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Biosynthesis of coprostanyl esters
ARCHIVES
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Biosynthesis
621-625
112,
(1965)
of Coprostanyl
Esters
R. S. ROSENFELD Institute
for Steroid
Research, Montefiore
Hospital
Received
and Medical
Center, Bronx,
New York
July 30, 1965
Portions of a fecal suspension were incubated in separate experiments with 7&Hcholesteryl palmitate and 14C-palmitic acid. It was found that: (1) The quantity of coprostanyl esters formed during incubation was approximately equivalent to the decrease in amount of free coprostanol. (2) A maximum of 0.1% of the radioactivity in 7a-SH-cholesteryl palmitate was present in coprostanyl palmitate at the conclusion of the experiment. (3) About 6% of %-palmitic acid appeared in coprostanyl esters. (4) From 5 to 7% of the 7&H-cholesteryl pdmitate was hydrolyzed. Since little radioactivity appeared in coprostanyl palmitate during the incubation with 7&Hcholesteryl palmitate when large amounts of saturated sterol esters were being produced, it may be concluded that the major route of formation of coprostanyl esters is from esterification of free coprostanol and not by reduction of cholesteryl esters. The data are in accord with the following sequence: (1) hydrolysis of cholesteryl palmitate; (2) reduction of cholesterol to coprostanol; and (3) esterification of coprostanol. There was no conclusive evidence for reduction of 7a-aH-cholesteryl palmitate without prior hydrolysis. It is probable that the reactions are carried out by fecal bacteria.
In a recent study it was found that from to 55% of the fecal sterols were esterified and that at least 60 % were coprostanyl esters; in some cases, no cholesteryl esters were detected (1). It was therefore of interest to establish whether coprostanyl esters arise by reduction of cholesteryl esters without prior hydrolysis or are formed by esterification of coprostanol. To this end, 7&Hcholesteryl palmitate was incubated with a suspension of feces, and the lipid products were fractionated into free sterols and sterol esters from which all unsaturated products, both sterol and fatty acid, had been removed. In a parallel experiment, 14C-palmitic acid was incubated with a portion of the same suspension and the saturated sterol esters were isolated. It is concluded that esterification of sterol occurs readily in stool and that coprostanyl esters are derived almost exclusively from free coprostanol rather than cholesteryl esters. 5
EXPERIMENTAL IT-Palmitic acid. The compound was over 99% pure by paper chromatography, thin-layer chro-
matography (TLC), gas-liquid chromatography of the methyl ester, and dilution analysis (Nuclear-Chicago Corp.). It was mixed with nonradioactive palmitic acid before use; specific activity, 3.14 X lo5 counts per minute (cpm) per milligram. ‘Yol-aH-Cholesteryl palmitate. Paper chromatography showed no trace of radioactivity other than that associated with cholesteryl palmitate. Neither free cholesterol nor palmitic acid could be detected by TLC (New England Nuclear Corp.). When a portion was mixed with free cholesterol and the mixture was chromatographed on alumina, 99% of the radioactivity was recovered in the ester fraction. The labeled ester was diluted with carrier cholesteryl palmitate for the study; specific activity, 1.35 X lo5 cpm per milligram. Radioactive measurements. Samples were dissolved in a toluene-scintillant mixture (2) and counted in an automated Packard Tri-Carb liquid scintillation spectrometer; the data are subject to a f596 counting error. Incubations. Feces from a normal subject were homogenized with an equal weight of 0.9% sodium chloride in a Waring Blendor at half speed for 3 minutes. Previous stool samples from this subject had been shown to contain coprostanyl esters [Subject D (l)]. To 250 cc of homogenate was 621
622
ROSENFELD
