Highly simplified method for gas-liquid chromatographic quantitation of bile acids and sterols in human stool

Transcription

1 Highly simplified method for gs-liquid chromtogrphic quntittion of bile cids nd sterols in humn stool Ashok Kumr Btt, 1, * Gerld Slen,*, Keshv R. Rpole,* Mnju Btt, Priti Btt, Dvid Alberts,** nd Dvid Ernest** Deprtment of Medicine,* Gstroenterology Division, University of Medicine nd Dentistry-New Jersey Medicl School, Newrk, NJ; Deprtment of Veterns Affirs Medicl Center, Est Ornge, NJ; Swrthmore College, Swrthmore, PA; nd University of Arizon Medicl Center,** Tucson, AZ Abstrct A simple method for the gs liquid chromtogrphic quntittion of humn fecl bile cids nd sterols is described where bile cids re subjected to n-butyl ester derivtiztion, without prior isoltion from the stool, followed by trimethylsilyltion of the sterols nd bile cids. Under these conditions, bile cid derivtives re well resolved from ech other nd from the trimethylsilyl ether derivtives of fecl sterols nd no overlp occurs. The method ws shown to be highly reproducible nd recoveries were similr to those obtined with other methods used for fecl bile cid nlysis. Appliction of the method for bile cid nd sterol nlysis in humn stool is described. Btt, A. K., G. Slen, K. R. Rpole, M. Btt, P. Btt, D. Alberts, nd D. Ernest. Highly simplified method for gs liquid chromtogrphic quntittion of bile cids nd sterols in humn stool. J. Lipid Res : Supplementry key words bile cids bile cid n-butyl ester-trimethylsilyl ethers cpillry gs liquid chromtogrphy humn fecl bile cids fecl sterols ftty cids Bile cids re the mjor ctbolic products of cholesterol nd fcilitte the excretion of bile lipids including cholesterol, nd the bsorption of dietry lipids including ft-soluble vitmins, vi their detergent ction. During their enteroheptic circultion, pproximtely 5% of bile cids escpe rebsorption nd seep into the colon where they re subjected to modifiction by intestinl bcteri to form secondry bile cids. There is significnt circumstntil evidence to suggest tht secondry bile cids, in prticulr the 7 -dehydroxylted bile cid, deoxycholic cid, ct s co- crcinogens in colon cncer. Thus, incresed mounts of deoxycholic cid hve been reported in ptients with colon cncer s compred with controls by some uthors, but not ll (1 3). However, fecl bile cid pttern is highly complex, due to extensive bcteril metbolism of bile cids during intestinl trnsit nd vrious mono-, di-, nd/or trioxo compounds nd iso- (3 hydroxy), urso- (7 -hydroxy), nd lgo- (12 -hydroxy) bile cids hve been reported (4 6). Thus, lthough incresed mounts of deoxycholic cid my be ssocited with colon polyp formtion, it is possible tht some of the other secondry bile cids my lso be co-crcinogenic. We hve recently shown tht ursodeoxycholic cid results in reduction of colon polyp in experimentl rts (7). We hve now extended this study to humns, nd s prt of the study, we needed to screen lrge number of hospitl out-ptients for fecl bile cid mesurements. Although, severl gs-chromtogrphic (GLC) methods hve been used for the purpose (4, 5, 8, 9), they re ll cumbersome nd time-consuming nd re not pplicble for routine screening purposes. We describe simple method for simultneous quntittion of fecl bile cids nd sterols, which ws found to be highly reproducible nd gives results comprble with other well estblished methods. EXPERIMENTAL Regents nd chemicls Ursodeoxycholic nd ursocholic cids were gifts from Tokyo Tnbe, Jpn. Nor-cholic cid ws synthesized in our lbortory ccording to literture (10). Other bile cids were purchsed from Sterloids (Wilton, NH). Cholesterol, cmpesterol, sito- Abbrevitions: The following bbrevitions nd trivil nmes hve been used: GLC, gs liquid chromtogrphy; TMS, trimethylsilyl; Silprep, hexmethyldisilzne: trimethylchlorosilne: pyridine, 3: 1: 9; lithocholic cid, 3 -hydroxy-5 -cholnoic cid; deoxycholic cid, 3, 12 -dihydroxy-5 -cholnoic cid; iso-deoxycholic cid, 3,12 -dihydroxy-5 -cholnoic cid; chenodeoxycholic cid, 3,7 -dihydroxy-5 cholnoic cid; cholic cid, 3,7,12 -trihydroxy-5 -cholnoic cid; ursodeoxycholic cid, 3,7 -dihydroxy-5 -cholnoic cid; ursocholic cid, 3,7,12 -trihydroxy-5 -cholnoic cid; nor-deoxycholic cid, 3,12 -dihydroxy-24-nor-5 -choln-23-oic cid; nor-cholic cid, 3,7, 12 -trihydroxy-24-nor-5 -choln-23-oic cid; 12-oxo-lithocholic cid, 12- oxo, 3 -hydroxy-5 -cholnoic cid; coprostnol, 5 -cholestn-3 -ol; cholesterol, 5-cholesten-3 -ol; 24-methylcoprostnol, 24-methyl-5 -cholestn- 3 -ol; cmpesterol; 24-methyl-5-cholesten-3 -ol; 24-ethylcoprostnol, 24-ethyl-5 -cholestn-3 -ol; stigmsterol, 24-ethyl-5,22-cholestdien-3 ol; sitosterol, 24-ethyl-5-cholesten-3 -ol, sitostnol, 24-ethyl-5 -cholestn- 3 -ol, fucosterol, stigmst-5,24(28)-dien-3 -ol; nd lnosterol, 4,4,14- trimethyl-cholest-8,24-dien-3 -ol. 1 To whom correspondence should be ddressed Journl of Lipid Reserch Volume 40, 1999

