73
2454
BILE ACID SULFATES IN SERUM BILE ACIDS DETERMINATION
J.F. PAGEAUX, B. DUPERRAY, D. ANRRR and M. DUBOIS
Service de Chimie Biologique, 406, Institut National des Sciences AppliquiZes,20 Avenue Albert Einstein, 69621 VILLEURBANNE CEDEX, France. Received 3-8-79 ABSTRACT
Some bile acid sulfates were synthesized and characterized. The configurationof sulfate groups at C-3, C-7 and C-12 positions was confirmed by Nuclear Magnetic Resonance analysis. These sulfates were utilized in a study of their chemical behaviour in different analytical procedures currently used for serum bile acids determination. Procedures for bile acids extraction from serum with ethanol or Amberlite XAD-2 result in sn important loss of the most polar sulfated bile acids. Complete separation of unsulfated from sulfated bile acids on Sephadex LH-20 is not achieved when deconjugationhas not been previously performed. Alkaline hydrolysis of some sulfated bile acids induces artifacts. Enzymatic deconjugationof the most polar bile acid sulfate is slow but does not produce artifacts. Enzymatic determination of bile acids gives positive response with some bile acid sulfates. The current procedures of serum bile acids determination are discussed in considerationof these results.
INTRODUCTION
As increasing interest is devoted to serum bile acid determination,many authors described methods for quantitation of unsulfated or sulfated bile acids in human serum (1,2,3,4). Unfortunately, they often lacked adequate reference substances to check the reliability of these methods in bile acid sulfates handling or in avoiding bile acid sulfates interference in unsulfated bile acid determination.As serum bile acid mono, di-and trisulfates levels a.re eften raised in patients with liver injury (4,5,6,7), precise assessment of bile acid sulfates interference in serum unsulfated bile acids determinationmay be worthwile. The present study was undertaken to specify the behaviour of some bile acid sulfates in existing procedures of serum bile acids analysis paying particular attention to the position of the sulfate
VoZwne 34, Number 1
fb
ZIImOX.DI
July,
1979
74
S
trD=OxDm
group on the steroid nucleus, and possibly to propose the modification of some analytical steps.
MATERIALS AND METHODS
Materials. Amberlite KAD-2 was obtained from Fluka (Switzerland), Sephadex LH-20 from Pharmacia (Uppsala, Sweden), reference bile acids from Steraloids (Pawling, N.Y., USA), (24.14~) lithocholic acid 50 mCi/mmol), (24-14~) chenodeoxycholicacid (52 mCi/mmol) and (24-ItC) taurocholic (50 mCi/mmol) acid, sodium salt, from the Radiochemical Centre (Amershsm, U.K.), cholylglycinehydrolase (Type III) from Sigma (St.Louis, Mo.,USA), 3& hydroxysteroid dehydrogenase (STDHP) from Worthington (Freehold, N.J., USA). Enzymatic bile acid deconjugation.Bile acid deconjugation was obtained according to Summerfield et -- al (8) (37'C, 4 h, pH 5-6) with 2 cholylglycine hydrolase units per assay ; under these conditions complete deconjugation of 2 )rmolestaurocholic acid occured within 1 he Bile acid determinationwith 3 o( hydroxysteroid dehydrogenase. Enzymatic bile acid determinationwas obtained according to Schwarz -et al (9) with U.V. spectrometry at 340 run. Bile acid thin-layer chromatography (TLC). TLC was carried out on precoated 250 p thick Silicagel G plates (Merck A.G., Darmstadt, Germany). Unsulfated and sulfated bile acids were analyzed in solvent system I (n.butanol-aceticacid-water, 10-l-l V/V) or in solvent system II (methyl ethyl ketone-chloroform-methanol-isopropanol-acetic acid-water, 20-6-2-2-4-l- V/V) (10). Methylated bile acids and methylated succinoyl derivatives of bile acids were analyzed with solvent system III (benzene-acetone,9-1 V/V), unmethylated succinoyl derivatives with solvent system IV (chloroform-acetone-aceticacid, 15-3-l V/V). Spots were detected by spraying the plates with 5% phosphoamlybdie acid in ethanol, then with 10X sulfuric acid and heating at 120°C+ Gas-liquid chromatography (GLC). Bile acids were chromatographed as methyl esters, methyl ester acetates, or methyl ester succinates on 4 ft columns packed with 3% OVI, 3% SP 2401 or 3% poly S 179 as stationary phase in a Girdel 3000 chromatograph at temperature varying from 230 to 260°C depending upon the type of derivatives. Methylation was achieved with diazomethane, acetylation was carried out with acetic anhydride-aceticacid-perchloricacid (7-5-0.01 V/V) (11). Gas-liquid chromatography- mass spectrometry (GLC-MS). Electron impact mass spectra were recorded with a VG 305 mass spectrometer. The ionization energy was 70 ev. Evaluation of data was performed on a VG multispec computer.
Nuclear magnetic resmance (NMQ. NMR spectra were obtained on a-Varian A 60 spectrometer. Chemical shift data are presented in parts per million (ppm) relative to tetramethylsilaneas an internal standard. Localisation of the signals of H-3, H-7 or H-12 protons was realized with a precision of l 0.08, * 0.03 and * 0.03 ppm respectively, signal width was estimated with a precision of k 5, l 3 and k 3 Hz. Solvents are indicated in table 2, trifluoraceticacid was added to each assay in order to eliminate the signals from hydroxyl groups. Radioactivity determination.Radioactivity determination was made by liquid scintillation counting (Tricarb 3320 scintillation spectrometer,Packard Instrumsnts) using Dnisolve I (Rock-LightLab.) as scintillation liquid. Infared spectrometry_(IR). IR spectra were determined on a Beckmann Acculab IV spectrometer using KBr pellets. Bile acids extraction from hunan serum. Bile acids were extracted using two different methods. a) 3 ml serum samples with added unlabeled bile acid standard were incubated for 1 h at 37'C, diluted with 9 volumes of 0.1 N NaOH in saline, then paured on XAD-2 coluxms (Ig, 8 cm high, 0.2 ml/min flow rate). The columns were washed with 10 ml water (pH 7) and bile acids were eluted with either 20 ml methanol or 20 ml ethanol or 10 ml ethanol containing 0.08 ml l2N HCl or 10 ml ethanol containing 0.8 ml 12 N HCl (12). The eluates were evaporated to dryness and the residues were submitted to solvolysis, alkaline hydrolysis, methylation and acetylation before GLC analysis (12). Similar experiments were conducted using radioactive bile acid standard : the dry residues of the eluates were counted for radioactivity determination. b) A known amount (5 ngr) of a radioactivebile acid standard was added to the serum sample (3 ml). After a one hour incubation, 50 pl 2N NaOH and 15 ml absolute ethanol were added ; the mixture was heated for 5 min at IOG'C, then centrifuged.The ssnm procedure was applied to the pellet ; the supernatantswere pooled, evaporated and counted for radioactivity determination. Sulfated and unsulfated bile acids chromatographyon Sephadax LB-20. Elution volume (X of the column volrmre)of each bile acid was determined using a Sephadex LH-20 column (6g Sephadex, 13 cm high, 1.6 cm ID, 0.30 ml/am flow rate) and chloroform - methanol (1-1 V/V) containing 0.01 H NaCl as eluting system ; 100 ng bile acid samples were used ; 2 ml fractions were collected then counted for radioactivity determinationor submitted to TLC analysis. Solvolvsis of bile acid sulfates. Dnconjugated sulfated bile acids were submitted to solvolysis according to Parmentier et al (13) and Van Berge Henegouwen et al-(14) procedures. When the fiztsmttmd was used, 1 mg sulfated%K acid and 1 mg GLC internal standard (cholic acid) were dissolved in 1 ml methanol ; 9 ml acetone and 0.1 ml 12N HCl were added, then the mixture was heated at 37'C. Aliquots
(1 ml) were diluted with 5 ml water, pH ajusted to 1 with 0.1 ml 12N HCl and free bile acids were extracted by diethyl ether (2 x 10 ml), methylated and quantitated by GLC. In order to check the second method, 0.1 mg sulfated bile acid and 0.1 mg GLC internal standard were introduced in the following medium : 2.5 ml 2.5N NaOH, 1.8 ml 12N HCl, 12 ml diethyl ether. The mixture was vigorously shaken, left at laboratory temperature for 0 (reference assays)to 3 h, the ethereal layer was taken off and the aqueous layer extracted again with 2 x 12 ml diethyl ether. Bile acids from the pooled organic layers were methylated and submitted to GLC analysis. As internal standard, we used chenodeoxycholic acid when deoxycholic acid 12-sulfatewas studied, otherwise we used deoxycholic acid. Sulfated bile acids stability during alkaline hydrolysis. Six samples of each sulfated bile acid under study were dissolved in 10 ml 215N NaOH and heated at 120°C in teflon containers for 8 h . The samples were cooled, pH ajusted to I with 6N HCl, and unsulfated bile acids extracted with 2 x 20 ml diethyl ether, The pooled organic phases were washed with 2 x IO ml water and evaporated to dryness. Bile acids were methylated ; from the 6 assays, 4 were analyzed without acetylation, by TLC with solvent system III (1 assay) or by GLC (3 assays) and 2 were analyzed by GLC after acetylation. Methyl esters and methyl ester acetates were then submitted to GLC-MS. Triplicate reference assays were not heated at 12O*C but carried out according to the same procedure. Sulfated bile acids stability during enzymatic hydrolysis. Litlwcholic acid sulfate. tauro and nlvcolithocholicacid sulfate, chenodeoxycholicacid 7-sulfate, deoxycholic acid 12-sulfate and taurocholic acid trisulfate were submitted to the above described enzymatic deconjugation procedure. The media containing bile acids were subsequently treated as after alkaline hydrolysis. Preparation of reference bile acid sulfates 11 Sodium salts of lithocholic acid sulfate, glycolithocholicacid sulfate, taurolithocholicacid sulfate and f24 14CJ lithocholic acid sulfate. They were prepared by reacting the corresponding unsulfated bile acids with the triethylamine - SO1 complex according to Tserng et al (15). As judged by IR: TLC and el&ntary analysis,the producczre identical to those obtained by these authors. 2/ Sodium salts of chenodeoxycholicacid disulfate (Ia) and deoxycholit acid disulfate (lb) Were prepared from 5 mm01 of unsulfated bNile acid and 12 mm01 triethylamine - SO3 complex in 10 ml dimethylformamide. Complete sulfation, as judged by TLC in solvent system II occured within 6 h . The disulfated bile acids were precipitated according to the procedure used by Tsemg et al (15) for taurolithocholicacid sulfate. Yields were 68% and 53%yeGectively for Ia and Ib. Both products decomposed over 240°C, both were pure according to TLC analysis (solvent systems I and II) (table 1). IR spectra showed absorption bands at 1555, 1210, 1070 and 970 cm-l. Anal.Calcd for C24H370H-$2Na3 ; 46.66% C, 5.99% H, 10.36% S, 11.17% Na. Found : 46.18% C, 6.22% H, 9.71% S, 33.68% Na for Ia and 45.72% C, 6.16% H, 9.88% S, 11.55% Na for Ib.
