THE JOURNAL OF BIOLOGICAL CHEMISTRY 0 1993 by The American Society for Biochemistry and Molecular Biology, Inc.

Vol. 268, No. 15, Issue of May 25, pp. 11239-11246,1993 Printed in U.S.A.

5P-Hydroxylation by the Liver IDENTIFICATION OF 3,5,7-TRIHYDROXY NOR-BILE ACIDS AS NEW MAJOR BIOTRANSFORMATION PRODUCTS OF 3,7-DIHYDROXY NOR-BILE ACIDS IN RODENTS*

(Received for publication, September 24, 1992, and in revised form, February 5, 1993)

Claudio D. SchteingartSG, Lee R. HageyS, Kenneth D. R. Setchelly, and Alan F. HofmannSII From the $Bile Acid Research Section, Division of Gastroenterology, Department of Medicine, University of California, San Diego. La Jolla. California 92093 and the (Deoartment of Pediatrics, Children’s Hospital Medical Center, Ci&nati, Ohzo 45229-2899

24-Norursodeoxycholic acid (nor-UDCA), when ad- ministered into the anesthetized biliary fistula hamster or injected into the perfusate of an isolated liver, was hydroxylated at C-5 to give 5~-hydroxynorursodeox- ycholic acid 2 (3~~,5,7B-trihydroxy-24-nor-5&cholan- 23-oic acid), which was secreted into bile mainly as such. Similarly, 24-norchenodeoxycholic acid (nor- CDCA) was 5B-hydroxylated to give 5B-hydroxynor- chenodeoxycholic acid 4 (3a,5,7a-trihydroxy-24-nor- 5j3-cholan-23-oic acid), which was also secreted into bile without appreciable further biotransformation. The site of hydroxylation was assigned by ”C and ‘H NMR and mass spectrometry. 5-Hydroxylation was a major biotransformation pathway at physiological bile acid loads. 5-Hydroxylation of UDCA also occurred in the perfused rat liver but to a lesser extent. 5-Hydrox- ylation of nor-UDCA was not observed in rabbit, dog, or man, indicating that its formation is species-specific. 5-Hydroxylation of nor-CDCA and nor-UDCA is the first reported example of hydroxylation of a tertiary carbon atom of bile acids. Nor-dihydroxy bile acids appear to be useful for the detection of minor hydrox- ylation pathways, because their prolonged hepatobili- ary retention exposes them repeatedly to hydroxylases present in the hepatobiliary system.

Nor-bile acids are homologues of the natural Cz4 bile acids in which the side chain has been shortened by one carbon atom. Such compounds occur naturally in trace proportions in bile (1) and urine (2) of healthy humans. For studies of their metabolism, such compounds are usually prepared syn- thetically by degradation of the side chain of natural bile acids; a convenient synthesis of these bile acids has been reported recently from this laboratory (3).

Nor-cholic acid, when injected parenterally into the rat, has been shown by several laboratories (4-7) to be secreted into bile in unchanged form; its metabolism thus differed strikingly

~

* Portions of this work were supported by National Institutes of Health Grants DK21506 and DK32130, as well as grants-in-aid from the Falk Foundation e.V., Freiburg, Germany. Secondary ion and high resolution mass spectra, which were performed at the University of California, San Francisco Biomedical Mass Spectrometry Re- source, were funded by Grant RR 01614 from the National Institutes of Health Division of Research Resources. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

3 To whom correspondence regarding chemical aspects of this paper should be addressed.

11 To whom correspondence regarding physiological aspects of this paper should be addressed.

from that of cholic acid, which like other common natural c24 bile acids, undergoes efficient N-acylation with taurine during hepatic transport (4-7). Subsequent studies in this laboratory of the metabolism in rodents of nor-ursodeoxycholate (nor- UDCA, 1; see Fig. 1)’ showed that this compound was similar to nor-cholate in undergoing little conjugation with taurine and in being secreted partly in unchanged form. However, in the hamster, it was biotransformed to an unknown metabolic product 2, as well as undergoing conjugation with glucuronic acid or sulfate (8). Similar observations were made for nor- chenodeoxycholate (nor-CDCA, 31, which was converted by the hamster liver into an analogous metabolite 4 (9).

In this paper, we describe the biotransformation of these nor-dihydroxy bile acids and the isolation and structure de- termination of the novel metabolites. A full characterization of these compounds by thin layer chromatography, mass spectrometry, and nuclear magnetic resonance spectrometry is presented.

EXPERIMENTAL PROCEDURES

Materials

Norursodeoxycholic acid, 1 (nor-UDCA, 3a,7P-dihydroxy-24-nor- 50-cholan-23-oic acid), and norchenodeoxycholic acid, 3 (nor-CDCA, 3a,7a-dihydroxy-24-nor-5~-cholan-23-oic acid), were prepared as de- scribed previously (3). [23-’4C]nor-UDCA (specific activity, 10 mCi/ mmol) and [23-I4C]nor-CDCA (specific activity, 10 mCi/mmol) were prepared by the method of Tserng and Klein (10); [2,4-3H]nor-CDCA (specific activity, 1 mCi/mmol) was prepared according to Ref. 11; all were purified by preparative TLC on silica gel and were more than 99% pure by radio-HPLC (12). Na235S04 was a generous gift of Dr. Ajit Varki, University of California, San Diego, D-[ l-’4C]glucose (specific activity, 50 mCi/mmol) and [U-14C]taurine (specific activity, 100 mCi/mmol) were obtained from Amersham Corp.

Isolated Perfused Hamster Livers

Isolated hamster livers were perfused as described by Anwer and Hegner (8) with minor modifications. For biotransformation studies an 8-pmol bolus of nor-UDCA or nor-CDCA, accompanied by 1 pCi of “C-labeled tracer, was added to the perfusate at 45 min. For identification of conjugates, the liver was preloaded at 20 min with 35SO:- (25 pCi), [’4C]glucose (35 pCi), or [I4C]taurine (10 pCi), and an 8-pmol bolus of nor-CDCA carrying 1 pCi of [3H]nor-CDCA was added to the perfusate at 45 min. Bile samples were collected at 15- min intervals and analyzed by TLC zonal scanning.