added 33.3 mg of 7&H-cholesteryl palmitate (4.5 X lo6 cpm) emulsified in 10 cc of 0.9% sodium chloride solution and stabilized with Tween 80; this was further homogenized for 1 minute. The mixture was incubated at 37” in a closed flask with stirring. After 48 hours, 750 cc of ethanol was thoroughly mixed with the homogenate, which was allowed to settle, and the supernatant solution was filtered through a large Soxhlet cup. The ethanol-insoluble residue was transferred to centrifuge bottles, washed 3 times with 200 cc of acetone, and centrifuged after each wash. The clear acetone supernatant fractions were added to the ethanol filtrate. The insoluble residue was finally mixed with ethanol, poured into the Soxhlet cup, and continuously extracted with hot ethanol for 24 hours. All washings were combined with the ethanol extract and the solvents were removed. The extract was acidified with 5’3, hydrochloric acid and partitioned between petroleum ether and 709;b ethanol; the ethanol solution was discarded. Fatty acids were removed from the petroleum ether solution by washing with 5a/, potassium hydroxide and were recovered after acidification and extraction of the aqueous phase with petroleum ether. Another 2.50.~~ portion of the same homogenate was incubated simultaneously with 29.7 mg of I%-palmitic acid (9.3 X 10” cpm). The procedure was identical with that described above. In order to measure the amount of free and esterified sterols present at the start of the experiment, 150 cc of the homogenate was immediately extracted and fractionated as described. Genera2 techniques. The neutral lipids were chromatographed on alumina (l), and a clear separation was achieved between the three main fractions: sterol esters, free coprostanol, and cholesterol. All TLC separations were carried out on plates coated with Silica Gel G. For sterol esters, solvent systems of cyclohexane-benzene (2:l) or benzene were used. Spots were visualized with a phosphomolybdic acid spray. In these systems, coprostanyl palmitate moved slightly behind cholesteryl palmitate. Free sterols were measured by gas-liquid chromatography (GLC) on a well-conditioned 3a/, &F-l phase on 100-140 mesh Gas Chrom-P. The column was maintained at 232”; similar analyses have been described (3). For analyses of fatty acids, the methyl esters were chromatographed on a 12yo diethyleneglycol succinate polyesterchromosorb-W column; oven temperature 180”. 7a-3H-Cholesterol palmitate incubation; sterol esters. Examination of the nonpolar eluates from the alumina chromatography by TLC permitted an approximate separation between coprostanyl esters and fractions containing cholesteryl esters.
The appropriate fractions were combined; there were 740 mg of coprostanyl esters where no traces of cholesteryl esters were visible (6.1yo of the radioactivity in the neutral fraction) and 145 mg of material largely coprostanyl esters that TLC showed to contain small amounts of cholesteryl esters (73.0% of the radioactivity). Each fraction was treated with m-chloroperbenzoic acid in chloroform (4), and the products were chromatographed on alumina. Model experiments had shown that the reagent oxidized double bonds in the fatty acid as well as the sterol portion of the ester. As before, individual eluates were examined by TLC for sterol esters. From the larger fraction (6.1% of the radioactivity), 142 mg of semicrystalline material was obtained with the same RI as coprostanyl palmitate or stearate. A second fraction contained 70 mg of material whose Rf was 0.4 that of coprostanyl palmitate. Further fractions afforded material which, on TLC, remained at the origin. The oxidized combined cholesteryl ester fraction (73.0yo of the radioactivity) yielded about 1 mg of nonpolar material which contained a substance that migrated identically with coprostanyl palmitate (TLC) and possessed less than 0.02yo of the counts in the eluates. Virtually all of the radioactivity was associated with more polar fractions. Saponification of 27 mg of coprostanyl esters of saturated acids (47 cpm per milligram) yielded 13.6 mg of coprostanol. The acid fraction was esterified with diazomethane and gas chromatographed. A