50% and for ¹⁴C ~70%.

Analysis of Ileocecal Content-- To recover different forms of sterols ranging in polarity from that of cholesterol esters to glucuronides of hydroxycholesterols, a combined solvent-solid phase extraction procedure was used, in which the alcoholic extract was cycled through a Sep-Pak tC₁₈ cartridge with a stepwise decrease of the alcohol concentration (17). This was performed at three different stages: after mild alkaline hydrolysis, after solvolysis, and after treatment with glucuronidase (cf. Fig. 1).

To an exact aliquot of the freeze-dried ileocecal content (20 mg) 1 ml of water and 400 ng of ²H₃-labeled 24-hydroxycholesterol were added, and the mixture was sonicated for 30 min. The mixture was then hydrolyzed with KOH as described previously (16) with the exception that ethanol was replaced with methanol. The alkaline hydrolysate was neutralized with 6 M HCl and centrifuged. The mixture, containing a concentration of methanol of ~80%, was passed through a Sep-Pak tC₁₈ bed (~0.3 g) that had been washed with chloroform/methanol (1:1), methanol, water, and methanol/water (4:1). The effluent from the bed was diluted with water to a concentration of 60% methanol. This mixture was passed through the same bed. Water was again added to the effluent to get a methanol concentration of 30%. This effluent was again passed through the same bed, and the effluent was adjusted with water to get a concentration of methanol 15%. After passage of this mixture the bed was washed with water. The steroids were then eluted from the column with methanol (5 ml). In some experiments part of this methanol phase, containing hydrolyzed steroids, was analyzed by combined gas chromatography/mass spectrometry (GC/MS) to study unconjugated steroids and by electrospray (ES) mass spectrometry to evaluate the presence of sulfates and glucuronides. The solvent was evaporated under vacuum, and the residue was redissolved in 200 µl of dried methanol. A mixture of tetrahydrofuran (1.8 ml) and trifluoroacetic acid (20 µl) was added, and the mixture was shaken at 45 °C for 45 min (18). The mixture was then neutralized with 2.6 ml of an aqueous solution of 0.1 M NaHCO₃. Water (3 ml) was added, and the mixture was concentrated under a stream of N₂ to remove the tetrahydrofuran. Methanol was then added to the aqueous mixture to a concentration of 80%. This mixture was passed through a Sep-Pak tC₁₈ bed equilibrated with 80% methanol, and the effluent was cycled through the C₁₈ bed as above. The hydrolyzed and solvolyzed steroids were eluted from the column with methanol (5 ml). In some experiments, part of this methanol phase was analyzed by GC/MS and/or ES mass spectrometry. After evaporation of the methanol, the above material was treated with H. pomatia digestive juice (equivalent to 30,000 international units of -glucuronidase) diluted in 5 ml of sodium acetate buffer (0.2 M), pH 4.5. This enzyme solution was purified by passage through a Sep-Pak tC₁₈ cartridge prior to use. After incubation for 1 h at 62 °C the hydrolysis mixture was centrifuged, and the supernatant was applied to a Sep-Pak tC₁₈ bed equilibrated with the same NaAc buffer. The mixture was centrifuged, and the supernatant was applied to a tC₁₈ bed and equilibrated with the NaAc buffer. Methanol was added to the effluent to a final concentration of 80%, and this mixture was recycled on the same column as above. After cycling with 60, 30, and 15% methanol as described above, the steroids were eluted in methanol and analyzed by mass spectrometry.

ES mass spectrometry was carried out using a Micromass Quattro 1 instrument (Micromass, Manchester, UK) at unit mass resolution. Optimal interface conditions for the recording of negative ion spectra were established with a solution containing a mixture of conjugated bile acids. The samples were injected in a stream of 50% aqueous methanol at a flow rate of 10 µl/min. The m/z range 200-800 was scanned for 2 min at a rate of 10 s/scan. GC/MS was performed under the same conditions as described previously (5, 12) with the use of deuterium-labeled 24-hydroxycholesterol as an internal standard.

Ethical Aspects-- The in vivo experiments with the two human volunteers as well as the experiments with the human hepatocytes were approved by the local ethics committee at Huddinge University Hospital (Huddinge, Sweden). The experiments with ileocecal contents were approved by the local ethics committee at Sahlgrenska University Hospital (Gothenburg, Sweden).

