STUDIES ON THE ES 'JROGENS OF THE DOMESTIC FOWL

by Herbert Farquhar MacRae

A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfilment of the requirements for the degree of Doctor of Philosophy.

Department of Ag~icultural Chemistry1 McGill University, Mont~eal. · April 1960 ACKNOWLEDGEMENTS

The author wishes to thank Proressor R.H. Common, who suggested the research described in this thesis and provided much encouragement throughout its course.

The author is also deeply indebted to the rollowing for their help: The lata Dr. w.s. Bauld, for many helpful discussions and Dr. J.A. Raeside, Ontario Veterinary Collage, for helpful advice on several occasions; Dr. w. Zaharia, Biochemistry Department, McGill University, for determining the infrared spectra of certain materials isolated during the course or the work; Dr. P.A. Anastassiadis, Macdonald Collage, for invaluable instruction in ultraviolet spectrophotometry; Dr. D.S. Layne, Worcester Foundation ror Experimental Biology, Shrewsbury, Maas., ror much helpful advice; The late Professor W.F. Oliver, for advice on radio­ autography; and Professors W.A. Maw and N. Nikolaiczuk, Macdonald Collage, for the provision of experimental birds.

The author is particularly indebted to Dr. D.G. Dale, Animal Pathology Department, Macdonald Collage, for oper­ ating on experimental birds which were used in the work. The author also wishes to thank the f'ollowing for providing samples of reference steroids, without which much of this work would have been impossible: Dr. D.A. McGinty, Park Davis & Co., New York; Dr. T.J. Gallagher, Sloan­ Kettering Institute, New York; Dr. W. Klyne, Postgraduate Medical School, London, England; Dr. W.L. Glen, Ayerst­ McKenna and Harrison, Montreal, and Professor G.F. Marrian, Imperial cancer Research Fund Laboratories, London, England.

In conclusion, the author wishes to thank canadian Industries Limited for financial support provided by a Fellowship held by him and to thank the National Research Council of canada for a grant which helped to defray part of the expanses. TABLE OF CONTENTS

GENERAL INTRODUCTION.. • • • • • • • • • • • • • • • • • • • • • • • • • • • • • 1 1. HISTORICAL INTRODUCTION ••••••••••••••••••••••••• 5 1.1. Some Effects of Estrogens in the Bird •••• 5 1.2. Isolation and Metabolism of the Estrogens 10 1.3. Methods of Extraction and Identification of the Estrogens from Animal Sources ••••• 22 2. EXPERIMENTAL METHODS •••••••••••••••••••••••••••• 35 2.1. General Material and Apparatus ••••••••••• 35 2.1.1. Hormones and Related Compounds •••• 35 2.1.2. Solvents •••••••••••••••••••••••••• 36 2.1.3. Color Reagents •••••••••••••••••••• 37 2.1.4. Chromatographie Apparatus ••••••••• 38 2.2. General Methods •••••••••••••••••••••••••• 41 2.2.1. Introduction •••••• ·•••••••••••••••• 41 2.2.2. Preparation of Final Extracts for Chromatography •••••••••••••••••••• 42 2.2.3. Chromatography ••• ~ •••••••••••• ; ••• 44 2.2.4. Detection of Steroids on Paper •••• 47 2.2.5. Spectrophotometry ••••••••••••••••• 48 2.2.6. Identification of Steroids by Radioautography ••••••••••••••••••• 49 3. EXPERIMENI'AL PROCEDURES AND RESULTS - PART I •••• 51 The Isolation and Characterization of Estrogens from the Excrement of Laying Hens. 3 .1. E!x:periment I...... 51 3.1.1. Object •••••••••••••••••••••••••••• 51 3.1.2. Method •••••••••••••••••••••••••••• 51 3.1.3. Resulta ••••••••••••••••••••••••••• 54

3.2. Experiment II~··••••••••••••••••••••••••• 57 3.2.1. Method •••••••••••••••••••••••••••• 57 3.2.2. Results •••••••••••••••••••••• ; •••• 59 3.3. Experiment III ••••••••••••••••••••••••••• 60 3.3.1. Object •••••••••••••••••••••••••••• 60 3.3.2. Method •••••••••••••••••••••••••••• 61 3.3.3. Resulta ••••••••••••••••••••••••••• 62 3.4. Experiment IV•••••••••••••••••••••••••••• 67 3.4.1. Method •••••••••••••••••••••••••••• 67 3.4.2. Resulta ••••••••••••••••••••••••••• 68 E:xperiment V ••••••••••••••••••••••••••••• P7fe 3.5.1. Object •••••••••••••••••••••••••••• 74 3.5.2. Method•••••••••••••••••••••••••••• 74 3.5.3. Resu1ts ••••••••••••••••••••••••••• 77 Discussion ••••••••••••••••••••••••••••••• 78 SUMMARY- PART I••••••••••••••••••••••••• 81 4• EXPERIMENrAL PROCEDURES AND RESULTS - PART II... 83 Studies on the Metabo11sm of the Estrogens in the Laying Hen. Experiment I ••••••••••••••••••••••••••••• 83 4.1.1. Object ••• ~•••••••••••••••••••••••• 83 4.1.2. Method•••••••••••••••••••••••••••• 83 4.1.3. Resu1ts and Discussion •••••••••••• 84 Experiment II•••••••••••••••••••••••••••• 90 4.2.1. Object•••••••••••••••••••••••••••• 90 4.2.2. Method•••••••••••••••••••••••••••• 90 4.2.3. Resu1ts and Discussion •••••••••••• 91 Experi.Inent III ••••••.•••••••••••••••••••• 102 4.3.1. Objeot••••••••••••••••••••••••••• 102 4.3.2. Method••••••••••••••••••••••••••• 103 4.3.3. Results and Discussion ••••••••••• 104 SUMMARY- PART II••••••••••••••••••••••• 124 CLAIMS TO ORIGINAL RESEARCH••••••••••••••••••••••• 127

REFE!RENCES•••••••••••••••••••••••••••••••••••••••• 129 .A.PPENI>IX •• • •. • •••••••••••••••••••••••.•••••••••••• • 140 LIST OF FIGURES FIGURE -PAGE 1. Schematic representation of reactions in estrogen metabolism in man •••••••••••• 17 2. Apparatus used for the separation ?f steroids by paper partition chroma tography ••.••••••••••••••••••••••••• 39 3. Photocopies of typical chromatograms showing separations obtained with reference steroids••••••••••••••••••••••• 46 4. Procedure for the extraction and purification of crude estrogen fractions •••••••••••••••••••••••••••••••• 53 5. Ultraviolet absorption of presumptive estriol and estrone from avian excreta ••• 56 6. Purification of orude estrogen fraction from avian excreta •••••••••••••• 58 7. Ultraviolet absorption of presumptive crystalline estradiol-17~ isolated from avian excreta••••••••••••••••••••••• 65 8. Infrared absorption of crystalline estradiol-17~•••••••••••••••••••••••••••• 66