’ The abbreviations used are: UDCA, ursodeoxycholic acid; CDCA, chenodeoxycholic acid; HPLC, high performance liquid chromatog- raphy; ‘H NMR, proton nuclear magnetic resonance; I3C NMR, I3C nuclear magnetic resonance; SIMS, secondary ion mass spectrometry; GC-MS, gas chromatography-mass spectrometry; TMS, trimethyl- silyl; DMES, dimethylethylsilyl; GASPE, gated spin echo; GC, gas liquid chromatography; COSY, correlated spectroscopy; EI, electron ionization; Py, ds-pyridine.

11239

11240 3,7-Dihydroxy Nor-bile Acids,

n O O R

1 R = H

1. R = CH3

3 R = H

3. R = CH3

2 R = H 4 R = H

2. R = CH3 4a R = CH3

FIG. 1. Structures of nor-bile acids and metabolites.

Acute Biliary Fistula Hamsters

Biotransformation Studies-Nor-UDCA or nor-CDCA was admin- istered intravenously, together with an appropriate amount of radio- active tracer, to anesthetized male Syrian golden hamsters a t doses ranging from 0.25 to 5 pmol/kg.min, and bile was collected from a cannula inserted in the common bile duct, as described previously (8, 9).

Metabolite Production-Nor-UDCA was administered to five ham- sters at a dose of 4 pmol/kg.min for 2.5 h, and bile samples were pooled. Nor-CDCA was administered to seven hamsters at a dose of 1.5 pmol/kg.min for 2.5 h, and bile samples were pooled.

Thin Layer Chromatography Zonal Scanning

Bile samples were spotted on silica gel TLC plates (E. Merck, Darmstadt, Germany) and the chromatogram developed with isoamyl acetate:propionic acidn-propyl alcohol:water, 4:3:2:1, v/v (13). After drying, 1-mm bands of absorbent were scraped from the glass plate into scintillation vials for single or dual counting, giving 179 fractions for each sample. Radioactivity present in each fraction was graphed and integrated by a computer program.

Purification of the Methyl Esters of the Unknown Metabolites

The pooled bile samples from administration of nor-UDCA 1 were diluted with 10 ml of water, the solution slowly acidified with 1 N HC1 to pH 1, and extracted with ethyl acetate (3 X 10 ml). The pooled organic layers were washed with 20% NaCl to neutrality, dried with sodium sulfate, and evaporated, to give 158 mg of residue. This was further purified by chromatography on a column of silica gel 60 H (E. Merck), 2.3 X 10 cm, eluting with ch1oroform:methanol mixtures of composition 85:15, 80:20, and 70:30, v/v. The more polar fraction, which contained mainly 2, was esterified with methanol using dia- zomethane in methanol-diethyl ether overnight. The crude product was purified by adsorption chromatography on a silica gel 60 H column, 1 X 10 cm, eluting with ch1oroform:methanol. 955 and 9010, v/v, to give 4.7 mg of pure methyl-3a,5,7P-trihydroxy-24-nor-5/3- cholan-23-oate 2a. High-resolution MS (EI, 70 eV) m/z:

C24H4005 (Mf) Calculated 408.2876 Found 408.2861

Bile from the administration of nor-CDCA 3 was-extracted as above to give 43 mg of residue which, after chromatography, gave a fraction containing mainly 4. Methyl esterification and chromatog- raphy as indicated above afforded 6.3 mg of pure methyl-3a,5,7a- trihydroxyy-24-nor-5P-chlan-23-oate 4a. High-resolution MS (EI, 70 eV) mlz:

S~-Hydroxylation by the Liver

Cz4H4006 (M+) Calculated 408.2876 Found 408.2861

Chemical Correlation of 4 with Nor-CDCA

4a (1 mg) was treated with 100 p1 of pyridine and 80 pl of acetic anhydride for 72 h at room temperature. The reagents were evapo- rated with a nitrogen stream to give methyl9a,7a-diacetyloxyy-5- hydroxy-24-nor-5/3-cholan-23-oate 4b, 99% pure by gas chromatog- raphy and 'H NMR. MS (EI, 70 eV) m/z (%): 414 (14, M - AcOH -

(25), 337 (231, 318 (100, M - 2 X AcOH - (C1-C4) (retro-Diels-Alder in ring A)), 281 (28), 271 (62, M - side chain - 2 X AcOH), 253 (28, M - side chain - H,O - 2 X AcOH), 226 (28, M - H,O - 2 x AcOH - side chain - (C1&7)), 211 (32, M - H20 - 2 X AcOH - side chain - (c1&17)). 'H NMR (CI3CD): 0.690 (s, 3H, Me-18), 0.914 (s, 3H, Me-191, 0.989 (d, 6.5 Hz, 3H, Me-21), 2.032 (s, 3H, CH3COO-),

7), 5.019 (m, lH, H-3). 4b (1 mg) was heated with 20 p1 of phosphorus oxychloride in 160

pl of pyridine at 60 "C for 6 h. The reagents were evaporated with a N, stream, and the residue was partitioned between 1 ml of ethyl ether and 1 ml of water. The organic phase was washed twice with 1 ml of water, dried with sodium sulfate, and evaporated. The product was purified by silica gel column chromatography (elution sol- vent:hexane-ethyl acetate, 9010) to give 0.9 mg of methyl-3a,7a- diacetyloxyy-24-norchol-4-en-23-oate 5. MS (EI, 70 eV) m/z (W): 354 (100, M - 2 X AcOH), 235 (12). 'H NMR (C13CD): 0.726 (s, 3H, Me- 181, 0.985 (d, 6.5 Hz, 3H, Me-21), 1.009 (s, 3H, Me-19), 2.01 (s, 3H, CHZCOO-), 2.028 (s, 3H, CH,COO-), 2.350 (dd, 10.0 and 3.0 Hz, lH, H-6), 2.423 (dd, 15.0 and 3.0 Hz, lH, H-22), 3.664 (s, 3H, -COOCH,), 4.876 (m, lH, H-7), 5.082 (m, lH, H-3), 5.385 (dd, 5 and 1 Hz, lH,

5 (0.5 mg) was dissolved in 600 p1 of ethyl acetate containing 10 p1 of acetic acid and hydrogenated (1 atmosphere) over PtOz at room temperature for 2 h. Analysis of the reaction mixture by GC-MS showed the presence of a 1.7:l mixture of methyl-7a-acetyloxy-24- nor-5P-cholan-23-oate 6 and methyl-3a, 7a-diacetyloxycy-24-nor-5P- cholan-23-oate 3b.

