- Email: [email protected]
Formation of Estrone, Estriol and an Unidentified Steroid from Estradiol in the Laying Hen
684
R. M. SMIBERT, M. FORBES, J. E. FABER, A. R. GABUTEN AND H. M. DEVOLT
Cornell Vet. XLVII: 112-126. Van Roekel, H., J. E. Gray, N. L. Shipkowitz, M. K. Clarke and R. M. Luchini, 1957. Etiology and pathology of the chronic respiratory disease complex in chickens. University of Massachusetts Experimental Station Bulletin No. 486: 1-94. White, F. H., G. D. Wallace and J. O. Alberts, 1954. Serological and electron microscope studies of chronic respiratory disease of chickens and of turkey sinusitis agent. Poultry Sci. 33: 500-507. Yamamoto, R., and H. E. Adler, 1958. Characterization of pleuropneumonia-like organisms of avian origin. I. Antigenic analysis of seven strains and their comparative pathogenicity for birds. J. Inf. Dis. 102: 143-152.
Formation of Estrone, Estriol and an Unidentified Steroid from Estradiol in the Laying Hen H. F. MACRAE, D. S. LAYNE AND R. H. COMMON
Department of Agricultural Chemistry, Macdonald College of McGill University, Quebec, Canada (Received for publication November 5, 1958)
EARD et al. (1956), Callow (1956) and Marrian (1958) have reviewed the metabolic inter-relationships of the estrogens. Among other conversions, the series estradiol ^ estrone —> estriol is well established for various mammalian species. The relationships of the more recently discovered estrogens have not been fully worked out. It seemed desirable to confirm whether or not the fowl also can produce estrone and estriol from estradiol. Estrone, estradiol and estriol have been detected in the excreta of the fowl by Hurst et al. (1957) and in extracts of the ovaries of laying hens by Layne et al. (1958).
H
EXPERIMENTAL
One mg. estradiol-17^, labelled with C14 at C-16 and containing 2.7 microcuries of radioactivity per mg. (Chas. Frosst & Co.,
Montreal) was dissolved in 1 ml. propylene glycol and injected into the wing vein of a laying hen. One hour later the bird- was killed by bleeding from the jugular vein. The blood was collected»in excess methanol, which was at once heated to inactivate enzymes. The entire gastrointestinal tract from the gizzard to the cloaca, inclusive of contents and excreta, was removed and quickly dispersed in methanol by use of a Waring Blendor. (It so happened that the bird did not void any excrement during the hour after injection.) The gall bladder was punctured and the bladder bile was also taken up in methanol. The blood was examined for estrogens and chromatograms were prepared as described by Layne and Common (1956). Radioautograms of the chromatograms revealed the presence of estradiol, but no other ra-
Downloaded from http://ps.oxfordjournals.org/ at UPVA on June 1, 2015
turkey sinusitis and chronic respiratory disease of chickens. Poultry Sci. 31: 902-904. Nelson, J. B., 1936. Studies on an uncomplicated coryza of the domestic fowl. VI. Coccobacilliform bodies in birds infected with the coryza of slow onset. J. Exper. Med. 63: 509-513. Price, K. E., E. Zolli, Jr., W. B. Hardie and M. J. Gallian, 1957. Respiratory tract flora in CRD and effect of antibiotics in the feed. Poultry Sci. 36: 219-225. Smibert, R. M., H. M. DeVolt and J. E. Faber, 1958. A study of the bacterial flora of the respiratory system of normal chickens. Poultry Sci. 37: 161-166. Taylor, J. R. E., and J. Fabricant, 1957. Studies on the isolation of pleuropneumonia-like organisms of chronic respiratory disease of fowls.