9. Ultraviolet abso~ption of compound HK •••••••• • ••••••••••••••••••••••••••••• • 70 10. Infrared absorption of compound HK ••••••• 71 11. Ultraviolet absorption of presumptive estriol isolated by high vacuum sublimation from âvian excreta ••••••••••• 73 12. Staining reactions with diazotized sulphanilic acid and radioautograms of ohromatograms of steroids from excreta of laying hen •• ~••••••••••••••••• 87 13. Chromatographie fingerprinting of presumptive estrone and estradiol from lipophilic fraction ••••••••••••••••• 93 FIGURE PAGE 14. Radioautogram of chromatogram of hydrophilic estrogen fraction from extract of droppings of a laying hen which bad been lnjected with estradiol-17~-16-cl~ •••••••••••••••••••••. 94 15. Chromatographie fingerprinting of presumptive estriol from hydrophilic fraction •••••••••••••••••••••••••••.••••• 95 16. Chromatographie separation of estriol and 16-!E!estriol in System I •••••••••••• 97 17. Chromatographie fingerprinting. of presumptive 16-~estriol •••••••••••••••• 18. Chromatography of lipophilic fraction of urinary estrogens of laying her · after injection with estriol-16-C 4 •••••• 105 19. Chromatographie fingerprinting of presumptive 16-~estriol from lipophilic fractiOn of urine ••••••••••••• 109 20. Ultraviolet absorption of presumptive equol after elution from chromatograms of extracts of avian excreta ••••••••••••• 111 21. Chromatography of equol obtained from avian excreta and reference equol in System II •••••••••••• ~•·•••••••••••••• 112 22. Chromatography of hydrophilic urinary estrogens of 1a14ng hen after injection of estriol-16-C ~··••••••••••••••••••••• 114 23. Chromatography of lipophilic fecal estrogens of lallng hen after injection of estriol-16-C 4 ...... 118 24. Chromatography of hydrophilic fecal estrogens of lallng hen after injection of estriol-16-C ~•••••••••••••••••••••••• 120 25. Distribution of radioactivity between lipophilic and hydrophilic estrogen fractions from both urine and feces after injection of estriol-16-cl~ •••••••• 121 26. Daily urine volumes for Hen A•••••••••••• 143 27. Daily urine volumes for Hen B•••••••••••• 145 GENERAL INTRODUCTION

Research with the domestic fowl bas played an important part in the development of knowledge in fundamental endocrinology. Tbè experimenta of Hunter with domestio fowl in 1749 appear to constitute the first experimental demonstra­ tion of what would now be described as ho~onal action by the gonad. Similarily, the experimenta of Berthold in 1849 on the transplantation of fowl testes are regarded as the first demonstration of an endocrine secretion. Later, in 1914, the experimenta of Goodale with domestic fowl played a large part in implicating the ovary as the source of the hormone or hormones that determine the female secondary sexual characters.

Studies on the affects of exogenous hormones have led to much information on the reproductive physiology and endocrinology of birds, particularly of the female domestio fowl. Exogenous estrogen has been shown to produce striking changes in immature pullets, both on the morphology of the secondary sex organe and on the composition of the blood and other tissues. The similarity of these changes to those which occur in the normal pullet at puberty clearly indicate the presence of endogenous estrogens in the bird.

In view of the importance plaoed on the ho~onal control of the laying oyole by many workers in this field, the - 2 -

identification of the gonadal hormones of the ben became a prerequisite to a more precise elucidation of their part in egg reproduction. Before 1957, chemical or physical evidence as to the exact nature of the naturally ocourring hormones was laoking. However, Dr. D.S. Layne, working at Macdonald College, identified progesterone in extracts of ovarian tissue from laying hens. The identification was based on chromatograpby, spectropbotometry and radioautographic lfingerprintingt. At the same ttme workers at Queen's University, Kingston, Ont., identified estriol and estrone in the droppings of male and female birds and estradiol in the droppings of male birds. Their identifications were based only on chromotatographio evidence and oolor tests. About the same time, Dr. Layne identified estradiol, estrone and estriol in extracts of ovaries from laying hens. His identifications were based on chromatography, Kober reactivity and radioautographic 'fingerprinting'. These observations provided the only information on the precise nature of the endogenous estrogens of avian species that waa known to the autbor before the present studies.

All the studies tbat have been carried out on the affect of exogenous eatrogens on birds have made use of mammalian or synthetic estrogens. The quantities of these hormones required to elicit. changes in an immature pullet comparable to those which occur at puberty are relatively large. - 3 -

Both androgen and progestogen are known to enhance the affect of estrogen on the oviduct when present in appro­ priate amounts and relative proportions. While these hormones are presumably present along with estrogen in the normal adult ben, the amount of exogenous estrogen necessary to produce puberal changes in the pullet are still large enough to suggest that the level of estrogenic activity in the laying ben must be extremely high. Consequently, a quantitative study of the excretion of the estrogens by the laying ben is of ultimate importance.

There is, at present no satisfactory supplement to the urinary assay for the purpose of assessing the secretory activity of estrogen-produoing tissue in any species, and practic·ally all the existing methods are designed accordingly. In this regard, the bird presents a serious complication in that the urine and feces are voided together and can only be collected separately after surgical modification of the birds. It sbould be borne in mind, however, that the import­ ance of fecal excretion of estrogens in various species has been increasingly recognized, and that there are considerable species differences in the nature of urinary and fecal

steroids, even as between mammals. It must be emphasized1 therefore, that a qualitative investigation of the excreted steroids should be made before their estimation is undertaken and that care must be exercised in applying to animal excreta, - 4 - whioh may oontain unknown interfering substances, methods originally designed for human urine.

This thesis is ooncerned with: (a) the isolation and identification of the steroid estrogens in the excreta of the laying hen; (b) studies on the metabolism of estrogens in the laying hen using c14 labelled oompounds; and (o) some preliminary observations on the amounts of estrogen exoreted by the laying hen. 1. HISTORICAL INTRODUCTION

1. 1. Some Effects of Estrogens in the Bird

The literature on early researches with birds on the basic concepts of endocrinology has been reviewed by Domm (1939). Studies on the relation of the gonads and their endocrine products to the development of somatic sexual characters, and to the functions of prtmary and accessory sex organs, have made much use of birds and especially of the domestic fow1. The avai1ability and convenience of the chicken embryo bas led to its extensive use in studies of genetic-endocrine interactions in the initiation of secondary sexua1 differentiation. Similar1y1 much use bas been made of the variety of secondary sax characters exhibited by birds and the pronounced sexual dimorphism displayed by some of them in evaluating auch concepts as hormone thresholds, hormone antagonisme and synergisms, and speoies specificity of ~esponse to ce~tain ho~mones.

Subsequent to 1940, many of the above mentioned problems have remained subjects of active research, though the main emphasis has shifted as has that of endocrinology generally, to studies of the metabolio affects of endocrine products. In particular, the administration of synthetio estrogens to poultry to increase the rate of fat deposition and to produoe additional cammercially desirable effeots, bas received much - 6 - attention. The efficacy of commercial estrogenization of poultry bas been for some time a matter of controversy, chiefly because of the possibility of a hazard to human health. · Nevertheless, commercial estrogenization of poultry had been legalized in the United States and canada for a time. The effects of estrogen on domestic fowl and the application of commercial estrogenization in the poultry industry has been exhaustively reviewed by Lorenz (1954), who in point of fact initiated the commercial application of estrogen treatment of poultry.

Juhn & Gustavson (1930) first showed that the accelerated growth of the oviduct, characteristic of puberty in the hen, could be stimulated in the immature female by administration of estrogen. This affect, together with other affects of estrogen on secondary sexual characters, bas been repeatedly confirmed. This aspect of avian physiology has been reviewed by Sturkie (1954) and no detailed discussion of the literature will be attempted here.

The relationsh1p of b1ood fat to egg-1aying in hens was first reported by Warner (1916) and by Lawrence & Riddle (1916). These workers ahowed that b1ood lipid levels are increased in the hen during periods of ovarian activity, a finding that has sinee been repeated1y confirmed and extended (Lorenz 1954). Str1k1ng increases in blood 1ipid leve1s were observed by Chaikoff, Entenman & Lorenz (1938) following - 7 - administration of crude gonadotrophin to immature females, and by Lorenz, Chaikoff & Entenman (1938) who administered "crude estrin11 to immature birds of both sexes. This affect of estrogen on the lipogenio meohanism has been confirmed for immature birds by many workers using a variety of estrogenic oompounds (Lorenz 1954; Sturkie 1954).

The blood serum contents of calcium, protein, vitamin A and riboflavin are inoreased by estrogen treatments. These, and other affects, have been reviewed by Riddle (1942),

Parkas & Emmena ( 19l.W.) and Lorenz: ( 1954) and no detailed citation of the literature will be given.