Methyl-7a-acetyloxy-24-nor-5~-cholan-23-oate 6-GC retention time relative to cholic acid methyl ester acetate: 0.275. MS (EI, 70 eV) m/z (%): 358 (100, M - AcOH), 343 (35, M - AcOH - CH3.1,

(51, M - AcOH - side chain), 230 (17, M - AcOH - side chain - (C16-Cl,)), 215 (35, M - AcOH - side chain - (c&&)). An authentic standard of 6 was prepared from 7a-hydroxy-5(3-cholan-24-oic acid as described in (3), followed by methylation and acetylation by standard methods.

Methyl-3a, 7a-diacetyloxy-24-nor-5~-cholan-23-oate 3b"GC re- tention time relative to cholic acid methyl ester acetate: 0.603. MS (EI, 70 eV) m/z (%): 416 (4, M - AcOH), 356 (100, M - 2 X AcOH), 341 (44, M - 2 X AcOH-CH3.), 315 (19, M - AcOH - side chain),

HzO), 372 (85, M - 2 X AcOH), 354 (75, M - Hz0 - 2 X AcOH), 339

2.071 (s, 3H, CH&OO-), 3.666 (s, 3H, -COOCH,), 4.928 (bs, 1H, H-

H-4).

327 (3, M - AcOH - CHaO.), 285 (6), 284 (4), 269 (4), 262 (14), 257

302 (9, M - 2 X AcOH-[C~-C~]), 283 (23, M - 2 X AcOH- .CH,COOCH,), 282 (8, M - 2 X AcOH - (CH,=C(OH)OCH,) (McLafferty rearrangement)), 267 (10, [ion 282]-CH3.), 255 (70, M - 2 X AcOH - side chain), 228 (27, M - 2 X AcOH - side chain - [C16-C17]), 213 (50, M - 2 X AcOH - side chain - (C1&17)).

Spectroscopic Methods

Proton nuclear magnetic resonance spectra ('H NMR) of la, 2a, 3a, and 4a were recorded on a 360-MHz instrument equipped with a modified Varian HR-220 console, an Oxford magnet and a Nicolet 1180-E computer system; 'H NMR spectra of 4b and 5 were obtained at 500 MHz on a Varian Unity 500 instrument. I3C NMR spectra were obtained on a Nicolet NT-200 wide-bore spectrometer with an Oxford magnet at 50.31 MHz; multiplicities were determined with the sequence-gated spin echo (GASPE) (14) with T = 7 ms. Chemical shifts are given in parts/million relative to tetramethylsilane; for I3C NMR the central peak of the signal of Cl3CD was used as reference 6 77.0 ppm. Secondary ion mass spectrometry (SIMS) was performed on a Kratos MS-50 mass spectrometer equipped with a cesium ion source operated at approximately 100 pA/cmZ beam flux in the negative mode. Glycerol was used as liquid matrix on a copper probe tip.

GC-MS analyses of 2 and 4 were performed on the methyl ester-

3,7-Dihydroxy Nor-bile Acids, 50-Hydroxylation by the Liver trimethylsilyl (Me-TMS) ether and the methyl ester-dimethylethyl- silyl (Me-DMES) ether derivatives of each compound (15-17). Fol- lowing methylation with freshly prepared ethereal diazomethane, the trimethylsilyl ether derivative was prepared by reaction with tri- methylsilylmidazole (50 pl) at 110 "C for 1 h, whereas the dimethyl- ethylilyl ether (Me-DMES) derivative was prepared using dimethyl- ethylsilylimidazole (50 pl) under the same conditions. The derivatiz- ing reagents were removed by passage of this sample through a small column (bed size 4 X 0.5 cm) of Lipidex 5000 gel prior to GC-MS analysis (18).

GC-MS of derivatives of 2 and 4 was carried out on a Finnigan 4635 GC-MS-DS instrument operated in repetitive scanning mode (2 s/cycle) over the mass range 50-1000 daltons and the positive ion mass spectra obtained from electron ionization (70 eV) were recorded using the Incos data system. Chromatographic separation of the Me- TMS ether derivatives was achieved on a 30-m DB-1 capillary column (J & W Scientific, Folsam, CA) using temperature-programmed op- eration from 225 to 295 "C with increments of 2 "C/min following initial and final isothermal periods of 5 and 30 min, respectively.

GC-MS analysis of the mixture of 3b and 6 and was performed on a Hewlett-Packard Series I1 model 5890 gas chromatograph equipped with a Hewlett-Packard 5970 Series Mass Selective Detector. Sepa- ration was carried out on a 30-m SPB-35 (Supelco, Bellefonte, PA) capillary column at 275 "C isothermal with a 1 ml/min flow of helium carrier gas.

RESULTS

Hepatic Metabolism-When nor-CDCA was infused into the isolated perfused hamster liver (8-pmol bolus) the bile acid was rapidly taken up but was recovered slowly in bile as reported previously (9). TLC analysis of bile (Fig. 2) showed a considerable amount of unchanged nor-CDCA 3 and in addition an unknown metabolite 4. Some very polar metab- olites (Fig. 2, a-f) were also produced.

A TLC zonal scan of bile after administration of ["Clnor- CDCA (Fig. 3, bottom) confirmed that the unknown spots were metabolites of the infused bile acid. A similar study after co-administration of ['Hlnor-CDCA and "SO:-, ["C]glucose (as a precursor of glucuronic acid), or ["CJtaurine identified the spots corresponding to metabolites containing both the nor-bile acid and each potential conjugating moiety. Fig. 3 shows the radioactive traces for glucose-derived, sulfate-de- rived, and taurine-derived radioactivity superimposed on the "C-nor-bile acid radioactivity profile (the ['Hlnor-CDCA trace is omitted for clarity but was used to precisely match the peaks in the three separate experiments). The results indicated that the very polar metabolites (but not 4) were mainly sulfates and glucuronides.