77
WR analysis of Sb showed that both hydroxyl groups were sulfated and that no stereochemicalmodification occured (table 2). 24 14C] chenodeoxycholicacid disulfate was prepared according to the same procedure using 50 ~1 [24 14~) chenodeoxycholicacid (1 mCi/nssol).By TLC analysis with solvent system I and II, the product exhibited only one spot with the same mobility as the unlabeled product. 41 Disodium deoxycholate 12-sulfate and disodium chenodeoxycholate 7-sulfate. Specific sulfation at C 12 or C 7 position was achieved after preliminary succinoylationof the 3-hydroxyl group. Succinoylationprocedure : Bile acid (12.5 mmol), succinic anhydride (250 mmol) and l-2 dimethoxyethane (25 ml} were mixed and refluxed. Progress of the reaction was observed by TLC in solvent system IV. After 12 h, 97% of the starting material was recovered as the monosuccinate and 3% as the disuccinate. Disuccinoylationat 95% yield required higher temperature (135°C) and much longer time (130 h). The solvent was evaporated, the residue extracted with 3 x 100 ml diethyl ether and succinic anhydride discarded by filtration. The diethyl ether was evaporated to dryness, the residue was dissolved in 100 ml methanol, heated at 10C°C for 2 h in asoppered flask, and further methylated with diasomethane.The solvent was evaporated, and dimethylsuccinatswas eliminated by distillation at 90°C under reduced pressure (0.1 sm Hg). Dimethyl chenodeoxycholate3-hemisuccinate (IIa) and dimethyl deoxycholate 3-hemisuccinate (IIb) were obtained from the dry residues after crystallizationas white crystals, the former after crystallization from diisopropyl ether-cyelohexane(1-3 V/V), the latter after double crystallizationfrom hexane; yields : 64% (IIaf, 62% (IIb) ; m-p 88-89OC (IIa), 99-100°C (IIb). All these products were pure according to TLC (solvent system III), CLC and elementary analysis. NMR analysis showed that hydroxyl and succinoyloxy groups were at the expected position and had the expected configuration (table 2 and discussion). Sulfation procedure : Dimethyl chenodeoxycholate3-hemisuccinate 7-sulfate (IIIa) was prepared by sulfation of compound IIa following the triethylamine- SO3 complex procedure. Complete derivatization was achieved within 6 h . Dimethyl deoxycholate 3-hemisuccinate 7-sulfate (IIIb) was prepared according-toHasiewood et al (16) from comnound IIb : the white crvstalline Product obtained?fGr overnisht cry‘stallisation from the aqkeous solution neutralized with N&X03,was washed with cold water and diethyl ether. C~po~ds IIIa and IIIb were pure according to TLC analysis (solvent system I and 11) ; the purity of compound IIIb was further confirmed by elementary analysis. Both compounds were submitted to mild alkaline hydrolysis (4 h , 70°C) in 50% aqueous methanol containing 5% NaOH. The mixture was partially neutralized by 2N DC1 (apparent pH g-10), the solvents were removed by evaporation ; the dry residue was suspended in water and pHadjusted to 7. After water evaporation, the bile acid sulfates were extracted from the dry residue by ethanol (2 x 50 ml),the solvent was filtered and evaporated to dryness. The crude bile acid sulfates were dissolved in 50 ml water and pH ajusted to 10 with O.lN NaOH. The solution was percolated through a column of XAD-2 (20g) and bile acid sulfates were eluted with methanol (250 ml) (13). Disodium chenodeoxycholate7-sulfa-
te (IVa) was recovered as the dry residue of the eluate ; disodium deoxycholate 12-sulfate (I!%) recovered as the dry residue of the eluate was further crystallized from methanol - ethylacetate (l-i2 V/V). Both compounds were pure according to TLC (table I), NMR analysis showed that the sulfate groups were at the expected positions and that no stereochemicalmodification oceured (table 2). By GLC analysis, after solvolysis and methylation, both compounds gave one peak with retention time identical to that of the correspondingmethylated bile acid. IR spectra : absorption bands at 1210, 1060, 970 cm-1 for IVa and IVb. Anal.Calcd. for C24R3807SNa2,B20 : 53.93% C, 7.12% H, 5.99% S, 8.61X Na. Found 54.31Z C, 7.351 H, 5.82% S, 9.01X Na (IVa) and 53.28% C, 7.29% 8, 5.85% S, 8.92% Na (IVb). 24 14C taurocholic acid - trisulfate sodium salt was prep_ared_ac_tording to the sulfation procedure of Fieser (17) with 16.1 mg [24 14C] taurocholate (0.8 mCi/mmol) as starting material for a period of 7 days at room temperature- The reaction mixture was terminated by the addition of IN NaOK, Pyridine was removed by evaporation, the residue was dissolved in 10 ml water and pH ajusted to 7. Due to its strong adsorption on XAD-2 the product was not desalted. The aqueous solution was analyzed in TLC with solvent I : after spraying with phosph~lybdic acid, only one spot (RF = 0.03) was detected, compatible with the trisup fate structure but not with the mono or disulfates oues (IO).
RESULTS
Extraction of bile acids from human serznn(table 3). When human serum with added radioactive bile acids was nercolated on Amberlite FAD-Z, no radioactivitywas found in the effluents. The same result was obtained after column washing with water. Ethanol and methanol failed to elute sulfated bile acids : with methanol only 10X of lithocholic acid sulfate, 6X of deoxycholic acid disulfate end traces of taurochotic acid trisulfate were eluted. With 10 ml ethanol containing 0.08 ml 12N HCl as eluant (table 3, column I), unsulfated and unconjugated rmnosulfated bile acid were recovered with yields higher than 90%. Recovery rate of lithocholic acid sulfate was slightly lowered by conjugation with glysine or taurine, DL and trisulfated bile acids were not satisfactorily extracted, but using 10 ml ethanol containing 0.8 ml 12N liC1as eluting system higher recovery rates were obtained (80% and 61X respectively). Partial solvolysis occured in ethanolic hydrochloric acid : from lithocholic acid sulfate, chenodeoxycholicacid 7-sulfate, deoxycholic acid 12-sulfate and deoxycholie acid disulfate, respectively 18%, 2W, 5% and 0.6% were recovered as unsulfated bile acids after 12 h in ethanol containing 8% 12N HCI. Serum treatment with ethanol failed to extra& taurocholic acid trisulfate but permitted satisfactory recovery of unconjugated chenodeoxycholic acid disulfate and lithocholic sulfate (table 3 , colunn~ II)‘ Unconjugated bile acid sulfates solvolysis (Fig. 1). lb solvolysis methods were tested : in both procedures, elimination of the
sulfate group is faster at C-3 that at C-12 or C-7 position. Van Berge Henegouwenle (14) method failed to remove the sulfate group from C-7 or C-12 positions whereas Parmentier'e procedure (13) achieved complete solvolysis within 12 h .
