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Excretion of steroid acids in man
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Excretion
97,
of Steroid
R. S. ROSENFELD From
the Division
406410
Acids
AND LEON
of Steroid Metabolism for Cancer Research, Received
(1962)
in Man’ HELLMAN
and Biochemistry, New York, New
December
Sloan-Kettering York
Institute
22, 1961
A procedure for the isolation of acids from defatted ethanolic extracts of human feces has been described which involves chromatography of their crude methyl ester acetates on silica gel. lo-Hydroxystearic acid, lithocholic acid, and deoxycholic acid have been shown to be the principal constituents of the alcohol-soluble acid fraction. It has been shown that the specific activity of deoxycholic acid in the feces is approximately equal to that of plasma ester cholesterol by the ninth day after the administration of cholesterol-4-C” to patients. A method has been presented which permits calculation of the fecal excretion of bile acids after the administration of a tracer dose of cholesterol+CF. This requires measurement of the total radioactivity in the stool acid fraction and determination of the specific activity of plasma cholesterol; by this method, an average excretion of 290 mg./day of bile acids has been found for the patients studied. INTRODUCTION
of the major steroid acids in feces and to present a method for calculating the daily fecal excretion of bile acids.
It has been well known for many years that the principal route of steroid excretion is in the feces. Although procedures have long been available for the isolation of sterols from feces, only comparatively recently have methods been developed for the separation, identification, and quantitation of the individual sterol components. Isolation of the steroid acids in stool has proceeded more slowly, but during the past few years several chromatographic methods (l-6) and a titrimetric procedure (7) have been reported for the separation and quantitation of bile acids in feces. Since there is evidence that the rate of fecal excretion of steroids may be related to the circulating sterol level (8-16), measurement of the quantity of both neutral and acidic steroids is important. It is the purpose of this communication to report a relatively simple procedure for the isolation and separation
EXPERIMENTAL
ISOLATION OF ETHANOL-SOLUBLE ACIDS FROM FECES Stool was homogenized in ethanol and dried in an evaporating dish on a steam bath for 24 hr. The residue was then continuously extracted with ethanol for 24 hr. in a Soxhlet apparatus. The extract was concentrated under reduced pressure and partitioned between petroleum ether and 70% ethanol in a three-funnel distribution. The petroleum ether fraction contained the sterols, neutral fats, and free fatty acids; the 70% ethanol contained the steroid acids and most of the hydroxylated fatty acids. Most of the ethanol was removed in vacua, sodium chloride was added, and the mixture was extracted with ethyl acetate. The ethyl acetate was washed with saturated sodium chloride solution and dried over sodium sulfate. After removal of the ethyl acetate in vacua, the residue was refluxed for 4 hr. with 10% potassium hydroxide in 80% ethanol. The alkaline solution was acidified with concentrated hydrochloric acid and extracted with ether. The ether was washed with saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The crude bile acids were esterified by stand-
‘This investigation was supported in part by a grant from the American Cancer Society and a research grant (CY-3207) from the National Cancer Institute of the National Institutes of Health, U. S. Public Health Service. 406
EXCRETIO?;
OF STEROID