The metabolites were isolated by preparative TLC and

9 . I . .

e e - 3

.e.. ....@.. 4

'.e. . . . . e a

FIG. 2. Thin layer chromatogram of bile from an isolated perfueed hamster liver. An 8-pmol bolus of nor-CDCA was added to the perfusate at 1 h (arrow), and samples were collected every 15 min. The metabolites are identified on the right: 3 and 4, see Fig. 1; a, nor-CDC-glycine; b, nor-CDCA sulfate(s); c and d, nor-CDCA glucuronides; e, nor-CDC-taurine; f, 4-glucuronide(s). The spots with RF similar to a and e which appear during the first hour are endoge- nous CDC-glycine and CDC-taurine, respectively.

* 14

* C-Taurine

3 5 ~ - ~ u ~ fate

I r * 11 i

14 C-61 ucose

11241

1 .o 0.8 0.6 0.4 0.2 0.0

FIG. 3. Thin layer chromatography radioactivity profilea of the metabolites of nor-CDCA produced by the isolated per- fused hamster liver (solvent front on the left). From fop to bottom, administration of: ["]nor-CDCA and ["Cltaurine. ['Hjnor- CDCA and ["Sjsulfate, ["]nor-CDCA and ["Cjgluco~e, and ["C] nor-CDCA alone. The 'H traces are not shown for clarity (see "Results"). The peaks labeled with a star were observed in control experiments and correspond to metabolites of endogenous origin. The vertical scales have been normalized and do not reflect actual pro- portions. Typical proportions 45 min after bolus addition were (in- tegration of the ["Clnor-CDCA trace): nor-CDCA, 10.4%. 4.48.9%, nor-CDC-Gly, 0.6%; nor-CDCA-sulfate, 3.0%; nor-CDCA-glucuro- nides. 24.4%; nor-CDC-taurine 1.3%, 4-ghcuroniden 11.4%.

analyzed by negative ion SIMS, which confirmed that b was a sulfate, that c and d were glucuronides of dihydroxy nor- bile acids (probably the 3-ether and 23-ester, respectively). and that f was a trihydroxy nor-bile acid glucuronide(s). In contrast, 4 gave a quasimolecular anion at m/z 393, consistent with an unconjugated trihydroxy nor-bile acid.

The pattern of biotransformation of nor-CDCA infused in the acute biliary fistula hamster in doses from 0.25 to 5 pmol/ kg. min was identical to that observed in the isolated perfused hamster liver. At high doses, however, the relative proportions of unchanged nor-bile acid and its glucuronides increased and

11242 3,7-Dihydroxy Nor-bile Acids,

the other metabolites decreased reciprocally. When nor- UDCA was administered in place of nor-CDCA, nearly iden- tical results were obtained both with regard to hepatic hand- ling and biotransformation products (8).

Isolation of Unknown Metabolites-Since the unknown me- tabolites constituted a significant proportion of the bile acids in bile, it was possible to obtain milligram amounts of metab- olites 2 and 4. Prolonged administration of a fairly high dose (4 pmollkg-min) of nor-UDCA 1 to five biliary fistula ham- sters afforded a mixture of bile acids containing the corre- sponding metabolite 2. This was fractionated by silica gel column chromatography, esterified with methanol to improve its solubility in organic solvents suitable for NMR spectros- copy, and repurified by silica gel column chromatography to give 4.7 mg of 2a. A similar procedure with nor-CDCA, infused at a lower dose (1.5 pmol/kg.min) to decrease the amount of recovered starting material in bile, gave 4, isolated as the methyl ester 4a (6.3 mg). High-resolution mass spec- trometry analysis of 2a and 4a showed molecular ions with the formula C24H4005 in both cases, corresponding to the methyl esters of the administered compounds plus one extra oxygen atom.

13C NMR Spectroscopy-The evidence gathered from the biotransformation studies and mass spectrometry studies sug- gested that the metabolites were the product of hydroxylation of the parent compounds. To determine the site of hydroxyl- ation, the metabolites were isolated and characterized using 13C NMR spectrometry, as this technique is the most suitable for complex molecules, and the appropriate substitution shift rules are well established (19). Table I gives 13C NMR spectral data for the methyl esters of the unknown compounds; the spectra of the methyl esters of nor-UDCA la and nor-CDCA 3a are also given for comparison. The spectral data indicated that the compounds were pure, contained 24 signals as ex- pected for methyl esters of nor-bile acids, and did not possess double bonds or ketone or amide carbonyls. The spectra showed three signals between 60 and 80 ppm, which corre- spond to three hydroxyl groups, confirming the preliminary findings.

TABLE I 13C NMR data of nor-bile acids and metabolites in C l X D

No. la

1 2 3 4 5 6 7 8 9

10 11 12 13 14 15 16 17 18 19 20 21 22 23

Me

34.7 30.1 71.0 37.1' 42.3 36.gb 71.0 43.5 39.0 33.9 21.0 39.9 43.6 55.6' 26.7 28.6 54.9' 12.0 23.2 33.5 19.5 41.3

173.8 51.2

29.7-b 29.8 29.4-b 29.9 67.5+ 67.8 42.9- 43.1 74.9- 76.2 46.0- 47.1 72.2+ 73.0 42.9+' 43.0 41.1+' 42.1 39.1- 39.3 21.1- 39.6- 43.5- 55.7+d 26.7-

54.8+d 28.6-

12.0+ 16.2+ 16.4 33.6+ 19.5+ 41.3-

173.8- 51.3+

3a

35.2 30.5 71.9 39.4b 41.3 34.6 68.3 39.3 32.7 34.9 20.4 39.7* 42.6 50.3 23.5 28.1 55.8 11.7 22.7 33.7 19.4 41.3

174.0 51.2

4a" Calculated, 3a.5B.1~1

29.8-' 29.9-' 67.7+ 44.0- 75.0- 45.1- 68.0+ 38.5+ 35.5+ 39.1- 20.7- 39.9- 42.4- 50.2+ 23.6-

55.7+ 28.1-

11.6+ 15.8+ 33.7+ 19.4+ 41.3-

173.8- 51.3+

29.8 30.0 68.6 44.9 75.2 45.5 69.9 38.6 35.6 39.6

15.6

+ or - indicate the GASPE sign. bd Assignments can be exchanged in each column.

