THE JOURNAL OF BIOLOGICAL CHEMISTRY

Prmted In U.S.A. Val. 256, No. 12, Issue of June 25, pp. 6155-6159. 1981

Bile Acid Synthesis METABOLISM OF 3P-HYDROXY-5-CHOLENOIC ACID IN THE HAMSTER*

(Received for publication, December 16, 1980)

Engeline Kokt, Shlomo Burstein, Norman B. Javitt, Marcel Gut, and Chang Yon Byon From the Division of Hepatic Diseases, The New York Hospital-Cornel1 Medical Center, New York, New York 10021 and The Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts 01545

Synthesis of 3~-hydroxy-5-[1,2-3~cholenoic acid has permitted a study of its metabolism in bile-fistula ham- sters that received the compound by intravenous infu- sion. Metabolites in bile were identified by reverse isotope dilution after their complete resolution by high pressure liquid chromatography using pporasil. Recov- ery of administered radioactivity ranged from 21-60% in three animals. In each study, lithocholic acid (0.8- 4.4%) and chenodeoxycholic acid (7.8-11.3%) were iden- tified as metabolites of 3P-hydroxy-5-cholenoate and can be considered primary bile acids in the side-chain pathway of bile acid synthesis beginning with the oxi- dation of cholesterol to 26-hydroxycholestero1.

Previous studies by members of our group reported in this journal demonstrated that 5-cholestene-3P,26-diol’ is metab- olized in the hamster (1) to both chenodeoxycholic and cholic acids. A minor metabolite, 3P-hydroxy-5-cholenoic acid, was also identified but the lack of a suitable tracer prevented further study of this compound. We have now succeeded in preparing 3P-hydroxy-5-[ 1,2,-3H]cholenoic acid and have de- termined its metabolism in uiuo in the hamster.

EXPERIMENTAL PROCEDURES

Synthesis (Fig. 1) of 3P-hydro~y-5-[1,2-~H]cholenoic acid followed established procedures previously reported in detail (2). Oppenauer oxidation of 1.0 g of the methyl ester of 3P-hydroxy-5-cholenoate (Steraloids) followed by reaction with 2,3-d~hloro-5,6-dicyano-1,4 benzoquinone (Aldrich) yielded 320 mg of the known methyl-3-oxo- chola-l,4-dien-24-oate (3). Tritiation (2.0 ml) of 20 mg of the dien dissolved in 1.0 ml of ethanol in the presence of 6.0 mg of Tris(tripheny1phosphine)chlororhodium (Pressure Chemical Co., Pittsburgh, PA) yielded 4 mg of methyl-3-[ 1,2-JH]oxochol-4-ene-24- oate with an RF identical with that of an authentic standard (4). The acetate was then formed by refluxing of 2 mg in a mixture of 0.5 ml of acetic anhydride and 1.0 ml acetyl chloride. After evaporation to dryness in uacuo, reduction with sodium borohydride was accom- plished by adding 3 mg to the acetate dissolved in 95% ethanol and reacting at 25 “C for 24 h. The product was then partitioned between methylene chloride and water and the organic phase was taken to dryness.

* This work was supported by Grants AM-16201 and AM-03419 from the National Institutes of Health and Grant PCM78-19594 from the National Science Foundation. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked “advertisement” in accord- ance with 18 U.S.C. Section 1734 solely to indicate this fact.

$ To whom reprint requests should be addressed. ’ Systematic names of steroids referred to in this paper by their

trivial names are: cholesterol, 5-cholestene-3P-01; 7a-hydroxycholes- terol, 5-cholestene-3P,7a-diok 26-hydroxycholesterol, 5-cholestene- 3b,26-diol; cholic acid, 3a,7a,l2a-trihydroxy-5~-cholanoic acid; che- nodeoxycholic acid, 3a,7a-dihydroxy-5~-cholanoic acid; lithocholic acid, 3a-hydroxy-5fi-cholanoic acid. The abbreviation used is: HPLC, high pressure liquid chromatography.