ing overnight in absolute methanol (concentration = 5 ml/g. of crude bile acids) to which concentrated sulfuric acid (10% of the weight of the acids) was added. After dilution with water, the methyl esters were extracted with ether, washed with 5% potassium hydroxide solution and water, and the solvent was removed. The product was refluxed for 3 hr. in a 1:l mixture of pyridine and acetic anhydride, poured on ice, and extracted with ether. After the ether was washed with 5% hydrochloric acid, 5% sodium hydroxide, and water and the solvent was removed, the mixture was chromatographed on 200-250 times its weight of silica gel. In a typical chromatogram three principal fractions were obtained as follows : Frnction 1. Eluted with petroleum ether-ether (19:1), 99 mg., it was shown to be the methyl loacetoxystearate. Fraction 2. Eluted with petroleum ether-ether (9: l), 126 mg. of crystalline material was obtained. This substance was recrystallized from acetone, m.p. 131-132”, and identified as methyl 3a-acetoxycholanate from the infrared spectrum (1150-800 cm.-‘, carbon disulfide), which was identical with that of an authentic specimen of the compound. Fraction, S. Eluted with petroleum ether-ether (7:3), 191 mg. was obtained. This substance was identified as methyl 3a, 12a-diacetoxycholanate from the infrared spectrum (1150-800 cm.-‘, carbon disulfide). Saponification yielded deoxycholic acid. Paper chromatography of the free acid in the isopropyl ether-heptane (20:80)-70% acetic acid system of SjGrall (17) showed a single spot with the mobility of authentic deoxycholic acid. lo-HYDROXTSTEARIC ACID FROM FRACTIOX 1 Elution of this fraction before methyl 3a-acetoxycholanate suggested that it might be a fatty acid derivative. Saponification and isolation of the free acid yielded a low-melting crystalline material which was recrystallized from ethyl acetate-petroleum ether, m.p. 80-82”, [a]~ = -0.62” (CHCI,); spectrum (KBr) : broad hydroxyl absorption from 3440 to 3290 cm.-‘; carbonyl absorption at 1705 cm.-‘. The tetranitromethane test was negative. The neutral equivalent was 308 (calculated neutral equivalent for a monohydroxy C,* acid = 300).
ACIDS
bazone was prepared and melted at 109-117” after several recrystallizations from methanol. Anal. Calcd. for C1PH370&3 : C, 64.3% ; H, 10.4% ; N, 11.8%. Found: C, 64.1%; H, 10.1%; N, 11.6%. Both 9-hydroxy- and lo-hydroxystearic acid have melting points comparable with that of the substance. Samples of 9-hydroxystearic acid’ and lohydroxystearic acida were obtained, but a comparison of their infrared spectra with that of the unknown hydroxy acid did not permit an unequivocal decision. The methyl ester of the keto acid obtained from oxidation of the hydroxy ester was analyzed in the mass spectrograph and found to be identical with that of methyl IO-ketostearate. Peaks at mass-charge ratio (m/e) = 312 and m/e = 281 were characteristic of a methyl ketooctadecanoate, and m/e = 156, 167, and 214 were characteristic of the cleavage products of the methyl ester of lOketostearic acid. Thus the unknown substance was lo-hydroxystearic acid. MEASUREMENT OF THE FECAL EXCRETION OF BILE ACIDS Fifty microcuries of cholesterol-4-C” (1 mg.) was administered to patients by vein as an emulsion in 0.9% saline. Blood samples were removed, and plasma-free and ester cholesterol were isolated as previously described (19). Stool collections for 24 hr. were processed for bile acids by the method described above. Radioactivity was measured in a windowless gasflow counter. The cholesterol was counted as the digitonide (19). An aliquot of the ethyl acetate solution of the crude bile acid from stool was pipetted onto lens paper, dried, and counted to obtain the total radioactivity in this fraction. The methyl ester acet,ates of the bile acids were assayed in the same manner. All counting was done in stainless steel planchets, and radioactivity was expressed as disintegrations per minute per milligram (d./min./ mg.) at infinite thickness. RESULTS
Anal. Calcd. for C18H3e03: C, 72.0%; H, 12.0%. Found: C, 71.7%; H, 11.7%.
Figure 1 illustrates a typical chromatographic pattern of the crude bile acids after esterification and acetylation; there is no overlapping of the substances. The three principal fractions comprise over 85% of the total of which the major portion is the de-
The acid was oxidized with chromic acid in 90% acetic acid. rl crystalline material was obtained which melted at 83” after recrystallization from acetone-petroleum ether. The infrared spectrum (KBr) showed no free hydroxyl group, and an isolated carbonyl group (1675 cm.-‘). The semicar-
‘9-Hydroxystearic acid was kindly supplied by Prof. F. D. Gunstone, University of Glasgow, Fife, Scotland, while the lo-hydroxy isomer was obtained through the courtesy of Dr. W. G. Bickford, U. S. Department of Agriculture, New Orleans, Louisiana.