2 sterol, coprostnol, fucosterol, stigmsterol, lnosterol, 4-cholesten-3-one, nd cholestnone were from Sterloids, Inc. 24-Methylnd 24-ethylcoprostnol were isolted from the sterol frction of humn stool by combintion of thin-lyer chromtogrphy nd high-performnce liquid chromtogrphy nd their structures were confirmed by mss spectr. All ftty cids used in the study were purchsed from Aldrich Chemicl Co. (Milwukee, WI). Methyl esters of the bile cids nd ftty cids were prepred by ddition of 0.1 ml of 3% nhydrous methnolic hydrochloric cid (Aldrich Chemicl Co.) to 5 20 g of the respective bile cid or ftty cid nd were kept t room temperture for 2 h followed by evportion of solvent t 55 C under N 2. All compounds were 98% pure s judged by GLC of the trimethylsilyl (TMS) ether derivtives nd exhibited mss spectrl frgmenttion ptterns comptible with their structures. Sil-prep (hexmethyldisilzne: trimethylchlorosilne: pyridine, 3: 1: 9) used for preprtion of TMS ether derivtives of the bile cid esters ws purchsed from Alltech Assocites (Deerfield, IL). Gs chromtogrphy A Hewlett-Pckrd model 6890 gs chromtogrph equipped with flme ioniztion detector nd n injector with split/splitless device for cpillry columns ws used for ll seprtions. The chromtogrphic column consisted of chemiclly bonded fused silic CP-Sil-5 CB (sttionry phse, 100% dimethylsiloxne) cpillry column (25 m 0.22 mm I.D.) (Chrompck, Rritn, NJ) nd helium ws used s the crrier gs. The GLC operting conditions were s follows: injector nd detector tempertures were 260 C nd 290 C, respectively. After injection, oven temperture ws kept t 100 C for 2 min, then progrmmed t rte of 35 C/min to finl temperture of 278 C (11). Gs chromtogrphy mss spectrometry Mss spetrometry of sterols nd bile cids, when needed, ws crried out on Hewlett-Pckrd model 5988 gs chromtogrph mss spectrometer using 25-m fused silic CP-Sil-5 CB cpillry column. Butyl ester formtion Bile cids or ftty cids (10 20 g ech) were tken in n-butnol (200 l) nd 50 l concentrted hydrochloric cid ws dded. The contents were heted t 60 C for 4 h nd solvents were removed t 60 C. Trimethylsilyltion The esterified bile cid or the sterol (5 10 g) ws rected with 100 l of Sil-prep for 30 min t 55 C. Solvents were evported t 55 C under N 2 nd the TMS ether derivtive formed ws tken in 100 l of hexne nd 1 l ws injected into the GLC column. The retention times of the vrious bile cids were clculted reltive to tht of nor-cholic cid. Quntittion of fecl bile cids nd sterols To mg freeze-dried stool (weighed exctly) ws dded internl stndrd (nor-cholic cid, 20 g) in 200 l of n-butnol followed by 50 l concentrted hydrochloric cid nd the contents were subjected to butyl ester formtion s described bove. The esterified product ws directly subjected to trimethylsilyltion. The TMS ether derivtives formed were tken in 200 l of hexne, centrifuged to seprte the stool debris, nd 1 2 l of the cler superntnt ws injected into the GLC column. Quntittion of fecl bile cids nd sterols by sodium hydroxide/solvent extrction method Freeze-dried stool (10 15 mg), to which 20 g of nor-cholic cid nd 20 g 5 -cholestne were dded, ws digested with 1 ml of 1 N sodium hydroxide for 1 h t 90 C in screw-cp tube (12). After cooling, the product ws diluted with wter (5 ml) nd repetedly extrcted with n-hexne (4 3 ml). The n-hexne ws evported to dryness nd the residue ws subjected to trimethylsilyltion. An liquot ws used for GLC to quntitte the neutrl sterols. The queous lyer fter extrction of neutrl sterols ws cidified to ph 1 with 5 N hydrochloric cid followed by extrction with ethyl cette (4 3 ml). Ethyl cette lyer ws wshed with wter to neutrlity, evported to dryness, nd the residue ws subjected to butyl ester formtion nd trimethylsilyltion. An liquot ws then used for GLC to quntitte bile cids. Quntittion of fecl bile cids nd sterols by Soxhlet extrction method Freeze-dried stool (10 15 mg) ws trnsferred into smll pper thimble, together with 20 g nor-cholic cid nd 20 g of 5 -cholestne, nd ws subjected to continuous extrction with 1% mmonicl ethnol for 16 h in Soxhlet extrctor (8). Ethnol ws evported to dryness nd