Purification of the reduced reaction products was achieved by thin layer and high pressure liquid chromatography. Aliquots were meth- ylated, placed on Silica Gel H, and run in a solvent system of hexane: ethyl acetate (41, v/v) using an authentic standard of 3P-hydroxy-5- cholenoate to locate the radioactivity. The radioactive eluate was then reduced in volume and injected on a stainless steel column (300 X 3.9 mm), packed with pPorasil (Waters Associates) rated at 3000 theoretical plates, and eluted with 7% ethyl acetate in hexane. As shown in Fig. 2, the methyl ester of 3/3-hydroxy-5-cholenoate is obtained as a single symmetrical peak separate from other monohy- droxy or keto compounds. It was then converted to the acetate with pyridine/acetic anhydride and again fractionated on the pPorasil column using a solvent system of 75% methylene chloride in hexane.

The methyl ester acetate was found to have a specific activity of 0.9 mCi/pmol. An aliquot taken for reverse isotope dilution showed no fall in specific activity from the initial value after recrystallization from methanol.

Verification of structure was done by preparation of the epoxide and digitonide. For epoxidation the methyl ester acetates of 3P- hydroxy-5-[1,2-’H]cholenoate and 3a-hydroxy-5@-cholanoate were re- acted with m-chloroperbenzoate and then analyzed by HPLC using pPorasil as described above. No change occurred in the retention time of 3a-hydroxy-58-cholanoate. The 3P-hydroxy-5-cholenoate deriva- tive now eluted as a broad symmetrical peak with a longer retention time than previously but with no change in specific activity. The finding indicates selective formation of a more polar compound, the epoxide, and verifies the presence of the ring unsaturation.

A digitonide was formed when 10 mg of the methyl ester of 3p- hydroxy-5-cholenoate (1037 cpm/pmol) in 3 ml of absolute ethanol was mixed with 30 mg of digitonin dissolved in 3.0 ml of 90% ethanol. The precipitate was washed with diethyl ether. After decomposition of the digitonide complex, the specific activity of the reisolated bile acid methyl ester was unchanged from the initial value (1037 cpm/ pmol). Under the conditions described, 10 mg of 3a-hydroxy-5P-cho- lanoate failed to yield a precipitate after the addition of digitonin. Thus the findings indicate, as expected, the presence of a 3P-oriented hydroxyl group.

The steps used in identification of metabolites were: (a) solvolysis; (6) hydrolysis; ( e ) extraction; (d ) methylation and thin layer chro- matography; (e) column chromatography, using Glycophase G, on controlled pore glass 80-100 mesh (Pierce Biochemical); ( f ) HPLC using pPorasil; and (g) reverse isotope dilution. Steps a, b, c, and d have been described in detail in previous publications (1,s-7), includ- ing the quantitation of radioactivity by liquid scintillation spectrom- etry using a Beckman CPM 200 instrument. Radioactive lithocholic, deoxycholic, chenodeoxycholic, and cholic acids were purchased from Amersham/Searle and/or New England Nuclear. Radioactive litho- cholic acid sulfate was prepared using chlorosulfonic acid/pyridine. Radioactive 3-keto-5P-cholanoate and 3P-hydroxy-5P-cholanoate were also prepared from lithocholic acid by chromate oxidation and sodium borohydride reduction. These compounds were purified by thin layer and column chromatography and used as standards.

For the purpose of these studies a new column chromatographic system was developed which permits complete separation into groups of mono-, di-, and trihydroxy bile acids as their methyl esters (Fig. 3). The flow rate is 1.8 ml/min, permitting a complete analysis within 3 h with no loss of radioactivity on the column. The column is prepared by weighing out 25 g of controlled pore glass/100 Glycophase G and making a slurry in ethyl acetate/hexane (1:3, v/v) which is then poured on a glass column (30 X 1.5 cm inside diameter).

6155

6156 3~-Hydroxy-5-cholenoic Acid Metabolism

FIG. 1. Synthesis of 3~-hydroxy-5-[1,2-3H]cholenoic acid. DDQ, 2,3-dichloro-5,6-dicyano-1,4-benzoquinone. The catalyst is Tris(tripheny1phosphine)chlororhodium.