ROSENFELD AND HELLMAN Table I in which it has been calculated that from 110 to 690 mg. of bile acids was excreted by these patients with an average of approximately 290 mg./day. DISCUSSION
FIG. 1. Chromatography of acetylated bile acid methyl esters from feces.
The isolat’ion and identification of lohydroxystearic acid confirms the recent work of James, Webb, and Kellock who reported this substance to be a major constituent of stool acids (20). Whether it is an intermediate in the transformation of stearic to oleic acid or is a product of bacterial action on stearic acid in the intestine is not known at the present time (21). The method described here permits the separation and identification of the principal bile acids in feces. Other investigations have also shown that deoxycholic acid is the main constituent of fecal bile acids (5) and that lithocholic acid can be present in significant amounts (6). It should be emphaTABLE I FECAL EXCRETION OF BILE ACIDS Patient
Days offer
administration
of cholesterol-4-C’4
Plasmaester cholesterol
Stool bile acid fraction
d./min./mg. d/min./day x lo-”
Bile acid excretion
mg./dayO
2. Radioactivity of deoxycholic acid from
A
rivative of deoxycholic acid. Figure 2 represents the specific activity of the deoxycholic acid fraction isolated from stool compared with the specific activity of plasma ester cholesterol taken as 100% on the same day and corrected for differences in molecular weights. By the 9th day, the specific activities of both substances are within 10% of one another. The specific activity of the minor bile acid component, lithocholic acid, is about SO-SO% of that of deoxycholic acid. A ratio3 similar to one reported earlier in measuring cholesterol excretion (8) has been used to calculate bile acid excretion in the feces 9 days or more after administration of the tracer. These data are reported in
3520 1900 1420 1140 1040 925 960 1050 975 975 980 980 980 980 866 866 866 866
4.68 1.00 0.82 0.91 0.41 0.32 0.33 0.23 0.32 0.33 0.21 0.26 0.68 0.22 0.095 0.15 0.28 0.26
440 175 190 260 395 345 345 215 325 335 220 265 690 220 110 171 328 302
B
6900 5200 8200
5.75 1.73 4.09
166 110 500
FIG.
feces.
3d./ min. of stool bile acid fraction = mg. of bile d./min./ms. plasma cholesterol acid in stool, since the specific activity of stool bile acid closely terol.
approximated
that
of plasma
o Calculation
: :$m?Fti
choles(see Resu2ts section).
EXCRETION
OF STEROID
sized that the bile acids secreted by the liver are cholic and chenodeoxycholic acids and are clearly different from those in feces. Norman and Sjiivall have demonstrated in animals that deoxycholic acid is a product of microbiological dehydroxylation in the intestine (22), and it is probable that such is the case in the human. Lithocholic acid has not been demonstrated to be a primary secretory product in bile, and it is possible that it is largely derived from chenodeoxycholic acid by bacterial dehydroxylation at C-7. Smaller quantities of other bile acids may also be present such as Sp-hydroxycholanic acid and 12-ketolithocholic acid (6)) and application of the elegant methods of Bergstrom, Norman, and Sjiivall (l-3) would undoubtedly show the presence of additional bile acid metabolites. Despite the qualitative differences between the steroid acids in bile and feces, the acids isolated in this study represent secretory products which, as shown by specific activity measurements, have been directly derived from circulat,ing cholesterol. This is in agreement with dynamic st,udies of the formation of bile acids in man which show them to be derived from plasma sterol (23j. In Fig. 2, it, can be seen that by the ninth day after administration of a tracer dose of cholesterol, the specific activity of deoxycholic acid approximated that of plasma cholesterol; the