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The isolation of coprostanol from sterol esters of human feces
ARCHIVES
OF
BIOCHEMISTRY
The Isolation
AND
BIOPHYSICS
108, 384385
of Coprostanol
from
(1964)
Sterol
Esters of Human
Feces
R. S. ROSENFELD From the Institute
for Steroid
Research, Montefiore
Received
Hospital,
Bronx,
New York
July 29, 1964
Coprostanol ]coprostane-38-o1(5~-cholestan-3,+ol)] had been isolated as the main sterol in the nonsaponifiable fraction of fecal sterol esters in five subjects. Coprostanol esters are probably formed either by microbiological reduction of cholesterol prior to esterification or by reduction of cholesterol esters. INTRODUCTION
During the course of cholesterol balance studies in man, it was found that a significant amount of the fecal sterol fraction was esterified and that the major portion consisted of coprostanol esters. The presence of sterol esters in feces was suggested by Cook et al. from analyses of total and free sterol (l), and Aylward and Wills demonstrated mixed sterol esters in the nonpolar eluates from chromatography of organic solvent extracts of feces (2). EXPERIMENTAL For reasons concerned with other studies, each subject had been administered labeled cholesterol either orally or intravenously. The isolation procedure outlined below was carried out with material obtained from a subject (D-2, Table I) who had received cholesterol-4.Cl4 by mouth 1 month prior to collection of the sample. A l-day stool collection was dried on a steam bath, and the residue (24 gm) was continuously extracted in a Soxhlet apparatus with absolute ethanol for 24 hours. Water was added to make a 7Oyc ethanolic suspension, and the lipids were extracted with petroleum ether, the ethanolic layer being discarded. The petroleum ethersoluble material, 3.67 gm, was chromatographed on 350 gm of alumina (Merck, acid-washed) deactivated by slurrying with 8% aqueous acetone, decanting the acetone solution, and, after airdrying, heating overnight at 60”. The chromatogram was developed with petroleum ether (A), petroleum ether-benzene mixtures (B), benzene (C), and ethyl acetate (D). The first 500 ml of A afforded 648 mg of nonpolar material which ran
at the solvent front in thin-layer chromatography (TLC) on silica gel, well ahead of authentic cholesterol palmitate and cholesterol oleate (system = cyclohexane-benzene, 2:l). The next 3.5 liters of A yielded 722 mg of oily ester fraction [191 counts per minute per milligram (cpm/mg)]r which on TLC moved with approximately the same mobility as cholesterol oleate in the cyclohexane-benzene system. From B (2.5 liters) 34 mg of oil were obtained with less than 0.4yc free sterol by gas chromatography (3) [SE-30 (3%) on Gas Chrom P at 235”]. Eluate C (6 liters) gave 593 mg of crystalline coprostanol identical with authentic material and showed no trace of cholesterol on TLC in cyclohexane-ethyl acetate (7:3). From 10 liters of D there was 150 mg of an oil which contained 27 mg of coprostanol and 16 mg of cholesterol by gas chromatography. The ester fraction (597 mg) was refluxed for 2 hours in 5% potassium hydroxide in 80% ethanol. After dilution with water and extraction with ether, 375 mg of crystalline material was obtained which was all coprostanol by gas chromatography. Recrystallization from acetone afforded needles, m.p. 99”101” (292 cpm/mg); infrared spectrum (potassium bromide dispersion) identical with authentic coprostanol. A portion of the acid fraction was esterified with diazomethane, the methyl esters being chromatographed at 175” on 12% diethyleneglycol succinate supported on Anachrom A. The major peaks (in decreasing amounts) had retention times identical with methyl linoleate, methyl palmitate, and methyl stearate. i Radioactive measurements were carried out through the courtesy of Dr. H. L. Bradlow. From 17 to 20 mg of material was assayed in a Packard Tri-Carb Scintillation Counter.
384
ESTER TABLE
COPROSTANOL
I
FREE AND ESTER C& STEROLS IN FECES Sterol Subject
A B C-l c-2 D-l D-2 E
from
Coprostanol
ester
(mg)
Cholesterol
Free stew1 Coprostanol
7 256 20 17 -a -a 2
157 794 128 108 360 375 24
(mg) Cholesterol
342
-O 400 loo 288 _a 16 -@
7iia 1700 539 626 217
a Less than 1% of the free sterol fraction.
