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An improved method for the isolation, quantitation, and identification of bile acids in rat feces
ANA,
WITAL
64. 567-577
BIOCHEMISTRY
An Improved Method and Identification B. 1. COHEN’, AND
Received
July
( I975
I
for the Isolation, Quantitation, of Bile Acids in Rat Feces R. F. RAICH?. G. E. H . MOSBACH’.’
IO. 1974;
accepted
SAI
November
EN:‘.
7. I974
An improved method for the isolation and quantitation of bile acids from rat feces was developed. This method employs an initial Soxhlet extraction of the solid fecal material. esteritication of the bile acid fraction with dry methanol/HCl and quantitation using a combination of tic and glc technique\. In addition. identification complished tion and ments.
of by
the individual tic and glc-ms.
identification
of
the
components This method fecal
bile
acid\
of the fecal has proven during
bile acid fraction is acuseful for the quantita-
sterol
metabolism
measure-
A method for accurately measuring fecal acidic steroids is necessary for sterol metabolism studies. Chromatographic methods for the isolation and quantitation of these fecal components have been reported for man (12). Certain modifications of these methods were required in the rat and a description of these modifications form the basis of this report. In addition, the fecal bile acids were isolated and identified (using a combination of tic and glc-ms) in order to assure accurate quantitation. The techniques described in this report provide a means for quantitation and identification of fecal acidic steroids in the rat. MATERIALS AND METHODS
I. Bile ucid stwtdur.ds. Cholic acid (Wedell Pharmaceuticals. London, England). deoxycholic acid (Ames Co., Elkhart, IN), chenodeoxycholic acid (IPDC. New Rochelle, NY). and lithocholic acid (I( & K Laboratory. Plainview. NY) were all better than 95% pure by glc on SE-30 and were used without further purification. Samples of U- and fl1 Department of Lipid Research of the New Yorh, Inc., 455 First Avenue. New L Manhattan V.A. Hospital. New York. East Orange V.A. Hospital., ’ Addres\ requests for reprints \titute. 455 Fit-St Avenue. New
Public York. NY
Health Research NY IOOlh. 10010.
East Orange. NJ 07 IO?. to: Dr. Erwin H. Mosbach. Yorh. NY 10016.
Institute
Public
Health
of the
Research
(‘it);
of
In-
568
COHEN ETAL.
muricholic acid were prepared as described by Hsia (3). 3cu,7cuDihydroxy-l?-keto-5/3-cholanoic acid ( I’-keto standard) was synthesized according to Fieser and Rajagopalan (4). 2. Su-Cholestrrne. (Applied Science Lab., State College, PA) was used as the internal standard for the glc analyses. 3. Thin layer chromatography. The methods used were essentially those of Miettinen. Grundy, and Ahrens (1.2). The bile acids were separated on 0.5-mm layers of silica gel HR (Brinkmann Instruments, Westbury, NY). Prior to use for bile acid analysis, impurities were removed by developing the plates in methanol-acetic acid (95:5, v/v) followed by activation at 100°C for I h. The plates were stored in a desiccator over drierite (Fisher Sci. Co.) prior to use. Samples were applied to the plates as a band using a semiautomatic sample streaker equipped with a 500 ~1 syringe (Applied Science Lab.). Solvents used for chromatographic separations were reagent grade or spectra grade quality and were used without further purification. 4. Gas-liquid clwotmtograplzy. Separations of the bile acids were carried out on a Hewlett Packard Gas Chromatograph 7610A equipped with hydrogen flame ionization detectors and a dual pen recorder. Ushaped columns (3 mm i.d., 6-ft length) were silanized and packed with 3% SE-30 on 100/l 20 mesh Supelcoport (Supelco Inc., Bellafonte. PA) or 1% HiEff 8BP on 100/l 20 Supelcoport. Prepurified nitrogen was used as a carrier gas at a flow rate of 20-30 ml/min and an inlet pressure of 40 psi. Measurements of peak areas were accomplished with a Hewlett Packard Automatic Integrator 3370B adjusted to record retention time in minutes (with automatic baseline correction). Operating conditions for the column, inlet, and detector were ?50”, 280”, and 280°C. respectively. All glc-ms measurements were carried out on a Varian MAT Ill. The column packings and operating conditions of the glc were similar to those of the 7610A. 5. Radiouctikity measurements. These were carried out using a Beckman LS-200 Liquid Scintillation system. The radioactivity of each sample was measured, after evaporation of solvent, by the addition of 10 ml of BBOT (2,5-bis-2-(5-tert-Butylbenzolyl))-thiophene solution (4 g/liter in toluene). Procedures 1. Extraction offeces. Fecal samples were collected and dried under vacuum over drierite for 48 h. The cholesterol pools of several rats were labeled with DL-[2-'%I mevalonolactone to obtain feces containing radioactive neutral and acidic steroids. An aliquot of 1.0-1.5 g of powdered fecal material was placed into an extraction thimble. The reservoir bottle for the Soxhlet extraction was a 125 ml reagent bottle (Corning No.
ANALYSIS
OF
FECAL
BILE
569
ACIDS
1500) which was charged with 80 ml of ethanol and 100 pg of the 13-keto standard. The thimble was placed into a Soxhlet extractor and the material was extracted for 48 h. The final volume was reduced to 20 ml under nitrogen. All subsequent reactions. centrifugations and extractions were carried out in the reservoir bottle. 2. Srpmution oj‘tlc~rrttwl stcwicis .f~otll acidic. stc~roicis. The procedures used were those of Miettinen et rrl. (3). 3.