Marlow & Riohert (1940) oarried out a quantitative study of the estrogenic aotivity of alooholio extraots of egg yolk and of various parts of the ovary of the laying hen. The alcoholio extraots were taken up in olive oil and injeoted into rats. The assay was carried out by the vaginal smear method of Kahnt & Doisy (1928) and the resulta

11 were reported in "rat units • Fraps & Sykes (1956) carried out tests for estrogenio potenoy in parts of the hents ovary. Alcoholio extracts of the walls of the follioles and of the remainder of the ovarian tissue were assayed for estrogenic potenoy by the method of Astwood (1938). The resulta of the assays were used to estimate the estrogen content of the total yolk-free ovary. Layne (1956) made a oomparison of the resulta obtained by Marlow & Richert (1940) - 8 - with those of Fraps & Sykes (1956). This comparison revealed that the values found by Fraps & Sykes (1956) were larger by a factor of several hundred than those reported by Marlow & Richert (1940) for comparable parts of the ovary. The values found by the latter indicate that the amount of estrogen present in the ovary is very small, while the findings of the former support the suggestion advanced by Riddle (1942) that the estrogen production of the ovary of the laying hen may be very large.

The work of Layne (1956) on the estrogens elaborated by the avian ovary are mainly of qualitative signifioanoe and this aspect of the work will be referred to later in this review. Nevertheless, the limited quantitative observations made by hlm are of value in that they indioate an estrogenio potenoy in the ovarian tissue much higher than that reported by Marlow & Ricbert (1940) and more olosely approximat1ng that reported by Fraps & Sykes (1956) using the Astwood {1938) bioassay.

Gustavson (1931) published a preliminary report on the estrogen content of the "feces" of hens as determined by b1oassay. No details as to the origin of the mater1al or the method of assay are given, but it may be assumed tbat the material assayed was actually an extraot of the total droppings (faces plus urine) of laying birds. Gustavson - 9 - commenta that "the hormone content of the feces of the hen compares favorably with that found in pregnancy urine". In a somewhat more detailed discussion, Gustavson (1932) reported an estrogenic potency of 0.7 to 1.0 Allen-Doisy rat units par gram of excreta from laying hens. Since the work seems to have been based on limited observations, it is questionable how far these figures are admissable as a basis for speculation about the rate of estrogen production by the ovary. These observations by Gustavson (1931, 1932), together with those of Hurst, Kuksis & Bendell (1957) appear to constitute the only published work on estrogens in the excreta of avian speoies previous to the present study.

When oonsidering the probable significance of these estrogen levels, it must be remembered that the reproductive cycle of the domestic ben, apart from its obvious differences from those of viviparous species, is notable among those of oviparous vertebrates in so far as the domestic ben produces eggs at short intervals over a long period of time. Most breeds of domestic hans have actually been rigorously selected over many generations for high reproductive activity as manifeated by high egg production. The metabolio activities of the liver and the ovary in elaborating the yolk material, and that of the oviduct in secreting the proteins of the egg white and shell membranes, and in mobilizing calcium and other minerals in shell formation, - 10 - are consequently very intensive. The laying ben i~ in fact, an animal in an extremely active reproductive state.

l. 2. Isolation and Metabolism of the Estrogens

The chemistry of the gonadal hormones came into being as part of the rapid, almost explosive, development of the chemistry of the sterols some 30 years ago. In 1929, two laboratories reported the isolation of estrone from the urine of pregnant women (Doisy, Veler & Thayer, 1929; Butenandt, 1929), and in the following year Marrian (1930a, 1930b) reported the isolation of estriol from the same source •

.At this time, work on the isolation of vitamin D (calciferol, ergocalciferol) was in progress in England and Germany, and the X-ray studies by Bernal (l932a, 1932b) on ergosterol and its irradiation products provoked Ro.senhe1m & King (l932a, l932b) to propose the perhydrocyclopentanophenanthrene formula for cholesterol. This new theoretical structure had an immediate and clarifying affect on the chemistry of a multitude of compounds including the sex hormones. Subsequently, Marrian and Haslewood (1932a) and Butenandt (1932) put forward the formulae for estrone and estriol.

A suspic1an arose that estrone was not the primary hormone when it was discovered that estradiol-17~, prepared by reduction of estrone, was physiologically more potent than - 11 - estrone. MacCorquoda1e, Thayer & Doisy (1935, 1936) succeeded in iso1ating soma 12 mg. of estradio1-17~ from four tons of sow•s ova!'ies, a y1e1d that accounted for about half of the estrogenic activity present. Eventua11y estradio1-17~ became established as the primai'y estrogen.

No new pheno1ic steroids were isolated from human pregnancy urine between 1940 and 1955-56, when Marrian and

Bauld (1955) 1solated 16-~estriol. The isolation of this

16~ substituted steroid indicated the possibi1ity of both

16a. - and 16~-hydroxy. lations in the metabo1ism of the estrogens.

Migeon (1953) obtained suggestive evidence for the p!'esence of 16-ketoestradiol-17~ in human urine by counter-current distribution and f1uorimetry. Watson & Marrian (1955) lata!' found in human urine extracts a new ketonic compound which gave the typical color in the estrogen­ specific Kober (1931) reaction and behaved like

16-ketoestradiol-17~ in counter-current distribution and partition chromatography. Marrian, Watson & Panattoni (1957) subsequent1y isolated their new Kober chromogen from enzymatical1y hydrolyzed urine in sufficient quantity to a1low chemical characterization. The properties of the crystalline material obtained clearly indicated that it was not identical with 16-ketoestradiol-17~. They suggested that this new compound was mainly 16a.-hydroxyestrone and that the - 12 -

16-ketoestradio1-17~ found in their ear1ier work (Watson & Marrian, 1955) had arisen by isomerization from 16a-hydroxyestrone during exposure to a1ka1i. The main component of the iso1ated crysta11ine materia1 was subsequent1y identified as 16a-hydroxyestrone by Marrian, Loke, Watson & Panattoni (1957).

Meanwhile, Levitz, Spitzer, & Twomb1y (1956) reported the detection, by reverse isotope dilution, of radioactive

16-ketoestradio1-17~ in the urine of human subjeots after the administration of estradio1-17~-16-c 14. This finding was, at first, subjected to adverse oriticism by Marriants group, however, in the light of subsequent work by Layne & Marrian (1958) this critioism was unreservedly withdrawn.

Marrian, Watson & Panattoni (1957) and Marrian, Loke, Watson & Panattoni (1957) suggested that 16a-hydroxyestrone, formed from estrone by 16a-hydraxylation, might be the metabolic precursor of estrio1 and that similarily,

16~-hydroxyestrone might be the precursor of 16-~estriol. At that time, their suggestion was entire1y speculative, but was considered sufficient1y plausible to warrant an intensive searoh for 16~-hydroxyestrone in pregnancy urine. Later, Layne & Marrian (1958) reported the isolation, in crystalline form, of both 16~-hydroxyestrone and 16-ketoestradiol-17~. Since the isolation procedues employed were un1ike1y to have caused artifactual formation of 16-ketoestradiol-17~, the work supported - 13 -

the view of Levitz, Spitzer & Twombly (1956) that

16-ketoestradiol-17~ is a natura1ly occurring estrogen metabolite.

Serchi (1953) reported the isolation of 16-ketoestrone from human urine. Later, Slaunwhite and Sandberg (1956) isolated 16-ketoestrone-16-c14 after administration of estrone-16-c14.

When considering the isolation of these newer estrogens, it must be remembered that the D-keto1ic compounds

(16a-hydroxyestrone, 16~-hydroxyestrone and

16-ketoestradiol-17~) are unatab1e. They are large1y destroyed by aoid hydrolyais. Consequently, enzymatic hydrolysis must be employed. Girard•s reaction for the separation of ketonic from non-ketonio materiels must be oarried out at room temperature. In alka1ine solution

(N NaOH), 16~-hydroxyestrone very rapidly rearranges to

16-ketoestradiol-17~, 16a-hydroxyestrone undergoes the

same rearrangement lesa rapidly, and 16-ketoestradiol-17~ itself becomes s1owly autoxidized to marrianolic aoid (Marrian, 1958).