ESTROGEN IN THE H E N
Tentative Zone DSA DNPH identification Ai most polar + — estriol Bi less than Ai + — estradiol B2 less than At but more than Bi — + unknown B3 least polar — — estrone
Since the amounts present were minute, the DSA staining, where perceptible, was too faint to permit of photography. However, the faintness or absence of perceptible staining was of advantage in so far as it offered the possibility of applying fingerprinting methodology in reverse in order to confirm the tentative identifications. Accordingly, the four radioactive zones were
cut out from the main unstained portions of the strips and eluted with dichloromethane-methanol (1:1 v/v). Twenty-five micrograms of non-radioactive reference steroid was added as carrier to the four eluates as follows: eluate Ai Bi B: Bs
non-radioactive steroid added estriol estradiol estrone estrone
The four eluates then were evaporated, chromatographed again on separate strips, stained with DSA, photographed through a blue filter and radioautographed. The amounts of radioactivity were such that two months' exposure was necessary to get sufficient blackening of the film. The results are presented in Figure 1. Strip Ax contained radioactive estriol and this identification was confirmed by the close correspondence in both shape and position of the radioautogram with the stained zone, the latter staining being due to the reference estriol added. Strip Ai also contained some radioactive estradiol, but insufficient to give any staining reaction. Strip Bi contained radioactive estradiol and this identification was confirmed by correspondence in both shape and position of the radioautogram with the staining of the reference estradiol added. It also contained some estriol, and there was enough estriol on this strip to give a perceptible stain with DSA even though carrier estriol had not been added. It is well known that estriol is particularly sensitive to staining with DSA. Strip B 3 contained radioactive estrone, confirmed by close correspondence of the radioautogram with the staining. It will be recalled that the radioactive estrone was in such minute amounts that it gave no stain with DSA. No other band was apparent
Downloaded from http://ps.oxfordjournals.org/ at UPVA on June 1, 2015
dioactive spot was observed. This observation appeared to discount the possibility that the ketosteroid observed by Layne and Common (1956) is a metabolite of estrogen. The bile was worked up by the method of Mitchell and Davies (1954) except that no attempt at separation into estriol and estrone-estradiol fractions was made. Chromatography was performed as described by Layne et al. (1958). Radioautograms of the chromatograms showed presence of estradiol and of faint traces of spots corresponding in position to estriol and estrone, but no other radioactive spots were detected. The intestinal tract and excreta were worked up by the same methods as were used for the bile. The first chromatograms yielded two broad radioactive zones, A and B. These were cut out, eluted and chromatographed separately. The more polar zone (A) now gave one narrow radioactive zone (Ai). The less polar zone (B) gave three radioactive zones (B1} B2, B 3 ). Narrow strips were cut from the papers and stained with diazotized sulphanilic acid (DSA) and with dinitrophenylhydrazine (DNPH). The results of staining and mobilities are summarized as follows:
685
686
H. F. MACRAE, D. S. LAYNE AND R. H. COMMON
S R
S R
Ai
Dl
D £.
S R
FIG. 1. Staining reactions with diazotized sulphanilic acid (S) and radioautograms (R) of chromatograms of steroids from excreta of laying hen. Al.—Presumptive estriol fraction with added carrier estriol. Note close correspondence of stained (DSA) region with uppermost radioactive region, confirming the identification of the latter as estriol. Note also presence of broad radioactive zone in presumptive position of estradiol (amount too small to give perceptible stain with DSA). The double nature of the estriol zone (seen also in Bl) probably arose from application of much contaminating material and relatively slight movement of estriol from point of application in the Bush system. Bl.—Presumptive estradiol fraction with added carrier estradiol. Note close correspondence of broad lowermost stained zone with broad radioactive zone, confirming identification of the latter as estradiol. Note presence also of some estriol, which was sufficient to give a staining reaction. B2.—Unidentified zone with added carrier estrone. Note that there was a faint trace of radioactive estrone, but that the strongly radioactive zone was neither estriol nor estradiol nor estrone. The staining at the point of application was due to some unknown constituent; it could not have been estriol since it was not radioactive. B3.—Presumptive estrone with added carrier estrone. Note close correspondence of staining and radioautogram, confirming identification as estrone. Note absence of any other radioactive zone, demonstrating cleanness of separation of the estrone fraction.