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Metabolism of Tritium-labeled 24S-Hydroxycholesterol in Human Primary Hepatocytes-- Table I summarizes the results of three experiments in which tritium-labeled 24S-hydroxycholesterol was incubated with human primary hepatocytes for 48 h under the conditions described under "Experimental Procedures." For reasons of comparison, equal amounts of tritium-labeled 24R-hydroxycholesterol and 7-hydroxycholesterol were incubated in parallel under the same conditions with the same number of cells.

View this table: [in this window] [in a new window]   Table I Conversion of tritium-labeled 7-hydroxycholesterol, 24R-hydroxycholesterol, and 24S-hydroxycholesterol into bile acids in human primary hepatocytes (n = 3)

The recovery of tritium in the acid fraction, expected to contain bile acids, was ~25% in the experiment with labeled 24S-hydroxycholesterol and ~65% in the experiments with labeled 7-hydroxycholesterol. In the experiment with 24R-hydroxycholesterol the recovery of tritium in the acid fraction was 19%. Analysis of the bile acid containing acid extracts by radio HPLC showed that in all the above experiments, 70-90% of the radioactivity corresponded to cholic acid and chenodeoxycholic acid (Fig. 2).

The amount of radioactivity recovered in the hepatocytes after incubation was about two times higher in the experiments with labeled 24S-hydroxycholesterol and 24R-hydroxycholesterol than in the experiments with 7-hydroxycholesterol. Almost all this radioactivity corresponded to unmetabolized substrate (results not shown). The amount of radioactivity recovered in the extract from the alkaline phase obtained directly after the hydrolysis was also about two times higher in the case of incubations with 24S- and 24R-hydroxycholesterol than with 7-hydroxycholesterol. Attempts to identify the radioactive steroids in this phase were not made because of the expected extensive degradation.

In addition to the three experiments documented in Table I, an additional experiment was made with a preparation of human hepatocytes that was considerably less active than the other preparations. In this experiment the degree of conversion of labeled 7-hydroxycholesterol into bile acids was less than 20%, and the degree of conversion of 24R- and 24S-hydroxycholesterol was less than 2%.

The above investigation gives accurate quantitative information with respect to the degree of conversion of the radioactive oxysterols into bile acids. The conditions needed for complete hydrolysis of the bile acid conjugates may, however, destroy and/or partially degrade conjugates of neutral steroids and steroids with a hydroxyl group in allylic position. To measure the degree of conversion of 24S-hydroxycholesterol into glucuronides and sulfates, half the medium from one of the incubations with the tritium-labeled 24S-hydroxycholesterol was subjected to weak alkaline hydrolysis followed by acid solvolysis and treatment with H. pomatia enzymes as shown in Fig. 1. This treatment is expected to give a complete cleavage of all conjugates of the neutral steroids but only a partial cleavage of bile acids. Part of the mixture obtained after the above treatment was analyzed by radio HPLC. There was a surprisingly high yield of cholic acid (corresponding to ~28% of total radioactivity) and relatively small amounts of more polar radioactive compounds corresponding to conjugated bile acids (~10%, cf. Fig. 3). The peak corresponding to chenodeoxycholic acid was low (less than 2%), and a similar low conversion into chenodeoxycholic acid was observed also in the corresponding incubation with tritium-labeled 7-hydroxycholesterol (results not shown). There was a prominent peak (corresponding to ~28% of total radioactivity) of 24S-hydroxycholesterol (Fig. 2). In addition, there was a radioactive peak of similar size with a mobility between that of bile acids and 24S-hydroxycholesterol (the peak with retention time 7.8 min in Fig. 3). Part of the material corresponding to this peak was converted into trimethylsilyl ether and analyzed by GC/MS. The retention time and mass spectrum of this compound was identical to that of 5-cholestene-3,24,27-triol trimethylsilyl ether ether (cf. below).

View larger version (17K): [in this window] [in a new window]   Fig. 1.   Overview of the different analytical steps in the analyses of conjugated and free metabolites of 24S-hydroxycholesterol in ileocecal fluid and in medium from an incubation with human hepatocytes.