In addition to the compounds described above, newer urinary estrogen metabolites have been isolated. Kraychy & Gal1agher (1957) isolated 2-methoxyestrone from human urine after administration of estradiol-17~-16-c14. This finding was confir.med by Engel, Baggett & Carter (1957). More - ~ - récently, Loke & Marrian (1958) isolated 2-methoxyestrone from human pregnancy urine. In all instances, enzymetic hydro1ysis was employed in the isolation procedure. Fisbman & Gallagher (1958) reported the formation in vivo of 2-methoxyestriol in human subjects following the injection of estradiol-17~-16-cl4. The discovery of these two methoxy oompounds points to the possible presence of other 2-methoxy metabolites in human urine.

The isolation of 18-hydroxyestrone from the urine of pregnant women by Loke, Marrian, Johnson, Meyer & Cameron (1958) is of great interest but its significance in estrogen metabolism is not yet c1ear. It seems possible that 18-hydroxyestrone may be an estrogen metabolite formed in the adrenal gland, for it was obtained when estrone was incubated with ox adrenal homogenates (Marrian, 1958).

The isolation of these newer estrogen metabolites has contributed much to the clarification of the prob1em of estrogen metabolism in humans. The work of Levitz, Spitzer & Twomb1y (1958) is of particular significance in that

16-ketoestradiol-17~ bas been shown to be an intermediate in the conversion of estriol to 16-!E!estriol. The administration of estriol-16-c14 to human subjects led to the excretion of radioactive 16-ketoestradio1 and 16-!E!estriol. Breuer, Nocke & Knuppen (1958) reported that the incubation of lba-hydroxyestrone with slices of buman liver yielded - 15 - estrio1 and 17-epiestrio1. Under the same conditions, 16- ketoestradio1-17~ was reduced to estriol and 16-!E!estriol. Breuer, Nocke & Knuppen (1959) also showed that the metabolic reduction of 16~-hydroxyestrone to estriol takes place in vitro in the 1iver of the rat and guinea-pig, but in contrast to the experimenta with human tissue, no 17-epiestriol could be detected.

In a later study, Breuer, Knuppen & Pangels (1959) incubated 16-ketoestrone with human liver and ovarian tissue. On the basis of their resulta, they assumed that the first reduction of 16-ketoestrone led to 16-ketoestradiol as well as to the isomers 16a-hydroxyestrone and 16~­ hydroxyestrone. In the course of the second reduction,

16-ketoestradiol-17~ was hydrogenated to estriol and 16- !E!estriol, while 16a-hydroxyestrone formed estriol and

17-epiestriol; similarily, 16~-hydroxyestrone was reduced to 16-epiestriol. These resulta are in agreement with those of Stimme1 (1958) 1 which indicated that estriol and

16-~estriol are metabolic products of 16-ketoestrone. While the metabolic statua of 16-ketoestrone in vivo is not certain, it may be assumed that its position lies between estrone and the D-ketolic estrogens.

Breuer & Nocke (1959) reported the identification of estriol-3, 16~, 17a, the fourth possible isomer of estriol, when 16a-hydroxyestrone and 16-ketoestradiol-17a - 16. - were incubated with normal human liver slices. They reported that estriol-3, 16~, 17a was a minor metabolite of

16~-hydroxyestrone but that 16-ketoestradiol-17a gave rise preferentially to estriol-3, 16~, 17a. However, they stated that the quantitative significance of this new epimer of estriol cannot be fu1ly estimated unti1 the position of 16-ketoestradiol•17a in the metabo1ic schema bas been elucidated. Later, Breuer & Pangels (1959) reported the isolation of estriol-3, 16~, 17a (10 micrograms) from late pregnancy urine in crystalline form.

On the basis of the evidence just presented, one may construct a schema that representa the possible sequence of reactions which have actually been shown to occur and those which arestill speculative in the metabolism of the estrogens in humans. Such a schema is presented in Figure I.

Both the in vivo and in vitro for.mation of estriol from estradiol-17~ and estrone are well known (Dorfman, 1957). Recently, a second distinct pathway for the forma­ tion of estriol in vitro was reported by Ryan (1958) when he demonstrated the aromatization of~5-androstene-3~, 16a,

17~-triol by human placenta. This observation, that estriol cou1d be produced by aromatization of a C-16 hydroxylated androgen without involving the olassic conversion from estrad­ iol, promoted a more extensive study by Ryan (1959) of the metabolism of C-16 oxygenated neutral and phenolic steroids by human placenta. This study demonstrated the conversion of - 17 -·

oH ~ estrucliol 0

r------P-~~~~------, 1 : 1 1

' 1 () . 1

· ~ . tr~etoestrone l p--OH-/ 1l ~ p-OH 1~-hydroxyestrone 16 ~-hydroxyestrone

"" OH ~ P-OH l6,17,epiestriol qH OH~ . ~ OH p--OH p---OH poH p=oO.H/

17-cpiestrio1 es triol 16-cpiestrio1 16-ketoe s tradio1-17~

FIGURE 1. Schematic representation of reactions in estrogen metabolism in man. Solid arrows indicate reactions which have been shown to occur either in vivo or in vitro. Broken arrows indicate reactions which are still speculative. · - 18 - three 16-oxygenated neutra1 steroids to estrio1 and a new source for estriol formation was estab1ished. Ryan (1959) also suggested a tentative intermediary role for 16a-hydroxyestrone between the 16-oxygenated neutra1 steroids and estrio1. It is generally oonceded that a major proportion of the estrio1 in human pregnanoy urine is of plaoental origin. Whether this fraction is produced by C-16- hydroxylation of estradiol or by aromatization of an already 16-hydroxylated androgen must await further study.

Other studies on the conversion in vitro of androgens to estrogena have been reported. Baggett, Engel, Savard & Dorfman (1956) reported the conversion of testosterone-3-c14 to estradiol-17~-16-cl4 by human ovarian tissue. Later, Baggett, Engel, Savard & Dort.man (1956) reported the conversion of testosterone-3-c14 to estradio1-17~-16-cl4 by human ovarian tissue. Later, Baggett, Engel, Balderas, Lanman, Savard & Dorfman (1959) demonstrated the conversion of testosterone-3-c14 to estradio1 by sta11ion•s testioular tissue and to estrone, estradiol and a substance similar to but not identica1 witb estrio1 by p1aoenta1 tissue. The overa11 conversion of androgens to estrogens, both in vivo and in vitro, bas been reviewed by Dorfman (1957).

While practically all the isolations or identifications of estrogen metabolites referred to above have been from human sources, the occurrence of estrogens in other species - 19 - has been reported. The literature on this aspect of the estrogens bas been reviewed by Dorfman (1957) and no detailed report will be attempted here.

El-Attar and Turner (1957) published an extensive report on the determination of estrogens by spectrophotofluorometric methods in the urine and feces of cows at different stages of pregnancy. They round that the non-ketonic fraction of the phenolio steroid extract was the major component in both urine and feces and that estrogen was excreted mainly by way of the feces. Pilot experimenta showed the absence of the non-ketonic, strongly phenolio estrogen (estriol) in both urine and feoes. The total amount of estrogen excreted in oows• urine was low as compared to the urine of the mare or that of the human female. The finding that estrogen is excreted mainly by the pregnant oow -via. the feoes has been reported by others (Pearlman, Rakoff, cantarow & Paschkis, 1947). The presence of estradiol as the major estrogen in cow feces while estrone is the major one in the cow bile may be due to the action of intestinal bacteria rather than to intermediate metabolic processes in the body (Levin, 1945). Klyne & Wright (1957) succeeded in isolating estradiol-17a from the urine of the goat and from the urine of the cow (Klyne &

Wright, 1959). Estradiol-17~ was not detected in the urine of either species by Klyne & Wright (loc.~.) although - 20 -

Gorsky & Erb (1959) have reported the presence of both estradiol-17a and estradiol-17~ in bovine placenta.