either by staining or radioautography. Strip B2 gave a strongly radioactive zone which did not correspond with the staining of the added estrone. This zone was distinctly more polar than estradiol and less polar than estriol. Strip B2 also yielded a minute trace of radioactive estrone, identi-
fiable by correspondence in position with the stained zone of carrier estrone. The identity of B2 has not been established; it was present in sufficient amounts to give a strong DNPH stain, and it was obviously responsible for more radioactivity than the other three radioactive zones. Its relative
Downloaded from http://ps.oxfordjournals.org/ at UPVA on June 1, 2015
5 R
687
ESTROGEN IN THE HEN
position on the chromatogram corresponds with that reported for the 16-hydroxyestrones in Bush chromatographic systems, but the zone did not yield a positive DSA stain. Furthermore, the instability of the 16-hydroxyestrones in alkali makes it improbable that they would survive the extraction procedures used in the work now reported. The possibility that it was formed secondarily by bacterial action in the intestine cannot be excluded, but equally, the possibility remains that it may represent a major metabolite of estrogens in the fowl. SUMMARY
The authors wish to thank the National Research Council of Canada for a grant which defrayed part of the expenses of this work, and Canadian Industries Ltd. for the Fellowship held by one of us (H. F. MacRae). They also wish to thank Dr. B. E. Baker for help in the photography, and the late Professor W. F. Oliver for advice in connection with tracer methodology. We wish also to thank Dr. D. A. McGinty, Parke Davis Co., Detroit, for kindly presenting a sample of reference estriol, and Professor W. A. Maw for providing experimental hens and for helpful advice. REFERENCES Callow, R. K., 1956. Metabolism of the steroids. Ann. Rep. Chem. Soc. 53: 305-318. Heard, R. D. H., E. C. Bligh, M. S. Cann, P. R. Jellinck, V. J. O'Donnell, B. C. Rao and J. L. Webb, 1956. Recent Progress in Hormone Research, 12: 45-71, New York: Academic Press. Hurst, R. O., A. Kuksis and J. F. Bendell, 1957. The separation of estrogens from avian droppings. Can. J. Biochem. Physiol. 35: 637-640. Layne, D. S., and R. H. Common, 1956. Detection of a presumptive keto-steroid in the ovaries and blood of the laying hen. Nature (London), 178: 419-420. Layne, D. S., R. H. Common, W. A. Maw and R. M. Fraps, 1958. Presence of estrone, estradiol and estriol in extracts of ovaries of laying hens. Nature (London), 181: 351-352. Marrian, G. F., 1958. The biochemistry of the estrogenic hormones. Proc. Fourth International Congress of Biochemistry Symposium No. IV. Vienna. Mitchell, F. L., and R. E. Davies, 1954. The isolation and estimation of the steroid estrogens in placental tissue. Biochem. J. 56: 690-698.
NEWS AND NOTES (Continued from page 667) been Director of the Department of Poultry Science Advancement of Science, the American Genetic at the University of Arizona. Association, and Sigma Xi. He is the author of a number of scientific and ONTARIO NOTES technical articles, and is a member of the Poultry The Department of Poultry Husbandry, Ontario Science Association, the American Society for the (Continued on page 700)
Downloaded from http://ps.oxfordjournals.org/ at UPVA on June 1, 2015
1. When radioactive estradiol was administered intraveneously to a laying hen, radioactive estradiol, estrone and estriol could be detected after one hour in the gut plus excreta by chromatographic and radioautographic methods. It has been shown therefore, that the fowl is able to convert estradiol into estrone and estriol. 2. The gut plus excreta also yielded a radioactive steroid other than estradiol, estrone or estriol. This steroid was less polar than estriol but more polar than estradiol in a methanol-petroleum ether chromatographic system, and gave a strong ketonic stain. It also contained more radioactivity than either the estradiol, the estriol or the estrone zone. 3. Radioactive estradiol and traces of radioactive estriol and estrone were detected chromatographically in the bile, but no other radioactive zones. Radioactive estradiol, but no other radioactive steroid, was detected in the blood.