FRACTIONNO. 4001u+~Luhu IO 14 IS 28 32 36 40 44 48 56 62 66 70

7% ETHYL ACETATE IN HEXANE

FIG. 2. HPLC separation of 3fi-hydroxy-5-cholenoate methyl ester and related compounds. The bile acids shown were injected together as the methyl ester derivatives on a column (300 X 3.9 mm) of pPorasi1 using solvent system of 7% ethyl acetate in hexane at 700 p.s.i. (flow rate, 2 ml/min). 70 fractions of 1.3 ml were collected. R = 0.65 represents the ratio of the peak tube number of 3P-OH-5-chole- noate/peak tube number of 3a-OH-5P-cholanoate.

MONOUYOROXY OlHYOROX? 3000 x.m-mr a

TRlUYOROXl ESTER-SULFMES

[..3#---5--. 0 D.dm.)rhOb. 0 A* Chohl G 0 . l i ~ l I O l .

- 3 : t d b 2 : 3 d L l : 9 + ~ l : I " s l -HEXANE : ETHYL ACETATE -MJ.ETAC:CH~CWI

FIG. 3. Chromatographic separation of bile acid methyl es- ters on CPG/100 Glycophase G (80-100 mesh). Group separation of mono-, di-, and trihydroxy bile acid standards as the methyl esters following a single injection of the mixture is obtained by using mixtures of solvents of increasing polarity. Partial resolution of de- oxycholate and chenodeoxycholate occurred. No attempt was made to resolve various ester sulfates. The flow rate was 1.8 ml/min which allows complete separation in 3 h.

G they were further identified by HPLC on yPorasil, initially as the Following separation of the bile acid methyl esters on Glycophase

methyl esters (Fig. 2), and then as the methyl ester acetates. As part of the procedure, the resolving power of the pPorasil column was determined before each analysis using authentic bile acid methyl esters alone and in combination. The equipment used was Waters Associates Model 6000A advanced design solvent delivery system and U6K universal liquid chromatograph injector coupled with a fraction collector.

It was found during HPLC chromatography that an isotope effect was detectable. This was used to advantage by determining the "H/

14 C ratio of mixtures of authentic bile acids methyl ester acetates as reference. Thus, final identification of a metabolite was established not only by an elution pattern identical to the known standard but also by the known isotope effect that occurs during elution of the peak. Reverse isotope dilution studies were done in the conventional manner and also by determination of the "H/I4C ratios after addition of authentic bile acid and crystallization. The latter procedure elim- inates errors in the drying and weighing of small amounts of crystal- line material.

For metabolic studies, the methyl ester acetate was hydrolyzed to the free acid which was dissolved in 0.05 ml of ethanol and then added, with vortexing, to a sterile solution of 5% dextrose in 0.458 NaCl containing 5% of human serum albumin.

Male Syrian hamsters weighing between 110 and 125 g were anes- thetized with intraperitoneal pentobarbital, underwent cannulation of the bile duct and femoral vein. The animals were then placed in restraining cages, offered food and water, and maintained on a con- stant intravenous infusion of 5% dextrose and 0.45% NaCl. After overnight drainage to deplete the bile acid pool, 3P-hydroxy-5-chole- noate varying in amounts from 2.9 nmol to 2.9 pmol were infused during a 1-h period. T o avoid the cholestatic effect of 3P-hydroxy-5- cholenoate, known to occur at the higher infusion rates (5), 29 pmol of sodium taurocholate were added to the infusion mixture to main- tain bile flow. Bile was collected for 20 h following the infusion.

RESULTS

Table I summarizes the results obtained with 3 infusions given to three hamsters. Recovery in bile of radioactivity derived from the lowest dose (2.9 nmol) was only 21% and significantly less than the radioactivity recovered from bile of hamsters that received the highest dose (2.9 pmol). In two studies, bile was collected separately at 4-h intervals and then pooled. It was noted that of the total amount recovered, more than 50% was excreted in bile during the initial 4 h.