5P-Hydroxylation by the Liver

The signals corresponding to the side chain and rings C and D of 2a and 4a were unchanged with respect to the appropriate parent compound, indicating that no modifica- tions had occurred in their vicinity. The chemical shifts of C- 14 and C-15 are known to be very sensitive to the orientation of the 7-hydroxyl group: 14-H bears a 1,3-diaxial relationship with 7a-H, producing a +4.5-ppm shift on the resonance of C-14 when present (an HC interaction as defined in (19)), whereas C-15 has a 6 configuration with respect to a 7P-OH group (a +2.5-ppm shift on C-15 when present). Since C-14 and C-15 had the same chemical shifts in 2a and 4a as in the parent compounds la and 3a, it seemed that the 7-hydroxyl group was in the same position and orientation as in the administered nor-bile acid.

Inspection of the GASPE spectra (multiplicity test that gives positive signals for methines and methyls and negative for quaternary carbons and methylenes) indicated that the new hydroxyl group in 2a and 4a was on a methyl group or on a quaternary carbon. Hydroxylation of 19-Me could easily be ruled out because it would have produced only minor changes on the signals of the carbons bearing the other two hydroxyl groups (later confirmed by the presence of three methyl groups on the 'H NMR spectrum). If the basic skeleton of the bile acid had not changed, the only suitable quaternary positions were C-5, C-8, and C-9. The fact that the signals of C-1 through C-10 and 19-Me were all altered and that the signals of the carbon atoms of ring C were unchanged sug- gested that the new hydroxyl group was located in a position central to rings A and B and was therefore on C-5.

To determine the orientation of the new hydroxyl group, we compared the chemical shifts of C-1 to C-10 and C-19 in 2a and 4a with the expected values for 5a- or 5P-hydroxyl- ation products. The substituent effects of introduction of a 7a- or 7P-hydroxyl group (20) were applied to the chemical shifts of the carbon atoms of rings A and B of 3a,5a- and 3a,SP-dihydroxycholestane (21, 22) (Table I). A 5a configu- ration could be excluded, because in this case the chemical shift of C-1 should be approximately 25 ppm (2l), whereas 2a and 4a did not show signals at such high fields among carbons corresponding to rings A or B. Instead, C-1 resonates at 29.7 ppm in 2a and at 29.8 ppm in 4a. The difference is due to the fact that in 3a,5a-dihydroxy steroids, C-1 has two HC interactions, through H1,-H9 and HIB-Hlg, and a CC gauche interaction (C2-Cl-Clo-Clg) for a total shift of +10.8 ppm with respect to a secondary six-member ring carbon; in contrast, in 3a,5@-dihydroxy steroids, C-1 has three HC in- teractions, H1S-H3B, Hla- H19, and Hla-Hlg, plus a CC gauche interaction (C2-Cl-Clo-Cg) for a total shift of +16.5 pprn (7 effects of the hydroxyl group are small in both cases).

The excellent agreement between the experimental data and the calculated values (Table I) indicate that in the me- tabolites 2 and 4 the new hydroxyl group has the configura- tion 50.

Similar calculations also excluded the possibility that con- current with the 5a- or 5P-hydroxylation the hydroxyl group on position 3 had been epimerized to 3P, a likely result if the metabolites had been formed through a 3-oxo-A' intermediate. In the 5/3 case, epimerization of the 3-hydroxyl to the P position would eliminate an HC interaction between 3P-H and 1P-H causing C-1 to resonate, once again, at 25 ppm (21). In the 5a series this epimerization creates a new HC inter- action (H3,-H1,), thus bringing the value of the chemical shift of C-1 to 31 ppm, reasonably close to the experimental values for 2a and 4a. However, the chemical shift of C-9, which is easy to distinguish by means of the GASPE experiment, provided a means to discriminate between 3a,5@,7a(P)- and

3,7-Dihydroxy Nor-bile Acids, 5P-Hydroxylation by the Liver 11243

3@,5a,7a(@)-trihydroxy structures. C-9 has one more HC in- teraction (H9-H1,) in the 5a isomer than in the 5P isomer, and therefore its chemical shift in the 5a isomer should be approximately 4.5 ppm higher. The calculated value for C-9 in the 3@,5a,7a isomer is 38.3 ppm (uersus 35.5 experimental) and in the 3P,5a,7@ isomer is 44.8 ppm (uersus 41.1 experi- mental). Considerable differences between the calculated val- ues for C-4 and C-6 are also present.

Thus, the most likely structure for the hepatic biotransfor- mation product of nor-UDCA is 5/3-hydroxynorursodeoxy- cholic acid (3a,5,7~-trihydroxy-24-nor-5/3-cholan-23-oic acid) 2 and that of the corresponding metabolite of nor-CDCA is 5~-hydroxynorchenodeoxycholic acid (3a,5,7a-trihydroxy-24- nor-5@-cholan-23-oic acid) 4.

' H NMR Spectroscopy-The spectrum of 2a in Cl&D (Table 11) showed signals for three methyl groups, a broad doublet for one 22-H, and the ester methyl group, all charac- teristic of methyl esters of nor-bile acids. Only two signals corresponding to protons geminal to hydroxyl groups were observed, 7-H and the septet for 3-H, which appeared de- shielded by approximately 0.5 ppm with respect to nor-UDCA methyl ester. This shift is in agreement with the 5P-OH structure. In decalin model compounds, an axial hydroxyl group deshields hydrogens in 1,3 diaxial relationship by about 0.37 ppm (23). In I@-hydroxy bile acids, where the 1P-OH group bears the same relationship to 3P-H as the 5P-OH group (24), 3-H appears at about 4.0 ppm, deshielded by 0.5 ppm. In the 'H NMR of the nor-CDCA metabolite 4a, the shift produced by the 5P-OH group causes 3-H to appear at 3.907 ppm, overlapping the signal of 7-H.

