isolation and quantitation of sulfated and unsulfated steroids in human feces

ANALYTICAL BIOCHEMISTRY 112, 371-377 (1981)

Isolation and Quantitation of Sulfated and Unsulfated Steroids in Human Feces

M. AMINUL ISLAM,ROBERT F. RAICHT,AND BERTRAM I. COHEN'

Veterans Administration Medical Center, 408 First Avenue, New York, New York 10010; and Department of Medicine, New York University Medical Center,

550 First Avenue. New York. New York IO016

Received December 15, 1980

An improved method for separation and quantitation of sulfated neutral and acidic steroids in human feces was developed. The procedure consists of separation of sulfated steroids on Sephadex LH-20 and hydrolysis by cholylglycine hydrolase followed by quantitation and identification of the trimethylsilylether derivatives by gas-liquid chromatography and gas-liquid chromatography-mass spectroscopy. Using this procedure, we detected no sulfated bile acids in human feces. However, sulfated cholesterol was detected in the sulfated bile acid fraction obtained from human fecal extracts. Analysis showed that cholesterol sulfate comprised 12.3, 11.2, and 31 .O% of the total neutral sterol fraction in the three fecal samples. Using our procedures, cholesterol sulfate and bile acid sulfates in a biological mixture can be quantitated and identified when they are present.

While using procedures to analyze and found to be formed from adenosine-3’-phos- quantitate the amount of sulfated and un- phate-5’phoso-[ 35S]sulfate when incubated sulfated bile acids in human feces, we with high-speed extracts of human breast found that cholesterol sulfate was present carcinoma tissue (5). Sulfation of lithocholic in the fecal extract supposedly containing acid is a major step in the metabolism of this the bile acid sulfates. As a result, we have bile acid in man (6). It has been suggested developed a method to quantitate this sterol that sulfation of cholesterol may play an im- in the feces. portant role in cholesterol excretion (1).

The occurrence of cholesterol sulfate (CS)2 in man was first described by Moser et al. (1) in 1966. Since then, this compound has been shown to be an active metabolite and has been detected in various tissues as well as human bile (2), feces (3), erythrocyte membranes (4), and urine. Cholesterol sulfate has been identified in patients with neu- rological disorders (1). This material was

1 Author to whom reprint requests should be ad- dressed.

2 Abbreviations used: CS, cholesterol sulfate; tic, thin-layer chromatography; glc, gas-liquid chromatography; glc-ms, gas-liquid chromatography-mass spectrometry; RRT, relative retention time; TMS, tri- methylsilyl; CDCA, [CPHlchenodeoxycholic acid.

Both sulfated bile acids (7) and cholesterol sulfate (3) along with other steroids (3) have been reported in human feces. To study the metabolism of sulfated and unsulfated steroids, an accurate method for esti- mation and separation of these two kinds of steroids is necessary. Recently, Cantafora et al. (8) has published a glc procedure for determination of sulfated and unsulfated bile acids in serum. Other procedures avail- able in the literature (1,3,5,9- 12) appear to be unsuitable for analyzing sulfated bile acids and cholesterol sulfate in feces and bile. The present paper provides an improved procedure for separation, quantitation, and identification of sulfated and un-

371 0003-2697/81/060371-07$02.00/0 Copyright Q 1981 by Academic Press, Inc. All rights of reproduction in any form reserved.

372 ISLAM, RAICHT. AND COHEN

TABLE 1

R, VALUES OF CHOLESTEROL AND CHOLESTEROL

SULFATE IN DIFFERENT SOLVENT SYSTEMS"**

Solvent system

R, values of cholesterol

R, values of cholesterol sulfate

64 0.97 0.73 (b) 0.91 0.70 (c) 0.92 0.60

a Solvent systems: (a) ethyl acetate : n-butanol : acetic acid : water (8 : 6 : 3 : 3); (b) n-propanol : ammonia : HZ0 (12 : 1: 2); (c) benzene : methanol (7 : 3).

* tic was carried out on silica gel G precoated plates, thickness 0.5 mm.

sulfated neutral and acidic steroids using column chromatography, tic, glc, and glc- ms techniques.