TABLE l-
TLC tmbilities (Rf) of bile acid sulfates
BILE SALT
Lithocholic acid Lithocholic acid sulfate Taurolithocholicacid Taurolithocholicacid sulfate Glycolithocholicacid Glycolithocholicacid sulfate Deoxycholic acid Dimethyl deoxycholate 3-hemieuccinate 12-sulfate (IIIb) Deoxycholic acid 12-sulfate (IVb) Deoxycholic acid dieulfate (Ib) Chenodeoxycholicacid Dimethyl chenodeoxycholate3-hemisuccinate 7-sulfate (IIIa) Ghenodeoxycholicacid 7-sulfate (IVa) Chenodeoxycholicacid diaulfate (Ia) Taurocholic acid trisulfate
SOLVENT SYSTEM I II 0.93
0.96 0.79
0.70 0.41 0.26 0.59 0.48 0.91
0.44 0.19 0.85 0.42 0.92
0.72 0.67 0.28 0.90
0.66 0.60 0.23 0.91
0.69
0.58 0.54 0.22 0.00
0.65 0.27 0.03
Bile acids fractionation on Sephadex I&l-20(Fig.2). Unconjugated bile acids were completely separated from bile acid sulfates but conjugated unsulfated and unconjugated sulfated bile acids overlapped. Bile acid determinationusing 3d,hydroxysteroid dehydrogenase (table 3). At 10m4 M concentration in the incubation medium, idenspectromatric responses were obtained with all uneulfated bile acids and with deoxycholic acid 12-sulfate.On the other hand, the enzyme failed to oxidize chenodeoxycholicacid 7-sulfate and, of course, bile acids bearing a sulfate group at C3 position. Bile acid sulfates stability during enzymatic hydrolysis. During enzymatic hydrolysis, lithocholic acid sulfate, chenodeoxycholic acid 7-sulfate, deoxycholic acid 12-sulfate suffered neither solvolysis nor degradation. Taurolithocholicacid sulfate and glycolithocholic acid sulfate were totally converted into lithocholic acid sulfate. When taurocholic acid trisulfate was submitted to enzymatic hydrolysis (4 h) and then to solvolysis, 25% of the starting material was recovered as cholic acid.
S
80
TABLE
-IYRR.OXD~
2 COOCH2
H-12:
H-12=
0.78
0.61
Effect of succinoylationand sulfation on the N.M.R chemical shift of H-3, H-7, and H-12 protons. a) solvent : deuterochloroformand trifluoracetic acid b) solvent : hexadeuterodimethylsulfoxideand trifluoraceticacid, in these compounds the C-24 methyl ester group is replaced by the sodium salt. ; Y : Na+ 0S020X : CH30CO-CH2-CH2-COO-
TABLE3:
Recovery rates (X) of bile acids after XAD-2* or ethanol extraction from luauanserum (columns L snd II) and bile acids response to treatment with NAD-3d hydroxysteroid dehydrogenase (optical density variation in 1 h at 340 nm with lo-4M bile acid concentration- column III) * eluant : 10 ml ethanol containing 0.08 ml 12N HCl
Bile acid Lithocholic acid Taurolithocholicacid Glycolithocholicacid Lithocholic acid sulfate Taurolithocholicacid sulfate Glycolithocholicacid sulfate Deoxycholic acid Deoxycholic acid 12-sulfate Deoxycholic acid disulfate Chenodeoxycholicacid Chenodeoxycholicacid Psulfate Chenodeoxycholicacid disulfate Taurochenodeoxycholicacid Taurocholic acid Taurocholic acid trisulfate
I
II
94.6
82.4
90.8
92.1
91.2 83.7 86.0 94.9 94.1 31.0 93.7 90.4 19.0 92.8 91.0 4.5
86.2
85.8 80.5 81.0 19.7
III 0.44 0.42 0.43 0 0 0 0.41 0.39 0 0.43 0.02 0 0.44 0.41
Bile acid sulfates stability during alkaline hydrolysis. From lithocholic acid sulfate submitted to the entire alkaline hydrolysis procedure, 15% were recovered as a compound showing the same properties as lithocholic acid as judged by TLC, GLC and GLC-MS analysis. When the alkaline medium was not heated but instantly treated with diethyl ether for free bile acids extraction, a smaller amOunt (8% of the starting material) of the lithocholic acid was recovered. Taurolithocholic acid sulfate submitted to alkaline hydrolysis gave also only lithocholic acid. The major constituent of the products from alkaline hydrolysis of chenodeoxycholic acid 7-sulfate was probably a ring B insaturated derivative of lithocholic acid. In fact, from chenodeoxycholicacid 'I-sulfate only 0.2% were recovered as chermdeoxycholicacid but 55% were recovered as compounds showing properties similar to those of lithocholic acid. TLC analysis exibited two spots : the weaker with Rf values identical to that of lithocholic acid, the major one with slightly smaller kf value. GLC analysis vith poly S 179 as stationary phase showed one shouldered peak with retention time slightly longer than that of lithocholic acid methyl ester. Mass spectrometric fragmentationshowed that the small shoulder was due to lithocholic acid methyl ester and, for the major constituent detected in GLC, resulted in a set of m/e values by 2 units smaller than the m/e values obtained with reference lithocholic acid
Characteristic fr~~tation ions were recorded at m/e 388 @I?, m!e 373 WCH3~, m/e 370 CM-%$@, m/e 316 (M-ringA), m/e 273 @-side chain), m/e 255
methyl ester.
m/e 249, m/e 228, m/e 213, m/e 201. When incubated with 3d hydroxysteroid d~dro~enase, the degradation products of cbenodeoxycholicacid 7-sulfate gave a positive response whereas no respoase was obtained with the starting material. When
Fig.I
: Time course of bile acid sulfates solvolysis. @ Lithocholie acid sulfate, m Deoxycholicacid 12-sulfate 0 Dsoxycholic acid disulfate, A Chenodeoxycholicacid Psul) Method A (Ref : la), (----) Method B (Ref : 14). fate. (A
[24 "4C3 ~~odeo~c~lic acid disulfate was sub~tted to the alkaline hydrolysis procedure, 13X of the radioactivity wes recovered in diethyl ether extract whereas the starting material not submitted to hydrolysis was not extrsotable. Neither sofvolysis nor degr8datio~ were observed when d~o~~~~ic a&d 12-sulfateand deoxycholicdisulfate were slitted to the alkaline bydro~~sia procedure. when duplicates of human serum samples receivedequal arts of taurb-
lithocbolic acid Bulfate, and then were percolated on Amberlite XAD-2 before being submitted to ansymatic or to alkaline hydrolysis, xnre lithocholic acid (or more similar undiatinguisbablamaterial) YBB found by GLC analysis after alkaline than after enzymatic hydrolyeia. The same result vas obtained whan ehenodeoxycbolicacid 7-sulfate vae added to serum samples. Uhen serum samples received chenodeoxycholicor deoxycholic acid Ii?-sulfate instead of taurolithocbolicacid sulfate or chenodeoxycholicacid 7-sulfate, the same aoPountsof lithocholic acid were found after alkaline hydrolysis or after enzymatic hydrolysis. In every case no difference was observed between chenodeoxycholicacid or deoxycholic acid amounts recovered after enzymatic or alkaline hydrolysis. These facts show that serum bile acid determination after alkaline hydrolysis results in erroneous quantitation of lithocholic acid but not of ~nodeo~~olic acid when the serum sample contains lithocholic acid sulfate or chenodeoxycholic‘I-sulfate; such false results are not induced by chanodeoxycholicacid or deoxycholic acid 12-sulfate.
2H I 0
I 100
I 200
I 300
1 400
, so0
, 600
1
Pig.2 : Chromatographyof bile ealts on Sephadex LIZ20 (coltrmn: 13 x I,5 cm containing 6g Sephadex LD-20, flow rate 0.3 ml/mn. solvent : methanol-chloroform(l/l V/V) containing 0.01 M NaCl NaCJ.) 1 : lithocholic acid, 2 : chenodaoxycholicacid, 3 : cholic acid, 4 : taurocholic acid, 5 : deoxycholic acid i2-sulfate,6 : cheuodeoxycholic acid 7-sulfate, 7 : lithochofic acid sulfate, 8 : taurolithocholicacid Bulfate, 9 : deoxycholic acid dieulfate.