The 'H NMR spectrum of the methyl ester of nor-CDCA 3a shows the signal of 4a-H (a quartet due to couplings to 4@-H, 3-H, and 5-H with similar coupling constants) at 2.22 ppm, deshielded by its close proximity to the 7a hydroxyl group. This proton resonates below 2.0 ppm in (5P)7@-hydroxy compounds (25) and in 3,5-dihydroxy compounds in any configuration (26). The 'H NMR spectrum of the methyl ester of the nor-CDCA metabolite, 4a, shows a triplet at 2.54 ppm, indicating a cis A/B junction with a 7a-OH group and the disappearance of one proton vicinal to 4a-H. The shift of this signal (from 2.22 to 2.54 ppm) is similar to the 0.33 ppm found for a proton p-trans to an axial OH in decalin model compounds (23).

The 'H NMR spectrum of 4a in &-pyridine showed re- solved signals for 3-H and 7-H (Table 11). Irradiation of 3-H simplified the signal corresponding to H-4a to a doublet, thus confirming the assignment.

In the 'H NMR spectrum of 2a in &-pyridine (Table 11),

19-Me appears shifted by 0.304 ppm relative to the spectrum in C13CD, and by 0.377 ppm in the case of 4a. Such large pyridine induced solvent shifts are not observed with the methyl esters of the substrates l a and 3a. For a methyl group vicinal to a hydroxyl group, the magnitude of the pyridine induced shift increases as the dihedral angle 0 subtended between the two decreases. If 0 is 180" (as would be the case in a 5a-OH bile acid), the induced shift is less than 0.1 ppm; whereas when 6' is 60", the shift is 0.2-0.3 ppm (27). The observed values for 2a and 4a are even higher, clearly showing the close spatial proximity of the 5-OH and 19-Me groups, thus supporting the 5@ configuration.

Mass Spectrometric Analysis-The mass spectrum of the Me-TMS ether derivatives of 2 and 4 are shown in Fig. 4. The two isomers were easily resolved by capillary column gas chromatography having relative retention indices of 32.31 mass units (3a,5/3,7a-trihydroxy isomer, 4) and 32.56 mass units (3a,5@,7P-trihydroxy isomer, 2), respectively, and this order of elution is consistent with the difference between the Me-TMS ethers of the 3,7-dihydroxy-5@-cholanoic isomers of chenodeoxycholic (3a,7a) and ursodeoxycholic (3a,7@) acids (17). The electron (70 eV) ionization spectra of the two isomers were virtually identical, although small differences were apparent in the relative intensities of the fragments. The molecular ions (m/z 624) in both spectra were absent and the ions of largest mass that were apparent were at m/z 534, indicating facile loss of a trimethylsilanol function (M - 90) from the molecular ion. Further cleavage of two additional derivatized hydroxyl groups was evident from the ions at m/ z 444 ("(2 x 90)) and m/z 355 (M - (2 x 90 + 89)). Loss of the methyl ester of the C4 carboxyl side chain (nor bile acid) was apparent from the loss of 101 daltons giving rise to the ABCD ring ion at m/z 253 and confirming the presence of three nuclear hydroxyl groups. Small low intensity ions occur at m/z 143 and 243 that may result from a 3-trimethylsiloxy- A4-structure arising after loss of the C-5 trimethylsiloxy group. The relatively intense ion at m/z 259 is unusual and difficult to explain. In the corresponding dimethylethylsilyl ether derivative (Fig. 4), this ion is shifted to m/z 287, repre- senting an increase of 28 daltons and confirming that this fragment contains two derivatized hydroxyl groups. It is sug- gested that the m/z 259 ion is analogous t o that of m/z 243 but contains an additional underivatized oxygen at C-5. This ion is more intense in the 3a,5&7@-trihydroxy isomer than in the 3a,5@,7a isomer. The ion at m/z 271 derives from the loss of the side chain (-101 daltons) from the fragment of m/z 372 (M - 162). The small, but significant, ion at m/z 318 confirms the C-3 hydroxyl group and is seen in C-3 hydrox-

TABLE 11 'H NMR data of nor-bile acids and metabolites

Chl, in C1,CD; Py, in d5-pyridine; chemical shifts are relative to tetramethylsilane; singlets are not indicated. bs, broad singlet; d, doublet; bd, broad doublet; t, triplet; q, quartet; m, multiplet. Coupling constants (in Hz) are indicated under the chemical shift values.

la, Chl la, Py 2a, Chl 2% PY 3a, Chl" 3% PY 4a, Chl 4% PY

H-3 3.59 (m) 3.865 (m) 4.032 (m) 4.695 (m) 3.46 (m) 3.808 (m) 3.907 (m) 4.698 (m) H-4a 2.22 (4) 3.096 (dt) 2.54 (t) 3.515 (dd)

H-7 12.0

3.59 (m) 3.783 (m) 3.503 (m) 3.757 (m) 3.86 (bs) 4.030 (bs) 3.907 (m) 4.166 (m) 11.7, 13.0 12.2 13.5, 11.8

Me-18 0.722 0.688 Me-19 0.957

0.706 0.692 0.72 0.966

0.686 0.903 1.207

0.696 0.719

Me-21 0.989 (d) 1.094 (dl 0.980 (d) 1.094 (dl 0.98 (d) 1.066 (d) 0.984 (d) 1.092 (d) 0.92 0.965 0.876 1.253

6.5 H-22b

6.1 6.1 6.1 5.7 5.8 6.0 6.1 2.44 (dd) 2.532 (dd) 2.428 (bd) 2.45 (bd) 2.48 (bd) 2.427 (bd) 2.498 (bd) 14.1, 2.9 14.4, 2.9

3.695 12.2 13.0 3.659 3.680

11.2 11.5 3.68 3.682 3.665 3.690

11.5 -COOCHa 3.668

Ref. 3. This signal corresponds to only one of the C-22 protons.