trace of methyl myristate was detected and the only other peaks corresponded to methyl esters of palmitic and stearic acid in a ratio of 53:47, respectively. From this ratio, the specific activity of coprostanol in the esters was calculated as 79 cpm per milligram, and the measured specific activity of the pure coprostanol isolated from these esters was 81 cpm per milligram. Free sterols. Amounts of coprostanone, coprostanol, and cholesterol in the fractions from chromatography of the neutral lipids were measured by GLC of the appropriate eluates. Isolation of the individual sterols and purification to constant specific activity were performed as described earlier (5). 14C-Palm&c acid incubation. Separation of the neutral lipids into sterol esters and free sterols was carried out as above. Thin-layer chromatography of the individual fractions showed no detectable cholesteryl esters. The combined ester fractions (7@& of incubated radioactivity) were treated with m-chloroperbenzoic acid and the product was isolated in the usual way. The epoxidation step was repeated and the esters were chromatographed as before. Over 88% of the radio-
BIOSYNTHESIS
OF COPROSTANYL
ESTERS
623
FIG. 1. Incubation of 7&H-cholesteryl palmitate with fecal homogenate. Fractionation of radioactivity in the neutral extract and isolation of saturated esters of coprostanol. Footnote explanations: (a) Bracketed numbers refer to percentage of radioactivity in the neutral fraction; (b) purified to constant specific activity; (c) MCPBA: m-chloroperbenzoic acid.
and re-esterification of labeled sterols. In our hands, it was impossible to separate coprostanyl esters of saturated acids; nevertheless, the amount of coprostanyl palmitate in this mixture could be determined by analysis of the fatty acids derived from the saturated mixed esters. Thus, the amount of radioactivity associated with coprostanyl palmitate, approximately 50% of the radioactivity in coprostanyl esters of saturated acids (Fig. 1) or 0.1% of the neutral fraction, RESULTS AND DISCUSSION represented a maximum value, and even this Figure 1 presents a flow diagram and sum- could have been formed by esterification of mary of data concerned with isolation and free coprostanol. For example (Table I), it can be calculated that at the beginning, inpurification of coprostanyl esters in incubated feces. To determine whether ~cx-~H- cubation 1 contained 530 mg of free coprostanol and at the end, there remained 196 mg; cholesteryl palmitate was reduced without cleavage of the ester linkage, it was necessary therefore about 334 mg was esterSed. The to measure the radioactivity that was solely specific activity of free coprostanol after inand specifically present in coprostanyl pal- cubation was 183 cpm per milligram (Fig. l), and if it is assumed that this is also the mitate. Radioactivity in any other sterol specific activity of coprostanol which was ester, except starting material, could only arise from cleavage of cholesteryl palmitate esterified, then 6.1 X lo4 cpm were incoractivity in the total ester fraction was recovered in material which behaved as coprostanyl palmitate or stearate on TLC. When this fraction was saponified, only coprostanol was obtained; no cholesterol could be detected by GLC. Free sterols from the neutral fraction were measured by GLC. Nonincubated control. The neutral fraction was chromatographed; sterol esters were saponified. The free and ester sterols were analyzed by GLC. Coprostanol was the only detectable sterol from the ester fraction.
624
ROSENFELD
TABLE I AMOUNTS OF FREE AND ESTER STEROLS BEFORE AND AFTER INCUBATION OF APPROXIMATELY EQUAL PORTIONS OF FECAL HOMOGENATES Sterols (mg) Experiment
Total
Control (not incubated) 7&H-Cholesteryl palmitate incubation 1-W-Palmitic acid incubation (II) a Not detectable. b Calculated from radioactivity NEUTRAL 6.26
(I)
FRACTION 1
, W’s I
I.
MC6SAb
2
A1203
3. Repeat
SATURATED
Coprostanol
530 196 409
Ester
Cholesterol
57 19 20
Coprostanol
100 435 300
Cholesterol
-a 10”
data.