M~~thods
,fi)r.
isolutiotl
mti
qutrtltittrtiotl
of’
acidic
steroids
,frottl
~~IIJ c,.t-trtrc’tiotl. The aqueous phase containing the acidic steroids in I N NaOH was evaporated at 5OYI to remove the ethanol. The volume was brought to 30 ml with distilled water and 2 ml of IO N NaOH was added (final normality = 3, N). The solution was autoclaved for 3 h at 120°C and 14-15 psi. The sample was cooled on ice and acidified with concentrated HCI to pH 1-3. Each sample was shaken with 80 ml of choloroform-methanol (2: I, v/v) and centrifuged immediately or stored overnight. The lower phase was removed and the aqueous phase was reextracted twice more with 50 ml of chloroform each time. The organic fractions were evaporated to dryness. The samples were dissolved in 30 ml of benzene-methanol (86: 14. v/v) and reevaporated to dryness. Estrrijicdon of’ Bile Acids. The dry bile acid residue was dissolved in 5 ml of 59% HCI (by titration) in dry methanol. The reaction mixture was sealed and allowed to remain at room temperature for 18 h. The solution was evaporated to dryness on an evaporator. Each sample was subsequently redissolved in 30 ml of the benzene-methanol solvent and reevaporated to remove any traces of HCI. The esterified bile acids were dissolved in chloroform-methanol (2: 1. v/v) and quantitatively transferred to test tubes. The samples were dried and redissolved in 2 ml of chloroform. tic. x/c. tit1tt glc-tns of’ tllr hilr uc,itl nlc~thyl cstrrs. A suitable aliquot of the esterified bile acid mixture was applied to a silica gel HR plate. The plate was developed first using benzene and then using isooctane-isopropanol-acetic acid (120: 40: I, v/v/v). The area from the origin to below the fatty acid band (visualized with iodine) contained all the bile acids and was removed from the plate using the procedures of Grundy (1). The bile acids in this region were quantitated by glc after making the trimethylsilylether (TMS) derivatives of the methyl esters. 5tr-Cholestane was added to all samples as an internal standard for the glc analyses on SE-30. In several cases, for the purpose of identifcation of the individual bile acid methyl esters, the mixture was plated on silica gel HR and developed in benzene-acetone (60:40, v/v). Bands were scraped corresponding to known samples of (a) methyl lithocholate. R, = 0.78: (b) methyl deoxycholate, R, = 0.50: and (c) methyl
,/i,c.c.\
-.\lr/‘c”li~ic,rrtic,~~
570
COHEN
ET AL.
cholate, R, = 0.22. In addition, materials at the origin, solvent front and between the known bands were analyzed using glc and glc-ms. RESULTS AND
DISCUSSION
This report describes a procedure for the extraction, isolation and quantitation of acidic steroids (bile acids) from rat feces. The method is similar to that previously described by Grundy and others (1 S-8) but was modified for use in sterol metabolism studies in the rat. An important modification of existing procedures consisted of the Soxhlet extraction of the fecal samples with ethanol. This step was necessary to prevent boilover of the samples during the hydrolysis of the acidic steroids in the autoclave either prior to or after removal of the neutral sterol fraction. Boilover of the sample frequently resulted in losses as high as 7040% of the total sample. Consequently, hot ethanolic extraction of the samples was employed to remove the steroids from the solid insoluble fecal material. No boilover of the samples occurred during hydrolysis after removal of the solid material. Completeness of this extraction was verified by combustion of “C-labeled fecal samples before and after extraction and determination of the amount of radioactivity present. Table 1 shows that the extraction step removes 95% of the neutral and acidic steroids from the feces. The I?-keto standard (3a,7cu-dihydroxy- I2-keto-5p-cholanoic acid) was added to the fecal samples to correct for losses of acidic steroids which occurred during the procedure. The glc detector response of the TMS ether of the 12-keto standard and the SLu-cholestane standard was I : I on a weight basis only on SE-30 ( 15). Previous studies showed that the 12-keto standard exhibited the same stability to rigorous saponification as the major fecal bile acids (I ). Recovery of this standard averaged 77% or better for 25 fecal samples. Losses of fecal acidic steroids were corrected accordingly. TABLE
I
VALIDA-IIONOFEXTRACTIONPROCEDURESFOR
Sample 1 2 3 4
Before extraction (dpmiO.75 16870 35300 22400 17200
g)
FECAL
After extraction (dpmi0.25 979 2260 1100 1300
g)
SAMPLES"
Completeness of extraction Y5r;“r 945; 95% 9x?
” Rats were labeled with DL-[2-'JC]mCVa10nOlaCtOne. Feces were collected. dried and ground. Aliquots of fecal material were cornbusted before and after ethanolic Soxhlet extraction and the amount of [“‘C]CO, counted. Duplicate samples were counted and the counts corrected for quench.
ANALYSIS
OF
FECAL
TABLE QUANTITATION
Lithocholic acid Deokycholic acid Chenodeoxycholic Cholic acid I?-Keto standard
acid
” The bile esterification
were esterified with the TMS derivatives
acids using
571
ACIDS
?