11244 3,7-Dihydroxy Nor-bile Acids, 5P-Hydroxylation by the Liver

FIG. 4. Electron ionization mass spectra of (from top to bottom): methyl ester TMS ether derivative of 4, methyl ester TMS ether deriv- ative of 2, methyl ester DMES ether derivative of 4, and methyl ester DMES ether derivative of 2.

ylated bile acids whether derivatized or not, resulting from a retro-Diels-Alder elimination of C-1 to C-4 after loss of the C-3 trimethylsilanol group.

In the Me-DMES ether derivatives (Fig. 4), similar frag- mentation patterns were evident for both compounds except for an increase of 14 daltons (-CH2) for all fragment ions containing a derivatized hydroxyl function. Thus these tri- hydroxy bile acids have molecular ions at m/z 666 (reflecting an increase of 3 x 14 daltons for the three derivatized hydroxyl groups) and losses of each functional group yielded analogous ions at m/z 562 (M - 104) and m/z 551 (M - 29 + 104). m/z 458 (M - (2 X 104)) and 355 (M - (2 X 104 + 103)).

Mass spectrometric analysis of both compounds served to confirm that both were isomers of trihydroxynorcholanoic acid. The fragmentation pattern was consistent with three hydroxyl groups in close proximity and present in the steroid rings and not the side chain. The mass spectra of both the Me-TMS ether and Me-DMES ether were unique and did not resemble any of the 111 mass spectra of bile acids recently compiled and published by Lawson and Setchell (17). On the basis of these spectra, and the known information of general fragmentation patterns of bile acids (15, 17), it was possible to exclude substitution of the nuclear hydroxyl groups from positions C-1, C-2, C-6, C-12, C-15, and C-16. Since these compounds are metabolites of nor-UDCA, and two of the three hydroxyl groups are assumed to be at the C-3 and C-7 positions, this leaves only C-4 and C-5 hydroxylation as possibilities for the position of the third hydroxyl group on rings A or B. Recently the mass spectrometric characteristics of a series of C-4 hydroxylated cholanoic acids were reported (28), including the Me-TMS ethers of 3,4,7-trihydroxycholan- oic acids; and the fragmentation patterns differed markedly from those of the two bile acids shown here.

Chemical Correlation of 4 with Nor-CDCA-In order to confirm that the new metabolites retained the original bile acid carbon squeleton and functionality, and that the new

hydroxyl group was on the 5 position, the most abundant metabolite, 4, was chemically correlated with its parent com- pound nor-CDCA (Fig. 5). Because direct elimination of the tertiary hydroxyl group of 4a by acid catalysis resulted in mixtures of extensively dehydrated products, the two second- ary hydroxyl groups were selectively protected by room tem- perature acetylation with acetic anhydride in pyridine to give 4b. Dehydration of 4b with phosphorus oxychloride in pyri- dine gave a reaction mixture that showed only one spot by TLC analysis. Since elimination of the tertiary hydroxyl group was likely to give a mixture of olefins with identical RF on TLC, the product corresponding to this single spot was purified by silica gel column chromatography. Analysis by both GC-MS and 'H NMR indicated that only one compound, 5, had been obtained. The 'H NMR spectrum of 5 showed two >CEJOAc signals at 4.876 and 5.082 ppm, and one doublet of doublets a t 5.385 ppm, which integrated for one proton, assigned to an olefinic hydrogen. Irradiation of the 5.082-ppm signal simplified the olefinic resonance to a broad singlet, thus showing that the double bond was vicinal to one of the acetate groups of 5 and that it was therefore located on positions 4(5) or 5(6). Irradiation of the olefinic proton mod- ified the signal at 5.082 ppm to a very broad singlet. This indicated that the resonance at 5.082 ppm corresponded to H-3, which still exhibited residual couplings to H-2a and H- 2p, whereas if the signal at 5.082 ppm was due to H-7, decoupling of the olefinic proton would have resulted in a doublet due to residual coupling with H-8. The double bond was therefore located in the 4(5) position. Moreover, in a model compound representative of the As isomer, 5-choles- tene-3a,7a-diol diacetate (29), the chemical shifts of H-3 and H-7 (both at 4.97 ppm) and of the olefinic proton (5.50 ppm) are clearly different from those of 5. The exclusive formation of the A4 isomer, 5, by dehydration of the 5-hydroxyl group is in full agreement with a 5p configuration for 4b, because in this compound only H-4a (and not H-6a or H-6P) has the

3,7-Dihydroxy Nor-bile Acids, 5/3-Hydroxylation by the Liver 11245

FIG. 5. Chemical correlation of 4a with 3b. i, AczO, Py, 23 "C, 12 h; ii, OPCla, Py, 60 "C, 6 h; iii, Hz (1 atmos- phere), PtOz, EtAcO-AcOH, 23 "C.

cn3cod

5

anti-periplanar orientation relative to the leaving group nec- essary for efficient elimination (30). Hydrogenation of 5 over platinum oxide in the presence of acetic acid gave a mixture which was analyzed by GC-MS. Two products were found the methyl ester diacetate of nor-CDCA 3b and methyl 7a- acetyloxy-24-nor-5~-cholan-23-oate 6, identified by their spectra and by comparison with authentic standards. Com- pound 6 is the result of allylic hydrogenolysis of the 3- acetyloxy group of 5 followed by hydrogenation of the double bond (31). The two products obtained had the 5P configura- tion; the presence of the 3a- and 7a-acetoxy groups in 5 , and the 7a-acetoxy group in its hydrogenolysis product2 prevented hydrogen addition from the a face, as has been observed for the hydrogenation of 3a-acetoxycholest-4-ene under similar conditions (31).

DISCUSSION

These data indicate that the two nor-dihydroxy bile acids, nor-UDCA 1 and nor-CDCA 3, were hydroxylated in the 5P position of the steroidal nucleus to give 2 and 4, respectively. In both cases, the final product was a more polar trihydroxy compound which was secreted into bile without undergoing appreciable additional hydroxylation or conjugation with glu- curonide, sulfate, or taurine.