I IOJcprn (1oo)a
687 660 729
FreEI
I and 2
ACIDS
Fra. 2. Incubation of 1-i4C-palmitic acid with fecal homogenate. Fractionation of radioactivity in the neutral extract. Footnote explanations: (a) Bracketed numbers refer to percentage of radioactivity in the neutral fraction; (b) MCPBA: m-chloroperbenzoic acid.
porated into coprostanyl esters. From the incubation, about 885 mg of esters was isolated where the sterol was almost entirely coprostanol. Therefore, the specific activity of this fraction, uncontaminated by high counting unreacted 7&H-cholesteryl palmitate, should be 6.1 X lo4 cpm/885 mg or 69 cpm per milligram; the specific activity of radiochemically pure coprostanyl esters was found to be 47 cpm per milligram (Fig. 1). Thus it can be concluded that at most a
negligible amount, and probably none, of 7a-3H-cholesteryl palmitate was reduced to coprostanyl palmitate without prior hydrolysis. In the same experiment, at least 5.7%, of the radioactivity in the neutral fraction was present in free sterols, and of this amount about 15% had been converted in vitro to coprostanol. That esterification of sterol occurred during incubation is shown in Fig. 2 in that 89 % of the radioactivity in the neutral lipids was associated with coprostanyl esters of saturated acids, or about 6 % of the total radioactivity of 14C-palmitic acid-l added. Esterification of sterol is demonstrated by the data in the table as well, where the quantities of cholesterol and coprostanol in free and ester sterols are compared before and after incubation. Within the limits of apportioning a homogenate of feces quantitatively, the total sterol in each experiment was the same. In both incubations, the quantity of esterified coprostanol increased greatly with a proportionate decrease of free sterol. Free fatty acids are the most probable substrate for esterification of coprostanol since little neutral fat was present while over 2 gm of free fatty acids was found in the alkaline washes of the lipid extracts. It is evident from these results that the enzyme system responsible for esterification of sterols in feces exhibits a different substrate specificity than that shown in mammalian sterol esterification. The latter is highly specific for sterol hydroxyl groups in the equatorial orientation at C-3 (6-8) ; in contrast, the fecal system probably of bac-
BIOSYNTHESIS
OF COPROSTANYL
terial origin readily esterifies the axially oriented hydroxyl group. Further, the results of this study show that esterases capable of hydrolyzing cholesteryl esters are present. The virtual absence of cholesteryl esters and the accumulation of coprostanyl esters suggest that these esterases are inactive on saturated, axially oriented ester 1inkage.s ACKNOWLEDGMENTS The interest and support of Dr. T. F. Gallagher are gratefully acknowledged. Radioactive measurements were carried out through the courtesy of Dr. H. Leon Bradlow. Thanks are due Miss Inge Paul for her skillful assistance in the laboratory. This work was supported by a grant from the American Cancer Society, and research grants CA 07364 and FR-73 from the National Cancer Institute.
ESTERS
625
REFERENCES 1. ROSENFELD,R. S., Arch. Biochem. Biophys. 108, 384 (1964). 2. FUKUSHIMA, D.K., BRADLOW,H.L., HELLMAN, L., AND GALLAGHER, T. F., J. Clin. Endocrinol. Metab. 23, 266 (1963). 3. ROSENFELD, R. S., LEBEAU, M. C., SHULMAN, S., AND SELTZER, J., J. Chromatog. 7, 293 (1962). 4. ROSENFELD, R. S., AND GALLAGHER, T. F., Steroids 4, 515 (1964). &yROSENFELD, R.S., ZUMOFF, B., AND HELLMAN, L., Arch. Biochem. Biophys. 96, 84 (1962). 6. ROSENFELD, R.S., ZUMOFF, B., AND HELLMAN, L., J. Lipid Res. 4, 337 (1963). 7. HERNANDEZ, H. H., AND CHAIKOFF, I. L., J. Biol. Chem. 2!28, 447 (1957). 8. SWELL, L., STUTZMAN, E., LAW, M. D., AND TREADWELL, C. R., Arch. Biochem. Biophys. 97, 411 (1962).