the residue ws tken up in 5 ml of 0.5 N sodium hydroxide. The neutrl sterols were then extrcted with n-hexne (4 5 ml). The n-hexne ws evported to dryness nd the residue ws subjected to trimethylsilyltion. An liquot ws used for GLC to quntitte the neutrl sterols. The queous solution fter removl of neutrl sterols ws cidified to ph 1 with 5 N hydrochloric cid nd bile cids were isolted nd derivtized s described bove. An liquot ws then used for GLC. Quntittion of fecl bile cids nd sterols fter extrction with chloroform methnol Freeze-dried stool (10 15 mg), to which nor-cholic cid (20 g) nd 5 -cholestne (20 g) were dded, ws heted with 5 ml chloroform methnol 2:1 t 65 C for 0.5 h in screw-cp tube. After centrifugtion, the superntnt ws collected nd the residul fecl mteril ws gin extrcted with chloroform methnol s described bove. The combined solution from four such extrctions ws evported to dryness, the residue ws tken up in 5 ml of 0.5 N sodium hydroxide, nd the neutrl sterols nd bile cids were extrcted s described bove under the Soxhlet extrction method. Aliquots were used for quntittion of neutrl sterols nd bile cids by GLC. RESULTS In n erlier publiction, Child, Aloe, nd Mee (13) showed tht the cette derivtives of the n-butyl esters of severl bile cids nd ftty cids nd of number of sterols were resolved from ech other on GLC nd the vrious compounds could be quntitted in presence of ech other. We recently showed tht the trimethylsilyl ether derivtives of the n-butyl esters of common bile cids were eluted lter thn ll plsm sterols, including lte eluting sitosterol, thus obviting the need for complete removl of sterols during plsm bile cid nlysis (14). In n extension of our method, we hve found tht when mixture of severl fecl ftty cids, sterols, nd bile cids ws subjected to the conditions of n-butyl ester followed by trimethylsilyl ether formtion nd injected into the gs chromtogrph, ech clss of compounds ws resolved from the others. Tble 1 shows the retention times of the n-butyl ester trimethylsilyl ether derivtives of number of ftty cids, bile cids, nd sterols tht re usully reported in Btt et l. Cpillry GLC of humn fecl bile cids nd sterols 1149

3 TABLE 1. GLC retention times of trimethylsilyl ether-n-butyl esters of bile cids, trimethylsilyl ethers of sterols, nd n-butyl esters of ftty cids on CP-Sil-5-CB cpillry column TMS Ether of RRT TMS Ether of RRT Nor-CA-n-butyl ester methylcoprostnol LCA-n-butyl ester ethylcoprostnol Oxo-5 -cholnoic cid-n-butyl ester cmpesterol cmpestnol DCA-n-butyl ester sitosterol CDCA-n-butyl ester sitostnol CA-n-butyl ester fucosterol UDCA-n-butyl ester lnosterol UCA-n-butyl ester plmitic cid Coprostnol heptdecnoic cid Cholesterol steric cid Cholestnol plmitoleic cid Cholestnone oleic cid Cholesten-3-one linoleic cid Stigmsterol rchidonic cid The n-butyl ester of ftty cid, n-butyl ester-tms ether of the bile cid, or the TMS ether of the sterol (0.1 g in 1 l hexne) ws injected onto fused-silic CP-Sil-5 CB cpillry column. The injector nd detector tempertures were kept t 260 C nd 290 C, respectively. After injection, oven temperture ws kept t 100 C for 2 min, then progrmmed t rte of 35 C/min to finl temperture of 278 C. Nor-CA, nor-cholic cid; LCA, lithocholic cid; DCA, deoxycholic cid; CDCA,chenodeoxycholic cid; CA, cholic cid; UDCA, ursodeoxycholic cid; UCA, ursocholic cid. Retention times re expressed reltive to tht of the n-butyl estertrimethylsilyl ether of nor-cholic cid (retention time, min). humn stool. Clerly, n-butyl esters of ftty cids re eluted from the GLC column s group well seprted from the trimethylsilyl ethers of sterols, which re in turn eluted before the n-butyl ester trimethylsilyl ethers of bile cids, nd there is no overlp between the vrious clsses of compounds. With pproprite temperture progrmming, better resolutions between individul ftty cids my be obtined nd more complex mixture of ftty cids my lso be resolvble. We hve lredy shown tht the retention times of the n-butyl ester trimethylsilyl ether derivtives of the bile cids re highly reproducible nd, for mounts of bile cids rnging from 0.02 to 0.2 g injected onto the column, the detector response, s shown by the integrtor, is liner (14). Even though the n-butyl ester trimethylsilyl ethers of chenodeoxycholic cid nd cholic cid did not show bseline resolution, when known mounts of these bile cid derivtives were injected together, the detector response s shown by the integrtor corresponded to the mounts injected in rnge of g of the bile