lower specific activity of the former during the initial phases of the experiment is probably the result of failure of the immedate precursor of bile acids to mix immediately wit,11 the plasma cholesterol as well as the time interval involved in the conversion of the bile acid to the fecal excretory product. Thus it is possible to calculate the weight of bile acid excreted in the feces 10 days or more after the plasma cholesterol pool has been labeled by (a) measurement of the total radioactivity in the crude bile acid fraction and (b) determination of the specific activity of plasma cholesterol at the time of stool collection. This calculation is valid as long as there is no destruction of the steroid nucleus which might remove significant quantities of radioactivity from the acid fraction. It is conceivable that a bacterial population in the intestine might possess this capability; how-
409
ACIDS
ever, no evidence of this has been observed in these laboratories. The measurement would be unaffected by the usual transformations of bile acids in the intestine where the cholanic acid structure remains int’act and only the functional groups may be altered. In this study, petroleum ethersoluble acidic substances derived from bile acids do not appear to be present since no steroid esters were det,ected in the methyl cst,ers of the acid fraction from the petroleum ether-soluble material when this fraction was analyzed by gas chromatography. This met,hod was capable of detecting less than 2% of methyl cholanate in fatty acid esters.” The weight of bile acid excreted per day (Table I: average = 290 mg.) calculated by the isotope procedure agrees reasonably well with data obtained by other methods in which 176715 mg. (241, 220580 mg. (13)) and 18&280 mg. ( 11) have been reported. It should be pointed out that the excretory rate of bile acids is lower than the secretory rate obtained from measurements in subjects with bile fistulas (23, 25j ; this would be expected since it is reasonable to assume that bile acid synthesis is maximal in the situation where there is no return through the enterohepatic circulation. In int,act subjects the fecal excretion of bile acids may well represent the normal synthetic rate of bile acids. ACKNOWLEDGMENTS The support and interest of Dr. T. F. Gallagher is gratefully acknowledged. The authors particularly wish to thank Docent Ragnar Ryhage, Mass Spectrometry Laboratory, Karolinska Institutet, Stockholm, Sweden, who carried out the mass spectrographic analysis of methyl IO-ketostearate. REFERENCES 1. BERGSTRO~~, S., .41i~ SJ~~V.~LL, J.,
Actn Chem. Stand. 5, 1267 (1951). 2. SJ~~VALL, J., Actn Physiol Scantl. 29, 232 (1953). 3. T\ToRM.~N, -4., Acta Chem. &and. 7, 1413 (1953). 4. MosBACH, E. H., ZO~LIZELY, C., .~ND KENDALL, F. E., Arch. Biochem. Biophys. 48, 95 (1954). 5. CAREY, J. C., JR., .4ND WATSON, C. J., J. Biol. Chem. 216, 847 (1955). 6. HEFT%lANs, E., WEISS, E., MILLER, H. K., ASD ‘Unpublished
observations
from this laboratory.
410
7. 8.
9. 10. 11. 12. 13. 14.
15. 16.
ROSENFELD
AND
MOSETTIG, E., Arch. Biochem. Biophys. 84, 324 (1959). LEWIS, B., S. African J. Lab. Clin. Med. 3, 316 (1957). HELLMAN, L., ROSENFELD, R. S., INSULL, W., JR., AND AHRENS, E. H., JR., J. Clin. Invest. 36, 898 (1957). GORDON, H., LEWIS, B., EALES, L., AND BROCK, J. F., Lancet 273, 1299 (1957). PORTMAN, 0. W., AND MURPHY, P., Arch. Biothem. Biophys. 76, 367 (1958). HAUST, H. L., AND BEVERIDGE, J. M. R., Arch. Biochem. Biophys. 78, 367 (1958). MERRILL, J. M., Cancer Research 7, 709 (1959). ENGELBERG, H., Proc. Sot. Exptl. Biol. Med. 102,365 (1959). ANDERSON, J. E., JR., CONIGLIO, J. G., AND BLOOD, F. R., Proc. Sot. Exptl. Biol. Med. 102, 155 (1959). CARROLL, K. K., J. Lipid Research 1,469 (1960). TENNENT, D. M., SIEGEL, H., ZANETTI, M. E.,
17. 18.