OF BII E ARID
No. of determination5
Sample
BILE
ESTERIFKXTION”
Average recovery with benzenemethanol evaporation (C)
5 5 5 5 8
Average recovery without benzenemethanol evaporation (“iI
9x YX 98 99 95 methanol/HCI of the methyl
and analyzed esters on pk.
for completeness
of
Esterification of the acidic steroids was carried out with dry methanol containing 5% HCl. It has been reported that this method of esterification is unsatisfactory since recoveries of cholic acid were as low as 18% (9). We have determined that traces of water present in the bile acid standards and fecal samples were responsible for the incomplete esterification. This problem was eliminated by evaporating the samples with a mixture of benzene-methanol (86:14, v/v) before adding the methylating reagent. The results of carrying out the methylation with and without the benzene-methanol evaporation step are summarized in Table 9. Nearly quantitative esterification was obtained when the evaporation step was used whereas incomplete esterification occurred when it was omitted. Quantitative methylation of the bile acids using methanolHCI was comparable to diazomethane (6). Methanol-HC1 has the advantage in that it is stable for periods up to 6 months when stored at -20°C. After esterification with methanol, the bile acid methyl esters were analyzed as the TMS ethers by glc on SE-30. For quantitation it was necessary to determine which glc peaks in the total fecal acidic steroid fraction were bile acids and which were not: therefore a detailed study using a combination of tic, glc. and glc-ms was carried out. A sample of methyl esters of the acidic steroid fraction (approximately 0.5 g) was applied to a preparative silica gel HR plate and developed in benzeneacetone (60:40, v/v). Standards of methyl lithocholate. methyl deoxycholate, and methyl cholate were applied to the plate. After development, the acidic steroids were made visible by spraying the plate with Bile-Spra (Supelco). Bands corresponding to the known bile acids were observed (Fig. 1). The bands were eluted with methanol and analyzed as the TMS ethers by glc. Peaks were observed in the various fractions (Fig. 1).
571
COHEN
ET
AL.
2A [I
I
L3 3A
FIG.
la.
tic,
and
glc-ms
analysis
of
fecal
bile
acids.
tic
of the
fecal
esters on silica gel HR using benzene-acetone (60:40, v/v) separated droxy (band I ). dihydroxy (band 2). and trihydroxy (band 3) cholanoic sponding to known samples of: (a) methyl lithocholate: (b) methyl methyl cholate H,= 1.00: Band
R, = 0.34; band
was
Peaks were are reported
Band made
were 1.
seen.
R,=0.78:
3. R, = 0.22: after addition
identified relative
The
plate Band
was IA,
divided
Band 3A. I’, = 0.00-o. of an internal standard
by their retention times and to the internal standard (actual
seven bands:
into
R,=O.h8:
bile
Band
2.
acid
R,=O.50:
Solvent Band
IO. glc and glc-ms analyses and preparation of the TMS glc-ms time
methyl
them into monohyacids. Bands corredeoxycholate: and (c)
data (Table = 5.16 min).
3).
Retention
front, ZA,
of each ethers. times
For preliminary identification, the retention time of each peak in each band was determined and compared to known bile acid standards. For positive identification of each peak in each band, analysis by glc-ms was employed using an SE-30 column in a Varian MAT 111mass spectrometer. The identification of each peak by means of mass spectrometry is shown in Table 3. The identified material amounted to 93% of the total bile acids in the fecal bile acid fraction. Material with a retention time shorter or longer than SLu-cholestane(Fig. 1) and which was found to be nonsteroidal (hydroxy fatty acids) was not used in the calculation of total bile acids. This material amounted to as much as 25% of the total bile acid fraction. Using this material in determining total daily bile acid output would have resulted in an overestimation by this amount (25%). All glc analyses were run for periods of time up to I h. In some cases peaks emerged after 45 min which were found to be nonsteroidal as well. The bile acids which were identified in the fecal material were: lithocholic, deoxycholic, IL!-ketolithocholic. cholic, LYmuricholic, p-muricholic, and w-muricholic acid. w-Muricholic acid has not been previously reported in rat feces. Its identification was made by mass-spectral data (Fragments at m/e 195 and m/e 38.5 are typical of this compound (11)) and by its retention time on SE-30 and HiEff 8BP (12). A representative glc pattern of the total fecal acidic steroid fraction, prior to separation into individual components by tic is shown in Fig. 2. Peaks 3-9 were used to calculate the total bile acids in the sample.
ANALYSIS
OF
FECAL
BILE
ACIDS
Sld I
:
ltl,KIlM
’
5
10
15 20 Mmutes
25
45
50 Minutes FIG
I.
Ih.
Peaks 1. 2, and 10 which did not have the steroid nucleus and peak 8 which was the I?-keto standard were not used in the calculation of total bile acids. In summary. we have reported a method for the isolation and quantitation of the fecal acidic steroids in the rat. The method permits accurate
5500 476(19) 684( 1)
55OY 564( 3)
5 6 7
8 9
IA
2
Band
Band
372(9) 46% 1) 462( 1) -
1 2 3 4
m/e Peak” M+’ .~._______~.__
Solvent Front Band 1
Band no.”
535(6) 549(3)
535(6) 461( 1) 669(5)
357(21) 453(22) 447(4) -
M-15
460(l) 474(30)
460( I) 386( 15) 579(12J
372(36) -
M-90
445(I) 459(3)
445(l) 371(5)
-
345(12) 359(16)
1
399(21)
489( 14) 370(15) 384(45)
345(12)
-
M-(115+ 90) -~-
-
-
-
M-(1 1.5+ (2X90)
ETHERS OF METHYL
370(15)
-
-
-
M-(3X90)
TABLE 3 STEROIDS AS THE TMS
M-(2X90)
ACIDIC
M-(90+ 15) ~-~ 357(23) -
MASS SPECTRA OF FECAL
255(100) 229( 100)
?55(100) 230( 100) 296( 100)
215(100) 73( 100) 215(100) -
Base peak
ESTERS
d
methyl deoxycholate” methyl 3Lu,7wdihydroxy-I?keto-5B-cholanoateP
methyl deoxycholate” methyl I?-ketolithocholatt? no steroid nucleus
no steroid nucleus no steroid nucleus methyl lithocholate” unknown bile acid
Compound identification
3
3A
Band
Band
-
-
identification
mass spectrum ma\ \prctrtlm detected.