cid (14). Under the derivtiztion conditions, dehydrtion of bile cids ws found to be miniml (13). In n effort to pply our method for quntittion of bile cids in fecl smples, we weighed pproximtely 10 mg of freeze-dried humn stool nd subjected it to the derivtiztion procedure; n liquot of the hexne extrct ws then subjected to GLC. The secondry bile cids, lithocholic cid nd deoxycholic cid, were the predominnt bile cids in the stool, with smller mounts of isodeoxycholic cid nd 12-ketolithocholic cid (Fig. 1). Sterols were completely resolved from the bile cids nd did not interfere with bile cid quntittion s shown in Fig 1. Thus, the mjor fecl sterols were eluted from the column between 14 nd 21 min. Sitosterol (retention time, min), the mjor lte eluting fecl sterol, ws eluted well before nor-cholic cid, the bile cid with the shortest retention time ( min) nd ws used s the internl stndrd. There ws no overlp between the two groups of compounds. In ddition to the bile cids, cholesterol nd its bcteril metbolite, coprostnol, nd plnt sterols, cmpesterol nd sitosterol, nd their bcteril metbolites, 24-methyl-5 -cholestn-3 -ol (24-methylcoprostnol), 24-ethyl-5 -cholestn-3 -ol (24-ethylcoprostnol), stigmsterol, nd sitostnol were ll present nd well seprted from ech other nd the bile cids (Fig. 1). All mjor peks seen in the chromtogrm were identified s due to bile cid derivtives or the trimethylsilyl ether derivtives of sterols, s confirmed by their mss spectrl frgmenttion pttern. Our method ws found to be reproducible for fecl sterol nd bile cid nlysis nd the recoveries of bile cids nd sterols on repet nlysis of fecl smples were found to be similr to those obtined by methods where rigorous solvent extrctions re used (Tble 2). Thus, the bile cids nd sterols were quntitted within 5% rnge in three different nlyses from given fecl smple. Also, the mounts of individul sterols nd bile cids obtined were highly comprble with those obtined when bile cids were first extrcted with ethnol in Soxhlet pprtus or with chloroform methnol, while ethyl cette extrction of the cidified stool yielded lower vlues for lithocholic cid, pprently due to incomplete extrction (Tble 2). A comprison of fecl bile cids nd sterols in five helthy individuls s obtined by our direct derivtiztion method nd three solvent extrction methods is given in Tble 3 nd Tble 4. Lithocholic cid nd deoxycholic cid were the predominnt fecl bile cids in ll subjects nd were found to be present in lmost equl mounts. Cholesterol nd coprostnol were the mjor fecl sterols nd significnt mounts of the plnt sterols, cmpesterol nd sitosterol, were lso present, but their corresponding 5 -H derivtives were not s bundnt. The mounts of both sterols nd bile cids obtined by our direct derivtiztion method were comprble to those obtined by solvent extrction methods except tht direct extrction of cidified stool showed somewht reduced mounts of lithocholic cid (Tble 3). DISCUSSION Becuse of efficient bcteril deconjugtion, bile cids in the stool re present lmost completely in the unconjugted form nd therefore the bile cid hydrolysis step cn usully be voided during fecl bile cid quntittion. However, mjor problem in fecl bile cid nlysis lies in their quntittive extrction from the stool nd highly cumbersome methods hve been used to extrct fecl bile cids. Furthermore, stool lso contins neutrl sterols, including cholesterol nd plnt sterols nd their bcteril metbolites, nd lso ftty cids, which my interfere in 1150 Journl of Lipid Reserch Volume 40, 1999

4 Fig. 1. GC chromtogrm of sterols nd bile cids present in stool from helthy control. Ten mgs of freeze-dried stool contining 20 g nor-cholic cid ws subjected to derivtiztion s described in the Experimentl section. After dissolving in 200 l hexne, 1 l ws injected into the GC column. Chromtogrphic conditions were s described in the Experimentl section. Pek identifiction: 1, nor-cholic cid; 2, lithocholic cid; 3, iso-deoxycholic cid; 4, deoxycholic cid; 5, chenodeoxycholic cid; 6, cholic cid; 9, 3- oxo,12 -hydroxy-5 -cholnoic cid; 10, 12-oxo-lithocholic cid;, coprostnol; b, cholesterol; c, 24-methylcoprostnol; d, cmpesterol; e, 24-ethylcoprostnol; f, stigmsterol; g, sitosterol, nd h, sitostnol. GLC nlysis. Eneroth, Hellstrom, nd Sjovll (8) used continuous extrction of liquots of homogenized stool for 16 h with chloroform methnol, hydrolyzed the extrct with strong lkli, nd removed neutrl compounds by solvent extrction followed by cidifiction nd isoltion of bile cids by