View larger version (13K): [in this window] [in a new window]   Fig. 2.   Radio HPLC of an acid extract obtained after alkaline hydrolysis of the medium of a culture of human hepatocytes together with tritium-labeled 24S-hydroxycholesterol (A) and tritium-labeled 7-hydroxycholesterol (B) for 48 h (experiment I in Table I). The solvent system used in the chromatography was methanol/potassium phosphate buffer 15 mM, pH 5.4 (3:1 (v/v)).

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Radio HPLC of the medium of a culture of human hepatocytes together with tritium-labeled 24S-hydroxycholesterol (24S-OH-cholesterol) for 48 h (experiment III in Table I). The medium had been treated by alkaline hydrolysis, acid solvolysis, and H. pomatia glucuronidase as described under "Experimental Procedures." The mobile phase used in the HPLC was methanol/water (85:15 (v/v)).

Conversion of Tritium-labeled 24S-Hydroxycholesterol into Bile Acids in Two Volunteers-- Intravenous administration of a mixture of tritium-labeled 24S-hydroxycholesterol and 4-¹⁴C-labeled 7-hydroxycholesterol in two volunteers led to significant incorporation of both isotopes in cholic acid and chenodeoxycholic acid in bile 24 and 48 h after the administration. As seen in Table II, the initial ratio between tritium and ¹⁴C had decreased by 25-50% during its conversion into cholic acid and by 50-60% during its conversion into chenodeoxycholic acid. This is consistent with a rate of conversion of 24S-hydroxycholesterol into bile acids that is about half that of 7-hydroxycholesterol.

View this table: [in this window] [in a new window]   Table II Conversion of 4-[14C]7-hydroxycholesterol and 1,2-[3H]24S-hydroxycholesterol into bile acids in two healthy volunteers

Quantitation of 24S-Hydroxycholesterol and Its Metabolites in Ileocecal Content. Identification of 5-Cholestene-3,24,27-triol-- Using the procedure summarized in Fig. 1, GC/MS and ES/MS, ileocecal content was shown to contain 24S-hydroxycholesterol in free and/or fatty acid esterified form, as sulfate ester, and as glucuronide or sulfate-glucuronide.

In preparations from different patients, the amount of the free and/or fatty acid esterified form varied between 1 and 4%, the amount of sulfate ester varied between 3 and 45%, and the amount of glucuronide varied between 40 and 50%. It should be emphasized that the glucuronide fraction includes a species of 24S-hydroxycholesterol that is both a glucuronide and a sulfate.

The above-mentioned figures were obtained by quantitation of 24S-hydroxycholesterol by GC/MS of the same ileocecal sample after alkaline hydrolysis, alkaline hydrolysis + solvolysis, or alkaline hydrolysis + solvolysis + treatment with H. pomatia enzymes. The efficiency of the solvolysis and the treatment with H. pomatia enzymes was confirmed by ES mass spectrometry. The ES mass spectrum of unsolvolyzed ileocecal content showed a peak at m/z 481, corresponding to a monosulfate of a monohydroxylated cholesterol species, most probably 24S-hydroxycholesterol. This peak disappeared completely after the solvolysis. Before treatment with H. pomatia enzymes, a peak was observed at m/z 577, corresponding to a monoglucuronide of a monohydroxylated species of cholesterol. This peak disappeared completely after the treatment. The results were analogous to those obtained in the previous identification of the doubly conjugated 24S-hydroxycholesterol present in the plasma of cholestatic infants (19).

Ileocecal contents subjected to alkaline hydrolysis, solvolysis, and treatment with H. pomatia enzymes were analyzed carefully for steroids with mass spectrometric characteristics expected for neutral steroids containing a 24-hydroxy group. In addition to 24S-hydroxycholesterol, a compound was found with a mass spectrum corresponding to 5-cholestene-3,24,27-triol (Fig. 4). The trimethylsilyl ether gave prominent peaks at m/z 544 (M-90), m/z 503 (M-131), m/z 413 (M-131-90), m/z 233 (side chain C22-C27 containing two silylated hydroxyl groups). The lack of a prominent peak at m/z 131 excludes the possibility that one of the two hydroxyl groups in the side chain is located in the C25 position, and the presence of hydroxyl groups in C24 and C27 is the only possible structure consistent with the mass spectrum. That mass spectra with hydroxyl groups at C24 and C27 give a prominent peak at m/z 233 has been shown previously (20, 21). The identity of the compound as 5-cholestene-3,24,27-triol was confirmed further by the incubation of racemic 24R,24S-hydroxycholesterol with reconstituted human CYP27, adrenodoxin, and adrenodoxin reductase under the conditions described previously for 27-hydroxylation of cholesterol (22). There was a significant conversion into the two 24 isomers of 5-cholestene-3,24,27-triol that both gave identical mass spectra with all the above characteristic peaks. The rate of conversion was, however, considerably lower than that with cholesterol as substrate.