19.
20. 21. 22. 23. 24. 25.
HELLMAN KURON, G. W., OTT, W. H., AND WOLF, F. J., J. Lipid Research 1, 171 (1960). SJ~VALL, J., Acta Chem. Stand. 8,339 (1954). VANDEN HEUVEL, W. J., SWEELEY, C. C., AND HORNING, E. C., Biochem Biophys. Research Communs. 3,33 (1960). ROSENFELD, R. S., HELLMAN, L., CONSIDINE, W. J., AND GALLAGHER, T. F., J. Biol. Chem. 208, 73 (1954). JAMES, A. T., WEBB, J. P. W., AND KELLOCK, T. D., Biochem. J. 78, 333 (1961). LENNARZ, W. J., AND BLOCH, K., J. Biol. Chem. 235, PC26 (1960). NORMAN, A., AND SJBVALL, J., J. Biol. Chem. 223, 872 ( 1958). ROSENFELD, R. S., AND HELLMAN, L., J. Clin. Znvest. 38, 1334 (1959). CURRAN, G. L., AZARNOFF, D. L., AND BOLINGER, R. E., J. &in. Invest. 38, 1251 (1959). SIPERSTEIN, M. D., AND MURRAY, A. W., J. Clin. Invest. 34, 1449 (1955).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Excretion
97,
of Steroid
R. S. ROSENFELD From
the Division
406410
Acids
AND LEON
of Steroid Metabolism for Cancer Research, Received
(1962)
in Man’ HELLMAN
and Biochemistry, New York, New
December
Sloan-Kettering York
Institute
22, 1961
A procedure for the isolation of acids from defatted ethanolic extracts of human feces has been described which involves chromatography of their crude methyl ester acetates on silica gel. lo-Hydroxystearic acid, lithocholic acid, and deoxycholic acid have been shown to be the principal constituents of the alcohol-soluble acid fraction. It has been shown that the specific activity of deoxycholic acid in the feces is approximately equal to that of plasma ester cholesterol by the ninth day after the administration of cholesterol-4-C” to patients. A method has been presented which permits calculation of the fecal excretion of bile acids after the administration of a tracer dose of cholesterol+CF. This requires measurement of the total radioactivity in the stool acid fraction and determination of the specific activity of plasma cholesterol; by this method, an average excretion of 290 mg./day of bile acids has been found for the patients studied. INTRODUCTION
of the major steroid acids in feces and to present a method for calculating the daily fecal excretion of bile acids.