DI.!XDSSION %a
present study was undertaken to specify the behaviour
of sulfated bile acids in the course of the analytical procedures intented for serum bile acids determination,As we could not perform this
work with the 45 sulfated derivatives of the most current bile acids, we restricted our study to a limited set of bile acids including the least and the most polar ones, some compounds with intermediary polarity, and two compounds specifically sulfated at C-7 or C-12 position. The bile acid sulfates we have synthesized were previously described (13-15-18-19) ; nevertheless we have shown that the sulfation procedure of Tserng -et al (15) is suitable for derivatization at C-7 and C-12 positions and that the protecting succinoxy group permits easy purification of the intermediary products. The position and the configuration of the sulfate groups were confirmed by NMB analysis. The NMR signals of H-3, H-7 and H-12 protons are distinctly separated from the signals of other protons : they are distinguished by differences in chemical shift (5 > and are identified by the signal width : using 3 the Karplus relationship giving the J coupling values and taking into consideration the fact that H-3 is axial and H-7 and H-12 equatorial, one can calculate theoretical values for the signal width of H-3 (24 to 34 Hz), H-7 (9 to 15 Hz) and H-12 (6 to IO Hz). As an example, with deoxycholic methyl ester these signal widthes are 35 and 12 Hz, and are unambiguously attributed to H-3 and H-12 respectively.Esterification induces deshielding effect of the proton attached to the same carbon atom as the esterified hydroxyl ; in cases of succinoylation and sulfation, deshielding effects (AS) are observed as large as 0.80 to 1.20 ppm and 0.55 to 0.67 ppm respectively (table 2), permitting unambiguous identificationof the group position. The signal width remains unmodified as long as the proton configuration does so ; if the configuration is inverted, the signal width should become 12 to 20 Hz for equatorial H-3, 24 to 34 Hz for axial H-7 and 12 to 17 Hz.for axial H-12. No change is induced in signal widthes by succinoylation or sulfation at any position ; this fact demonstrates that in all intermediary or final products under study the hydroxyl group is in the ~4 configuration. Bile acids extraction from serum is currently performed either by serum treatment with ethanol or by adsorption on Amberlite XAD-2 and subsequent elution with methanol or ethanol. The former method results in poor recovery (20%) of taurocholic acid trisulfate. Concerning the latter method, one may first emphasize the fact that,
contrarily to the opinion of Tserng _et al (20), all bile acids, including the most polar ones, are firmly adsorbed on XAD-2. This suggests that the commonly used solvents are not adequate to elute bile acid sulfates : in our hands, neither methanol nor ethanol did perform satisfactory elution of sulfates. This fact could explain difficulties encountered by some authors : Eyssen et al (Zl), extracting bile acids TzC] cholic acid, found that 5 from feces of mice injected with [24 to 7% of the radioactivitywas lost during the desalting step on XAD-2, using methanol as eluting solvent. May be, the radioactivity lost on XAD-2 was that of di and trisulfates not detected in significant smount by these authors at the end of the entire procedure. On the other hand, we showed that monosulfates are eluted at 80-90% recovery rate with 10 ml ethanol containing 0.08 ml. 12N KC1 snd that satisfactory elution of the most polar sulfates necessitates 10 ml ethanol containing 0.8 ml 12N HCL. However a drawback of this procedure consists in partial solvolysis of bile acid sulfates. So, our results suggest that the currently used extraction procedures don't permit bile acid sulfates quantitation unless labelled internal standards are at hands to determine the recovery rates of the most polar bile acid sulfates, Some authors used Amberlite XAR-7 instead of XAD-2 and obtained higher recovery rates of bile acid sulfates with the former than with the latter (22-23) but they had no standard to determine the recovery rates of the most polar sulfates. Alma -et al (24) claimed that complete extraction of urinary bile acid suffates was obtained by percolation of acidified urine on XAD-2 and subsequent elution with ethanol containing ammonium hydroxide. Such a procedure doesn't seem to be suitable for serum samples ; nevertheless, it is worth studyinS. Bradlow (25) recently devised a new procedure to elute steroid hormone monosulfates and glucuronides from XAD-2 columns as their triethylammoniumsalts. Such a procedure might be convenient for elution of di and trisulfatedbile acids but even so, Bradlow's method (25) is lon8er and does not result in higher yields thsn the procedure xe just proposed above. In our hands, the solvolysis procedure of Parmentier -et al (13) achieved complete desulfation of unconjugated bile acid sulfates without artifact formation, which confirms the results obtained by these
authors. Now, further work is needed to study the solvolysis of conjugated bile acid sulfates. Van Berge Henegouwen -et al (14) designed an analytical procedure involving an alkaline hydrolysis step but no specified solvolysis step, claiming that spontaneous desulfation occured during acidification of the media and extraction with diethyl ether. Our results show that spontaneous solvolysis occurs only for sulfate groups located at C-3 position ; this fact together with the undermentionned degradation of some sulfates during alkaline hydrolysis casts doubts on the reliability of this procedure. Class separation of sulfated from unsulfated bile acids on Sephadex LB-20 is not SC complete as claimed by authors using this fractionation procedure (1,3,4) ; figure 2 shows mutual overlapping of taurocholic acid and deoxycholic acid 12-sulfate.Clean separation necessitates enzymatic hydrolysis prior to fractionation on Sephadex LH-20. No degradation is observed when bile acid sulfates are submitted to enzymatic hydrolysis but complete deconjugation of taurocholic acid trisulfate was not obtained within 4 hours. Exhaustion of our [24 "CJ taurocholic acid trisulfate standard prevents us from determining the conditions necessary for total hydrolysis ; nevertheless, these results show that bile acid sulfates are deconjugated by cholyglycine hydrolase even if deconjugation proceeds more slowly. Performing enzymatic hydrolysis on serum sample would offer two advantages : first, bile acid sulfate elution from XAD-2 would be easier ; next, separation of sulfated from unsulfated bile acids on Sephadex LH-20 would be improved. Alkaline hydrolysis carried out under conditions currently used for bile acids determination, results in the transformationof lithocholic acid sulfate into lithocholic acid and induces formation of an unsulfated monohydroxylated compound from chenodeoxycholicFsulfate. This compound was tentatively identified as 34
-hydroxy-5p
-chol-6-
en-24 oic acid. In TLC and GLC it exhibited nobilities very similar to those of lithocholic acid. It gave a positive response with 3di hydroxysteroid dehydrogenase. The MS spectrum exhibited signals with the m/e values expected for a 3-hydroxy cholenoic acid methyl ester particular7 at m/e 249 indicating the presence of a double bond at C6-C7 or CS-
~6 position (26) : in the present case, C6-C7 position is more likely. Nevertheless, the presence of 3 dhydroxy-5 p -chol-6-en-24 oic acid, evidenced by the m/e 249 signal, does not exclude the presence of 3d hydroxy-lie-chol-7~~1-24 oic acid : the obtained mass spectrum may be that of a mixture of these two isomers. Such a mixture of zwnoineaturated compounds with the double bond located either a C6-C7 or at C7-Cg position was found by Eyssen et al (21) and Parmentier et al (13) after alkaline hydrolysis of cholic acid 7-sulfate. When cbenodeoxycholicacid 7-sulfate or lithocholic acid sulfate were added to serum sample from which duplicates were treated in the ssme way, one by alkaline hydrolysis, and the other by enzymatic hydrolysis, we always found more lithocholic acid in the first thsn in the second. Hence, as human serum always contains large amounts of bile acid sulfate (2,3,4,5,6),it becomes obvious that serum lithocholic acid level may be systematicallyoverestimatedby some analytical procedures in unsulfated bile acid determination involving an alkaline hydrolysis step. T.nother respects, some authors performing alkaline hydrolysis on serum samples prior to CLC analysis, found "abnormal" bile acids as, for example, 3d hydroxy cholenoic or 3 t hydroxy cholenoic acids (27). It seems to us that at least a part of these abnormal bile acids are artifacts from alkaline hydrolysis. Our first conclusion is that further studies are needed to design analytical procedures permitting satisfactory extraction of the most polar serum bile acid sulfates and quantitative separation of the less polar ones. Our second conclusion concerns the procedures intended to uneulfated bile acids determination : alkaline hydrolysis, elution from XAD-2 with acidified solvents and extraction with diethyl ether at low pH must be avoided when lithocholic acid determination is wanted. The last conclusion regards the enzymatic determinationof serum bile acids : as some bile acid sulfates with free 3Mhydroxyl
group give
positive response whereas some others don't, systematic work is needed to evaluate the influence of these facts on the reliability of the results obtained by enzymatic determination.
88
REFERENCES
This research was supported by the Institut National de la SantC et de la Recherche sdicale (F.R.A. ~'5) and by the Centre National de la Recherche Scientifique (E.R.A.n' 560).