MATERIALS AND METHODS

Reference compounds. Standard bile acids were obtained from Supelco and their purity (95%) was checked by tic and glc. Cholesterol (98% pure) was purchased from Sigma Chemical Company. Sa-Cholestane (Applied Science Laboratories) was used as an internal standard for all glc. All other re- agents were of analytical grade. Sulfolitho- cholylglycine and sulfolithocholyltaurine were purchased from Calbiochem.

Synthesized compounds. [24J4C]Litho- cholic acid-3-sulfate and [3H]chenodeoxycholic acid-3-sulfate were synthesized and purified according to the procedure of Palmer and Bolt (13). CS and [4-14C]CS were synthesized according to the procedure of Anderson et al. (14) with certain modifications and purified by preparative tic on silica gel G in a solvent system of ethyl acetate : n-butanol : acetic acid : water (8:6:3:3) (v:v:v:v).

The purity of the synthesized cholesterol sulfate was checked by tic (Table 1). It was further characterized by glc and glc-ms (Table 2).

Analytical techniques. The tic analyses of the steroids were carried out using silica gel G precoated plates (Analtech). The

plates were prerun in methanol to remove any impurities. The plates were reactivated at 110°C for I h. Samples were applied to the plate as a band using a semiautomatic plate streaker (Applied Science Laborato- ries).

Total bile acid measurements were made on a Hewlett-Packard 5830A gas chromatograph according to the procedure pre- viously described (15,16). For this study, individual steroids (Table 2) were analyzed on 3% QF-1 (on 80/100 Gas Chrom Q) column using a Hewlett-Packard 7610A gas chromatograph. The following conditions were employed: column length, 6 ft.; id, 2 mm; oven temperature, 245°C; injector and detector temperature, 265°C.

The individual neutral sterols and bile acids were identified using a Hewlett- Packard 5992B glc-ms. The compounds were analyzed as their trimethylsilylether derivatives on 2% SP 2250 (Supelco) (simi- lar to SE-30). The following conditions were employed : helium flow, 20 ml/min; source temperature, 220°C; analyzer temperature 130°C; ion-source pressure, 2 to 3 x lop6 Torr; and ion-source electron en- ergy, 70 eV.

The radioactivity was measured, after evaporation of solvent whenever necessary, by addition of 10 ml of (2,5-bis[2-(S-tertbu-

TABLE 2

COMPARISON OF RELATIVE RETENTION TIMES (RRT) OF CHOLESTEROL AND LITHOCHOLIC ACID

AS TMS-ETHER DERIVATIVES ON 3% SE-30 AND 3% QF-I COLUMNS'

Compound

Cholesterol TMS Methyl

lithocholate-TMS Free cholesterol

RRT values RRT values on 3% SE-30 on 3% QF-1

2.10 1.53

2.10 2.51 1.86 2.67

a Sa-Cholestane was used as an internal standard. Actual retentions of So-cholestane on 3% SE-30 and 3% QF-1 columns were 2.50 and 1.80 mm, respectively. For glc conditions, see Materials and Methods.

QIJANTITATION OF SULFATED STEROIDS IN HUMAN FECES 373

Human feces

Freeze dried and ground

Ethanol extraction of acidic end neutral sterols Extraction with hexene to 'remwe free neutral sterols

Chromatography on Sephader U-20 column

/ 1 Unsulfated bile acids Sulfated steroids

1 c Enzymatic hydrolysis Enzymatic hydrolysis

I c Solvolysis Solvolysis

Methyl&ion Methyl&ion

Purification by TLC Purification by TLC

I TMS ether derivative

1 TMS ether derivative

c GLC and GLC-MS GLC and GLC-MS

FIG. I. Analytical procedure for sulfated and unsulfated steroids. The outline of methodology developed for the separation and quantitation of sulfated and unsulfated steroids is given in the figure. The steroids are extracted from the feces by ethanol. Free neutral sterols are removed by hexane extraction. Column chromatography separated sulfated from unsulfated steroids. Enzymatic hydrolysis deconjugated the steroids. This was followed by several steps including solvolysis, methylation, tic, glc, and glc-ms to quantitate and identify the compounds which were present in the fecal sample.

tylbenzoxazolyl)]) thiophene solution (4 g/liter in toluene).