It has been well known for many years that the principal route of steroid excretion is in the feces. Although procedures have long been available for the isolation of sterols from feces, only comparatively recently have methods been developed for the separation, identification, and quantitation of the individual sterol components. Isolation of the steroid acids in stool has proceeded more slowly, but during the past few years several chromatographic methods (l-6) and a titrimetric procedure (7) have been reported for the separation and quantitation of bile acids in feces. Since there is evidence that the rate of fecal excretion of steroids may be related to the circulating sterol level (8-16), measurement of the quantity of both neutral and acidic steroids is important. It is the purpose of this communication to report a relatively simple procedure for the isolation and separation
EXPERIMENTAL
ISOLATION OF ETHANOL-SOLUBLE ACIDS FROM FECES Stool was homogenized in ethanol and dried in an evaporating dish on a steam bath for 24 hr. The residue was then continuously extracted with ethanol for 24 hr. in a Soxhlet apparatus. The extract was concentrated under reduced pressure and partitioned between petroleum ether and 70% ethanol in a three-funnel distribution. The petroleum ether fraction contained the sterols, neutral fats, and free fatty acids; the 70% ethanol contained the steroid acids and most of the hydroxylated fatty acids. Most of the ethanol was removed in vacua, sodium chloride was added, and the mixture was extracted with ethyl acetate. The ethyl acetate was washed with saturated sodium chloride solution and dried over sodium sulfate. After removal of the ethyl acetate in vacua, the residue was refluxed for 4 hr. with 10% potassium hydroxide in 80% ethanol. The alkaline solution was acidified with concentrated hydrochloric acid and extracted with ether. The ether was washed with saturated sodium chloride solution, dried over sodium sulfate, and concentrated. The crude bile acids were esterified by stand-
‘This investigation was supported in part by a grant from the American Cancer Society and a research grant (CY-3207) from the National Cancer Institute of the National Institutes of Health, U. S. Public Health Service. 406
EXCRETIO?;
OF STEROID
ing overnight in absolute methanol (concentration = 5 ml/g. of crude bile acids) to which concentrated sulfuric acid (10% of the weight of the acids) was added. After dilution with water, the methyl esters were extracted with ether, washed with 5% potassium hydroxide solution and water, and the solvent was removed. The product was refluxed for 3 hr. in a 1:l mixture of pyridine and acetic anhydride, poured on ice, and extracted with ether. After the ether was washed with 5% hydrochloric acid, 5% sodium hydroxide, and water and the solvent was removed, the mixture was chromatographed on 200-250 times its weight of silica gel. In a typical chromatogram three principal fractions were obtained as follows : Frnction 1. Eluted with petroleum ether-ether (19:1), 99 mg., it was shown to be the methyl loacetoxystearate. Fraction 2. Eluted with petroleum ether-ether (9: l), 126 mg. of crystalline material was obtained. This substance was recrystallized from acetone, m.p. 131-132”, and identified as methyl 3a-acetoxycholanate from the infrared spectrum (1150-800 cm.-‘, carbon disulfide), which was identical with that of an authentic specimen of the compound. Fraction, S. Eluted with petroleum ether-ether (7:3), 191 mg. was obtained. This substance was identified as methyl 3a, 12a-diacetoxycholanate from the infrared spectrum (1150-800 cm.-‘, carbon disulfide). Saponification yielded deoxycholic acid. Paper chromatography of the free acid in the isopropyl ether-heptane (20:80)-70% acetic acid system of SjGrall (17) showed a single spot with the mobility of authentic deoxycholic acid. lo-HYDROXTSTEARIC ACID FROM FRACTIOX 1 Elution of this fraction before methyl 3a-acetoxycholanate suggested that it might be a fatty acid derivative. Saponification and isolation of the free acid yielded a low-melting crystalline material which was recrystallized from ethyl acetate-petroleum ether, m.p. 80-82”, [a]~ = -0.62” (CHCI,); spectrum (KBr) : broad hydroxyl absorption from 3440 to 3290 cm.-‘; carbonyl absorption at 1705 cm.-‘. The tetranitromethane test was negative. The neutral equivalent was 308 (calculated neutral equivalent for a monohydroxy C,* acid = 300).