(1) Makino, I., Shinosaki, K., Nakagawa, S., and Mashimo, K., J.Lipid. RI%.,
g,
132 (1974)
(2) Van Berge Henegouwen, G.P., Brandt, K.H., Eyssen, H., and Parmentier, G ., Gut, _II,861 (1976) (3) Stiehl, A., Eur.J.Clin.Inves.,4, 59 (1974) (4) Campbell, C.B., McGuffie, C., and Powell, L.W., Clin.Chim.Acta,63, 249 (1975) (5) Makino, I., Hashimoto, H., Shinozaki, K., Yoshino, K., and Nakagawa, S Gastroenteroloty,68, 545 (1975) (6) Dekrs, L.M., and Hepner, G.W., Amer.J.Clin.Pathol.,66, 831 (1976) (7) Campbell, C.B., McGUFFIE, C., Powell, L.W., Roberts, R.K., and Stewart, A.W., Amer.J.Dig.Dis.,2, 599 (1978) J.A., Billing, B.H., and Shackleton, C.H.L., Biochem. (8) Surmaerfield, J ., 54, 507 (1976) (9) Schwarz, H.P., Von Bergmann, K.,and Paumgartner, G., Clin.Chim. Acta, 50, 197 (1974) (10) Parmenxer, G., and Eyssen, H., J.Chromatog., 152, 285 (1978) (11) Roovers, J., Evrard, E., and Vanderhaeghe, H.,Tin.Chim.Acta, 2, 449 (1969) (12) Pageaux, J.F., Duperray, B., Dubois, M., Pacheco, Y., Pacheco, H., Herne, N., and Hauteville, D., Clin.Chim.Acta,E, 131 (1978) (13) Parmentier, G., and Eyssen, H., Steroids, 6, 721 (1975) (14) Van Berge Henegouwen, G.P., Allan, R.N., Hofmann, A.F., and Yu, P.Y.S., J.Lipid.Res., 18, 118 (1977) (15) Tserng, K.Y., and Kleiz P.D., J.Lipid.Res.,s, 491 (1977) (16) Haslewood, E.S., and Haslewood, G.A.D., Biochem.J., 155, 401 (1976) (17) Fieser, L.F., J.Amer.Chem.Soc.,70, 3232 (1948) (18) Parmentier, G., and Eyssen, H., Steroids, 30, 583 (1977) (19) Summerfield, J.A., Gollan, J.L., and Billi
2454
BILE ACID SULFATES IN SERUM BILE ACIDS DETERMINATION
J.F. PAGEAUX, B. DUPERRAY, D. ANRRR and M. DUBOIS
Service de Chimie Biologique, 406, Institut National des Sciences AppliquiZes,20 Avenue Albert Einstein, 69621 VILLEURBANNE CEDEX, France. Received 3-8-79 ABSTRACT
Some bile acid sulfates were synthesized and characterized. The configurationof sulfate groups at C-3, C-7 and C-12 positions was confirmed by Nuclear Magnetic Resonance analysis. These sulfates were utilized in a study of their chemical behaviour in different analytical procedures currently used for serum bile acids determination. Procedures for bile acids extraction from serum with ethanol or Amberlite XAD-2 result in sn important loss of the most polar sulfated bile acids. Complete separation of unsulfated from sulfated bile acids on Sephadex LH-20 is not achieved when deconjugationhas not been previously performed. Alkaline hydrolysis of some sulfated bile acids induces artifacts. Enzymatic deconjugationof the most polar bile acid sulfate is slow but does not produce artifacts. Enzymatic determination of bile acids gives positive response with some bile acid sulfates. The current procedures of serum bile acids determination are discussed in considerationof these results.
INTRODUCTION
As increasing interest is devoted to serum bile acid determination,many authors described methods for quantitation of unsulfated or sulfated bile acids in human serum (1,2,3,4). Unfortunately, they often lacked adequate reference substances to check the reliability of these methods in bile acid sulfates handling or in avoiding bile acid sulfates interference in unsulfated bile acid determination.As serum bile acid mono, di-and trisulfates levels a.re eften raised in patients with liver injury (4,5,6,7), precise assessment of bile acid sulfates interference in serum unsulfated bile acids determinationmay be worthwile. The present study was undertaken to specify the behaviour of some bile acid sulfates in existing procedures of serum bile acids analysis paying particular attention to the position of the sulfate
VoZwne 34, Number 1
fb
ZIImOX.DI
July,
1979
74
S
trD=OxDm
group on the steroid nucleus, and possibly to propose the modification of some analytical steps.
MATERIALS AND METHODS
Materials. Amberlite KAD-2 was obtained from Fluka (Switzerland), Sephadex LH-20 from Pharmacia (Uppsala, Sweden), reference bile acids from Steraloids (Pawling, N.Y., USA), (24.14~) lithocholic acid 50 mCi/mmol), (24-14~) chenodeoxycholicacid (52 mCi/mmol) and (24-ItC) taurocholic (50 mCi/mmol) acid, sodium salt, from the Radiochemical Centre (Amershsm, U.K.), cholylglycinehydrolase (Type III) from Sigma (St.Louis, Mo.,USA), 3& hydroxysteroid dehydrogenase (STDHP) from Worthington (Freehold, N.J., USA). Enzymatic bile acid deconjugation.Bile acid deconjugation was obtained according to Summerfield et -- al (8) (37'C, 4 h, pH 5-6) with 2 cholylglycine hydrolase units per assay ; under these conditions complete deconjugation of 2 )rmolestaurocholic acid occured within 1 he Bile acid determinationwith 3 o( hydroxysteroid dehydrogenase. Enzymatic bile acid determinationwas obtained according to Schwarz -et al (9) with U.V. spectrometry at 340 run. Bile acid thin-layer chromatography (TLC). TLC was carried out on precoated 250 p thick Silicagel G plates (Merck A.G., Darmstadt, Germany). Unsulfated and sulfated bile acids were analyzed in solvent system I (n.butanol-aceticacid-water, 10-l-l V/V) or in solvent system II (methyl ethyl ketone-chloroform-methanol-isopropanol-acetic acid-water, 20-6-2-2-4-l- V/V) (10). Methylated bile acids and methylated succinoyl derivatives of bile acids were analyzed with solvent system III (benzene-acetone,9-1 V/V), unmethylated succinoyl derivatives with solvent system IV (chloroform-acetone-aceticacid, 15-3-l V/V). Spots were detected by spraying the plates with 5% phosphoamlybdie acid in ethanol, then with 10X sulfuric acid and heating at 120°C+ Gas-liquid chromatography (GLC). Bile acids were chromatographed as methyl esters, methyl ester acetates, or methyl ester succinates on 4 ft columns packed with 3% OVI, 3% SP 2401 or 3% poly S 179 as stationary phase in a Girdel 3000 chromatograph at temperature varying from 230 to 260°C depending upon the type of derivatives. Methylation was achieved with diazomethane, acetylation was carried out with acetic anhydride-aceticacid-perchloricacid (7-5-0.01 V/V) (11). Gas-liquid chromatography- mass spectrometry (GLC-MS). Electron impact mass spectra were recorded with a VG 305 mass spectrometer. The ionization energy was 70 ev. Evaluation of data was performed on a VG multispec computer.
Nuclear magnetic resmance (NMQ. NMR spectra were obtained on a-Varian A 60 spectrometer. Chemical shift data are presented in parts per million (ppm) relative to tetramethylsilaneas an internal standard. Localisation of the signals of H-3, H-7 or H-12 protons was realized with a precision of l 0.08, * 0.03 and * 0.03 ppm respectively, signal width was estimated with a precision of k 5, l 3 and k 3 Hz. Solvents are indicated in table 2, trifluoraceticacid was added to each assay in order to eliminate the signals from hydroxyl groups. Radioactivity determination.Radioactivity determination was made by liquid scintillation counting (Tricarb 3320 scintillation spectrometer,Packard Instrumsnts) using Dnisolve I (Rock-LightLab.) as scintillation liquid. Infared spectrometry_(IR). IR spectra were determined on a Beckmann Acculab IV spectrometer using KBr pellets. Bile acids extraction from hunan serum. Bile acids were extracted using two different methods. a) 3 ml serum samples with added unlabeled bile acid standard were incubated for 1 h at 37'C, diluted with 9 volumes of 0.1 N NaOH in saline, then paured on XAD-2 coluxms (Ig, 8 cm high, 0.2 ml/min flow rate). The columns were washed with 10 ml water (pH 7) and bile acids were eluted with either 20 ml methanol or 20 ml ethanol or 10 ml ethanol containing 0.08 ml l2N HCl or 10 ml ethanol containing 0.8 ml 12 N HCl (12). The eluates were evaporated to dryness and the residues were submitted to solvolysis, alkaline hydrolysis, methylation and acetylation before GLC analysis (12). Similar experiments were conducted using radioactive bile acid standard : the dry residues of the eluates were counted for radioactivity determination. b) A known amount (5 ngr) of a radioactivebile acid standard was added to the serum sample (3 ml). After a one hour incubation, 50 pl 2N NaOH and 15 ml absolute ethanol were added ; the mixture was heated for 5 min at IOG'C, then centrifuged.The ssnm procedure was applied to the pellet ; the supernatantswere pooled, evaporated and counted for radioactivity determination. Sulfated and unsulfated bile acids chromatographyon Sephadax LB-20. Elution volume (X of the column volrmre)of each bile acid was determined using a Sephadex LH-20 column (6g Sephadex, 13 cm high, 1.6 cm ID, 0.30 ml/am flow rate) and chloroform - methanol (1-1 V/V) containing 0.01 H NaCl as eluting system ; 100 ng bile acid samples were used ; 2 ml fractions were collected then counted for radioactivity determinationor submitted to TLC analysis. Solvolvsis of bile acid sulfates. Dnconjugated sulfated bile acids were submitted to solvolysis according to Parmentier et al (13) and Van Berge Henegouwen et al-(14) procedures. When the fiztsmttmd was used, 1 mg sulfated%K acid and 1 mg GLC internal standard (cholic acid) were dissolved in 1 ml methanol ; 9 ml acetone and 0.1 ml 12N HCl were added, then the mixture was heated at 37'C. Aliquots