View larger version (17K): [in this window] [in a new window]   Fig. 4.   Mass spectrum of trimethylsilyl ether of the neutral metabolite of 24S-hydroxycholesterol present in ileocecal fluid from a patient with an ileocecal fistula. The material had been subjected to alkaline hydrolysis, acid solvolysis, and treatment with H. pomatia enzymes before the analysis.

The concentration of 5-cholestene-3,24,27-triol in ileocecal fluid was similar to that of 24S-hydroxycholesterol. Similar to the latter steroid most of the triol was present in glucuronidated and/or sulfated forms. No other C-27 or C-26 steroid metabolite with a 24-hydroxy group could be found.

When reanalyzing the above mixtures after the addition of one additional final solvolysis step, the yield of 24S-hydroxycholesterol did not change. The yield of 5-cholestene-3,24,27-triol, however, increased significantly with ~20%. Because of this, an additional solvolysis step was added in all the subsequent analyses.

The total content of 24S-hydroxycholesterol and 5-cholestene-3,24,27-triol was measured as described above in the ileocecal content from five patients. The quantitations were made with deuterium-labeled 24-hydroxycholesterol as internal standard after alkaline hydrolysis, solvolysis, treatment with H. pomatia enzymes, and resolvolysis. The yield of 24S-hydroxycholesterol was 1.6 ± 0.3 mg/24 h, whereas the yield of 5-cholestene-3,24,27-triol was 1.9 ± 0.4 mg/24 h (mean ± S.E.). The total excretion of the two steroids in the five patients was thus 3.5 ± 0.5 mg/24 h.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

There are several mechanisms by which the liver may eliminate 24S-hydroxycholesterol. The oxysterol may be excreted as such or in conjugated form. Another possibility is that the steroid may be hydroxylated and then excreted as such or as a conjugate. Finally the oxysterol may be converted into cholic or chenodeoxycholic acid before excretion in bile. According to the present investigation all these mechanisms are operating in humans.

The rate of conversion of 24S-hydroxycholesterol into bile acids was found to be ~40% of that of 7-hydroxycholesterol in most of the experiments with human hepatocytes and ~50% of that of 7-hydroxycholesterol in the in vivo experiment with the two volunteers. In the experiments with human hepatocytes, the major bile acids formed were cholic acid and chenodeoxycholic acid in proportions almost identical to those obtained in experiments with labeled 7-hydroxycholesterol. The rate of conversion of 24R-hydroxycholesterol, an isomer not normally present in human tissues, was similar to that of 24S-hydroxycholesterol, with a similar pattern of products.

Interestingly, 27-hydroxylated 24S-hydroxycholesterol (5-cholestene-3,24S,27-triol) was found to be a quantitatively important metabolite in the experiments with the hepatocytes. This triol may be formed by the action of sterol 27-hydroxylase (CYP27) on 24S-hydroxycholesterol, and it was confirmed here that 24-hydroxycholesterol is a substrate for human CYP27. The rate of 27-hydroxylation of 24-hydroxycholesterol by reconstituted CYP27, however, was relatively low: lower than the rate of 27-hydroxylation of cholesterol by the same enzyme. The possibility can thus not be completely excluded that an enzyme other than CYP27 may be involved in the conversion. Another possibility is that it is a conjugate of 24S-hydroxycholesterol rather than the free steroid that is a substrate for CYP27 under in vivo conditions. In any case, it is evident that a substantial portion of 24S-hydroxycholesterol is converted into 5-cholestene-3,24,27-triol as a first step, possibly as a consequence of a relatively inefficient 7-hydroxylation. In contrast to 25-hydroxycholesterol and 27-hydroxycholesterol, both of which are 7-hydroxylated efficiently by astrocytes and Schwann cells, 24-hydroxycholesterol is not 7-hydroxylated by these cells (9). We have shown previously that 24S-hydroxycholesterol is not a substrate for the oxysterol 7-hydroxylase (CYP7B) (7), and this is probably the case also with 5-cholestene-3,24S,27-triol. This may increase the biological half-life of the latter triol and may explain its relatively high excretion in ileocecal fluid. The extent to which 5-cholestene-3,24S,27-triol is a natural intermediate in the conversion of 24S-hydroxycholesterol into bile acids cannot be evaluated from the present work.