An aliquot of bile obtained from each animal was solvolyzed, hydrolyzed, extracted with ethyl acetate, and then methyl- ated. Losses during each procedure were less than 7%. The

TABLE I Metabolism of 3~-hydro~y~5-[1,2-~H]cholenoic acid in the hamster

Study Amount adminis- Amount recovered in bile

tered Total 3fi-OH" Lithocholic c h e ~ { ~ ~ ~ y -

pmol sCi B 1 2.9 x lo-,' 2.6 21 10.8 0.8 9.4 2 2.9' 4.5 58 46.5 3.7 7.8 3 2.9' 4.5 60 44.3 4.4 11.3

3p-OH, 3~-hydroxy-5-[1,2-RH]cholenoic acid. Nonradioactive 3/3-hydroxy-5-cholenoic acid was added to the

radioactive compound.

7oor MONOHYDROXY DIHYDROXY

~ H E X A Y E / E T A C 3 : I 4 k H E X A H E / E T K 2 : 3 i

FIG. 4. Radioactive peaks in hameter bile after administra- tion of 3P-hydroxy-5-cholenoic acid. An aliquot of the methyl

tionated on CPG/100 Glycophase G (see Fig. 3). ester of bile acids obtained after solvolysis and hydrolysis was frac-

3/3-Hydroxy-5-cholenoic Acid Metabolism 6157

methyl esters were then fractionated on Glycophase G (Fig. 4). In each instance, a relatively broad peak of tritiated com- ponents was obtained in the monohydroxy region and a sym- metrical peak in the dihydroxy region, No radioactivity was detected in the trihydroxy region. Except for 4-5% of the radioactivity which was recovered by washing the column with methanol, all the radioactivity (>95%) was found in either the monohydroxy or dihydroxy regions. Although it is possible that the 4-5% of radioactivity represents compounds that failed to hydrolyze or solvolyze, no further study of this more polar fraction was done. Elimination of the solvolysis step in studies 2 and 3 (Table I) greatly reduced the proportion recovered in the monohydroxy region (~15%) and increased the proportion of radioactivity in the methanol wash (>40%).

HPLC analysis of the radioactivity obtained from the mo- nohydroxy region using PPorasil indicates that it represented mostly the infused 3P-hydroxy-5-cholenoate but, also, in each

fRu7mm36 40 44 46 52 56 60 64 68 7% ETHYL ACETATE IN HEXANE

FIG. 5. Radioactivity in hamster bile analyzed by HPLC on a porasil column. The bulk of th e radioactivity was identified as 3P-hydroxy-5-cholenoate. A small peak of “H (tubes 63-65) was always found and proved to be lithocholic acid.

3H and “C-~~~~~~t+~~~ &XI “H-~c+do~roRoXY BILEPCIO YETHYL ESTER METHYL ESTER CCETATE XETATES

4500 ‘V-LITHOCHOLIC Pa0

r r METWL ESTER ACETATE

FRACTIONNo. 20 24 28 22 24 26 28 METHYLENE CHLOR,OE.HEXANE 73 (V/VI

FIG. 6. Comparison of putative lithocholic acid derived from 3/?-hydroxy-5-[1,2-3Hlcholenoate to authentic [3H]- and [‘“Cl- lithocholic acid. Using HPLC on microporasil a slight isotope effect was noted with authentic compounds (left). The radioactivity ob- tained from hamster bile and previously separated by glycophase and HPLC was fractionated together with [‘4C]lithocholic acid and an isotope effect comparable to the standards was found (right).

,!+C,V,W&NOL: ETHYL IIC~AlE.HEX,WE I:IO:39WV)

FIG. 7. Resolution of deoxycholic acid chenodeoxycholic acids as methyl esters by HPLC on pporasil.