The urine of pregnant mares bas provid.ed a variety of interesting phenolic steroids. In addition to the wall known ketonic steroids, viz., estrone, aquilin and equilenint which differ only in the degree of saturation in ring B, the corresponding diols in whicb the hydroxyl group at carbon 17 has the a-configuration, i.e.,estradiol-17a, dihydroequilin-17a, dihydroequilenin-17a are also present, (Glen, 1958). It would appear that the 17-a configuration is characteristic of the diols present in pregnant mares• urine. It should be noted that the ring B benzenoid steroids have not been recognized as either endogenous or exogenous estrogen metabolites in species other than the horse, except for the reported isolation of equilenin from an adrenal femininizing tumor by Salhanick & Berliner (1957). The steroidal ketoneA5,7,9_estratrienol-3-one-17 is not a well-known compound, but it was found by Glen (1958) in pregnant marest urine in greater quantity than was equilenin. The only other isolation of this compound recorded in the literature is that of Heard & Hoffman (1940). Until about 1948, when satisfaotory methods for the aromatization of ring A were developed, all the estrogens required commercially were obtained from pregnant marest urine. Consequently, mucb detailed study has been devoted to this materiel. The - 21 - isolation of steroids from the urine of the pregnant mare bas been described by many research groups and no attempt will be made to review this literature.

As regards avian species, Hurst, Kuksis & Bendell (1957) reported the presenee of estrone and estriol in the droppings of male and female domestic fowl. In addition, they observed the presence of estradiol in very small amounts in the droppings of the rooster. Their identifications were based on chromatographie evidence and color tests.

Layne, Common, Maw & Fraps (1958) identified estrone and estradiol in the avaries of laying hena. Their identifications of these two estrogens were based on chromatographie evidence, ultraviolet spectrophotometry and radioautographic fingerprinting. In addition, they identified estriol by chromatography and ultraviolet speotrophotometry from the same source.

The occurrence of estriol in avian species is of particular significance. Except for its reported isolation from the female pussy willow and from the placentae of the chimpanzee, estriol has been isolated only from human sources and is generally regarded as characteristic of the human species (Merrill, 1958). Dorfman, (1957), however, discusses the reported identifications of estriol in species - 22 - other than man and states that "these studies remove the myth that estriol is an exclusive product of man". Recently, Mitchell & Hobkirk (1959) have shown that avian liver slices can convert estradiol-17~ to estriol.

1. 3. Methods of Extraction and Identification or the Estrogens from Animât sources

The investigation of the steroid hormones in animal tissues or excreta involves the extraction of the hormones rrom the particular material under study and the purification and identification of the individual steroids or their metabolites. The amount of steroid present is often minute and experimental losses are almost always considerable. It is often necessary, therefore, that the method employed should be capable of detecting and estimating amounts of the order of 5 to 100 micrograms.

The techniques used will depend on the kind of material being studied and on the type of steroid or group of steroids that is being sought. Mitchell & Davies (1954) have reported an extraction procedure applicable to the isolation of estrogens from human placenta. Bauld & Greenway (1957) reported a detailed procedure for the determination of estrogens in human urine. Both these methods have been adapted in the present work to the investigation of estrogens in avian excreta. - 23 -

Estrogena, as well as other steroids, are excreted in the urine mainly in the form of water-soluble conjugates of glucuronic acid or sulphuric acid. Much of the literature on the nature and formation of these conjugates has been reviewed by Roberts & Szego (1953) and specifically on estrogen conjugates by Bauld & Greenway (1957). Hydrolysis of these compounds is desirable for two ressons. First, the chemical nature of all conjugates is not known, so that all methods of assay have been developed using the readily available free estrogens. Second, extraction with organic solvants is a convenient method for the separation of estrogens from the highly water-soluble compounds whioh conatitute the bulk of urinary solutes. Suoh an extraction is facilitated by hydrolysis, since free estrogens have much more favorable partition coefficients for extraction by organic solvants than their conjugates. Considerably greater purification is thus achieved when the extraction is preceded by hydrolysis. In the past, it has been the practice to carry out a preliminary removal of the free steroids before hydrolysis. Indeed, Bauld & Greenway (1957) have considered it to be q:uestionable whether free estrogens are ever excreted in the urine.

A suocessful recovery of steroids from their conjugates is dependant on the establishment of conditions of hydrolysis which give optimal breakdown of the conjugates - 24 - with little destruction of the steroids themselves. Bauld & Greenway (1957) have discussed the optimal conditions for hydrolysis of urinary estrogens. The problem of destruction of estriol, estrone and estradiol-17~ during hot acid hydrolysis was investigated by Brown (1956). He found two indications of destruction of estrogens when these were boiled with urine acidified with 15 volumes per cent hydrochloric acid. First, the amounts of the three estrogens were diminished when boiling was continued for more than 60 minutes. Second, the recovery of free estrogens added to urine before hydrolysis under these conditions was 10-20% less than when they had been added after hydrolysis. Brown (1956) found that neither the removal of ether-soluble material or proteine before hydrolyais nor the addition of a variety of oxidizing or reducing agents afforded an increaaed yield of eatrogens from urine or ohanged the recovery of free estrogens added to urine. Decrease in acid concentration and over-laying with solvant increased the recovery of added free estrogena but lowered the yield obtained by simple reflux of the urine specimen for 60 minutes after the addition of 15 volumes percent of hydrochloric acid. On the other band, ten-fold dilution of the urine specimen with water increased the yield of estrogens and prevented losa of added free eatrogens. - 25 -

The advantage of enzymatic hydrolysis of urinary estrogens over acid hydrolysis is a lasser destruction of estrogens. The disadvantages are the additional time required, the expanse of suitable enzyme preparations, their sensitivity to inhibition and their tendency to cause emulsification in the extraction procedure. Bauld & Greenway (1957) have reviewed the various types of enzyme preparations used. It should be noted that enzymatic hydrolysis is essential for the determination of 16-hydroxyestrone (see section 1. 2.).

The purification procedures applied to extracts are designed to separate particular groups of steroids and to remove certain contaminants. The most difficult problem in the case of urinary extraots is the elimination of pigments which are fluorogenic or chromogenic and hence may interfere with color reactions which are employed to detect or estimate the steroids. Cohen & Marrian (1934) introduced a procedure for the extraction, purification and separation of estrogens from acid hydrolyzed human pregnancy urine ·based on simple solvant partition. The

ether extracts of the urine were washed with 10% Na2co3 to remove the acidic fraction. This acid fraction includes various organic aoids, traces of hydroxyaryloarboxylio aoids and substituted hydantoins formed by condensation of urea with a-keto acids during aoid hydrolysis. The - 26 - neutral fraction includes most non-phenolic steroids, indigoids and cholesterol (Bauld & Greenway, 1957).

Bachman & Petit (1941) and Mather (1942) showed that losses of estriol occur on washing ether with 10% Na co • 2 3 Engel, Slaunwhite, Carter & Nathanson (1950) and Stevenson & Marrian (1947) suggested that sodium bicarbonate be substituted for sodium carbonate but this gave considerably less pure fractions than the original procedure of Cohen & Marrian ( 1934).

Brown (1955) introduoed some very effective steps in the purification of urinary estrogen fractions. He made the important observation that the distribution of estriol between ether and w.eak alkali depended more upon the ionie strength than upon the pH of the aqueous phase. Thus he found the partition coefficient of estriol between ether and concentrated carbonate buffer of pH 10.5 to be practically the same as that between ether and saturated NaHco3 solution. This permitted remova1 of almost as much impurity as the original 10% Na c 0 wash. 2 3 • Bauld (1956) observed that brown pigments were formed when the ether extract from urine was shaken with 2N NaOH following a wash with concentrated carbonate buffer. These pigments remained in the aqueous phase when the pH was brought to about 10. by the addition of NaHco • The estrogens 3 - 27 -

(estriol, estrone and estradiol) were not deoomposed by the alka1ine treatment and could be re-extracted by ether from the aqueous phase at this pH.