Analytical procedure. An outline of our analytical procedures employed for separation of unsulfated neutral and acidic steroids is shown in Fig. 1.

Extraction and analysis of feces. Fresh human fecal samples were obtained from three patients without history of gastroin-

testinal disorders. The feces were freeze dried at - 40°C for 48 h. One sample in duplicate was analyzed from each patient. To one sample we added [4-14C]cholesterol-3- sulfate (3.9 pg) and to the other we added [24J4C]lithocholic acid-3-sulfate (2.7 Fg). The fecal aliquots (1 .O- 1.5 g) were extracted with ethanol according to the method of Cohen ef al. (15,16). One frac-

tion was used to determine neutral and acids. Hydrolysis of the samples eluted acidic steroids using procedures described from the LH-20 column was accomplished earlier (16). A portion of the fecal extract using cholylglycine hydrolase EC 3.5.1.24 was refluxed 1 h with 20 ml of 1 N NaOH. (Sigma Chemical Co.) at 37°C for 12 h (8). [4J4C]Cholesterol was added to correct for The following reaction mixture was used losses of neutral sterols. The neutral sterols for this purpose: acetate buffer, pH 5.6, 0.4 were extracted with hexane while the acidic M (0.5 ml); EDTA 0.2 M (0.1 ml), 2-mercap- steroids remained in the aqueous phase. toethanol, 0.2 M (0.1 ml), and cholylglycine

The aqueous phase containing acidic hydrolase (0.1 ml containing 47 U), in a steroids was evaporated under N, to re- total volume of 2 ml. It was found that cho- move ethanol. Water (20 ml) and 2 ml of 10 lylglycine hydrolase does not affect the sul- N NaOH were added to a final concentra- fate group of bile acids. This enzyme com- tion of 2 N. The solution was autoclaved for pletely hydrolyzed the taurine and glycine 3 h at 120°C (14-15 psi), cooled and ad- groups from sulfolithocholylglycine and justed to pH l-2 with concentrated HCl. sulfolithocholyltaurine standards. Each sample was then quickly shaken with Solvolysis of the bile acids. After enzy- 80 ml of chloroform: methanol (2: 1) and matic hydrolysis of the conjugated bile stored overnight. The lower phase was re- acids, the samples were diluted with water moved and the aqueous phase was reex- and acidified with HCl at 0°C to pH l-2. tracted with chloroform. The organic phase Each sample was extracted with chloro- was evaporated to dryness. Benzene : meth- form: methanol, 2 : 1, and allowed to stand anol (86 : 14) was added and the sample was for 4 h at room temperature. The aqueous reevaporated. This procedure was done phase was reextracted twice with chloro- twice. Methyl esters of bile acids were pre- form. The organic fractions were evap- pared by dissolving the residue in 5 ml of orated to dryness, redissolved in ben- 5% HCl in methanol and left at room tem- zene : methanol (86 : 14), and reevaporated. perature for 18 h. The methyl esters were The dry residue was dissolved in 5 ml of 5% separated on tic and analyzed by glc as methanolic HCl to form the methyl esters TMS-ether derivatives on 3% SE-30. which were purified by tic (16).

Another fraction was extracted twice with 80 ml of hexane to remove any unsul- RESULTS fated neutral sterols. The ethanol was evaporated and the dry residue redissolved in 1

Separation of Sulfated and Unsulfated

ml of chloroform : methanol (60 : 40) mixture Steroids

saturated with dry sodium chloride. This Sulfated and unsulfated steroids were material was chromatographed on 3 g Seph- separated on Sephadex LH-20. A typical adex LH-20 (Pharmacia) in a column chromatographic separation of a mixture of (1 x 28.5 cm). The column was equili- [G-3H]chenodeoxycholic acid (CDCA) and brated with 75 ml of chloroform : methanol [24J4C]lithocholic acid-3-sulfate is shown (60 : 40) saturated with sodium chloride. in Fig 2. The unsulfated steroid [G- Unsulfated steroids were eluted with the 3H]CDCA was eluted in tubes 6-12 with same solution (Fig. 1) whereas the sulfated chloroform : methanol while the sulfated steroids were subsequently eluted with steroid [24-14C]lithocholic acid-3-sulfate methanol. The flow rate of the column was was eluted in tubes 22-30 using methanol 0.5 ml/min. as solvent. Cholesterol sulfate was also