OF
BIOCHEMISTRY
AND
BIOPHYSICS
Biosynthesis
621-625
112,
(1965)
of Coprostanyl
Esters
R. S. ROSENFELD Institute
for Steroid
Research, Montefiore
Hospital
Received
and Medical
Center, Bronx,
New York
July 30, 1965
Portions of a fecal suspension were incubated in separate experiments with 7&Hcholesteryl palmitate and 14C-palmitic acid. It was found that: (1) The quantity of coprostanyl esters formed during incubation was approximately equivalent to the decrease in amount of free coprostanol. (2) A maximum of 0.1% of the radioactivity in 7a-SH-cholesteryl palmitate was present in coprostanyl palmitate at the conclusion of the experiment. (3) About 6% of %-palmitic acid appeared in coprostanyl esters. (4) From 5 to 7% of the 7&H-cholesteryl pdmitate was hydrolyzed. Since little radioactivity appeared in coprostanyl palmitate during the incubation with 7&Hcholesteryl palmitate when large amounts of saturated sterol esters were being produced, it may be concluded that the major route of formation of coprostanyl esters is from esterification of free coprostanol and not by reduction of cholesteryl esters. The data are in accord with the following sequence: (1) hydrolysis of cholesteryl palmitate; (2) reduction of cholesterol to coprostanol; and (3) esterification of coprostanol. There was no conclusive evidence for reduction of 7a-aH-cholesteryl palmitate without prior hydrolysis. It is probable that the reactions are carried out by fecal bacteria.
In a recent study it was found that from to 55% of the fecal sterols were esterified and that at least 60 % were coprostanyl esters; in some cases, no cholesteryl esters were detected (1). It was therefore of interest to establish whether coprostanyl esters arise by reduction of cholesteryl esters without prior hydrolysis or are formed by esterification of coprostanol. To this end, 7&Hcholesteryl palmitate was incubated with a suspension of feces, and the lipid products were fractionated into free sterols and sterol esters from which all unsaturated products, both sterol and fatty acid, had been removed. In a parallel experiment, 14C-palmitic acid was incubated with a portion of the same suspension and the saturated sterol esters were isolated. It is concluded that esterification of sterol occurs readily in stool and that coprostanyl esters are derived almost exclusively from free coprostanol rather than cholesteryl esters. 5
EXPERIMENTAL IT-Palmitic acid. The compound was over 99% pure by paper chromatography, thin-layer chro-
matography (TLC), gas-liquid chromatography of the methyl ester, and dilution analysis (Nuclear-Chicago Corp.). It was mixed with nonradioactive palmitic acid before use; specific activity, 3.14 X lo5 counts per minute (cpm) per milligram. ‘Yol-aH-Cholesteryl palmitate. Paper chromatography showed no trace of radioactivity other than that associated with cholesteryl palmitate. Neither free cholesterol nor palmitic acid could be detected by TLC (New England Nuclear Corp.). When a portion was mixed with free cholesterol and the mixture was chromatographed on alumina, 99% of the radioactivity was recovered in the ester fraction. The labeled ester was diluted with carrier cholesteryl palmitate for the study; specific activity, 1.35 X lo5 cpm per milligram. Radioactive measurements. Samples were dissolved in a toluene-scintillant mixture (2) and counted in an automated Packard Tri-Carb liquid scintillation spectrometer; the data are subject to a f596 counting error. Incubations. Feces from a normal subject were homogenized with an equal weight of 0.9% sodium chloride in a Waring Blendor at half speed for 3 minutes. Previous stool samples from this subject had been shown to contain coprostanyl esters [Subject D (l)]. To 250 cc of homogenate was 621
622
ROSENFELD