In spite of the effectiveness of this step in purification, it was found (Bauld, 1956) that further exposure to alkali resu1ted in formation of more brown · pigment. Saponification in aqueous N NaOH effective1y removed these ohromogens. Diczfalusy (1953) first demonstrated the stability of estrone to boiling in dilute

NaOH. The stabi1ity of estriol, estrone and estradiol-17~ to refluxing 30 minutes in N NaOH has since been shown (Bauld, 1956). It will be recalled (see section 1.2.) that purification of estrogen extracts by alkali treatments must be avoided in the determination of the 16-hydroxyestrones. The final steps in the procedure of Bauld (1956) for the determination of urinary estrogens involve the partition of the partially purified ether extract between benzene (dissolves estrone and estradio1) and water (dissolves estriol). Eaoh fraction is then submitted to partition chromatography on Celite columns.

In addition to the simple partition methods discussed above, tbere are several ohemical methods for the purification of estrogen fractions. All these chemical methods involve the formation of derivatives with partition coefficients differing from those of the parent estrogens. - 28 -

The use of Girard's Reagent T is very effective for the purification of the ketonic {estrone) estrogen fraction. The ketones form water-soluble derivatives with this reagent and can be separated from ether soluble impurities by simple partition. The non-ketonic estrogens {estriol and estradiol) may be further purified by condensing them with succinic or pbthalic anhydrides to form the corresponding hemiesters (Pincus & Pearlman, 1942).

The principle of purification by derivative formation was applied in a most efficient form by Brown (1955).

After separation of estriol, estrone and estradiol-17~ from "acids" and"neutral" contaminants by simple partition procedures, he formed the methyl ethers of the phenolic groups by treatment with dimethyl sulphate in alkaline solution. These derivatives were extractable from aqueous solution with non-polar solvants and separation from residual polar contaminants wasacbieved in this way. Moreover, the increased stability conferred on the molecules by methylation of the phenolic groups permitted the use of hydrogen peroxide to oxidize various impurities to polar end products.

The steroids and steroid metabolites which have been extracted and partially purified must be separated and identified. In earlier work, each compound was isolated - 29 - in the crystalline state and estimated gravimetrically, but chromatographie methode are now universally employed and have made possible the separation of small amounts of closely related steroids. However, in spite of the development of newer methods auch as chromatography, crystallization is still a most important method of isolation. In sorne cases this may involve the processing of large amounts of materiel by paper chromatography before crystallization can be carried out (see Layne & Marrian, 1958).

A very useful review of the methode for separating estrogens from hydrolyzed urine is given by Bauld & Greenway (1957). The methods reviewed by them include adsorption chromatography, ion exchange ohromatography, countercurrent distribution, oolumn partition chromatograpby and paper partition chromatography. Another useful review on the methode of adsorption and partition ohromatography of steroids and related oompounds bas been given by Bush (1954).

Filter paper partition methods are of two main types. That introduced by Zaffaroni, Burton and Keutman (1949) uses paper impregnated with a high-boiling polar solvant, usually propylene glycol or formamide, as the stationary phase. The mobile phase is a volatile, non-polar solvent - 30 - saturated with the stationary solvant. Bush (1954) used a second type of system, in which both phases consisted of volatile solvents, the stationary polar phase being preferentially adsorbed onto the paper during equilibration at a slightly elevated temperature (32° to 34°c) before the start of the run. Reineke (1956) has discussed the relative advantages and limitations of the two types of system.

The detection of steroids on paper is achieved from a knowledge of the behaviour of pure reference substances in the system and by chemical tests with reagents specifie for particular grpups of steroids. Soma steroids are visible on the paper under ultraviolet light. It must be emphasized that no one technique is capable of isolating and identifying more than a ltmited group of compounds, so that chromatographie systems and methods of detection employed vary with the characteristics of the steroid being sought. Bush (1954) has tabulated appropriate methods of detection and identification for many types of steroid material.

The mere number of known steroids of closely similar structure and the possibilities for isomerism which exist Pender identification by chromatographie mobility and reaction to chemical tests inconclusive. The mobility of steroid material is subject to change when non-steroid - 31 - materials are present, although the relative positions of the individual steroids with respect to one another is almost always constant (Bush, 1954). Mobility values in a given system are usually expressed in relation to that of some standard reference compound instead of in relation to distance travelled by the solvant front (Kochakian & Stidworthy, 1952; Savard, 1954). Reineke (1956) has compiled mobility values for a large number of steroids in several different chromatographie systems. Bush (1954) pointed out the confusion that may arise from the fact that many of the chemical reagents used for detecting steroids may react with non-steroidal material.

Ultraviolet absorption analysis bas been widely applied to the investigation of steroids. Dorfman (1953) published an extensive and useful tabulation of the major ultraviolet absorption peaks of a large number of steroids and steroid derivatives in various organic solvants. Zaffaroni (1950) and Bernstein & Lenhard (1953) studied the ultraviolet absorption spectra of steroids in ooncentrated sulphuric acid. The spectra of steroids in phosphoric acidhas also been investigated (Nowaczynski & Steyermark, 1955). Infrared spectrophotometry bas become increasingly useful in providing a great deal of information on the structure of steroid materials (Jones & Herling, 1954). Two volumes of a comprehensive atlas of the infrared spectra - 32 - of steroids have been published (Dobriner, Katzenellenbogen & Jones, 1953; Roberts, Ga1lagher & Jones, 1958). The application of infrared analysis to materials obtainab1e from paper chromatograms is rather limited due to technical difficulties arising as a consequence of interfering materia1s from the paper (Layne, 1956).

The so-called "fingerprint" method of radioautography can be applied to the identification of steroids provided that labelled reference samples of the steroids are available. The chromatographie fingerprint technique is described by Roberts, Abelson, Cowie, Bolton & Britten (1955) and by Layne, Common, Maw & Fraps (1958) for the identification of estrone, and estradiol in avian ovarian extracts. The isolated compound and the labelled sample are mixed and chromatographed together. The chromatogram is stained with a chemical reagent which bas previous1y been shown to react in a similar fashion with the reference steroid. A radioautograph of the stained strip is then made. Detailed correspondance in position and shape as between the material as located by radioautography and by the staining reaction constitutes strong evidence for the identity of the unknown with the reference substance.

The quantitative assay of isolated estrogens can be accompliahed chiefly by colorimetrie, fluorimetric, spectrophotometric and biological methods. The most widely - 33 - used colorimetrie method for the estrogens is the Kober (1931) reaction which is surprisingly specifie for the estrogens. An excellent review on the development of this reaction is given by Bauld & Greenway (1957). Recently, Kadis & Salhanick (1959) reported a new colorimetrie method for the determination of the estrogens using piperonal chloride in trifluoroacetio acid. The method appears to be considerably more convenient than the Kober reaction in that it requires a single reagent for the three classical estrogens and the reaction may be carried out in a much shorter time than tbat required for the Kober reaction. The authors claim the reaction to be highly specifie for the estrogens and considerably more sensitive than the Kober reaction. The chief disadvantages are the necessity for the synthesis of the piperonal chloride and the corrosive and volatile characteristics of trifluoroacetic acid.

In recent years, considerable progress bas been made in the rluorimetrio deterinination of estrogens. This method has at least one-hundred times the sensitivity of the Kober reaction. However, in the estimation of estrogens in urinary extracts, the method is rendered inaccurate by self­ absorption caused by impurities which are colored in hot sulphuric acid. Rigorous purification is required not only for specificity but also for accuracy. Bauld & Greenway (1957) have reviewed the development of this method for - 34 - the estimation of urinary estrogens.