(1 ml) were diluted with 5 ml water, pH ajusted to 1 with 0.1 ml 12N HCl and free bile acids were extracted by diethyl ether (2 x 10 ml), methylated and quantitated by GLC. In order to check the second method, 0.1 mg sulfated bile acid and 0.1 mg GLC internal standard were introduced in the following medium : 2.5 ml 2.5N NaOH, 1.8 ml 12N HCl, 12 ml diethyl ether. The mixture was vigorously shaken, left at laboratory temperature for 0 (reference assays)to 3 h, the ethereal layer was taken off and the aqueous layer extracted again with 2 x 12 ml diethyl ether. Bile acids from the pooled organic layers were methylated and submitted to GLC analysis. As internal standard, we used chenodeoxycholic acid when deoxycholic acid 12-sulfatewas studied, otherwise we used deoxycholic acid. Sulfated bile acids stability during alkaline hydrolysis. Six samples of each sulfated bile acid under study were dissolved in 10 ml 215N NaOH and heated at 120°C in teflon containers for 8 h . The samples were cooled, pH ajusted to I with 6N HCl, and unsulfated bile acids extracted with 2 x 20 ml diethyl ether, The pooled organic phases were washed with 2 x IO ml water and evaporated to dryness. Bile acids were methylated ; from the 6 assays, 4 were analyzed without acetylation, by TLC with solvent system III (1 assay) or by GLC (3 assays) and 2 were analyzed by GLC after acetylation. Methyl esters and methyl ester acetates were then submitted to GLC-MS. Triplicate reference assays were not heated at 12O*C but carried out according to the same procedure. Sulfated bile acids stability during enzymatic hydrolysis. Litlwcholic acid sulfate. tauro and nlvcolithocholicacid sulfate, chenodeoxycholicacid 7-sulfate, deoxycholic acid 12-sulfate and taurocholic acid trisulfate were submitted to the above described enzymatic deconjugation procedure. The media containing bile acids were subsequently treated as after alkaline hydrolysis. Preparation of reference bile acid sulfates 11 Sodium salts of lithocholic acid sulfate, glycolithocholicacid sulfate, taurolithocholicacid sulfate and f24 14CJ lithocholic acid sulfate. They were prepared by reacting the corresponding unsulfated bile acids with the triethylamine - SO1 complex according to Tserng et al (15). As judged by IR: TLC and el&ntary analysis,the producczre identical to those obtained by these authors. 2/ Sodium salts of chenodeoxycholicacid disulfate (Ia) and deoxycholit acid disulfate (lb) Were prepared from 5 mm01 of unsulfated bNile acid and 12 mm01 triethylamine - SO3 complex in 10 ml dimethylformamide. Complete sulfation, as judged by TLC in solvent system II occured within 6 h . The disulfated bile acids were precipitated according to the procedure used by Tsemg et al (15) for taurolithocholicacid sulfate. Yields were 68% and 53%yeGectively for Ia and Ib. Both products decomposed over 240°C, both were pure according to TLC analysis (solvent systems I and II) (table 1). IR spectra showed absorption bands at 1555, 1210, 1070 and 970 cm-l. Anal.Calcd for C24H370H-$2Na3 ; 46.66% C, 5.99% H, 10.36% S, 11.17% Na. Found : 46.18% C, 6.22% H, 9.71% S, 33.68% Na for Ia and 45.72% C, 6.16% H, 9.88% S, 11.55% Na for Ib.
77
WR analysis of Sb showed that both hydroxyl groups were sulfated and that no stereochemicalmodification occured (table 2). 24 14C] chenodeoxycholicacid disulfate was prepared according to the same procedure using 50 ~1 [24 14~) chenodeoxycholicacid (1 mCi/nssol).By TLC analysis with solvent system I and II, the product exhibited only one spot with the same mobility as the unlabeled product. 41 Disodium deoxycholate 12-sulfate and disodium chenodeoxycholate 7-sulfate. Specific sulfation at C 12 or C 7 position was achieved after preliminary succinoylationof the 3-hydroxyl group. Succinoylationprocedure : Bile acid (12.5 mmol), succinic anhydride (250 mmol) and l-2 dimethoxyethane (25 ml} were mixed and refluxed. Progress of the reaction was observed by TLC in solvent system IV. After 12 h, 97% of the starting material was recovered as the monosuccinate and 3% as the disuccinate. Disuccinoylationat 95% yield required higher temperature (135°C) and much longer time (130 h). The solvent was evaporated, the residue extracted with 3 x 100 ml diethyl ether and succinic anhydride discarded by filtration. The diethyl ether was evaporated to dryness, the residue was dissolved in 100 ml methanol, heated at 10C°C for 2 h in asoppered flask, and further methylated with diasomethane.The solvent was evaporated, and dimethylsuccinatswas eliminated by distillation at 90°C under reduced pressure (0.1 sm Hg). Dimethyl chenodeoxycholate3-hemisuccinate (IIa) and dimethyl deoxycholate 3-hemisuccinate (IIb) were obtained from the dry residues after crystallizationas white crystals, the former after crystallization from diisopropyl ether-cyelohexane(1-3 V/V), the latter after double crystallizationfrom hexane; yields : 64% (IIaf, 62% (IIb) ; m-p 88-89OC (IIa), 99-100°C (IIb). All these products were pure according to TLC (solvent system III), CLC and elementary analysis. NMR analysis showed that hydroxyl and succinoyloxy groups were at the expected position and had the expected configuration (table 2 and discussion). Sulfation procedure : Dimethyl chenodeoxycholate3-hemisuccinate 7-sulfate (IIIa) was prepared by sulfation of compound IIa following the triethylamine- SO3 complex procedure. Complete derivatization was achieved within 6 h . Dimethyl deoxycholate 3-hemisuccinate 7-sulfate (IIIb) was prepared according-toHasiewood et al (16) from comnound IIb : the white crvstalline Product obtained?fGr overnisht cry‘stallisation from the aqkeous solution neutralized with N&X03,was washed with cold water and diethyl ether. C~po~ds IIIa and IIIb were pure according to TLC analysis (solvent system I and 11) ; the purity of compound IIIb was further confirmed by elementary analysis. Both compounds were submitted to mild alkaline hydrolysis (4 h , 70°C) in 50% aqueous methanol containing 5% NaOH. The mixture was partially neutralized by 2N DC1 (apparent pH g-10), the solvents were removed by evaporation ; the dry residue was suspended in water and pHadjusted to 7. After water evaporation, the bile acid sulfates were extracted from the dry residue by ethanol (2 x 50 ml),the solvent was filtered and evaporated to dryness. The crude bile acid sulfates were dissolved in 50 ml water and pH ajusted to 10 with O.lN NaOH. The solution was percolated through a column of XAD-2 (20g) and bile acid sulfates were eluted with methanol (250 ml) (13). Disodium chenodeoxycholate7-sulfa-
te (IVa) was recovered as the dry residue of the eluate ; disodium deoxycholate 12-sulfate (I!%) recovered as the dry residue of the eluate was further crystallized from methanol - ethylacetate (l-i2 V/V). Both compounds were pure according to TLC (table I), NMR analysis showed that the sulfate groups were at the expected positions and that no stereochemicalmodification oceured (table 2). By GLC analysis, after solvolysis and methylation, both compounds gave one peak with retention time identical to that of the correspondingmethylated bile acid. IR spectra : absorption bands at 1210, 1060, 970 cm-1 for IVa and IVb. Anal.Calcd. for C24R3807SNa2,B20 : 53.93% C, 7.12% H, 5.99% S, 8.61X Na. Found 54.31Z C, 7.351 H, 5.82% S, 9.01X Na (IVa) and 53.28% C, 7.29% 8, 5.85% S, 8.92% Na (IVb). 24 14C taurocholic acid - trisulfate sodium salt was prep_ared_ac_tording to the sulfation procedure of Fieser (17) with 16.1 mg [24 14C] taurocholate (0.8 mCi/mmol) as starting material for a period of 7 days at room temperature- The reaction mixture was terminated by the addition of IN NaOK, Pyridine was removed by evaporation, the residue was dissolved in 10 ml water and pH ajusted to 7. Due to its strong adsorption on XAD-2 the product was not desalted. The aqueous solution was analyzed in TLC with solvent I : after spraying with phosph~lybdic acid, only one spot (RF = 0.03) was detected, compatible with the trisup fate structure but not with the mono or disulfates oues (IO).