added 33.3 mg of 7&H-cholesteryl palmitate (4.5 X lo6 cpm) emulsified in 10 cc of 0.9% sodium chloride solution and stabilized with Tween 80; this was further homogenized for 1 minute. The mixture was incubated at 37” in a closed flask with stirring. After 48 hours, 750 cc of ethanol was thoroughly mixed with the homogenate, which was allowed to settle, and the supernatant solution was filtered through a large Soxhlet cup. The ethanol-insoluble residue was transferred to centrifuge bottles, washed 3 times with 200 cc of acetone, and centrifuged after each wash. The clear acetone supernatant fractions were added to the ethanol filtrate. The insoluble residue was finally mixed with ethanol, poured into the Soxhlet cup, and continuously extracted with hot ethanol for 24 hours. All washings were combined with the ethanol extract and the solvents were removed. The extract was acidified with 5’3, hydrochloric acid and partitioned between petroleum ether and 709;b ethanol; the ethanol solution was discarded. Fatty acids were removed from the petroleum ether solution by washing with 5a/, potassium hydroxide and were recovered after acidification and extraction of the aqueous phase with petroleum ether. Another 2.50.~~ portion of the same homogenate was incubated simultaneously with 29.7 mg of I%-palmitic acid (9.3 X 10” cpm). The procedure was identical with that described above. In order to measure the amount of free and esterified sterols present at the start of the experiment, 150 cc of the homogenate was immediately extracted and fractionated as described. Genera2 techniques. The neutral lipids were chromatographed on alumina (l), and a clear separation was achieved between the three main fractions: sterol esters, free coprostanol, and cholesterol. All TLC separations were carried out on plates coated with Silica Gel G. For sterol esters, solvent systems of cyclohexane-benzene (2:l) or benzene were used. Spots were visualized with a phosphomolybdic acid spray. In these systems, coprostanyl palmitate moved slightly behind cholesteryl palmitate. Free sterols were measured by gas-liquid chromatography (GLC) on a well-conditioned 3a/, &F-l phase on 100-140 mesh Gas Chrom-P. The column was maintained at 232”; similar analyses have been described (3). For analyses of fatty acids, the methyl esters were chromatographed on a 12yo diethyleneglycol succinate polyesterchromosorb-W column; oven temperature 180”. 7a-3H-Cholesterol palmitate incubation; sterol esters. Examination of the nonpolar eluates from the alumina chromatography by TLC permitted an approximate separation between coprostanyl esters and fractions containing cholesteryl esters.
The appropriate fractions were combined; there were 740 mg of coprostanyl esters where no traces of cholesteryl esters were visible (6.1yo of the radioactivity in the neutral fraction) and 145 mg of material largely coprostanyl esters that TLC showed to contain small amounts of cholesteryl esters (73.0% of the radioactivity). Each fraction was treated with m-chloroperbenzoic acid in chloroform (4), and the products were chromatographed on alumina. Model experiments had shown that the reagent oxidized double bonds in the fatty acid as well as the sterol portion of the ester. As before, individual eluates were examined by TLC for sterol esters. From the larger fraction (6.1% of the radioactivity), 142 mg of semicrystalline material was obtained with the same RI as coprostanyl palmitate or stearate. A second fraction contained 70 mg of material whose Rf was 0.4 that of coprostanyl palmitate. Further fractions afforded material which, on TLC, remained at the origin. The oxidized combined cholesteryl ester fraction (73.0yo of the radioactivity) yielded about 1 mg of nonpolar material which contained a substance that migrated identically with coprostanyl palmitate (TLC) and possessed less than 0.02yo of the counts in the eluates. Virtually all of the radioactivity was associated with more polar fractions. Saponification of 27 mg of coprostanyl esters of saturated acids (47 cpm per milligram) yielded 13.6 mg of coprostanol. The acid fraction was esterified with diazomethane and gas chromatographed. A trace of methyl myristate was