‘H and “c~CMENooEOXYCHCUC 3H-~~~YOROXY SILE ACID METHYL ESTER METHYL ESTER STPINDAROS

“C-CHE,,00EOXYCHOM BCtlD

5000 r r METHYL ESTER STANMRO

6 2 E 2000 I

LOO0 c

L

FRACTION NO ‘q ,SOWOPWOL ETHYL ACEThTE “EXANE L 10 39 rl”

FIG. 8. Comparison of authentic chenodeoxycholic acid standards with putative [3H]chenodeoxycholic acid isolated from hamster bile following administration of 3,&hydroxy-5-cholenoate.

instance, a small amount of 3Lu-hydroxy-5/Scholanoic acid (Fig. 5) was detected. Confirmation of the 3/3-hydroxy-5-chole- noate was obtained by recrystallization with added nonradio- active compound. No reduction in calculated specific activity occurred. The radioactivity obtained in the lithocholic acid methyl ester region was mixed with an authentic “‘C standard of methyl 3a-hydroxy-5P-cholanoate and then analyzed by HPLC as both the methyl ester and methyl ester acetate derivative. Comparison of elution patterns of the acetate on pPorasil to the elution pattern of a mixture of authentic [“HI- and [‘4C]lithocholic acid methyl ester acetates indicated similar patterns (Fig. 6). In some studies, chromatography of the methyl ester was repeated after addition of [‘?]lithocholic acid methyl ester. In each study, the ‘H/‘*C chromatographic peaks were added to nonradioactive lithocholic acid methyl ester or its acetate and the isotope ratio compared before and after crystallization. No significant changes in isotope ratio occurred (Table II).

The consistent finding of small amounts of lithocholic acid derived from 3/I-hydroxy-Scholenoate led to an additional study in which [?J]chenodeoxycholate was given intrave- nously together with the tracer. Although [‘4C]chenodeoxy- cholate together with [“Hlchenodeoxycholate was found in

6158 3P-Hydroxy-5-cholenoic Acid Metabolism

TABLE I1 Zdentification of PHJlithocholic and PHJchenodeoxycholic acids

as metabolites of 3~-hydro~y-5-[I,Z-~HJCholenoic acid "H/"C ratios Specific ac-

Lithocholic acid tivity of

Chenodeoxycholic acid chenodeox- ycholic acid

Methyl ester Acetate Methyl ester Diacetate ter diace- methyl es-

tate c p m / p o l

Study

1 1.77" 1.32 1.28 2195' 1.83' 1.67' 1974b

1792b 1792'

2 1.77" 1.60 0.49 0.42' 0.39' 0.40' 0.40'

3 1.50" 1.49 1.54' 1.48'

1.50' 1.54b

a Ratio determined before pPorasil HPLC. ' Successive crystallizations from methanol. Ratio determined after pPorasil HPLC.

bile, there was no 14C found in the monohydroxy region when the methyl esters were separated on a glycophase column. Also, addition of 3P-hydroxy-5-[ 1,2-3H]cholenoate to control hamster bile and carrying it through all the procedures failed to generate any radioactivity in the lithocholate or cheno- deoxycholate fractions obtained during chromatography on glycophase.

Analysis by HPLC of the radioactivity obtained from the dihydroxy region of the glycophase column indicated that it had the retention time of chenodeoxycholic acid methyl ester which could be separated from deoxycholic acid (Fig. 7). Addition of authentic ['4C]chenodeoxycholic acid methyl ester and HPLC analysis as both the methyl ester and methyl ester diacetate (Fig. 8) also indicated that the [3H]dihydroxy bile acid was chenodeoxycholic acid. Reverse isotope dilution fur- ther confirmed these findings (Table 11).

DISCUSSION

These studies were undertaken to complete the metabolic pathway of bile acid synthesis beginning with 26-hydroxyl- ation of cholesterol. In our previous studies in humans (6), hamsters, and rats (1, 7), chenodeoxycholic and cholic acids were always identified as metabolites. In addition, except for the human studies in which there was insufficient radioactiv- ity to characterize fully the monohydroxy bile acids, we were also able to identify 3P-hydroxy-5-cholenoate as a metabolite of 26-hydroxycholesterol. The present findings in the hamster, together with previous studies in the rat by Mitropoulos and Myant (8), indicate that during the further in vivo degradation of the side-chain of 26-hydroxycholesterol to 3P-hydroxy-5- cholenoic acid the capacity for 12a-hydroxylation is lost. Therefore, cholic acid is not found as a metabolite of 3P- hydroxy-5-cholenoate. Further study of this aspect of cholic acid synthesis will require the preparation of the appropriate intermediates in the side-chain oxidation pathway of 26-hy- droxycholesterol to 3P-hydroxy-5-cholenoate.