ACKNOWLEDGMENTS
R. M. SMIBERT, M. FORBES, J. E. FABER, A. R. GABUTEN AND H. M. DEVOLT
Cornell Vet. XLVII: 112-126. Van Roekel, H., J. E. Gray, N. L. Shipkowitz, M. K. Clarke and R. M. Luchini, 1957. Etiology and pathology of the chronic respiratory disease complex in chickens. University of Massachusetts Experimental Station Bulletin No. 486: 1-94. White, F. H., G. D. Wallace and J. O. Alberts, 1954. Serological and electron microscope studies of chronic respiratory disease of chickens and of turkey sinusitis agent. Poultry Sci. 33: 500-507. Yamamoto, R., and H. E. Adler, 1958. Characterization of pleuropneumonia-like organisms of avian origin. I. Antigenic analysis of seven strains and their comparative pathogenicity for birds. J. Inf. Dis. 102: 143-152.
Formation of Estrone, Estriol and an Unidentified Steroid from Estradiol in the Laying Hen H. F. MACRAE, D. S. LAYNE AND R. H. COMMON
Department of Agricultural Chemistry, Macdonald College of McGill University, Quebec, Canada (Received for publication November 5, 1958)
EARD et al. (1956), Callow (1956) and Marrian (1958) have reviewed the metabolic inter-relationships of the estrogens. Among other conversions, the series estradiol ^ estrone —> estriol is well established for various mammalian species. The relationships of the more recently discovered estrogens have not been fully worked out. It seemed desirable to confirm whether or not the fowl also can produce estrone and estriol from estradiol. Estrone, estradiol and estriol have been detected in the excreta of the fowl by Hurst et al. (1957) and in extracts of the ovaries of laying hens by Layne et al. (1958).
H
EXPERIMENTAL
One mg. estradiol-17^, labelled with C14 at C-16 and containing 2.7 microcuries of radioactivity per mg. (Chas. Frosst & Co.,
Montreal) was dissolved in 1 ml. propylene glycol and injected into the wing vein of a laying hen. One hour later the bird- was killed by bleeding from the jugular vein. The blood was collected»in excess methanol, which was at once heated to inactivate enzymes. The entire gastrointestinal tract from the gizzard to the cloaca, inclusive of contents and excreta, was removed and quickly dispersed in methanol by use of a Waring Blendor. (It so happened that the bird did not void any excrement during the hour after injection.) The gall bladder was punctured and the bladder bile was also taken up in methanol. The blood was examined for estrogens and chromatograms were prepared as described by Layne and Common (1956). Radioautograms of the chromatograms revealed the presence of estradiol, but no other ra-
Downloaded from http://ps.oxfordjournals.org/ at UPVA on June 1, 2015
turkey sinusitis and chronic respiratory disease of chickens. Poultry Sci. 31: 902-904. Nelson, J. B., 1936. Studies on an uncomplicated coryza of the domestic fowl. VI. Coccobacilliform bodies in birds infected with the coryza of slow onset. J. Exper. Med. 63: 509-513. Price, K. E., E. Zolli, Jr., W. B. Hardie and M. J. Gallian, 1957. Respiratory tract flora in CRD and effect of antibiotics in the feed. Poultry Sci. 36: 219-225. Smibert, R. M., H. M. DeVolt and J. E. Faber, 1958. A study of the bacterial flora of the respiratory system of normal chickens. Poultry Sci. 37: 161-166. Taylor, J. R. E., and J. Fabricant, 1957. Studies on the isolation of pleuropneumonia-like organisms of chronic respiratory disease of fowls.