detected and the only other peaks corresponded to methyl esters of palmitic and stearic acid in a ratio of 53:47, respectively. From this ratio, the specific activity of coprostanol in the esters was calculated as 79 cpm per milligram, and the measured specific activity of the pure coprostanol isolated from these esters was 81 cpm per milligram. Free sterols. Amounts of coprostanone, coprostanol, and cholesterol in the fractions from chromatography of the neutral lipids were measured by GLC of the appropriate eluates. Isolation of the individual sterols and purification to constant specific activity were performed as described earlier (5). 14C-Palm&c acid incubation. Separation of the neutral lipids into sterol esters and free sterols was carried out as above. Thin-layer chromatography of the individual fractions showed no detectable cholesteryl esters. The combined ester fractions (7@& of incubated radioactivity) were treated with m-chloroperbenzoic acid and the product was isolated in the usual way. The epoxidation step was repeated and the esters were chromatographed as before. Over 88% of the radio-
BIOSYNTHESIS
OF COPROSTANYL
ESTERS
623
FIG. 1. Incubation of 7&H-cholesteryl palmitate with fecal homogenate. Fractionation of radioactivity in the neutral extract and isolation of saturated esters of coprostanol. Footnote explanations: (a) Bracketed numbers refer to percentage of radioactivity in the neutral fraction; (b) purified to constant specific activity; (c) MCPBA: m-chloroperbenzoic acid.
and re-esterification of labeled sterols. In our hands, it was impossible to separate coprostanyl esters of saturated acids; nevertheless, the amount of coprostanyl palmitate in this mixture could be determined by analysis of the fatty acids derived from the saturated mixed esters. Thus, the amount of radioactivity associated with coprostanyl palmitate, approximately 50% of the radioactivity in coprostanyl esters of saturated acids (Fig. 1) or 0.1% of the neutral fraction, RESULTS AND DISCUSSION represented a maximum value, and even this Figure 1 presents a flow diagram and sum- could have been formed by esterification of mary of data concerned with isolation and free coprostanol. For example (Table I), it can be calculated that at the beginning, inpurification of coprostanyl esters in incubated feces. To determine whether ~cx-~H- cubation 1 contained 530 mg of free coprostanol and at the end, there remained 196 mg; cholesteryl palmitate was reduced without cleavage of the ester linkage, it was necessary therefore about 334 mg was esterSed. The to measure the radioactivity that was solely specific activity of free coprostanol after inand specifically present in coprostanyl pal- cubation was 183 cpm per milligram (Fig. l), and if it is assumed that this is also the mitate. Radioactivity in any other sterol specific activity of coprostanol which was ester, except starting material, could only arise from cleavage of cholesteryl palmitate esterified, then 6.1 X lo4 cpm were incoractivity in the total ester fraction was recovered in material which behaved as coprostanyl palmitate or stearate on TLC. When this fraction was saponified, only coprostanol was obtained; no cholesterol could be detected by GLC. Free sterols from the neutral fraction were measured by GLC. Nonincubated control. The neutral fraction was chromatographed; sterol esters were saponified. The free and ester sterols were analyzed by GLC. Coprostanol was the only detectable sterol from the ester fraction.
624
ROSENFELD
TABLE I AMOUNTS OF FREE AND ESTER STEROLS BEFORE AND AFTER INCUBATION OF APPROXIMATELY EQUAL PORTIONS OF FECAL HOMOGENATES Sterols (mg) Experiment
Total
Control (not incubated) 7&H-Cholesteryl palmitate incubation 1-W-Palmitic acid incubation (II) a Not detectable. b Calculated from radioactivity NEUTRAL 6.26
(I)
FRACTION 1
, W’s I
I.
MC6SAb
2
A1203
3. Repeat
SATURATED
Coprostanol
530 196 409
Ester
Cholesterol
57 19 20
Coprostanol
100 435 300
Cholesterol
-a 10”
data.