ACIDS
bazone was prepared and melted at 109-117” after several recrystallizations from methanol. Anal. Calcd. for C1PH370&3 : C, 64.3% ; H, 10.4% ; N, 11.8%. Found: C, 64.1%; H, 10.1%; N, 11.6%. Both 9-hydroxy- and lo-hydroxystearic acid have melting points comparable with that of the substance. Samples of 9-hydroxystearic acid’ and lohydroxystearic acida were obtained, but a comparison of their infrared spectra with that of the unknown hydroxy acid did not permit an unequivocal decision. The methyl ester of the keto acid obtained from oxidation of the hydroxy ester was analyzed in the mass spectrograph and found to be identical with that of methyl IO-ketostearate. Peaks at mass-charge ratio (m/e) = 312 and m/e = 281 were characteristic of a methyl ketooctadecanoate, and m/e = 156, 167, and 214 were characteristic of the cleavage products of the methyl ester of lOketostearic acid. Thus the unknown substance was lo-hydroxystearic acid. MEASUREMENT OF THE FECAL EXCRETION OF BILE ACIDS Fifty microcuries of cholesterol-4-C” (1 mg.) was administered to patients by vein as an emulsion in 0.9% saline. Blood samples were removed, and plasma-free and ester cholesterol were isolated as previously described (19). Stool collections for 24 hr. were processed for bile acids by the method described above. Radioactivity was measured in a windowless gasflow counter. The cholesterol was counted as the digitonide (19). An aliquot of the ethyl acetate solution of the crude bile acid from stool was pipetted onto lens paper, dried, and counted to obtain the total radioactivity in this fraction. The methyl ester acet,ates of the bile acids were assayed in the same manner. All counting was done in stainless steel planchets, and radioactivity was expressed as disintegrations per minute per milligram (d./min./ mg.) at infinite thickness. RESULTS
Anal. Calcd. for C18H3e03: C, 72.0%; H, 12.0%. Found: C, 71.7%; H, 11.7%.
Figure 1 illustrates a typical chromatographic pattern of the crude bile acids after esterification and acetylation; there is no overlapping of the substances. The three principal fractions comprise over 85% of the total of which the major portion is the de-
The acid was oxidized with chromic acid in 90% acetic acid. rl crystalline material was obtained which melted at 83” after recrystallization from acetone-petroleum ether. The infrared spectrum (KBr) showed no free hydroxyl group, and an isolated carbonyl group (1675 cm.-‘). The semicar-
‘9-Hydroxystearic acid was kindly supplied by Prof. F. D. Gunstone, University of Glasgow, Fife, Scotland, while the lo-hydroxy isomer was obtained through the courtesy of Dr. W. G. Bickford, U. S. Department of Agriculture, New Orleans, Louisiana.
ROSENFELD AND HELLMAN Table I in which it has been calculated that from 110 to 690 mg. of bile acids was excreted by these patients with an average of approximately 290 mg./day. DISCUSSION
FIG. 1. Chromatography of acetylated bile acid methyl esters from feces.
The isolat’ion and identification of lohydroxystearic acid confirms the recent work of James, Webb, and Kellock who reported this substance to be a major constituent of stool acids (20). Whether it is an intermediate in the transformation of stearic to oleic acid or is a product of bacterial action on stearic acid in the intestine is not known at the present time (21). The method described here permits the separation and identification of the principal bile acids in feces. Other investigations have also shown that deoxycholic acid is the main constituent of fecal bile acids (5) and that lithocholic acid can be present in significant amounts (6). It should be emphaTABLE I FECAL EXCRETION OF BILE ACIDS Patient
Days offer
administration
of cholesterol-4-C’4
Plasmaester cholesterol
Stool bile acid fraction
d./min./mg. d/min./day x lo-”
Bile acid excretion
mg./dayO
2. Radioactivity of deoxycholic acid from
A
rivative of deoxycholic acid. Figure 2 represents the specific activity of the deoxycholic acid fraction isolated from stool compared with the specific activity of plasma ester cholesterol taken as 100% on the same day and corrected for differences in molecular weights. By the 9th day, the specific activities of both substances are within 10% of one another. The specific activity of the minor bile acid component, lithocholic acid, is about SO-SO% of that of deoxycholic acid. A ratio3 similar to one reported earlier in measuring cholesterol excretion (8) has been used to calculate bile acid excretion in the feces 9 days or more after administration of the tracer. These data are reported in
3520 1900 1420 1140 1040 925 960 1050 975 975 980 980 980 980 866 866 866 866
4.68 1.00 0.82 0.91 0.41 0.32 0.33 0.23 0.32 0.33 0.21 0.26 0.68 0.22 0.095 0.15 0.28 0.26
440 175 190 260 395 345 345 215 325 335 220 265 690 220 110 171 328 302
B
6900 5200 8200
5.75 1.73 4.09
166 110 500
FIG.
feces.