Enzymatic hydrolysis of conjugated bile chromatographed on the same column and

314 ISLAM, RAICHT, AND COHEN

QUANTITATION OF SULFATED STEROIDS IN HUMAN FECES 375

its elution pattern is shown in Fig. 2. It was eluted with methanol in the same fraction as the sulfated bile acids.

Recoveries of Sulfated Steroids Using the Present Procedure

In order to study the different steps of the analytical procedure described in Fig. 1, we analyzed known amounts of purified cholesterol-3-sulfate and lithocholic acid-3- sulfate both individually and as mixtures without feces. The samples were chromatographed on the Sephadex LH-20 column, followed by elution of the samples with methanol. These were then subjected to enzymatic hydrolysis. The standard samples of sulfolithocholylglycine and sulfolithocholyltaurine were found to be completely deconjugated but not desulfated using the conditions described earlier. The samples were subsequently acidified, extracted, methylated, and the methyl esters were purified by tic. Finally, TMS-ether derivatives of both cholesterol and methyl litho-

FIG. 2. LH-20 chromatography of sulfated and unsulfated steroids. The figure shows the LH-20 chromatography of [WHjchenodeoxycholic acid ([WCDCA), [24J4C]lithocholic acid sulfate ([WILCA-S), and [4-Ylcholesterol sulfate ([V]CS). The labeled chenodeoxycholic acid (r3H]CDCA) was eluted in tubes 6- 12 using chloroform: methanol (saturated with sodium chloride) as solvent. The labeled sulfated lithocholic acid ([Y]LCA-S) was eluted in tubes 22-30 using methanol as solvent. A second column was run with labeled cholesterol sulfate ([YJCS) which was eluted in the same fraction as the lithocho- lit acid sulfate (fractions 22-30 using methanol as solvent).

TABLE 3

RECOVERY OF CHOLESTEROL SULFATE AND

LITHOCHOLIC ACID SULFATE BY

PRESENT PROCEDURE’

Sample

Choleslerol sulfate Lithocholic acid sulfate Mixture of cholesterol

sulfate and lithocholic acid sulfate

Amounts Amounts used recovered I mg) (ms)

5.00 4.59 5.00 4.49 5.00 4.80 5.00 4.34

PtXentage recovery

91 89 96 87

D Known amounts of cholesterol sulfate and lithocholic acid sulfate were analyzed using all the steps in the methodology described in the text. The amounts reported represent the final recoveries using glc on 3% SE-30 after preparation of the TMS-ether derivatives.

cholate were analyzed by glc on a 3% QF-1 column. The results of these analyses are shown in Table 3. Recoveries ranged from 87 to 96%.

Quantitation of Cholesterol and Lithocholic Acid by Gas -Liquid Chromatography

Our analyses of human feces indicated that cholesterol sulfate and/or lithocholic acid sulfate were present in the sulfated fraction. Therefore, we developed a procedure to identify and estimate cholesterol in pres- ence of lithocholic acid. Table 2 shows the comparison of relative retention times of the TMS-ether derivatives of cholesterol and methyl lithocholate on 3% SE-30. Both of these compounds have the same RRT values (2.1) on 3% SE-30 column. However, cholesterol TMS-ether, and methyl lithocholate TMS-ether derivatives have different RRT values on 3% QF-1 (1.53 vs 2.51, respectively). It is possible to identify and estimate those compounds from a mixture using a 3% QF- 1 column. Free cholesterol has RRT values of 1.86 and 2.67 on 3% SE-30 and 3% QF-1 column, respectively, and would not, therefore, interfere with the analysis.