Reference bas been made to the use of ultraviolet absorption in the investigation of steroids. Friedgood, Garat & Haagen-Smit (1948) applied ultraviolet spectrophotometry to the quantitative estimation of estrone, estradiol-17~ and estriol. Absorption maxima for all three estrogens were at 280 mp with a secondary peak at 288 ~· Beerst law was obeyed and reproducibility was excellent.

However, estimation required 12-15 ~g. par ml. so that the method is insufficiently sensitive for most urinary assays. Moreover, the method is highly non-specifie because phenols absorb at 270 mp and consequently any phenolic impurities in urinary fractions may cause marked interference (Bauld & Greenway, 1957).

In all analyses of steroid material obtained from animal sources, it is important to bear in mind that many steroids are unstable, being sensitive to light, oxidizing agents, strong acids and bases, heat and strong adsorbants (Bush, 1954). Savard (1954) bas described the changes which oocur in several ketosteroids when they are exposed to ultraviolet light, and has pointed out the dangers of oxidative decomposition of steroids on paper atrips where a small amount of material ia spread over a relatively large area. 2. EXPERIMENTAL ME'lliODS

2. 1. General Material and Apparatus

2. 1. 1. Hormones and Related Compounds.

Crys·talline estrone, estradiol-17(3 obtained from Steroid Laboratories Ltd., Montreal.

Crystalline estriol, obtained from Parka, Davis and Co., Detroit.

Crystalline 2-methoxyestrone, 16-epiestriol and 16-ketoestradiol-17(3, presented by Dr. T.J. Gallagher, Sloan-Kettering Institute, New York.

Crystalline 16-ketoestradiol-17(3, presented by Dr. R. Hobkirk, Montreal General Hospital.

Crystalline equilenin, presented by Dr. D.A. McGinty, Parke, Davis and Co., Detroit.

Cryatalline aquilin, preaented by Dr. W. Klyne, Postgraduate Medical School, London, England.

Crystalline ~5, 7 ,9-Eatratrienolone and A.5, 7 ,9_ Estratrienol-3-one 17-acetate preaented by Dr. W.L. Glen,

Ayerat~McKenna and Harrison, Montreal.

Equol, prepared from goata• urine by W. Klyne and presented by Profeasor G.F. Marrian, Imperial Cancer Reaearch Fund, London. - 36 -

Crystalline androsterone, dehydroisoandrosterone, pregnane-3a, 20a-diol and allo-pregnane-3a, 20a-diol, obtained from Sigma Chemioals, St. Louis, Missouri.

Crystalline pregnanedione, obtained from Nutritional Bioohemioals, Cleveland, Ohio.

Cholesterol, Eastman Organio Chemicals, Rochester, N,Y.

Estradiol-17~-16-c14 (specifie aotivity, 2.7 micro­ curies per mgm.), obtained from Charles E. Frosst and Co., Montreal.

Estriol-16-cl4, obtained from Dr. M. Levitz, Bellevue Medical Centre, New York University, New York.

2. 1. 2. Solvants.

Methanol, A.c.s. reagent; absolute ethanol; ethyl acetate, U.S.P.; petroleum ether, boiling range 30 0 -60 0 C; benzene, A.c.s. reagent (thiophene-free), obtained from Nichols Chemioal Co.

. 0 0 Petroleum ether, boiling range 80 - lOO c, analytioal reagent, free from aromatio hydrocarbons, obtained from British Drug Houses Ltd.

Chloroform, U.S.P., redistilled, from Nichols Chemioal Co. - 37 -

Dichloromethane, from Eastman Kodak Co.

Ethyl ether, U.S.P., Nichols ;Chemical Co., freed from peroxides according to the procedure of Bauld (1956) and stored over a zinc-copper couple.

Ethylene dichloride, from Baker & Adamson, purified according to the procedure of Bauld (1956).

Formamide, technical grade, from Fischer Scientifio Co., purified for chromatographie use as follows: a 20% suspension of charcoal in formamide was shaken at regular intervals for four hours. The charcoal was removed by filtration.

2. 1. 3. Color Reagents.

Diazotized sulphanilic acid, prepared immediately before use according to the directions of Block & Bolling (1957).

A 1 per cent solution of ferric chloride, mixed just before use with an equal volume of 1 per cent potassium ferricyanide, as described by Barton, Evans & Gardner, (1952).

A saturated solution of 2,4-dinitrophenylhydrazine in 2 N hydrochloric acid.

A 2 par cent w/v solution of recrystallized - 38 - m-dinitrobenzene mixed just before use with an equal volume of 30 per cent w/v potassium hydroxide in ethyl alcohol (Kochakian & Stidworthy, 1952).

2. 1. 4. Chromatographie Apparatus.

Two chromatographie solvant systems were used in this work and these will henceforward be referred to as

System I mù System II.

System I consisted of petroleum ether, Analar reagent (B.D.H.), free from aromatic hydrocarbona (boiling

range, 80°-l00°C) 1 as the mobile phase and absolute methanol as the stationary phase. This system is essentially that described by Layne (1956). The chromatographie apparatus used in this system consisted of an air tight chamber capable of accommodating a descending run of 40 cm. The chamber, shown in Fig. 2, was made from a glass bell jar 35 cm. tall and 15 cm. in diameter, with a ground glass base. The jar was set on a pyrex desiccator 10 cm. deep by 15 cm. in diameter. The joint between the jar and the desiccator was lightly smeared with vacuum grease to provide an airtight seal. The opening in the top of the bell jar was fitted with a rubber stopper bearing a small funnel, the end of which protruded just into the top of the chromatography trough, The funnel was closed - 39 -

:fiG-UHi~ 2. Apparatus Used for the SerJarution of Steroids by Pape r Partition Chromatography in Solvent Systems of the Bush Type - 40 - with a small cork. Whatman No. 1 filter paper strips were used. The width of the strip varied depending on the amount and the nature of the material being chromatographed. The paper strips were first boiled in methanol and then in petroleum ether (Analar, boiling range 8o0 -l00°C). The strips should be dried in an air oven at 100°C immediately before use. The strips being chromatographed were suspended from glass roda supported on a metal stand as shown in Fig. 2. The ends of the paper dipped into the trough and were held in place by a shorter glass rod in the bottom of the v. The length of the strip, up which the mobile solvant travelled from the trough to the suspending rod before beginning the descending run, was used to control the running time, and this could be varied either by altering the distance of the trough from the top of the stand or by varying the height of the liquid in the trough. The material to be separated was applied to the strip at a point 8 cm. from the end of the strip. Saturation of the chromatographie chamber was accomplished by filling the bottom of the desiccator to a depth of 3 cm. with the stationary phase and placing a beaker containing 300-500 ml. of the mobile phase in the desiccator. The entire apparatus was placed in an insulated, double-walled plywood cabinet maintained at 34°C±l0 c by means of a chromel wire heater connected in series with a - u -

DeKhotinsky thermoregulator. The cabinet was provided with a glass inner door so that the progress of a run could be checked without altering the temperature.

System II was that described by Layne & Marrian (1958), using chloroform (redistilled) as the mobile phase and purified formamide (see section 2.1.2) as the stationary phase. The chromatographie apparatus used in this system was essentially the same as that used in System I, except that the chromatographie runs were carried out at room temperature. Whatman No. 42 paper was used. The paper strips were extracted in a Soxhlet apparatus for 3 days with a mixture of methanol anddhloroform. Before a chromatographie run, the paper strips were impregnated with the stationary phase by immersing them in a 2:1 (v/v) methanol-formamide mixture, blotted with filter paper between two sheets of plate glass and dried in a horizontal position at 37°C for 45 minutes. Saturation of the chromatographie chamber was aocomplished by placing a beaker containing 150 ml. of chloroform (previously saturated with formamide) on the bottom of the chamber.

2. 2. General Methode

2. 2. 1. Introduction.