continuous extrction with ethyl ether. The bile cids were further purified by preprtive TLC s the methyl esters nd prepred for GLC. In nother elborte method developed by Grundy, Ahrens, nd Miettinen (9), stool ws homogenized with n equl volume of wter nd n internl stndrd of [ 14 C]deoxycholic cid ws dded followed by mild lkline hydrolysis. The neutrl sterols were extrcted out with petroleum ether nd the queous suspension ws subjected to rigorous lkline hydrolysis. After cidifiction, bile cids were extrcted with chloroform methnol 2:1, subjected to chromtogrphy over Florisil, nd the purified bile cid frction ws subjected to methyl ester formtion followed by preprtive TLC to remove the ftty cids. Bile cid frction ws then isolted nd liquots were subjected to rdioctivity mesurement, to correct for recovery, nd to GLC. In lternte methods, stool hs been extrcted with mmonicl lcohol, methnol hydrochloric cid, cetic cid toluene, nd bile cids were extrcted fter removl of neutrl sterols (15 18). As ftty cids re less strongly retined on the cpillry GLC columns, bile cids cn usully be quntitted in the presence of ftty cids. However, sterols still need to be seprted, s they cn interfere with bile cids during GLC nlysis. Thus, the GLC retention time of the TMS ether of cholesterol is close to tht of the methyl ester of lithocholic cid on CP-Sil-5 CB cpillry column, while the plnt sterols, cmpesterol nd sitosterol, which re usully present in the stool, re eluted in the generl bile cid region (14). Becuse the retention times of bile cids increse when their esters with higher homologs re used insted of the methyl ester trimethylsilyl ethers (19), Tscons et l. (20) prepred the isobutyl ester TMS ethers of bile cids nd found tht most common bile cids were well resolved from sterols on cpillry GLC. However, we found tht lthough the isobutyl ester TMS ethers of bile cids were eluted lter thn the TMS ether of cholesterol on CP-Sil-5 CB cpillry column, the plnt sterol, sitosterol, mjor fecl sterol, ws eluted lter thn lithocholic Btt et l. Cpillry GLC of humn fecl bile cids nd sterols 1151

5 TABLE 2. Reproducibility of quntittion of fecl sterols nd bile cids TABLE 3. Bile cid nlysis in humn stool by gs-chromtogrphy: comprison of different methods Compound Current Method Soxhlet Extrction b Folsch Extrction c NOH/ Extrction d Bile Acid Current Method Soxhlet Extrction b Folsch Extrction c NOH/ Extrction d g/mg dry stool LCA Iso-DCA DCA Coprostnol Cholesterol Methylcoprostnol Cmpesterol Ethylcoprostnol Sitosterol Men SD of nlysis performed on 3 seprte smples of the freeze-dried humn fecl smple used in Fig. 1. Gs-chromtogrphic conditions re described in Tble cholestne ws dded, ws directly subjected to n-butyl ester formtion followed by TMS ether derivtiztion. After ddition of hexne (200 l), 2 l ws used for GC. Sterols were quntitted ginst 5 cholestne nd bile cids were quntitted ginst nor-cholic cid s the internl stndrd. b 5 -cholestne ws dded, ws continuously extrcted for 18 h with mmonicl ethnol in Soxhlet extrctor. Sterols were extrcted with n- hexne nd bile cids were then extrcted with ethyl cette. Sterol frction ws subjected to silyltion nd the bile cid frction ws subjected to n-butyl ester formtion followed by TMS ether derivtiztion. After ddition of hexne (200 l) to ech frction, 2 l ws used for GC s described bove. c 5 -cholestne ws dded, ws extrcted with chloroform methnol 2:1 (4 5 ml). Sterols were extrcted with n-hexne fter which bile cids were extrcted with ethyl cette. Sterols nd bile cids were quntitted by GC exctly s described bove under the Soxhlet extrction method. d 5 -cholestne ws dded, ws digested with 1 N sodium hydroxide t 90 C for 1 h, diluted with wter (5 ml), nd sterols were extrcted with n-hexne. Bile cids were then extrcted with ethyl cette. Sterols nd bile cids were quntitted by GC exctly s described bove under the Soxhlet extrction method. g/mg dry stool LCA e IsoDCA DCA Other f Totl Men SD from five helthy subjects. Gs-chromtogrphic conditions re described in Tble 1. Freeze-dried stool (10 15 mg), to which 20 g of ech of norcholic cid nd 5 -cholestne ws dded, ws directly subjected to n- butyl ester formtion followed by TMS ether derivtiztion. After ddition of hexne (200 l), 2 l ws injected into the gs-chromtogrph. Bile cids were quntitted ginst nor-cholic cid s internl stndrd. b Freeze-dried stool (10 15 mg), to which 20 g of ech of norcholic cid nd 5 -cholestne ws dded, ws continuously extrcted for 18 h with mmonicl ethnol in