” The
’ The ‘. The ‘i Not
of’ thi\ of this
i\ made
’ M + i\ the moleculw ion. theses repI-esent the relative
obtained in each
-
1)
on silica tic h;md
533(
-
4590)
533(i) 533( 1)
100)
gel tic (Fig. by glc analysis
-
458(29) 458(h)
458(6) X34(421
458(
I) for the bile of the TMS
368(41) 368t17)
-
368( 14) 368(4)
the
,, ,,
compound compound
using
dau
wa\ identical corresponded
combined to that to that
of tic-K,
plc
retention
of a known srnndard. reported in the literature
values
16)
I)
(1 I. 12).
:\nd
-90).
acid methyl ethers.
-
JU(3) -
35%
43%
time\.
m/e represent loss of various fragment\ CH St - 15). TMS-OH( intensity of each peak w\ a percent of the parent peak (100).
IO the bands peaks obtained
-
548(3) 538( I)
-
638”
638g
14 I.(
-
-
-
474130)
53W)
I.3
548(4) 548(Z)
638 cl 5640)
II I2
-
638 cl
I0
” Etlch band corresponded b Numbers rcprc\ent the
2A
Band
data
steroid
chain( fol- abundance
side
-
ester\.
195( 100)
@c-m\
and
-
100)
153(100)
XYt
35X( 100) 195(100)
343(5)
343QOI
-
-
343(3) -
fragments.
numbers
cholatc” w-muricholate’
of v;ll-iou\
- I I5 ). The
no peak5
methyl methyl
methyl 3a.7a-dihydroxy-12. keto-S/3-cholanoate’ unknown bile acid
methyl a-muricholate’ methylp-murichc~laTe’
in paren-
576
COHEN
ET AL.
Std
9
-lion5
10
15
20
25 Minutes
Peak
R?-
3 4 5 4 7 8 9 IO
I 25 I 63 2 12 251 2 78 3 37 3 67 4 42 4 84 8 93
30
45
50
55
FIG. 2. Analysis of total fecal bile acids by glc. glc of a mixture of fecal bile acids as the TMS ethers was run on SE-30. Retention times are reported relative to the internal standard Sa-cholestane (actual time = 5.16 min). Peaks I. 2, and IO are not bile acids (corresponding to peaks I, 2, and 7 in Table 3). Peak 8 represents the methyl ester of 3~. 7adihydroxy-l2-keto-S@holanoate standard added to each sample. Peaks 3-7 and peak 9 are the fecal bile acids used for calculations of the total bile acid output. The bile acids in the peaks are: peak 3, lithocholate: peak 4. deoxycholate: peak 5. cholate and LYmuricholate; peak 6, 12.ketolithocholate; peak 7. P-muricholate: and peak 9. Wmuricholate. Conditions for the glc analysis are given in the text.
determination of the total fecal bile acids as well as subsequent identification of the individual bile acids. ACKNOWLEDGMENTS We wish to thank Dr. G. S. Tint for determining the mass spectra and Miss Cathy Ross of Dr. Zilversmit’s laboratory for the combustion of the fecal samples. This research was supported in part by grants HL 10894 and AM 05222 from the U.S. Public Health Service, GB 3 19 19X from the National Science Foundation. and a grant from the I.P.D. Corporation.
REFERENCES I. Grundy,
S. M., Ahrens, IO. 3. Miettinen. T. A.. Ahrens.
E. H.. Jr.,
and
Miettinen.
T.
A. (1965)
J. Lipid
Res.
6,
J. Lipid
Rcs.
6,
397-4
E. H.,
Jr..
and
Grundy.
S. M.
(1965)
411-424.
Hsia. S. L. (1971) ilr The Bile Acids: Chemistry, Physiology, and Metabolism (Nair, P. P., and Kritchevsky. D., eds.). I st ed.. Vol. I, pp. 95-120. Plenum Press, New York. 4. Fieser. L., and Rajagopalan. S. (I 950) J. Amer. Cltenr. So<,. 72. 5530-5536.
3.
ANALYSIS
OF
FECAL
BlLE
ACIDS
577
5. SjBvall. J. (1964) irl Methods of Biochemical Analysis (Glick, D.. ed.). Vol. I?. pp. 97-141, Interscience, New York. 6. Eneroth. P.. and Sjavall. J. (1969) in Methods in Enzymology (Clayton. R. B., ed.). Vol. 15. pp. 237-280. Academic Press. New York. 7. Horning, E. C., Brooks, C. J. W.. and Vandenheuvel. W. J. (1968) irl Advances in Lipid Research (Paoletti. R., and Kritchevshy. D.. eds.). Vol. 6, pp. 3-73-391. Academic Press. New Yorh. 8. Makita. M.. and Wells, M. (1963) .4rlcl/. Birwhrm. 5, 523-530. 9. Grundy. S. M.. and Metzger, A. L. t 1972) G[r.srrr)c,r~fero/o~~ 62, 1100-l 2 17. 0. Feher. T.. Papp, J.. and Kazik. M. H. (1973) C‘/i,r. Chim. Acrc~. 44, 409-418. I. Elliott. W. H. (1972) in Biochemical Applications of Mass Spectrometry (Wailer. G. R.. ed.), 1st ed., pp. 391-311. John Wiley and Sons. New York. ?. Elliott. W. H.. Walsh. L.. B.. Mui. M. M., and Thornem. M. A. ( 1969) ./. CI~~~,rt~rrtog. 44,45z!-464.