or ester sterol
In five other studies, the ester fraction was similarly separated from free sterol by column chromatography, and the esters were examined by TLC to insure the absence of free sterol. Saponification of the ester fraction yielded sterol which was analyzed by gas chromatography (3). In addition, with the exception of B, the nonsaponifiable fractions from the sterol esters were chromatographed on alumina to separate coprostanol and cholesterol (4). RESULTS
AND
DISCUSSION
The sterol analyses of seven 24-hour collections from five subjects are presented in Table I. The quantities as well as the composition of ester sterols were variable, but the major sterol in the ester fraction, as in the free, was coprostanol. Although all of the sterol esters were radioactive, it was considered unnecessary to demonstrate their presence by radiochemical means, as was done in D-2. A wide separation was achieved between esterified and free sterols when the fecal lipids were chromatographed on alumina, and sufficient material was available from the ester fraction for identification of the sterol components by conventional procedures. Radioactive coprostanol obtained from saponification of the sterol esters indicates that a significant portion of this material was derived
front
biliary
excretion,
endogenous
sources
intestinal
by
either
secretion,
or
385
both. Even though D-2 received cholesterol4-Cl4 orally, all radioactivity due to unabsorbed sterol should have been eliminated in the month prior to collection of the sample. In addition, the subjects to whom labeled cholesterol was administered intraveneously all had labeled fecal sterol esters; it is thus probable that coprostanol esters resulted from microbiological action. Circulating cholesterol excreted via the bile is in the free form, and after reduction to coprostanol (3p-hydroxyl = axial) could be esterified by bacterial sterol esterases. In man, tissue sterol esterases appear to esterify only C-3 hydroxyl groups with the equatorial conformation (5). Alternatively cholesterol esters might be directly secreted into the lumen from the intestinal wall or cholesterol might be esterified at this site prior to reduction. To determine whether coprostanol itself is esterified or whether direct reduction of cholesterol esters occurs needs further investigation; nevertheless, it is noteworthy that coprostanol, ordinarily considered to be an end product of sterol metabolism, was found in the esterified form. ACKXOWLEDGMENTS The interest and support of Dr. T. F. Ga.llagher are gratefully acknowledged. Miss Inge Paul, to whom thanks are also due, rendered skillful assistance in the laboratory. The work was supported by a grant from the American Cancer Society, and research grants CA 07304 and FIi-73 from the National Cancer Institute. REFERENCES 1. COOK, 1~. P., EDWARDS, D. C., AND RIDDELL,
C., Biochem J. 62, 255 (1956). 2. AYLWM~, F., AND WILLS, P.; Xature 191, 1397 (1961). 3. ROSENFELD, It. S., LEBEIU, M. C., SHULMAN, S., AND SELTZER, J., J. Chromatog. ‘7, 293 (1962). 4. ROSENFELL), IL. S., FUKUSHIMA, D. K., HELLM.4N, L., AND GALLAGHER, T. F., J. &Ol. Chem. 211, 301 (1954). 5. ROSENFELD,R.S., ZUMOFF, B., AND HELLMAN, L., J. Lipid Res. 4, 337 (1963).
OF
BIOCHEMISTRY
The Isolation
AND
BIOPHYSICS
108, 384385
of Coprostanol
from
(1964)
Sterol
Esters of Human
Feces
R. S. ROSENFELD From the Institute
for Steroid
Research, Montefiore
Received
Hospital,
Bronx,
New York
July 29, 1964
Coprostanol ]coprostane-38-o1(5~-cholestan-3,+ol)] had been isolated as the main sterol in the nonsaponifiable fraction of fecal sterol esters in five subjects. Coprostanol esters are probably formed either by microbiological reduction of cholesterol prior to esterification or by reduction of cholesterol esters. INTRODUCTION
During the course of cholesterol balance studies in man, it was found that a significant amount of the fecal sterol fraction was esterified and that the major portion consisted of coprostanol esters. The presence of sterol esters in feces was suggested by Cook et al. from analyses of total and free sterol (l), and Aylward and Wills demonstrated mixed sterol esters in the nonpolar eluates from chromatography of organic solvent extracts of feces (2). EXPERIMENTAL For reasons concerned with other studies, each subject had been administered labeled cholesterol either orally or intravenously. The isolation procedure outlined below was carried out with material obtained from a subject (D-2, Table I) who had received cholesterol-4.Cl4 by mouth 1 month prior to collection of the sample. A l-day stool collection was dried on a steam bath, and the residue (24 gm) was continuously extracted in a Soxhlet apparatus with absolute ethanol for 24 hours. Water was added to make a 7Oyc ethanolic suspension, and the lipids were extracted with petroleum ether, the ethanolic layer being discarded. The petroleum ethersoluble material, 3.67 gm, was chromatographed on 350 gm of alumina (Merck, acid-washed) deactivated by slurrying with 8% aqueous acetone, decanting the acetone solution, and, after airdrying, heating overnight at 60”. The chromatogram was developed with petroleum ether (A), petroleum ether-benzene mixtures (B), benzene (C), and ethyl acetate (D). The first 500 ml of A afforded 648 mg of nonpolar material which ran
at the solvent front in thin-layer chromatography (TLC) on silica gel, well ahead of authentic cholesterol palmitate and cholesterol oleate (system = cyclohexane-benzene, 2:l). The next 3.5 liters of A yielded 722 mg of oily ester fraction [191 counts per minute per milligram (cpm/mg)]r which on TLC moved with approximately the same mobility as cholesterol oleate in the cyclohexane-benzene system. From B (2.5 liters) 34 mg of oil were obtained with less than 0.4yc free sterol by gas chromatography (3) [SE-30 (3%) on Gas Chrom P at 235”]. Eluate C (6 liters) gave 593 mg of crystalline coprostanol identical with authentic material and showed no trace of cholesterol on TLC in cyclohexane-ethyl acetate (7:3). From 10 liters of D there was 150 mg of an oil which contained 27 mg of coprostanol and 16 mg of cholesterol by gas chromatography. The ester fraction (597 mg) was refluxed for 2 hours in 5% potassium hydroxide in 80% ethanol. After dilution with water and extraction with ether, 375 mg of crystalline material was obtained which was all coprostanol by gas chromatography. Recrystallization from acetone afforded needles, m.p. 99”101” (292 cpm/mg); infrared spectrum (potassium bromide dispersion) identical with authentic coprostanol. A portion of the acid fraction was esterified with diazomethane, the methyl esters being chromatographed at 175” on 12% diethyleneglycol succinate supported on Anachrom A. The major peaks (in decreasing amounts) had retention times identical with methyl linoleate, methyl palmitate, and methyl stearate. i Radioactive measurements were carried out through the courtesy of Dr. H. L. Bradlow. From 17 to 20 mg of material was assayed in a Packard Tri-Carb Scintillation Counter.