Soxhlet extrctor. Sterols were extrcted with n-hexne nd then bile cids were extrcted with ethyl cette nd subjected to n-butyl ester formtion followed by TMS ether derivtiztion. After ddition of hexne (200 l), 2 l ws used for GC s described bove. c Freeze-dried stool (10 15 mg) ws extrcted with chloroform methnol 2:1 (4 5 ml). After evportion of solvents, the residue ws tken in 0.5 N sodium hydroxide nd sterols were extrcted with n-hexne. Bile cids were then extrcted with ethyl cette nd subjected to n-butyl ester formtion followed by TMS ether derivtiztion. After ddition of hexne (200 l), 2 l ws used for GC s described bove. d Freeze-dried stool (10 15 mg) ws digested with 1 N sodium hydroxide t 90 C for 1 h, diluted with wter (5 ml), nd sterols were extrcted with n-hexne. Bile cids were then extrcted with ethyl cette nd subjected to n-butyl ester formtion followed by TMS ether derivtiztion. After ddition of hexne (200 l), 2 l ws used for GC s described bove. e LCA, 3 -hydroxy-5 -cholnoic cid; DCA, 3,12 -dihydroxy-5 cholnoic cid; Iso-DCA, 3,12 -dihydroxy-5 -cholnoic cid. f Includes chenodeoxycholic cid, cholic cid, ursodeoxycholic cid, ursocholic cid, 3-oxo,12 -hydroxy-5 -cholnoic cid, nd 12- ketolithocholic cid. cid. Furthermore, nor-deoxycholic cid nd nor-cholic cid, routinely used s internl recovery stndrds, were lso eluted in the sme region s the sterols. In nother method, Child et l. (13) prepred the n- butyl ester cette derivtives of bile cids nd showed tht these bile cid derivtives were resolved from sterols commonly present in stool. They used the method for fecl bile cid nlysis fter solvent extrction of stool but without prior seprtion of fecl sterols. Fced with severl drwbcks of cette-n-butyl ester derivtives of bile cids, including reduced sensitivity of cette derivtives (11) nd incresed retention times, we recently prepred the n-butyl ester TMS ether derivtives of bile cids nd found tht the method could be conveniently used to quntitte plsm bile cids even with incomplete removl of plsm sterols (14). It ws lso found tht the n-butyl ester TMS ether derivtive of nor-cholic cid ws eluted lter thn the mjor fecl sterols nd this bile cid could sfely be used s n internl recovery stndrd. In our ttempt to pply this derivtiztion method for fecl bile cid nlysis, nd in the process to further simplify the method for routine screening of fecl smples, we digested freeze-dried stool (together with nor-cholic cid s internl stndrd) with sodium hydroxide nd extrcted out neutrl sterols followed by extrction of bile cids which were directly derivtized for GLC. A comprison of this method with erlier methods tht used more rigorous solvent extrction for isoltion of fecl bile cids (8, 9) showed lower levels of lithocholic cid by sodium hydroxide/solvent extrction of the stool (Tble 2) thus suggesting tht this bile cid needed more rigorous solvent extrction. All methods were, however, generlly comprble for quntittion of fecl sterols (Tble 2). Thus, the non-polr fecl sterols re quntittively isolted by repeted extrction with hexne, wheres the more polr bile cids need rigorous extrction procedures. It is lso likely tht the bile cids re tightly bound to the bcteril debris in the stool nd re, therefore, difficult to extrct quntittively (21). Of the severl other methods reported for extrction of fecl bile cids, we were ttrcted by the method of vn den Ende et l. (17) where they used methnol concentrted hydrochloric cid to extrct bile cids from fecl smples. As the use of methnol hydrochloric cid would result in t lest prtil methyl ester formtion of bile 1152 Journl of Lipid Reserch Volume 40, 1999

6 TABLE 4. Sterol nlysis in humn stool by gs-chromtogrphy: comprison of different methods Sterol Current Method Soxhlet Extrction b Folsch Extrction c NOH/ Extrction d g/mg dry stool Coprostnol Cholesterol Methylcoprostnol Cmpesterol Ethylcoprostnol Stigmsterol Sitosterol Totl Men SD from five helthy subjects. Gs-chromtogrphic conditions re described in Tble 1. Freeze-dried stool (10 15 mg), to which 20 g of ech of nor-cholic cid nd 5 -cholestne ws dded, ws directly subjected to n-butyl ester formtion followed by TMS ether derivtiztion. After ddition of hexne (200 l), 2 l ws used for GLC. Sterols were quntitted ginst 5 -cholestne s internl stndrd. b Freeze-dried stool (10 15 mg) ws continuously extrcted for 18 h with mmonicl ethnol, in Soxhlet extrctor; sterols were extrcted with n-hexne nd subjected to TMS ether derivtiztion. After ddition of hexne (200 l), 