It has recently been shown that there are two enzymes in the liver that can initiate oxidation of the C27-steroid side- chain (9). A mitochondrial enzyme hydroxylates the 25-pro- S-methyl group, to form 25-R-26-hydroxycholesterol. A mi- crosomal enzyme shows no activity toward cholesterol, but hydroxylates the 25-pro-R-methyl group of 5P-cholestane- 3a,7a-diol and SP-cholestane-3a,7a,l2n-triol to form the re- spective 25-S-26-triol or -tetrol. Thus, initiation of bile acid

synthesis by microsomal 7a-hydroxylation would give rise to intermediates with a different stereospecificity than those formed when the initial step is mitochondrial hydroxylation. The possible significance of the presence of these two types of intermediates has not been evaluated. It is of interest to note however, that the proportion of 25-R-26-hydroxycholestero1, the natural intermediate found in kryptogenin (10) and used in our previous studies, metabolized to chenodeoxycholic and cholic acids is apparently greater than when a racemic mixture synthesized from 3~-hydroxy-5-cholestenoic acid was used in man (11).

Our findings indicate that there are three pathways for the further metabolism of 3P-hydroxy-5-cholenoate formed in the liver. Although the ester sulfates of the monohydroxy bile acids were not isolated specifically in these studies, the marked reduction in the radioactivity in the monohydroxy fraction when chemical solvolysis was omitted together with recovery of an increased proportion of radioactivity as more polar compounds implies their presence in bile as ester sulfates.

The lowest dose administered, 2.9 nmol, did not expand significantly the bile acid pool of the hamster, which is ap- proximately 25 pmol(12). It is of interest to note that recovery in bile was proportionally less when a tracer amount was given, implying distribution in an in vivo pool.

A second pathway appears to be metabolism to lithocholic acid which was found in relatively small amounts in all three studies. That lithocholic acid represents a metabolite of 3P- hydroxy-5-cholenoate was further established by eliminating the possibility that it could be accounted for by bacterial dehydroxylation of ["H]chenodeoxycholic acid which might occur if contamination occurred during the collection of bile. Also, addition of 3P-hydroxy-5-[ 1,2-"HJcholenoate to bile fol- lowed by all the chemical procedures used in these studies failed to generate radioactive compounds that might mimic lithocholic acid in its chromatographic behavior. Thus, we conclude that lithocholic acid can be synthesized in the liver from 26-hydroxycholestero1 via 3P-hydroxy-5-cholenoate.

The findings of chenodeoxycholic acid as a metabolite of 3/3-hydroxy-5-cholenoate indicates further that multiple path- ways exist for its synthesis from cholesterol (13). Presumably the major route is via 7a-hydroxylation of cholesterol. Other pathways probably include 7a-hydroxylation of a variety of other intermediates including 26-hydroxycholesterol. In ad- dition, the hamster has been shown previously to synthesize chenodeoxycholic acid from lithocholic acid (14). Thus, in these studies it is reasonable to consider that the chenodeox- ycholic acid synthesized from 3P-hydroxy-5-cholenoate oc- curred via lithocholic acid as an intermediate. However, we cannot exclude the possibility proposed by Yamasaki (15) that 7a-hydroxylation of 3P-hydroxy-5-cholenoate occurs prior to transformation of the sterol ring.

Using 3P-hydroxy-5-cholenoate, prepared by catalytic tri- tiation, it has been reported previously that it is metabolized in-the rat to both lithocholic and chenodeoxycholic acids (8). Only 1.4 mCi of compound was obtained after purification of the 284 mCi of reaction product. Our attempt to prepare 3p- hydroxy-5-cholenoate by cataiytic tritiatiod also indicated progressive loss of radioactivity on repeated crystallizations. Because a specific activity could not be established and the possibility of tritium exchange could not be excluded in vivo, we considered it important to further ascertain the metabolic fate of 3,%hydroxy-5-cholenoate.

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