ESTROGEN IN THE H E N
Tentative Zone DSA DNPH identification Ai most polar + — estriol Bi less than Ai + — estradiol B2 less than At but more than Bi — + unknown B3 least polar — — estrone
Since the amounts present were minute, the DSA staining, where perceptible, was too faint to permit of photography. However, the faintness or absence of perceptible staining was of advantage in so far as it offered the possibility of applying fingerprinting methodology in reverse in order to confirm the tentative identifications. Accordingly, the four radioactive zones were
cut out from the main unstained portions of the strips and eluted with dichloromethane-methanol (1:1 v/v). Twenty-five micrograms of non-radioactive reference steroid was added as carrier to the four eluates as follows: eluate Ai Bi B: Bs
non-radioactive steroid added estriol estradiol estrone estrone
The four eluates then were evaporated, chromatographed again on separate strips, stained with DSA, photographed through a blue filter and radioautographed. The amounts of radioactivity were such that two months' exposure was necessary to get sufficient blackening of the film. The results are presented in Figure 1. Strip Ax contained radioactive estriol and this identification was confirmed by the close correspondence in both shape and position of the radioautogram with the stained zone, the latter staining being due to the reference estriol added. Strip Ai also contained some radioactive estradiol, but insufficient to give any staining reaction. Strip Bi contained radioactive estradiol and this identification was confirmed by correspondence in both shape and position of the radioautogram with the staining of the reference estradiol added. It also contained some estriol, and there was enough estriol on this strip to give a perceptible stain with DSA even though carrier estriol had not been added. It is well known that estriol is particularly sensitive to staining with DSA. Strip B 3 contained radioactive estrone, confirmed by close correspondence of the radioautogram with the staining. It will be recalled that the radioactive estrone was in such minute amounts that it gave no stain with DSA. No other band was apparent
Downloaded from http://ps.oxfordjournals.org/ at UPVA on June 1, 2015
dioactive spot was observed. This observation appeared to discount the possibility that the ketosteroid observed by Layne and Common (1956) is a metabolite of estrogen. The bile was worked up by the method of Mitchell and Davies (1954) except that no attempt at separation into estriol and estrone-estradiol fractions was made. Chromatography was performed as described by Layne et al. (1958). Radioautograms of the chromatograms showed presence of estradiol and of faint traces of spots corresponding in position to estriol and estrone, but no other radioactive spots were detected. The intestinal tract and excreta were worked up by the same methods as were used for the bile. The first chromatograms yielded two broad radioactive zones, A and B. These were cut out, eluted and chromatographed separately. The more polar zone (A) now gave one narrow radioactive zone (Ai). The less polar zone (B) gave three radioactive zones (B1} B2, B 3 ). Narrow strips were cut from the papers and stained with diazotized sulphanilic acid (DSA) and with dinitrophenylhydrazine (DNPH). The results of staining and mobilities are summarized as follows:
685
686
H. F. MACRAE, D. S. LAYNE AND R. H. COMMON
S R
S R
Ai
Dl
D £.
S R
FIG. 1. Staining reactions with diazotized sulphanilic acid (S) and radioautograms (R) of chromatograms of steroids from excreta of laying hen. Al.—Presumptive estriol fraction with added carrier estriol. Note close correspondence of stained (DSA) region with uppermost radioactive region, confirming the identification of the latter as estriol. Note also presence of broad radioactive zone in presumptive position of estradiol (amount too small to give perceptible stain with DSA). The double nature of the estriol zone (seen also in Bl) probably arose from application of much contaminating material and relatively slight movement of estriol from point of application in the Bush system. Bl.—Presumptive estradiol fraction with added carrier estradiol. Note close correspondence of broad lowermost stained zone with broad radioactive zone, confirming identification of the latter as estradiol. Note presence also of some estriol, which was sufficient to give a staining reaction. B2.—Unidentified zone with added carrier estrone. Note that there was a faint trace of radioactive estrone, but that the strongly radioactive zone was neither estriol nor estradiol nor estrone. The staining at the point of application was due to some unknown constituent; it could not have been estriol since it was not radioactive. B3.—Presumptive estrone with added carrier estrone. Note close correspondence of staining and radioautogram, confirming identification as estrone. Note absence of any other radioactive zone, demonstrating cleanness of separation of the estrone fraction.