WITAL
64. 567-577
BIOCHEMISTRY
An Improved Method and Identification B. 1. COHEN’, AND
Received
July
( I975
I
for the Isolation, Quantitation, of Bile Acids in Rat Feces R. F. RAICH?. G. E. H . MOSBACH’.’
IO. 1974;
accepted
SAI
November
EN:‘.
7. I974
An improved method for the isolation and quantitation of bile acids from rat feces was developed. This method employs an initial Soxhlet extraction of the solid fecal material. esteritication of the bile acid fraction with dry methanol/HCl and quantitation using a combination of tic and glc technique\. In addition. identification complished tion and ments.
of by
the individual tic and glc-ms.
identification
of
the
components This method fecal
bile
acid\
of the fecal has proven during
bile acid fraction is acuseful for the quantita-
sterol
metabolism
measure-
A method for accurately measuring fecal acidic steroids is necessary for sterol metabolism studies. Chromatographic methods for the isolation and quantitation of these fecal components have been reported for man (12). Certain modifications of these methods were required in the rat and a description of these modifications form the basis of this report. In addition, the fecal bile acids were isolated and identified (using a combination of tic and glc-ms) in order to assure accurate quantitation. The techniques described in this report provide a means for quantitation and identification of fecal acidic steroids in the rat. MATERIALS AND METHODS
I. Bile ucid stwtdur.ds. Cholic acid (Wedell Pharmaceuticals. London, England). deoxycholic acid (Ames Co., Elkhart, IN), chenodeoxycholic acid (IPDC. New Rochelle, NY). and lithocholic acid (I( & K Laboratory. Plainview. NY) were all better than 95% pure by glc on SE-30 and were used without further purification. Samples of U- and fl1 Department of Lipid Research of the New Yorh, Inc., 455 First Avenue. New L Manhattan V.A. Hospital. New York. East Orange V.A. Hospital., ’ Addres\ requests for reprints \titute. 455 Fit-St Avenue. New
Public York. NY
Health Research NY IOOlh. 10010.
East Orange. NJ 07 IO?. to: Dr. Erwin H. Mosbach. Yorh. NY 10016.
Institute
Public
Health
of the
Research
(‘it);
of
In-
568
COHEN ETAL.
muricholic acid were prepared as described by Hsia (3). 3cu,7cuDihydroxy-l?-keto-5/3-cholanoic acid ( I’-keto standard) was synthesized according to Fieser and Rajagopalan (4). 2. Su-Cholestrrne. (Applied Science Lab., State College, PA) was used as the internal standard for the glc analyses. 3. Thin layer chromatography. The methods used were essentially those of Miettinen. Grundy, and Ahrens (1.2). The bile acids were separated on 0.5-mm layers of silica gel HR (Brinkmann Instruments, Westbury, NY). Prior to use for bile acid analysis, impurities were removed by developing the plates in methanol-acetic acid (95:5, v/v) followed by activation at 100°C for I h. The plates were stored in a desiccator over drierite (Fisher Sci. Co.) prior to use. Samples were applied to the plates as a band using a semiautomatic sample streaker equipped with a 500 ~1 syringe (Applied Science Lab.). Solvents used for chromatographic separations were reagent grade or spectra grade quality and were used without further purification. 4. Gas-liquid clwotmtograplzy. Separations of the bile acids were carried out on a Hewlett Packard Gas Chromatograph 7610A equipped with hydrogen flame ionization detectors and a dual pen recorder. Ushaped columns (3 mm i.d., 6-ft length) were silanized and packed with 3% SE-30 on 100/l 20 mesh Supelcoport (Supelco Inc., Bellafonte. PA) or 1% HiEff 8BP on 100/l 20 Supelcoport. Prepurified nitrogen was used as a carrier gas at a flow rate of 20-30 ml/min and an inlet pressure of 40 psi. Measurements of peak areas were accomplished with a Hewlett Packard Automatic Integrator 3370B adjusted to record retention time in minutes (with automatic baseline correction). Operating conditions for the column, inlet, and detector were ?50”, 280”, and 280°C. respectively. All glc-ms measurements were carried out on a Varian MAT Ill. The column packings and operating conditions of the glc were similar to those of the 7610A. 5. Radiouctikity measurements. These were carried out using a Beckman LS-200 Liquid Scintillation system. The radioactivity of each sample was measured, after evaporation of solvent, by the addition of 10 ml of BBOT (2,5-bis-2-(5-tert-Butylbenzolyl))-thiophene solution (4 g/liter in toluene). Procedures 1. Extraction offeces. Fecal samples were collected and dried under vacuum over drierite for 48 h. The cholesterol pools of several rats were labeled with DL-[2-'%I mevalonolactone to obtain feces containing radioactive neutral and acidic steroids. An aliquot of 1.0-1.5 g of powdered fecal material was placed into an extraction thimble. The reservoir bottle for the Soxhlet extraction was a 125 ml reagent bottle (Corning No.
ANALYSIS
OF
FECAL
BILE
569
ACIDS
1500) which was charged with 80 ml of ethanol and 100 pg of the 13-keto standard. The thimble was placed into a Soxhlet extractor and the material was extracted for 48 h. The final volume was reduced to 20 ml under nitrogen. All subsequent reactions. centrifugations and extractions were carried out in the reservoir bottle. 2. Srpmution oj‘tlc~rrttwl stcwicis .f~otll acidic. stc~roicis. The procedures used were those of Miettinen et rrl. (3). 3.