A 5P-trihydroxy compound is also formed from UDCA in small amounts by the perfused rat liver (32). However, a 5P- hydroxy metabolite is not formed when UDCA is administered to the dog (33), to the guinea pig (8), to the rabbit (34), or to humans (35); nor was the 5P-hydroxy derivative of CDCA detected in gallbladder bile of cholesterol gallstone patients receiving CDCA chronically to induce gallstone dissolution (36,37). In patients with cholestasis, CDCA has been reported to be hydroxylated at the 1 or 6 positions; but 5-hydroxylation has not been observed (38-40). In general, 3-hydroxy bile acids undergo 6- or 7-hydroxylation rather than 5-hydroxyl- ation in most rodents (41). Thus, 5-hydroxylation appears to be specific for 3,7-dihydroxy bile acids and also to be rather species-specific.

Studies of biliary bile acid composition and bile acid bio- transformation in many vertebrate species have shown that bile acids can be hydroxylated by hepatic enzymes in all secondary positions in the nucleus with the possible exception of Cll and Cls (41). Our work presents the first example of hydroxylation of bile acids by the rodent liver on a tertiary (5P) position of the steroid n u c l e u ~ . ~ Indeed, hydroxylation of

A trace amount of a compound provisionally identified as the 501- epimer of 6 was found in the GC-MS analysis of the hydrogenation mixture, but the Sa-epimer of 3b was not detected.

The only known 5-hydroxy bile acid, 5~-hydroxydeoxycholic acid, has been found among the photoproducts of solid state irradiation of an inclusion complex of diethyl ketone in deoxycholic acid (42).

6 3b

the nucleus of steroids on tertiary positions by vertebrate tissues appears to have been reported only once previously, namely, the 5P-hydroxylation of digitoxigenin by rabbit liver homogenates (43).

Chemical Aspects-The structures of the metabolites 2 and 4 were determined solely on the basis of I3C NMR chemical shifts and multiplicity experiments. Since the new hydroxyl group was on a tertiary position, 'H NMR spectrometry could only give indirect clues to the structures. The more readily accessible two-dimensional experiment, 'H,'H COSY, af- forded no new information, displaying only cross-peak pat- terns similar to the original nor-bile acids (data not shown). One of the metabolites could be correlated with its parent compound by a sequence of well defined chemical reactions which by itself pointed to a structure where a 5P-hydroxyl group had been added to the original bile acid.

There are no published studies of mass spectra of C-5 hydroxy bile acids, although mass spectra of TMS ethers of cholestane-3,5-diols have been reported (44, 45). In these sterols, intense ions of m/z 143 and 243 were present. In the mass spectra of the two bile acids isolated from bile, these ions were found, but they were not prominent features. In- stead an ion of m/z 259 was a striking feature of the spectrum of both compounds; and to our knowledge, this ion has not been associated previously with any known bile acid deriva- tive. These mass spectrometric data, combined with the NMR studies, would suggest that this fragment ion may be specifi- cally associated with the fragmentation of the AB rings, resulting from the presence of hydroxyl groups at positions C-3, C-5, and C-7. In a recent report, a 5-hydroxylated me- tabolite of UDCA was tentatively identified by MS from the urine of a healthy subject receiving oral UDCA (46). However, the published mass spectrum of that compound does not resemble that of the C-5 hydroxylation nor-bile acids shown here. The definitive confirmation of the structures of 2 and 4, and of the mass spectrometric fragmentation pattern of C- 5-hydroxylated c24 bile acids, requires the chemical synthesis of pure standards that are presently unavailable.

Physiological Aspects-Nor-dihydroxy bile acids are known to be recovered slowly in bile compared with most other conjugated and unconjugated bile acids (8, 9). In contrast to their CZ4 homologues, nor-dihydroxy bile acids are ineffi- ciently esterified with coenzyme A (47) on entering the hep- atocyte and as a consequence regurgitate out of the cell into sinusoidal blood (48). They are thus presented to both peri- portal and pericentral zones of the liver during hepatic trans- port. Bile acid hydroxylases are active in pericentral cells (49), and this might explain the formation of the 5-hydroxy bile acid. In addition, these molecules are secreted in part in unconjugated form in bile and are reabsorbed from the epi- thelial cells and return to the hepatocyte to be secreted once

11246 3,7-Dihydroxy Nor-bile Acids, S(3-Hydroxylation by the Liver again in bile (8, 9). Such 'Lcholehepatic shunting77 exposes the 21. Bull, J. R., and Chalmer, A. A. (1977) s. Afr. J . Chem. 30,105-116

same molecule repeatedly to hepatocyte (and cholangiocyte) hydroxylases. Nor-dihydroxy bile acids, because of their in- 23. Schneider, H.-J., and Jung, M. (1988) M a n . Reson. Chem. 26,679-682

C. (1977) j. Org.'Chem. h2, 789-793

24. Tohma, M., Mahara, R., Takeshita, H., Kurosawa, T., and Ikegawa, S. efficient elimination from the hepatobiliary system, appear to (1986) Chem. & Pharm. Bull. (Tokyo) 34,2890-2899 be useful for amplifying trace biotransformation pathways in 25. Waterhous, D. V., Barnes, S., and Muccio, D. D. (19%) J. Lipid Res. 2 6 ,

the hepatobiliary system.

22. VanAntwerp C. L. Eggert H., Meakins, G. D., Miners, J. O., and Djerassi,

1068-1078

1032-1039 26. Mihailovic, M. L., Lorene, L., and Pavlovic, V. (1981) Helu. Chim. Acta 6 4 ,

27. Demarco, P. V., Farkas, E., Doddrell, D., Mylari, B. L., and Wenkert, E. Acknowledgments-We thank Drs. Y. B. Yoon, L. M. Clayton, and (1968) J. Am. Chem. SOC. 90,5480-5486

K. J, Lambed for bile samples and Vicky L. Huebner for editorial 28. Dumaswala, R., Setchell, K. D. R., Zimmer-Nechemias, L., Iida, T., Goto,

assistance. We gratefully acknowledge David Maltby for secondary 29. Stary, I., and Kocovsky, p. (1985) Collect. CzechoskJuak Chem. Commun. ion and high resolution mass spectra. 60,1227-1238

30. Kocovsky, P. (1983) Collect. Czechoslovak Chem. Commun. 48,3643-3659 31. Shoppee, C. W., Agashe, B. D., and Summersk, G. H. R. (1957) J. Chem.

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