RESULTS
Extraction of bile acids from human serznn(table 3). When human serum with added radioactive bile acids was nercolated on Amberlite FAD-Z, no radioactivitywas found in the effluents. The same result was obtained after column washing with water. Ethanol and methanol failed to elute sulfated bile acids : with methanol only 10X of lithocholic acid sulfate, 6X of deoxycholic acid disulfate end traces of taurochotic acid trisulfate were eluted. With 10 ml ethanol containing 0.08 ml 12N HCl as eluant (table 3, column I), unsulfated and unconjugated rmnosulfated bile acid were recovered with yields higher than 90%. Recovery rate of lithocholic acid sulfate was slightly lowered by conjugation with glysine or taurine, DL and trisulfated bile acids were not satisfactorily extracted, but using 10 ml ethanol containing 0.8 ml 12N liC1as eluting system higher recovery rates were obtained (80% and 61X respectively). Partial solvolysis occured in ethanolic hydrochloric acid : from lithocholic acid sulfate, chenodeoxycholicacid 7-sulfate, deoxycholic acid 12-sulfate and deoxycholie acid disulfate, respectively 18%, 2W, 5% and 0.6% were recovered as unsulfated bile acids after 12 h in ethanol containing 8% 12N HCI. Serum treatment with ethanol failed to extra& taurocholic acid trisulfate but permitted satisfactory recovery of unconjugated chenodeoxycholic acid disulfate and lithocholic sulfate (table 3 , colunn~ II)‘ Unconjugated bile acid sulfates solvolysis (Fig. 1). lb solvolysis methods were tested : in both procedures, elimination of the
sulfate group is faster at C-3 that at C-12 or C-7 position. Van Berge Henegouwenle (14) method failed to remove the sulfate group from C-7 or C-12 positions whereas Parmentier'e procedure (13) achieved complete solvolysis within 12 h .
TABLE l-
TLC tmbilities (Rf) of bile acid sulfates
BILE SALT
Lithocholic acid Lithocholic acid sulfate Taurolithocholicacid Taurolithocholicacid sulfate Glycolithocholicacid Glycolithocholicacid sulfate Deoxycholic acid Dimethyl deoxycholate 3-hemieuccinate 12-sulfate (IIIb) Deoxycholic acid 12-sulfate (IVb) Deoxycholic acid dieulfate (Ib) Chenodeoxycholicacid Dimethyl chenodeoxycholate3-hemisuccinate 7-sulfate (IIIa) Ghenodeoxycholicacid 7-sulfate (IVa) Chenodeoxycholicacid diaulfate (Ia) Taurocholic acid trisulfate
SOLVENT SYSTEM I II 0.93
0.96 0.79
0.70 0.41 0.26 0.59 0.48 0.91
0.44 0.19 0.85 0.42 0.92
0.72 0.67 0.28 0.90
0.66 0.60 0.23 0.91
0.69
0.58 0.54 0.22 0.00
0.65 0.27 0.03
Bile acids fractionation on Sephadex I&l-20(Fig.2). Unconjugated bile acids were completely separated from bile acid sulfates but conjugated unsulfated and unconjugated sulfated bile acids overlapped. Bile acid determinationusing 3d,hydroxysteroid dehydrogenase (table 3). At 10m4 M concentration in the incubation medium, idenspectromatric responses were obtained with all uneulfated bile acids and with deoxycholic acid 12-sulfate.On the other hand, the enzyme failed to oxidize chenodeoxycholicacid 7-sulfate and, of course, bile acids bearing a sulfate group at C3 position. Bile acid sulfates stability during enzymatic hydrolysis. During enzymatic hydrolysis, lithocholic acid sulfate, chenodeoxycholic acid 7-sulfate, deoxycholic acid 12-sulfate suffered neither solvolysis nor degradation. Taurolithocholicacid sulfate and glycolithocholic acid sulfate were totally converted into lithocholic acid sulfate. When taurocholic acid trisulfate was submitted to enzymatic hydrolysis (4 h) and then to solvolysis, 25% of the starting material was recovered as cholic acid.
S
80
TABLE
-IYRR.OXD~
2 COOCH2
H-12:
H-12=
0.78
0.61
Effect of succinoylationand sulfation on the N.M.R chemical shift of H-3, H-7, and H-12 protons. a) solvent : deuterochloroformand trifluoracetic acid b) solvent : hexadeuterodimethylsulfoxideand trifluoraceticacid, in these compounds the C-24 methyl ester group is replaced by the sodium salt. ; Y : Na+ 0S020X : CH30CO-CH2-CH2-COO-
TABLE3:
Recovery rates (X) of bile acids after XAD-2* or ethanol extraction from luauanserum (columns L snd II) and bile acids response to treatment with NAD-3d hydroxysteroid dehydrogenase (optical density variation in 1 h at 340 nm with lo-4M bile acid concentration- column III) * eluant : 10 ml ethanol containing 0.08 ml 12N HCl
Bile acid Lithocholic acid Taurolithocholicacid Glycolithocholicacid Lithocholic acid sulfate Taurolithocholicacid sulfate Glycolithocholicacid sulfate Deoxycholic acid Deoxycholic acid 12-sulfate Deoxycholic acid disulfate Chenodeoxycholicacid Chenodeoxycholicacid Psulfate Chenodeoxycholicacid disulfate Taurochenodeoxycholicacid Taurocholic acid Taurocholic acid trisulfate
I
II
94.6
82.4
90.8
92.1
91.2 83.7 86.0 94.9 94.1 31.0 93.7 90.4 19.0 92.8 91.0 4.5
86.2
85.8 80.5 81.0 19.7
III 0.44 0.42 0.43 0 0 0 0.41 0.39 0 0.43 0.02 0 0.44 0.41
Bile acid sulfates stability during alkaline hydrolysis. From lithocholic acid sulfate submitted to the entire alkaline hydrolysis procedure, 15% were recovered as a compound showing the same properties as lithocholic acid as judged by TLC, GLC and GLC-MS analysis. When the alkaline medium was not heated but instantly treated with diethyl ether for free bile acids extraction, a smaller amOunt (8% of the starting material) of the lithocholic acid was recovered. Taurolithocholic acid sulfate submitted to alkaline hydrolysis gave also only lithocholic acid. The major constituent of the products from alkaline hydrolysis of chenodeoxycholic acid 7-sulfate was probably a ring B insaturated derivative of lithocholic acid. In fact, from chenodeoxycholicacid 'I-sulfate only 0.2% were recovered as chermdeoxycholicacid but 55% were recovered as compounds showing properties similar to those of lithocholic acid. TLC analysis exibited two spots : the weaker with Rf values identical to that of lithocholic acid, the major one with slightly smaller kf value. GLC analysis vith poly S 179 as stationary phase showed one shouldered peak with retention time slightly longer than that of lithocholic acid methyl ester. Mass spectrometric fragmentationshowed that the small shoulder was due to lithocholic acid methyl ester and, for the major constituent detected in GLC, resulted in a set of m/e values by 2 units smaller than the m/e values obtained with reference lithocholic acid
Characteristic fr~~tation ions were recorded at m/e 388 @I?, m!e 373 WCH3~, m/e 370 CM-%$@, m/e 316 (M-ringA), m/e 273 @-side chain), m/e 255
methyl ester.
m/e 249, m/e 228, m/e 213, m/e 201. When incubated with 3d hydroxysteroid d~dro~enase, the degradation products of cbenodeoxycholicacid 7-sulfate gave a positive response whereas no respoase was obtained with the starting material. When
Fig.I
: Time course of bile acid sulfates solvolysis. @ Lithocholie acid sulfate, m Deoxycholicacid 12-sulfate 0 Dsoxycholic acid disulfate, A Chenodeoxycholicacid Psul) Method A (Ref : la), (----) Method B (Ref : 14). fate. (A
[24 "4C3 ~~odeo~c~lic acid disulfate was sub~tted to the alkaline hydrolysis procedure, 13X of the radioactivity wes recovered in diethyl ether extract whereas the starting material not submitted to hydrolysis was not extrsotable. Neither sofvolysis nor degr8datio~ were observed when d~o~~~~ic a&d 12-sulfateand deoxycholicdisulfate were slitted to the alkaline bydro~~sia procedure. when duplicates of human serum samples receivedequal arts of taurb-
lithocbolic acid Bulfate, and then were percolated on Amberlite XAD-2 before being submitted to ansymatic or to alkaline hydrolysis, xnre lithocholic acid (or more similar undiatinguisbablamaterial) YBB found by GLC analysis after alkaline than after enzymatic hydrolyeia. The same result vas obtained whan ehenodeoxycbolicacid 7-sulfate vae added to serum samples. Uhen serum samples received chenodeoxycholicor deoxycholic acid Ii?-sulfate instead of taurolithocbolicacid sulfate or chenodeoxycholicacid 7-sulfate, the same aoPountsof lithocholic acid were found after alkaline hydrolysis or after enzymatic hydrolysis. In every case no difference was observed between chenodeoxycholicacid or deoxycholic acid amounts recovered after enzymatic or alkaline hydrolysis. These facts show that serum bile acid determination after alkaline hydrolysis results in erroneous quantitation of lithocholic acid but not of ~nodeo~~olic acid when the serum sample contains lithocholic acid sulfate or chenodeoxycholic‘I-sulfate; such false results are not induced by chanodeoxycholicacid or deoxycholic acid 12-sulfate.
2H I 0
I 100
I 200
I 300
1 400
, so0
, 600
1
Pig.2 : Chromatographyof bile ealts on Sephadex LIZ20 (coltrmn: 13 x I,5 cm containing 6g Sephadex LD-20, flow rate 0.3 ml/mn. solvent : methanol-chloroform(l/l V/V) containing 0.01 M NaCl NaCJ.) 1 : lithocholic acid, 2 : chenodaoxycholicacid, 3 : cholic acid, 4 : taurocholic acid, 5 : deoxycholic acid i2-sulfate,6 : cheuodeoxycholic acid 7-sulfate, 7 : lithochofic acid sulfate, 8 : taurolithocholicacid Bulfate, 9 : deoxycholic acid dieulfate.