M~~thods
,fi)r.
isolutiotl
mti
qutrtltittrtiotl
of’
acidic
steroids
,frottl
~~IIJ c,.t-trtrc’tiotl. The aqueous phase containing the acidic steroids in I N NaOH was evaporated at 5OYI to remove the ethanol. The volume was brought to 30 ml with distilled water and 2 ml of IO N NaOH was added (final normality = 3, N). The solution was autoclaved for 3 h at 120°C and 14-15 psi. The sample was cooled on ice and acidified with concentrated HCI to pH 1-3. Each sample was shaken with 80 ml of choloroform-methanol (2: I, v/v) and centrifuged immediately or stored overnight. The lower phase was removed and the aqueous phase was reextracted twice more with 50 ml of chloroform each time. The organic fractions were evaporated to dryness. The samples were dissolved in 30 ml of benzene-methanol (86: 14. v/v) and reevaporated to dryness. Estrrijicdon of’ Bile Acids. The dry bile acid residue was dissolved in 5 ml of 59% HCI (by titration) in dry methanol. The reaction mixture was sealed and allowed to remain at room temperature for 18 h. The solution was evaporated to dryness on an evaporator. Each sample was subsequently redissolved in 30 ml of the benzene-methanol solvent and reevaporated to remove any traces of HCI. The esterified bile acids were dissolved in chloroform-methanol (2: 1. v/v) and quantitatively transferred to test tubes. The samples were dried and redissolved in 2 ml of chloroform. tic. x/c. tit1tt glc-tns of’ tllr hilr uc,itl nlc~thyl cstrrs. A suitable aliquot of the esterified bile acid mixture was applied to a silica gel HR plate. The plate was developed first using benzene and then using isooctane-isopropanol-acetic acid (120: 40: I, v/v/v). The area from the origin to below the fatty acid band (visualized with iodine) contained all the bile acids and was removed from the plate using the procedures of Grundy (1). The bile acids in this region were quantitated by glc after making the trimethylsilylether (TMS) derivatives of the methyl esters. 5tr-Cholestane was added to all samples as an internal standard for the glc analyses on SE-30. In several cases, for the purpose of identifcation of the individual bile acid methyl esters, the mixture was plated on silica gel HR and developed in benzene-acetone (60:40, v/v). Bands were scraped corresponding to known samples of (a) methyl lithocholate. R, = 0.78: (b) methyl deoxycholate, R, = 0.50: and (c) methyl
,/i,c.c.\
-.\lr/‘c”li~ic,rrtic,~~
570
COHEN
ET AL.
cholate, R, = 0.22. In addition, materials at the origin, solvent front and between the known bands were analyzed using glc and glc-ms. RESULTS AND
DISCUSSION
This report describes a procedure for the extraction, isolation and quantitation of acidic steroids (bile acids) from rat feces. The method is similar to that previously described by Grundy and others (1 S-8) but was modified for use in sterol metabolism studies in the rat. An important modification of existing procedures consisted of the Soxhlet extraction of the fecal samples with ethanol. This step was necessary to prevent boilover of the samples during the hydrolysis of the acidic steroids in the autoclave either prior to or after removal of the neutral sterol fraction. Boilover of the sample frequently resulted in losses as high as 7040% of the total sample. Consequently, hot ethanolic extraction of the samples was employed to remove the steroids from the solid insoluble fecal material. No boilover of the samples occurred during hydrolysis after removal of the solid material. Completeness of this extraction was verified by combustion of “C-labeled fecal samples before and after extraction and determination of the amount of radioactivity present. Table 1 shows that the extraction step removes 95% of the neutral and acidic steroids from the feces. The I?-keto standard (3a,7cu-dihydroxy- I2-keto-5p-cholanoic acid) was added to the fecal samples to correct for losses of acidic steroids which occurred during the procedure. The glc detector response of the TMS ether of the 12-keto standard and the SLu-cholestane standard was I : I on a weight basis only on SE-30 ( 15). Previous studies showed that the 12-keto standard exhibited the same stability to rigorous saponification as the major fecal bile acids (I ). Recovery of this standard averaged 77% or better for 25 fecal samples. Losses of fecal acidic steroids were corrected accordingly. TABLE
I
VALIDA-IIONOFEXTRACTIONPROCEDURESFOR
Sample 1 2 3 4
Before extraction (dpmiO.75 16870 35300 22400 17200
g)
FECAL
After extraction (dpmi0.25 979 2260 1100 1300
g)
SAMPLES"
Completeness of extraction Y5r;“r 945; 95% 9x?
” Rats were labeled with DL-[2-'JC]mCVa10nOlaCtOne. Feces were collected. dried and ground. Aliquots of fecal material were cornbusted before and after ethanolic Soxhlet extraction and the amount of [“‘C]CO, counted. Duplicate samples were counted and the counts corrected for quench.
ANALYSIS
OF
FECAL
TABLE QUANTITATION
Lithocholic acid Deokycholic acid Chenodeoxycholic Cholic acid I?-Keto standard
acid
” The bile esterification
were esterified with the TMS derivatives
acids using
571
ACIDS
?