either by staining or radioautography. Strip B2 gave a strongly radioactive zone which did not correspond with the staining of the added estrone. This zone was distinctly more polar than estradiol and less polar than estriol. Strip B2 also yielded a minute trace of radioactive estrone, identi-
fiable by correspondence in position with the stained zone of carrier estrone. The identity of B2 has not been established; it was present in sufficient amounts to give a strong DNPH stain, and it was obviously responsible for more radioactivity than the other three radioactive zones. Its relative
Downloaded from http://ps.oxfordjournals.org/ at UPVA on June 1, 2015
5 R
687
ESTROGEN IN THE HEN
position on the chromatogram corresponds with that reported for the 16-hydroxyestrones in Bush chromatographic systems, but the zone did not yield a positive DSA stain. Furthermore, the instability of the 16-hydroxyestrones in alkali makes it improbable that they would survive the extraction procedures used in the work now reported. The possibility that it was formed secondarily by bacterial action in the intestine cannot be excluded, but equally, the possibility remains that it may represent a major metabolite of estrogens in the fowl. SUMMARY
The authors wish to thank the National Research Council of Canada for a grant which defrayed part of the expenses of this work, and Canadian Industries Ltd. for the Fellowship held by one of us (H. F. MacRae). They also wish to thank Dr. B. E. Baker for help in the photography, and the late Professor W. F. Oliver for advice in connection with tracer methodology. We wish also to thank Dr. D. A. McGinty, Parke Davis Co., Detroit, for kindly presenting a sample of reference estriol, and Professor W. A. Maw for providing experimental hens and for helpful advice. REFERENCES Callow, R. K., 1956. Metabolism of the steroids. Ann. Rep. Chem. Soc. 53: 305-318. Heard, R. D. H., E. C. Bligh, M. S. Cann, P. R. Jellinck, V. J. O'Donnell, B. C. Rao and J. L. Webb, 1956. Recent Progress in Hormone Research, 12: 45-71, New York: Academic Press. Hurst, R. O., A. Kuksis and J. F. Bendell, 1957. The separation of estrogens from avian droppings. Can. J. Biochem. Physiol. 35: 637-640. Layne, D. S., and R. H. Common, 1956. Detection of a presumptive keto-steroid in the ovaries and blood of the laying hen. Nature (London), 178: 419-420. Layne, D. S., R. H. Common, W. A. Maw and R. M. Fraps, 1958. Presence of estrone, estradiol and estriol in extracts of ovaries of laying hens. Nature (London), 181: 351-352. Marrian, G. F., 1958. The biochemistry of the estrogenic hormones. Proc. Fourth International Congress of Biochemistry Symposium No. IV. Vienna. Mitchell, F. L., and R. E. Davies, 1954. The isolation and estimation of the steroid estrogens in placental tissue. Biochem. J. 56: 690-698.
NEWS AND NOTES (Continued from page 667) been Director of the Department of Poultry Science Advancement of Science, the American Genetic at the University of Arizona. Association, and Sigma Xi. He is the author of a number of scientific and ONTARIO NOTES technical articles, and is a member of the Poultry The Department of Poultry Husbandry, Ontario Science Association, the American Society for the (Continued on page 700)
Downloaded from http://ps.oxfordjournals.org/ at UPVA on June 1, 2015
1. When radioactive estradiol was administered intraveneously to a laying hen, radioactive estradiol, estrone and estriol could be detected after one hour in the gut plus excreta by chromatographic and radioautographic methods. It has been shown therefore, that the fowl is able to convert estradiol into estrone and estriol. 2. The gut plus excreta also yielded a radioactive steroid other than estradiol, estrone or estriol. This steroid was less polar than estriol but more polar than estradiol in a methanol-petroleum ether chromatographic system, and gave a strong ketonic stain. It also contained more radioactivity than either the estradiol, the estriol or the estrone zone. 3. Radioactive estradiol and traces of radioactive estriol and estrone were detected chromatographically in the bile, but no other radioactive zones. Radioactive estradiol, but no other radioactive steroid, was detected in the blood.
ACKNOWLEDGMENTS