Methyl lithocholate TMS-ether was identified by glc-ms. Major fragments were

376 ISLAM. RAICHT, AND COHEN

TABLE 4

ANALYSIS OF HUMAN FECAL SAMPLES AND RECOVERY OF THE LABELED CHOLESTEROL SUL- FATE AND LITHOCHOLIC ACID SULFATE ADDED TOTHE FECES

RWWEry Recowy of Cholesterol Human Cholesterol of labeled labeled sulfate

fecal sulfate cholesterol lithocholic corrected samples (&g fecesp sulfate* acid sulfatee (Pgk feces)

1 (A) 1688 67% - 2519

(W 1539 - 83% 2297 2 (A) 1254 12% - 1741

(B) 1208 - 81% 1677

3 (A) 626 73% - 857

(W 603 - 82% 826

a Cholesterol sulfate was determined using glc analyses of duplicate samples.

b 60.000 dpm (2.7 @g) of [4-‘%]cholesterol sulfate was added to the samples et the beginning of the procedure.

’ 52,000 dpm (3.9 ~8) of [24-‘%]lithocholic acid sulfate was added to the samples at the beginning of the procedure.

identical to a known standard [m/e (abundance %)I 215 (80.0), 216 (33.5), 217 (12.6), 230 (20.4), 257 (36.7), 357 (27.7), 358 (7.0), 372 (loo), 373 (27.9), 462 (6.2). Cholesterol TMS-ether was identified by its mass spec- trum [m/e (abundance%)] 247 (21.8), 255 (21.3), 328 (28.5), 329 (IOO), 330 (28.4), 353 (37.9), 368 (99.3), 458 (47.3), 459 (17.7), also identical to a known standard.

Quantitation of Sulfated and Unsulfated Steroids in Human Feces and Reproducibility of Analyses

The method shown in Fig. 1 was employed to analyze three human fecal samples for sulfated and unsulfated steroids. The unsulfated sterols (neutral sterols ex- cept cholesterol sulfate) were analyzed according to our previous procedure ( 15). The remaining sterols containing sulfated bile acids (if present), cholesterol sulfate and unsulfated bile acids were separated on LH- 20 column and worked up as described in Fig. 1. Cholesterol was quantitated on a 3% QF-1 column and identified by glc-ms. The recoveries of cholesterol sulfate and lithocholic acid sulfate were established after add- ing labeled cholesterol sulfate and lithoch-

olic acid sulfate to the fecal samples at the start of the procedure. The radioactive compounds were carried through all the steps of the procedure. The average recoveries of cholesterol sulfate and lithocholic acid sulfate in three samples were found to be 70.6 f 3.2% and 82.0 f I%, respectively. Cor- rections were made for losses. The results obtained by replication (n = 2) of the analysis of a fecal sample are shown in Table 4.

Cholesterol sulfate was found to be 12.3, 11.2, and 31 .O% of the total neutral sterols determined using standard methods (15). Sulfated bile acids were found to be neg- ligible.

DISCUSSION

Podesta et al. (7) reported that sulfated bile acids comprised as much as 30-40% of the bile acids in human feces. Conse- quently, we examined human fecal extracts to determine the amounts of these compounds in our samples. We found one major component in the bile acid fraction by glc. Subsequent analyses by glc-ms showed this compound to be cholesterol and not a bile acid. In fact, we detected no sulfated bile acids. Thus, we modified our procedure (15,16) to isolate and quantitate both sulfated and unsulfated neutral sterols as well as acidic steroids in fecal extracts.

Our studies showed that no sulfated acidic steroids were present in the feces obtained from patients with no gastrointestinal disorder. However, cholesterol sulfate (a neutral sterol sulfate) comprised 12.3, 11.2, and 3 1 .O% of the total neutral sterols in three fecal samples. It is anticipated that our modified procedure will be useful for studying the metabolism of sulfated bile acids and cholesterol sulfate in patients with various gastrointestinal disorders.

ACKNOWLEDGMENTS

This research was supported by Grant CA27438 from The National Large Bowel Project through the National Cancer Institute (NIH) and grants to Robert

QUANTITATION OF SULFATED STEROIDS IN HUMAN FECES 377

F. Raicht and Bertram I. Cohen from the Veterans Ad- ministration. We wish to thank Miss Rubell Smith for her expert secretarial effort.

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isolation and quantitation of sulfated and unsulfated steroids in human feces

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