3d./ min. of stool bile acid fraction = mg. of bile d./min./ms. plasma cholesterol acid in stool, since the specific activity of stool bile acid closely terol.
approximated
that
of plasma
o Calculation
: :$m?Fti
choles(see Resu2ts section).
EXCRETION
OF STEROID
sized that the bile acids secreted by the liver are cholic and chenodeoxycholic acids and are clearly different from those in feces. Norman and Sjiivall have demonstrated in animals that deoxycholic acid is a product of microbiological dehydroxylation in the intestine (22), and it is probable that such is the case in the human. Lithocholic acid has not been demonstrated to be a primary secretory product in bile, and it is possible that it is largely derived from chenodeoxycholic acid by bacterial dehydroxylation at C-7. Smaller quantities of other bile acids may also be present such as Sp-hydroxycholanic acid and 12-ketolithocholic acid (6)) and application of the elegant methods of Bergstrom, Norman, and Sjiivall (l-3) would undoubtedly show the presence of additional bile acid metabolites. Despite the qualitative differences between the steroid acids in bile and feces, the acids isolated in this study represent secretory products which, as shown by specific activity measurements, have been directly derived from circulat,ing cholesterol. This is in agreement with dynamic st,udies of the formation of bile acids in man which show them to be derived from plasma sterol (23j. In Fig. 2, it, can be seen that by the ninth day after administration of a tracer dose of cholesterol, the specific activity of deoxycholic acid approximated that of plasma cholesterol; the lower specific activity of the former during the initial phases of the experiment is probably the result of failure of the immedate precursor of bile acids to mix immediately wit,11 the plasma cholesterol as well as the time interval involved in the conversion of the bile acid to the fecal excretory product. Thus it is possible to calculate the weight of bile acid excreted in the feces 10 days or more after the plasma cholesterol pool has been labeled by (a) measurement of the total radioactivity in the crude bile acid fraction and (b) determination of the specific activity of plasma cholesterol at the time of stool collection. This calculation is valid as long as there is no destruction of the steroid nucleus which might remove significant quantities of radioactivity from the acid fraction. It is conceivable that a bacterial population in the intestine might possess this capability; how-
409
ACIDS
ever, no evidence of this has been observed in these laboratories. The measurement would be unaffected by the usual transformations of bile acids in the intestine where the cholanic acid structure remains int’act and only the functional groups may be altered. In this study, petroleum ethersoluble acidic substances derived from bile acids do not appear to be present since no steroid esters were det,ected in the methyl cst,ers of the acid fraction from the petroleum ether-soluble material when this fraction was analyzed by gas chromatography. This met,hod was capable of detecting less than 2% of methyl cholanate in fatty acid esters.” The weight of bile acid excreted per day (Table I: average = 290 mg.) calculated by the isotope procedure agrees reasonably well with data obtained by other methods in which 176715 mg. (241, 220580 mg. (13)) and 18&280 mg. ( 11) have been reported. It should be pointed out that the excretory rate of bile acids is lower than the secretory rate obtained from measurements in subjects with bile fistulas (23, 25j ; this would be expected since it is reasonable to assume that bile acid synthesis is maximal in the situation where there is no return through the enterohepatic circulation. In int,act subjects the fecal excretion of bile acids may well represent the normal synthetic rate of bile acids. ACKNOWLEDGMENTS The support and interest of Dr. T. F. Gallagher is gratefully acknowledged. The authors particularly wish to thank Docent Ragnar Ryhage, Mass Spectrometry Laboratory, Karolinska Institutet, Stockholm, Sweden, who carried out the mass spectrographic analysis of methyl IO-ketostearate. REFERENCES 1. BERGSTRO~~, S., .41i~ SJ~~V.~LL, J.,
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