OF BII E ARID
No. of determination5
Sample
BILE
ESTERIFKXTION”
Average recovery with benzenemethanol evaporation (C)
5 5 5 5 8
Average recovery without benzenemethanol evaporation (“iI
9x YX 98 99 95 methanol/HCI of the methyl
and analyzed esters on pk.
for completeness
of
Esterification of the acidic steroids was carried out with dry methanol containing 5% HCl. It has been reported that this method of esterification is unsatisfactory since recoveries of cholic acid were as low as 18% (9). We have determined that traces of water present in the bile acid standards and fecal samples were responsible for the incomplete esterification. This problem was eliminated by evaporating the samples with a mixture of benzene-methanol (86:14, v/v) before adding the methylating reagent. The results of carrying out the methylation with and without the benzene-methanol evaporation step are summarized in Table 9. Nearly quantitative esterification was obtained when the evaporation step was used whereas incomplete esterification occurred when it was omitted. Quantitative methylation of the bile acids using methanolHCI was comparable to diazomethane (6). Methanol-HC1 has the advantage in that it is stable for periods up to 6 months when stored at -20°C. After esterification with methanol, the bile acid methyl esters were analyzed as the TMS ethers by glc on SE-30. For quantitation it was necessary to determine which glc peaks in the total fecal acidic steroid fraction were bile acids and which were not: therefore a detailed study using a combination of tic, glc. and glc-ms was carried out. A sample of methyl esters of the acidic steroid fraction (approximately 0.5 g) was applied to a preparative silica gel HR plate and developed in benzeneacetone (60:40, v/v). Standards of methyl lithocholate. methyl deoxycholate, and methyl cholate were applied to the plate. After development, the acidic steroids were made visible by spraying the plate with Bile-Spra (Supelco). Bands corresponding to the known bile acids were observed (Fig. 1). The bands were eluted with methanol and analyzed as the TMS ethers by glc. Peaks were observed in the various fractions (Fig. 1).
571
COHEN
ET
AL.
2A [I
I
L3 3A
FIG.
la.
tic,
and
glc-ms
analysis
of
fecal
bile
acids.
tic
of the
fecal
esters on silica gel HR using benzene-acetone (60:40, v/v) separated droxy (band I ). dihydroxy (band 2). and trihydroxy (band 3) cholanoic sponding to known samples of: (a) methyl lithocholate: (b) methyl methyl cholate H,= 1.00: Band
R, = 0.34; band
was
Peaks were are reported
Band made
were 1.
seen.
R,=0.78:
3. R, = 0.22: after addition
identified relative
The
plate Band
was IA,
divided
Band 3A. I’, = 0.00-o. of an internal standard
by their retention times and to the internal standard (actual
seven bands:
into
R,=O.h8:
bile
Band
2.
acid
R,=O.50:
Solvent Band
IO. glc and glc-ms analyses and preparation of the TMS glc-ms time
methyl
them into monohyacids. Bands corredeoxycholate: and (c)
data (Table = 5.16 min).
3).
Retention
front, ZA,
of each ethers. times
For preliminary identification, the retention time of each peak in each band was determined and compared to known bile acid standards. For positive identification of each peak in each band, analysis by glc-ms was employed using an SE-30 column in a Varian MAT 111mass spectrometer. The identification of each peak by means of mass spectrometry is shown in Table 3. The identified material amounted to 93% of the total bile acids in the fecal bile acid fraction. Material with a retention time shorter or longer than SLu-cholestane(Fig. 1) and which was found to be nonsteroidal (hydroxy fatty acids) was not used in the calculation of total bile acids. This material amounted to as much as 25% of the total bile acid fraction. Using this material in determining total daily bile acid output would have resulted in an overestimation by this amount (25%). All glc analyses were run for periods of time up to I h. In some cases peaks emerged after 45 min which were found to be nonsteroidal as well. The bile acids which were identified in the fecal material were: lithocholic, deoxycholic, IL!-ketolithocholic. cholic, LYmuricholic, p-muricholic, and w-muricholic acid. w-Muricholic acid has not been previously reported in rat feces. Its identification was made by mass-spectral data (Fragments at m/e 195 and m/e 38.5 are typical of this compound (11)) and by its retention time on SE-30 and HiEff 8BP (12). A representative glc pattern of the total fecal acidic steroid fraction, prior to separation into individual components by tic is shown in Fig. 2. Peaks 3-9 were used to calculate the total bile acids in the sample.
ANALYSIS
OF
FECAL
BILE
ACIDS
Sld I
:
ltl,KIlM
’
5
10
15 20 Mmutes
25
45
50 Minutes FIG
I.
Ih.
Peaks 1. 2, and 10 which did not have the steroid nucleus and peak 8 which was the I?-keto standard were not used in the calculation of total bile acids. In summary. we have reported a method for the isolation and quantitation of the fecal acidic steroids in the rat. The method permits accurate
5500 476(19) 684( 1)
55OY 564( 3)
5 6 7
8 9
IA
2
Band
Band
372(9) 46% 1) 462( 1) -
1 2 3 4
m/e Peak” M+’ .~._______~.__
Solvent Front Band 1
Band no.”
535(6) 549(3)
535(6) 461( 1) 669(5)
357(21) 453(22) 447(4) -
M-15
460(l) 474(30)
460( I) 386( 15) 579(12J
372(36) -
M-90
445(I) 459(3)
445(l) 371(5)
-
345(12) 359(16)
1
399(21)
489( 14) 370(15) 384(45)
345(12)
-
M-(115+ 90) -~-
-
-
-
M-(1 1.5+ (2X90)
ETHERS OF METHYL
370(15)
-
-
-
M-(3X90)
TABLE 3 STEROIDS AS THE TMS
M-(2X90)
ACIDIC
M-(90+ 15) ~-~ 357(23) -
MASS SPECTRA OF FECAL
255(100) 229( 100)
?55(100) 230( 100) 296( 100)
215(100) 73( 100) 215(100) -
Base peak
ESTERS
d
methyl deoxycholate” methyl 3Lu,7wdihydroxy-I?keto-5B-cholanoateP
methyl deoxycholate” methyl I?-ketolithocholatt? no steroid nucleus
no steroid nucleus no steroid nucleus methyl lithocholate” unknown bile acid
Compound identification
3
3A
Band
Band
-
-
identification
mass spectrum ma\ \prctrtlm detected.