DI.!XDSSION %a
present study was undertaken to specify the behaviour
of sulfated bile acids in the course of the analytical procedures intented for serum bile acids determination,As we could not perform this
work with the 45 sulfated derivatives of the most current bile acids, we restricted our study to a limited set of bile acids including the least and the most polar ones, some compounds with intermediary polarity, and two compounds specifically sulfated at C-7 or C-12 position. The bile acid sulfates we have synthesized were previously described (13-15-18-19) ; nevertheless we have shown that the sulfation procedure of Tserng -et al (15) is suitable for derivatization at C-7 and C-12 positions and that the protecting succinoxy group permits easy purification of the intermediary products. The position and the configuration of the sulfate groups were confirmed by NMB analysis. The NMR signals of H-3, H-7 and H-12 protons are distinctly separated from the signals of other protons : they are distinguished by differences in chemical shift (5 > and are identified by the signal width : using 3 the Karplus relationship giving the J coupling values and taking into consideration the fact that H-3 is axial and H-7 and H-12 equatorial, one can calculate theoretical values for the signal width of H-3 (24 to 34 Hz), H-7 (9 to 15 Hz) and H-12 (6 to IO Hz). As an example, with deoxycholic methyl ester these signal widthes are 35 and 12 Hz, and are unambiguously attributed to H-3 and H-12 respectively.Esterification induces deshielding effect of the proton attached to the same carbon atom as the esterified hydroxyl ; in cases of succinoylation and sulfation, deshielding effects (AS) are observed as large as 0.80 to 1.20 ppm and 0.55 to 0.67 ppm respectively (table 2), permitting unambiguous identificationof the group position. The signal width remains unmodified as long as the proton configuration does so ; if the configuration is inverted, the signal width should become 12 to 20 Hz for equatorial H-3, 24 to 34 Hz for axial H-7 and 12 to 17 Hz.for axial H-12. No change is induced in signal widthes by succinoylation or sulfation at any position ; this fact demonstrates that in all intermediary or final products under study the hydroxyl group is in the ~4 configuration. Bile acids extraction from serum is currently performed either by serum treatment with ethanol or by adsorption on Amberlite XAD-2 and subsequent elution with methanol or ethanol. The former method results in poor recovery (20%) of taurocholic acid trisulfate. Concerning the latter method, one may first emphasize the fact that,
contrarily to the opinion of Tserng _et al (20), all bile acids, including the most polar ones, are firmly adsorbed on XAD-2. This suggests that the commonly used solvents are not adequate to elute bile acid sulfates : in our hands, neither methanol nor ethanol did perform satisfactory elution of sulfates. This fact could explain difficulties encountered by some authors : Eyssen et al (Zl), extracting bile acids TzC] cholic acid, found that 5 from feces of mice injected with [24 to 7% of the radioactivitywas lost during the desalting step on XAD-2, using methanol as eluting solvent. May be, the radioactivity lost on XAD-2 was that of di and trisulfates not detected in significant smount by these authors at the end of the entire procedure. On the other hand, we showed that monosulfates are eluted at 80-90% recovery rate with 10 ml ethanol containing 0.08 ml. 12N KC1 snd that satisfactory elution of the most polar sulfates necessitates 10 ml ethanol containing 0.8 ml 12N HCL. However a drawback of this procedure consists in partial solvolysis of bile acid sulfates. So, our results suggest that the currently used extraction procedures don't permit bile acid sulfates quantitation unless labelled internal standards are at hands to determine the recovery rates of the most polar bile acid sulfates, Some authors used Amberlite XAR-7 instead of XAD-2 and obtained higher recovery rates of bile acid sulfates with the former than with the latter (22-23) but they had no standard to determine the recovery rates of the most polar sulfates. Alma -et al (24) claimed that complete extraction of urinary bile acid suffates was obtained by percolation of acidified urine on XAD-2 and subsequent elution with ethanol containing ammonium hydroxide. Such a procedure doesn't seem to be suitable for serum samples ; nevertheless, it is worth studyinS. Bradlow (25) recently devised a new procedure to elute steroid hormone monosulfates and glucuronides from XAD-2 columns as their triethylammoniumsalts. Such a procedure might be convenient for elution of di and trisulfatedbile acids but even so, Bradlow's method (25) is lon8er and does not result in higher yields thsn the procedure xe just proposed above. In our hands, the solvolysis procedure of Parmentier -et al (13) achieved complete desulfation of unconjugated bile acid sulfates without artifact formation, which confirms the results obtained by these
authors. Now, further work is needed to study the solvolysis of conjugated bile acid sulfates. Van Berge Henegouwen -et al (14) designed an analytical procedure involving an alkaline hydrolysis step but no specified solvolysis step, claiming that spontaneous desulfation occured during acidification of the media and extraction with diethyl ether. Our results show that spontaneous solvolysis occurs only for sulfate groups located at C-3 position ; this fact together with the undermentionned degradation of some sulfates during alkaline hydrolysis casts doubts on the reliability of this procedure. Class separation of sulfated from unsulfated bile acids on Sephadex LB-20 is not SC complete as claimed by authors using this fractionation procedure (1,3,4) ; figure 2 shows mutual overlapping of taurocholic acid and deoxycholic acid 12-sulfate.Clean separation necessitates enzymatic hydrolysis prior to fractionation on Sephadex LH-20. No degradation is observed when bile acid sulfates are submitted to enzymatic hydrolysis but complete deconjugation of taurocholic acid trisulfate was not obtained within 4 hours. Exhaustion of our [24 "CJ taurocholic acid trisulfate standard prevents us from determining the conditions necessary for total hydrolysis ; nevertheless, these results show that bile acid sulfates are deconjugated by cholyglycine hydrolase even if deconjugation proceeds more slowly. Performing enzymatic hydrolysis on serum sample would offer two advantages : first, bile acid sulfate elution from XAD-2 would be easier ; next, separation of sulfated from unsulfated bile acids on Sephadex LH-20 would be improved. Alkaline hydrolysis carried out under conditions currently used for bile acids determination, results in the transformationof lithocholic acid sulfate into lithocholic acid and induces formation of an unsulfated monohydroxylated compound from chenodeoxycholicFsulfate. This compound was tentatively identified as 34
-hydroxy-5p
-chol-6-
en-24 oic acid. In TLC and GLC it exhibited nobilities very similar to those of lithocholic acid. It gave a positive response with 3di hydroxysteroid dehydrogenase. The MS spectrum exhibited signals with the m/e values expected for a 3-hydroxy cholenoic acid methyl ester particular7 at m/e 249 indicating the presence of a double bond at C6-C7 or CS-
~6 position (26) : in the present case, C6-C7 position is more likely. Nevertheless, the presence of 3 dhydroxy-5 p -chol-6-en-24 oic acid, evidenced by the m/e 249 signal, does not exclude the presence of 3d hydroxy-lie-chol-7~~1-24 oic acid : the obtained mass spectrum may be that of a mixture of these two isomers. Such a mixture of zwnoineaturated compounds with the double bond located either a C6-C7 or at C7-Cg position was found by Eyssen et al (21) and Parmentier et al (13) after alkaline hydrolysis of cholic acid 7-sulfate. When cbenodeoxycholicacid 7-sulfate or lithocholic acid sulfate were added to serum sample from which duplicates were treated in the ssme way, one by alkaline hydrolysis, and the other by enzymatic hydrolysis, we always found more lithocholic acid in the first thsn in the second. Hence, as human serum always contains large amounts of bile acid sulfate (2,3,4,5,6),it becomes obvious that serum lithocholic acid level may be systematicallyoverestimatedby some analytical procedures in unsulfated bile acid determination involving an alkaline hydrolysis step. T.nother respects, some authors performing alkaline hydrolysis on serum samples prior to CLC analysis, found "abnormal" bile acids as, for example, 3d hydroxy cholenoic or 3 t hydroxy cholenoic acids (27). It seems to us that at least a part of these abnormal bile acids are artifacts from alkaline hydrolysis. Our first conclusion is that further studies are needed to design analytical procedures permitting satisfactory extraction of the most polar serum bile acid sulfates and quantitative separation of the less polar ones. Our second conclusion concerns the procedures intended to uneulfated bile acids determination : alkaline hydrolysis, elution from XAD-2 with acidified solvents and extraction with diethyl ether at low pH must be avoided when lithocholic acid determination is wanted. The last conclusion regards the enzymatic determinationof serum bile acids : as some bile acid sulfates with free 3Mhydroxyl
group give
positive response whereas some others don't, systematic work is needed to evaluate the influence of these facts on the reliability of the results obtained by enzymatic determination.
88
REFERENCES
This research was supported by the Institut National de la SantC et de la Recherche sdicale (F.R.A. ~'5) and by the Centre National de la Recherche Scientifique (E.R.A.n' 560).
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