The results of the above experiments are consistent with a relatively inefficient conversion of 24S-hydroxycholesterol into bile acids and show that glucuronidation, sulfation, and 27-hydroxylation of the steroid side chain are important metabolic steps in the elimination of the steroid. This is in agreement with our previous finding of the sulfated and glucuronidated form of 24S-hydroxycholesterol as a major oxysterol in the plasma of infants with severe intrahepatic cholestasis (19). Doubly conjugated cholestanetriols were also present in these samples. It is difficult, however, to quantitate the relative importance of the different metabolic events with use of these experimental models. Quantitative data could be obtained from the patients with an ileostomy, in which all steroid metabolites excreted in bile should be present in the ileocecal content. It was shown clearly that the amount of 24S-hydroxycholesterol excreted as free, sulfated, and/or glucuronidated steroid with or without an additional hydroxyl group in the 27-position was ~3.5 mg/24 h. Because we have shown that the daily production of 24S-hydroxycholesterol in humans is 6-7 mg (3), it can be concluded that the normal conversion of 24S-hydroxycholesterol into of bile acids is about 3 mg/24 h.

Approximately 10% of the 24S-hydroxycholesterol present in circulation and brain has been reported to be sulfated (1, 23), and sulfated 24S-hydroxycholesterol has also been reported to be present in human feces (24). The present study shows that glucuronidation is a most important reaction in the metabolism of 24S-hydroxycholesterol in humans without liver disease. Thus, the formation of the double conjugate is not limited to patients with severe cholestatic conditions (19).

The sites of conjugation have not been determined and neither has the order in which conjugation occurs. However, it is reasonable to assume that the double conjugate is a 3-sulfate,24-glucuronide, as was found to be the case in cholestatic infants (19).

The relatively slow hepatic metabolism of 24S-hydroxycholesterol may explain the relatively high concentration of this oxysterol in the circulation. We have shown previously that the half-life of deuterium-labeled racemic 24-hydroxycholesterol is 10-14 h, whereas the corresponding half-life of 7-hydroxycholesterol and 27-hydroxycholesterol is less than 1 h (3, 5). Despite the low production of 24S-hydroxycholesterol, its plasma concentration is higher than that of any other oxysterol in infants and only slightly lower than that of 27-hydroxycholesterol in adults (12).

Based on its inhibitory action on hydroxymethylglutaryl-CoA reductase in isolated cells and the concentration of this oxysterol in mouse liver, 24S-hydroxycholesterol has been suggested to be an important physiological regulator of cholesterol homeostasis (25). In this connection it is of interest that 24S-hydroxycholesterol has a relatively high affinity to the nuclear receptor LXR (26), which is known to mediate cholesterol-induced effects on cholesterol degradation (27). The biological half-life of a regulator of cholesterol turnover would be expected to be short. The relatively slow rate of elimination of 24S-hydroxycholesterol demonstrated here does not support the contention that 24S-hydroxycholesterol is an important regulator of cholesterol homeostasis in humans, at least not for short-term regulation. However, at the present state of knowledge the possibility can not be excluded that the rates of conjugation might be of some importance in keeping the intracellular concentration of the free sterol at a regulatory level.

	ACKNOWLEDGEMENTS

The skillful technical assistance of Anita Lövgren-Sandblom and Manfred Held is gratefully acknowledged.

	FOOTNOTES

^* This work was supported by grants from the Swedish Medical Research Council, The Strategic Foundation, and the Swedish Heart-Lung Foundation.The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

^§ To whom correspondence should be addressed. Tel.: 46-8-58581235; Fax: 46-8-58581260; E-mail: Ingemar.Bjorkhem@chemlab.hs.sll.se.

Published, JBC Papers in Press, July 19, 2001, DOI 10.1074/jbc.M103828200

	ABBREVIATIONS

The abbreviations used are: HPLC, high pressure liquid chromatography; GC, gas chromatography; MS, mass spectrometry; ES, electrospray.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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