I IOJcprn (1oo)a
687 660 729
FreEI
I and 2
ACIDS
Fra. 2. Incubation of 1-i4C-palmitic acid with fecal homogenate. Fractionation of radioactivity in the neutral extract. Footnote explanations: (a) Bracketed numbers refer to percentage of radioactivity in the neutral fraction; (b) MCPBA: m-chloroperbenzoic acid.
porated into coprostanyl esters. From the incubation, about 885 mg of esters was isolated where the sterol was almost entirely coprostanol. Therefore, the specific activity of this fraction, uncontaminated by high counting unreacted 7&H-cholesteryl palmitate, should be 6.1 X lo4 cpm/885 mg or 69 cpm per milligram; the specific activity of radiochemically pure coprostanyl esters was found to be 47 cpm per milligram (Fig. 1). Thus it can be concluded that at most a
negligible amount, and probably none, of 7a-3H-cholesteryl palmitate was reduced to coprostanyl palmitate without prior hydrolysis. In the same experiment, at least 5.7%, of the radioactivity in the neutral fraction was present in free sterols, and of this amount about 15% had been converted in vitro to coprostanol. That esterification of sterol occurred during incubation is shown in Fig. 2 in that 89 % of the radioactivity in the neutral lipids was associated with coprostanyl esters of saturated acids, or about 6 % of the total radioactivity of 14C-palmitic acid-l added. Esterification of sterol is demonstrated by the data in the table as well, where the quantities of cholesterol and coprostanol in free and ester sterols are compared before and after incubation. Within the limits of apportioning a homogenate of feces quantitatively, the total sterol in each experiment was the same. In both incubations, the quantity of esterified coprostanol increased greatly with a proportionate decrease of free sterol. Free fatty acids are the most probable substrate for esterification of coprostanol since little neutral fat was present while over 2 gm of free fatty acids was found in the alkaline washes of the lipid extracts. It is evident from these results that the enzyme system responsible for esterification of sterols in feces exhibits a different substrate specificity than that shown in mammalian sterol esterification. The latter is highly specific for sterol hydroxyl groups in the equatorial orientation at C-3 (6-8) ; in contrast, the fecal system probably of bac-
BIOSYNTHESIS
OF COPROSTANYL
terial origin readily esterifies the axially oriented hydroxyl group. Further, the results of this study show that esterases capable of hydrolyzing cholesteryl esters are present. The virtual absence of cholesteryl esters and the accumulation of coprostanyl esters suggest that these esterases are inactive on saturated, axially oriented ester 1inkage.s ACKNOWLEDGMENTS The interest and support of Dr. T. F. Gallagher are gratefully acknowledged. Radioactive measurements were carried out through the courtesy of Dr. H. Leon Bradlow. Thanks are due Miss Inge Paul for her skillful assistance in the laboratory. This work was supported by a grant from the American Cancer Society, and research grants CA 07364 and FR-73 from the National Cancer Institute.
ESTERS
625
REFERENCES 1. ROSENFELD,R. S., Arch. Biochem. Biophys. 108, 384 (1964). 2. FUKUSHIMA, D.K., BRADLOW,H.L., HELLMAN, L., AND GALLAGHER, T. F., J. Clin. Endocrinol. Metab. 23, 266 (1963). 3. ROSENFELD, R. S., LEBEAU, M. C., SHULMAN, S., AND SELTZER, J., J. Chromatog. 7, 293 (1962). 4. ROSENFELD, R. S., AND GALLAGHER, T. F., Steroids 4, 515 (1964). &yROSENFELD, R.S., ZUMOFF, B., AND HELLMAN, L., Arch. Biochem. Biophys. 96, 84 (1962). 6. ROSENFELD, R.S., ZUMOFF, B., AND HELLMAN, L., J. Lipid Res. 4, 337 (1963). 7. HERNANDEZ, H. H., AND CHAIKOFF, I. L., J. Biol. Chem. 2!28, 447 (1957). 8. SWELL, L., STUTZMAN, E., LAW, M. D., AND TREADWELL, C. R., Arch. Biochem. Biophys. 97, 411 (1962).