2 l ws used for GLC s described bove. c Freeze-dried stool (10 15 mg) ws extrcted with chloroform methnol 2:1 (4 5 ml). After evportion of solvents, the residue ws tken in 0.5 N sodium hydroxide nd sterols were extrcted with n-hexne nd subjected to TMS ether derivtiztion. After ddition of hexne (200 l), 2 l ws used for GLC. d Freeze-dried stool (10 15 mg) ws digested with 1 N sodium hydroxide t 90 C for 1 h, diluted with wter (5 ml), nd sterols were extrcted with n-hexne nd subjected to TMS ether derivtiztion. After ddition of hexne (200 l), 2 l ws used for GLC s described bove. cids in the presence of smll mounts of wter (17), we considered tht this would enhnce extrction of bile cids, prticulrly lithocholic cid, from the feces, bile cid methyl esters being more soluble in orgnic solvents. Thus, hydrochloric cid my free the bile cids bound to the bcteri with simultneous esterifiction. With this in mind, we considered treting the freeze-dried stool directly with n-butnol/hydrochloric cid with the im to extrct fecl bile cids nd convert them into their n-butyl esters. Tretment of the stool contents with Sil-prep would now form the TMS ethers of the bile cid esters nd of the neutrl sterols, which could be solubilized in n-hexne nd used for GLC in the usul wy. As we hve lredy shown tht the n-butyl ester TMS ethers of bile cids re well resolved from the TMS ethers of sterols, including sitosterol, on cpillry GLC (14), nd s ftty cids re known to elute significntly erlier from the column thn sterols, fecl sterols nd bile cids could be esily nlyzed in single chromtogrphic run (Fig. 1). When compred with two methods tht used rigorous solvent extrction for isoltion of bile cids, our method ws found to be highly comptible for both fecl sterol nd bile cid nlysis (Tbles 3 nd 4) nd lithocholic cid, the highly insoluble nd most difficult bile cid to isolte quntittively from the stool, ws t lest s well extrcted by the present method s by the solvent extrction methods. On the other hnd, our direct derivtiztion procedure is very simple nd ll extrctions nd pre-purifiction of bile cid frctions re eliminted. In fct, the whole procedure mounts to the direct derivtiztion of stool for GLC nlysis s if bile cids were not tightly bound to the bcteril debris. The method is thus extremely convenient for screening purposes when lrge number of smples need to be nlyzed. The wekness of the method is tht it cnnot be used for fecl bile cid nlysis under certin pthologicl conditions, where bile cids in the stool my remin conjugted with glycine nd/ or turine. An dded dvntge of n-butyl ester derivtiztion with hydrochloric cid t elevted tempertures is tht ny sterol esters re lso hydrolyzed. Thus, in n experiment when cholesterol sterte nd cholesterol heptdecnote were subjected to the bove derivtiztion procedure, both compounds were completely hydrolyzed to produce the TMS ether of cholesterol nd the n-butyl ester of the corresponding ftty cid. Ironiclly, tretment with methnol hydrochloric cid t room temperture for 4 h filed to hydrolyze these ftty cid esters of cholesterol while complete hydrolysis ws observed t 60 C. It is likely tht polymerized or esterified deoxycholic cid, suspected to be present in feces (22, 23), is lso converted into deoxycholic cid under the cidic conditions used for derivtiztion. In summry, we hve developed highly simplified method for fecl sterol nd bile cid nlysis where the bile cids re directly converted into their n-butyl ester TMS ether derivtives nd sterols re converted to the TMS ether derivtives, without prior isoltion from the stool. In this method, the elborte extrction of bile cids from stool, removl of neutrl sterols, nd chromtogrphic purifiction of bile cids prior to derivtiztion for GLC re voided. This not only renders the method esy to perform but the usul errors tht occur during multiple extrctions re lso eliminted. Under most circumstnces, the method is pplicble to stool nlysis, where more thn 98% bile cids re present in unconjugted form nd their n-butyl ester TMS ethers cn be directly formed. However, this method for fecl bile cid nlysis is not pplicble in certin pthologicl conditions or in germ-free nimls, where conjugted bile cids re expected in the stool. We hve used this method for quntittion of bile cids in humn stool nd obtined mounts of bile cids com- Btt et l. Cpillry GLC of humn fecl bile cids nd sterols 1153