384
ESTER TABLE
COPROSTANOL
I
FREE AND ESTER C& STEROLS IN FECES Sterol Subject
A B C-l c-2 D-l D-2 E
from
Coprostanol
ester
(mg)
Cholesterol
Free stew1 Coprostanol
7 256 20 17 -a -a 2
157 794 128 108 360 375 24
(mg) Cholesterol
342
-O 400 loo 288 _a 16 -@
7iia 1700 539 626 217
a Less than 1% of the free sterol fraction.
or ester sterol
In five other studies, the ester fraction was similarly separated from free sterol by column chromatography, and the esters were examined by TLC to insure the absence of free sterol. Saponification of the ester fraction yielded sterol which was analyzed by gas chromatography (3). In addition, with the exception of B, the nonsaponifiable fractions from the sterol esters were chromatographed on alumina to separate coprostanol and cholesterol (4). RESULTS
AND
DISCUSSION
The sterol analyses of seven 24-hour collections from five subjects are presented in Table I. The quantities as well as the composition of ester sterols were variable, but the major sterol in the ester fraction, as in the free, was coprostanol. Although all of the sterol esters were radioactive, it was considered unnecessary to demonstrate their presence by radiochemical means, as was done in D-2. A wide separation was achieved between esterified and free sterols when the fecal lipids were chromatographed on alumina, and sufficient material was available from the ester fraction for identification of the sterol components by conventional procedures. Radioactive coprostanol obtained from saponification of the sterol esters indicates that a significant portion of this material was derived
front
biliary
excretion,
endogenous
sources
intestinal
by
either
secretion,
or
385
both. Even though D-2 received cholesterol4-Cl4 orally, all radioactivity due to unabsorbed sterol should have been eliminated in the month prior to collection of the sample. In addition, the subjects to whom labeled cholesterol was administered intraveneously all had labeled fecal sterol esters; it is thus probable that coprostanol esters resulted from microbiological action. Circulating cholesterol excreted via the bile is in the free form, and after reduction to coprostanol (3p-hydroxyl = axial) could be esterified by bacterial sterol esterases. In man, tissue sterol esterases appear to esterify only C-3 hydroxyl groups with the equatorial conformation (5). Alternatively cholesterol esters might be directly secreted into the lumen from the intestinal wall or cholesterol might be esterified at this site prior to reduction. To determine whether coprostanol itself is esterified or whether direct reduction of cholesterol esters occurs needs further investigation; nevertheless, it is noteworthy that coprostanol, ordinarily considered to be an end product of sterol metabolism, was found in the esterified form. ACKXOWLEDGMENTS The interest and support of Dr. T. F. Ga.llagher are gratefully acknowledged. Miss Inge Paul, to whom thanks are also due, rendered skillful assistance in the laboratory. The work was supported by a grant from the American Cancer Society, and research grants CA 07304 and FIi-73 from the National Cancer Institute. REFERENCES 1. COOK, 1~. P., EDWARDS, D. C., AND RIDDELL,
C., Biochem J. 62, 255 (1956). 2. AYLWM~, F., AND WILLS, P.; Xature 191, 1397 (1961). 3. ROSENFELD, It. S., LEBEIU, M. C., SHULMAN, S., AND SELTZER, J., J. Chromatog. ‘7, 293 (1962). 4. ROSENFELL), IL. S., FUKUSHIMA, D. K., HELLM.4N, L., AND GALLAGHER, T. F., J. &Ol. Chem. 211, 301 (1954). 5. ROSENFELD,R.S., ZUMOFF, B., AND HELLMAN, L., J. Lipid Res. 4, 337 (1963).