” The
’ The ‘. The ‘i Not
of’ thi\ of this
i\ made
’ M + i\ the moleculw ion. theses repI-esent the relative
obtained in each
-
1)
on silica tic h;md
533(
-
4590)
533(i) 533( 1)
100)
gel tic (Fig. by glc analysis
-
458(29) 458(h)
458(6) X34(421
458(
I) for the bile of the TMS
368(41) 368t17)
-
368( 14) 368(4)
the
,, ,,
compound compound
using
dau
wa\ identical corresponded
combined to that to that
of tic-K,
plc
retention
of a known srnndard. reported in the literature
values
16)
I)
(1 I. 12).
:\nd
-90).
acid methyl ethers.
-
JU(3) -
35%
43%
time\.
m/e represent loss of various fragment\ CH St - 15). TMS-OH( intensity of each peak w\ a percent of the parent peak (100).
IO the bands peaks obtained
-
548(3) 538( I)
-
638”
638g
14 I.(
-
-
-
474130)
53W)
I.3
548(4) 548(Z)
638 cl 5640)
II I2
-
638 cl
I0
” Etlch band corresponded b Numbers rcprc\ent the
2A
Band
data
steroid
chain( fol- abundance
side
-
ester\.
195( 100)
@c-m\
and
-
100)
153(100)
XYt
35X( 100) 195(100)
343(5)
343QOI
-
-
343(3) -
fragments.
numbers
cholatc” w-muricholate’
of v;ll-iou\
- I I5 ). The
no peak5
methyl methyl
methyl 3a.7a-dihydroxy-12. keto-S/3-cholanoate’ unknown bile acid
methyl a-muricholate’ methylp-murichc~laTe’
in paren-
576
COHEN
ET AL.
Std
9
-lion5
10
15
20
25 Minutes
Peak
R?-
3 4 5 4 7 8 9 IO
I 25 I 63 2 12 251 2 78 3 37 3 67 4 42 4 84 8 93
30
45
50
55
FIG. 2. Analysis of total fecal bile acids by glc. glc of a mixture of fecal bile acids as the TMS ethers was run on SE-30. Retention times are reported relative to the internal standard Sa-cholestane (actual time = 5.16 min). Peaks I. 2, and IO are not bile acids (corresponding to peaks I, 2, and 7 in Table 3). Peak 8 represents the methyl ester of 3~. 7adihydroxy-l2-keto-S@holanoate standard added to each sample. Peaks 3-7 and peak 9 are the fecal bile acids used for calculations of the total bile acid output. The bile acids in the peaks are: peak 3, lithocholate: peak 4. deoxycholate: peak 5. cholate and LYmuricholate; peak 6, 12.ketolithocholate; peak 7. P-muricholate: and peak 9. Wmuricholate. Conditions for the glc analysis are given in the text.
determination of the total fecal bile acids as well as subsequent identification of the individual bile acids. ACKNOWLEDGMENTS We wish to thank Dr. G. S. Tint for determining the mass spectra and Miss Cathy Ross of Dr. Zilversmit’s laboratory for the combustion of the fecal samples. This research was supported in part by grants HL 10894 and AM 05222 from the U.S. Public Health Service, GB 3 19 19X from the National Science Foundation. and a grant from the I.P.D. Corporation.
REFERENCES I. Grundy,
S. M., Ahrens, IO. 3. Miettinen. T. A.. Ahrens.
E. H.. Jr.,
and
Miettinen.
T.
A. (1965)
J. Lipid
Res.
6,
J. Lipid
Rcs.
6,
397-4
E. H.,
Jr..
and
Grundy.
S. M.
(1965)
411-424.
Hsia. S. L. (1971) ilr The Bile Acids: Chemistry, Physiology, and Metabolism (Nair, P. P., and Kritchevsky. D., eds.). I st ed.. Vol. I, pp. 95-120. Plenum Press, New York. 4. Fieser. L., and Rajagopalan. S. (I 950) J. Amer. Cltenr. So<,. 72. 5530-5536.
3.
ANALYSIS
OF
FECAL
BlLE
ACIDS
577
5. SjBvall. J. (1964) irl Methods of Biochemical Analysis (Glick, D.. ed.). Vol. I?. pp. 97-141, Interscience, New York. 6. Eneroth. P.. and Sjavall. J. (1969) in Methods in Enzymology (Clayton. R. B., ed.). Vol. 15. pp. 237-280. Academic Press. New York. 7. Horning, E. C., Brooks, C. J. W.. and Vandenheuvel. W. J. (1968) irl Advances in Lipid Research (Paoletti. R., and Kritchevshy. D.. eds.). Vol. 6, pp. 3-73-391. Academic Press. New Yorh. 8. Makita. M.. and Wells, M. (1963) .4rlcl/. Birwhrm. 5, 523-530. 9. Grundy. S. M.. and Metzger, A. L. t 1972) G[r.srrr)c,r~fero/o~~ 62, 1100-l 2 17. 0. Feher. T.. Papp, J.. and Kazik. M. H. (1973) C‘/i,r. Chim. Acrc~. 44, 409-418. I. Elliott. W. H. (1972) in Biochemical Applications of Mass Spectrometry (Wailer. G. R.. ed.), 1st ed., pp. 391-311. John Wiley and Sons. New York. ?. Elliott. W. H.. Walsh. L.. B.. Mui. M. M., and Thornem. M. A. ( 1969) ./. CI~~~,rt~rrtog. 44,45z!-464.