7 prble to those obtined by estblished methods tht used rigorous solvent extrction of bile cids nd removl of neutrl sterols before derivtiztion nd quntittion by GLC. This work ws supported by U.S. Public Helth Service grnt HL-17818, grnt from the Veterns Affirs Reserch Service, Wshington, DC, nd Ntionl Cncer Institute grnt PO1-CA The contents of this publiction re solely the responsibility of the uthors nd do not necessrily represent the officil views of the Ntionl Cncer Institute. Mnuscript received 21 September 1998 nd in revised form 5 Februry REFERENCES 1. Hill, M. J., nd V. C. Aries Fecl steroid composition nd its reltionship to cncer of the lrge bowel. J. Pthol. 104: Reddy, B. S., J. H. Weisburger, nd E. L. Wynder Colon cncer: bile slts s tumor promoters. In Mechnisms of Tumor Promotion nd Crcinogenesis. Vol. 2. T. J. Slg, A. Sivk, nd R. K. Boutwell, editors. Rven Press, New York Mudd, D. G., S. T. D. McKelvey, W. Norwood, D. T. Elmore, nd A. D. Roy Fecl bile cid concentrtions of people with crcinom or incresed risk of crcinom in the lrge bowel. Gut. 21: Eneroth, P., B. Gordon, R. Ryhge, nd J. Sjovll Identifiction of mono- nd dihydroxy bile cids in humn feces by gs liquid chromtogrphy nd mss spectrometry. J. Lipid Res. 7: Eneroth, P., B. Gordon, nd J. Sjovll Chrcteriztion of trisubstituted cholnoic cids in humn feces. J. Lipid Res. 7: Tohm, M., R. Mhr, H. Tkeshit, nd T. Kurosz A convenient synthesis of 3,1 -, 3,7 -, nd 3,7 -dihydroxy-5-cholen- 24-oic cids: unusul bile cids in humn biologicl fluids. Steroids. 48: Ernest, D. L., H. Holubec, R. K. Wli, C. S. Jolley, M. Bissonnette, A. K. Bhttchryy, H. Roy, S. Khre, nd T. A. Brsitus Chemoprevention of zoxymethne-induced colonic crcinogenesis by supplementl dietry ursodeoxycholic cid. Cncer Res. 54: Eneroth, P., K. Hellstrom, nd J. Sjovll A method for quntittive determintion of bile cids in humn feces. Act Chem. Scnd. 22: Grundy, S. M., E. H. Ahrens, nd T. A. Miettinen Quntittive isoltion nd gs liquid chromtogrphic nlysis of totl fecl bile cids. J. Lipid Res. 6: Schteingrt, C. D., nd A. F. Hofmnn Synthesis of 24-nor- 5 -choln-23-oic cid derivtives: convenient nd efficient onecrbon degrdtion of the side chin of nturl bile cids. J. Lipid Res. 29: Btt, A. K., G. Slen, M. Btt, D. Ernest, nd D. Alberts Cpillry gs liquid chromtogrphy of cette-methyl esters of bile cids. J. Chromtogr. (A). 766: Btt, A. K., G. Slen, H. Holubec, T. A. Brsitus, D. Alberts, nd D. Ernest Enrichment of the more hydrophilic bile cid ursodeoxycholic cid in the fecl wter-soluble frction fter feeding to rts with colon polyps. Cncer Res. 58: Child, P., M. Aloe, nd D. Mee Seprtion nd quntittion of ftty cids, sterols, nd bile cids in feces by gs chromtogrphy s the butyl ester-cette derivtives. J. Chromtogr. 415: Btt, A. K., G. Slen, K. R. Rpole, M. Btt, D. Ernest, nd D. Alberts Cpillry gs chromtogrphic nlysis of serum bile cids s the n-butyl ester-trimethylsilyl ether derivtives. J. Chromtogr. (B). 706: Setchell, K. D. R., A. M. Lwson, N. Tnid, nd J. Sjovll Generl methods for the nlysis of metbolic profiles of bile cids nd relted compounds in feces. J. Lipid Res. 24: Trjumn, N., nd C. Jcob Diet, nutrition intke, nd metbolism in popultions t high nd low risk for colon cncer. Am. J. Clin. Nutr. 40: vn den Ende, A., C. E. Rdecker, W. M. Miruhu, nd A. P. vn Znten Improved extrction procedure for determintion of bile cids in feces. Clin. Chim. Act. 121: Evrd, E., nd G. Jnssen Gs liquid chromtogrphic determintion of humn fecl bile cids. J. Lipid Res. 9: Miyzki, H., M. Ishibshi, nd K. Ymshit Use of new silylting gent for seprtion of bile cids nd cholesterol by selected ion monitoring with the computer controlled intensity mtching technique. Biomed. Mss Spectrom. 5: Tscons, C., P. Pdieu, G. Mume, M. Chessebeuf, N. Hussein, nd N. Pitoizet Gs chromtogrphy mss spectrometry of isobutyl ester trimethylsilyl ether derivtives of bile cids nd ppliction to the study of bile sterol nd bile cid biosynthesis in rt liver epithelil cell lines. Anl. Biochem. 157: Gustfsson, B. E., nd A. Normn Comprison of bile cids in intestinl content of germ-free nd conventionl rts. Proc. Soc. Exp. Biol. Med. 110: Korpel, J. T., T. Fotsis, nd H. Adlercreutz Multicomponent nlysis of bile cids in feces by nion exchnge nd cpillry column gs-chromtogrphy: ppliction in oxytetrcycline-treted subjects. J. Steroid Biochem. 25: Benson, G. M., N. J. Hskins, C. Eckers, P. J. Moore, D. G. Reid, R. C. Mitchell, S. Wghmre, nd K. E. Suckling Polydeoxycholte in humn nd hmster feces: mjor product of cholte metbolism. J. Lipid Res. 34: Journl of Lipid Reserch Volume 40, 1999