The method of Mitchell & Davies (1954) for the - 42 - extraction and purification of estrogen fractions from placental tissue bas been adapted to the identification of estrogens in avian excreta in the course of the present work. The method described by Bauld & Greenway (1957) for the extraction and purification of estrogen fractions from human urine has been similarly adapted. Various modifications of these two methods have been used, and for this reason, discussion of these modifications will not be given in this section on general methods but will be outlined in detail in the appropriate sections under experimental procedures. Consequently, this section will deal ohiefly with descriptions of chromatographie techniques and methods of identification.

2. 2. 2. Preparation of Final Extracts for Chromatography.

The final extraots, obtained by the various procedures to be described later, were prepared for chromatograpby by first reducing the solvant to a volume of about 1 ml. in a 5o ml. round bottomed flask. The flask was rinsed with four successive 2 ml. volumes of a 1:1 mixture of dichloromethane and methanol. The washings were transferred with a dropper to a 15 ml. oonical centrifuge tube, and reduoed to a volume of a few drops under a stream of nitrogen. The walls of the tube were washed down with a further 0.5 ml. of dichloromethane-methanol and the solution - 43 - in the bottom of' the tube was again reduoed to a few drops. The solution was applied to the paper strips with a 5o miorolitre pipette. The tube was washed twice with a f'ew drops of the solvant and the wasbings applied to the paper a trip.

Wben extraots were being ohromatographed the materiel was applied in narrow horizontal bands aoross the width of the paper str.ip. It was possible, theref'ore, to out narrow strips from the aides of the ohromatogram. These narrow strips oould be stained witb certain oolor reagents in order to identify the oomponents • When reference oompounds were ohromatographed, either alone or with extracts, the materiel was applied as spots.

When it was deoided to elute a partioular portion of the chromatogram in order to subjeot it to further chromatography, the relevant portion was out from the paper strip and out into small pieoes with a sharp pair of stainless steel soissors. These pieoes were oompletely immersed in diohloromethane-methanol and left for 5 hours. The solvant was then transferred with a dropper to a 15 ml. conioal centrifuge tube. The paper was left for one hour in a fresh portion of solvant which was then transferred to the centrifuge tube. After two further washings with small portions of solvant, the combined eluate was reduced under nitrogen and the material transferred to a paper strip as previously desoribed. - 44-

2. 2. 3. Chromatography.

System I was the only chromatographie system used in the first part of the work described in this thesis (see Experimental Procedures and Resulta, Part I). This system, as described by Layne (1957), is capable of separating estriol, estradiol-17~, estrone and other relatively polar material. The paper strips bearing the material to be chromatographed were placed in the chambers and the whole apparatus allowed to equilibrate in the constant temperature cabinet (34°~1°C) overnight, or for at least 5 hours before the run was started. At the start of the run, 30 ml. of the mobile solvant was rapidly poured into the trough through the funnel, which was immediately restoppered to prevent desaturation of the atmosphere in the chamber. During the run, the outer door of the cabinet was only opened for short periods for occasional observation. Care was taken in all experimenta to avoid the prolonged exposure of cbromatogramsbeaErng steroid material to strong light, and in particular to fluorescent lighting. In this system, a running time· of 8 hours for 10 microgram samples of the reference steroids, gave Rf values of the following order: estrone, o.6o, estradiol-1713, 0.41 and estriol, 0.15. It was possible to increase the Rf values by increasing the running time to 10-12 hours. - 45 -

In the second part of the work described in this thesis {see Experimental Procedures and Resulta, Part II) both System I and System II were used. System II is capable of separating estriol, 16-~estrio1, 16~­ hydroxyestrone, 16-ketoestradio1-17~ and 16a-hydroxyestrone.

Lesa polar compounds, auch as estrone, estradio1-17~, 2-methoxyestrone and 16-ketoestrone, move fairly near the solvant front in this system, but are also separated to an appreciable degree.

When extracts from avian excreta or extracts from feces and urine were subjected to chromatography, the extracts were first run in System I for about 8 hours. This preliminary chromatographie run separated much contam­ inating non-polar materiels from the extract. The main portion of the chromatogram could then be run in System II to separate auch compounds as are listed above.

Figure 3 shows typical separations obtained with the two solvant systems. The Rf values are reproducible with pure ateroida, but the non-steroidal materiel present in extracts from tissues caused changes ln the mobility of the steroida. This was particularly true of System I. System II gave better reproducibility of Rf values even in the presence of contaminating materials. After several chromatographie purifications, the steroids obtained from extracts usually attained the same mobilitiea as the reference ES TRIOL

BSTRIOL

ESTRADIOt

ESTRONE 16-EPIESTRIOL

16-KETOESTRADIOL

A

FIGURE 3. Photocopies of typical chromatograms showing separations obtained with reference steroids. A. Estrogen spots on chromatograms deve1oped in System II ror 6.5 hourg and stained with diazotized su1phani1ic acid (DSA).

H. Estro·gen spots on chromatograms deve1oped in System I for 10 hours and stained with OSA. - 47 - compounds. In order to obtain reproducible resulta in System I, it was necessary to dry the paper strips in an air oven at l00°C for at least 20 minutes before applying the material to be separated. The poor séparation of the estrogens in System I consequent on the presence of hygroscopie moisture was first pointed out by Layne (1957).

2. 2. 4. Detection of Steroids on Paper. The position of steroids on paper strips was established by cutting a strip, 2 to 3 mm. wide, from the edge of the chromatogram and then dipping it in an appropriate color reagent. The estrogens gave blue spots with a mixture of equal volumes of 1 per cent potassium ferricyanide and 1 per cent ferric chloride. These spots could be made permanent by immersing the strip in dilute hydrochloric acid (Layne, 1956). Henceforward, in this

thesis th~ expression 11 stained with DSA" will imply the immersion of the chromatogram in the diazotized sulphanilic acid reagent, followed by treatment with sodium carbonate. The yellow spots obtained with this reagent could be

stabilized by re-immersing the strip in the sulphanilic acid. This reagent was capable of detecting the estrogens in concentrations as low as 5 micrograms par sq. cm. of paper, and was used more frequently in this work than the potassium ferricyanide-ferric chloride reagent. The latter stain was slightly more sensitive, but lesa convenient to use, and the blue spots obtàined were difficult to photograph - 48 -

for permanent record.

Ketosteroids and other ketonic materials were located on the paper by immersing the strip in an acid solution of 2, 4-dinitrophenylhydrazine. This procedure will henceforward be referred to as "staining with DNPH". Subsequent immersion of the strip in 5 per cent sodium

hydroxide produced a fairly permanent spot. The Z~erman reagent was used to detect ketosteroids on some of the chromatograms. The paper strip was immersed in the reagent and blotted with filter paper to remove excess reagent. The color was quickly developed by gently warming (65°c) .the paper strip.

2. 2. 5. Spectrophotometry.

The ultraviolet speotra of samples in ethanol was obtained in a Warren 11 Spectrochord11 recording spectrophoto-

mater over the range from 220 - 360 millimicrons (m~). When the spectrum of an eluate from a chromatogram was determined, the eluate from an equal area of paper eut from the oorresponding region of a blank chromatogram was used as the blank solution. Spots were eluted into 3.5 ml. of ethanol.

While solution of estrone, estradiol and estriol obey the Beer-Lambert law, their optical density is - 49 -

~elatively low, and accu~ate quantitative measu~ement by ultraviolet spectrophotomet~y is possible only with concent~ations greater t.han 12 to 15 mic~ograms par ml. (Friedgood, Garst & Haagen-Smit, 1948).

Infrared absorption spectra over the range from 1700 -1 to Boo cm. were determined on soma of the isolated materials by Dr. W. Zaharia, Department of Biochemistry, McGill University.

2. 2. 6. Identi~ication of Steroids by Radioautography.

The so-called fingerprint method of radioautography can be used to provide a ~inal identification of steroids isola ted by paper chromatography (Layne, ·Common, Maw & Fraps, 1958). The spot to be tested is eluted from the chromatogram and mixed witb a relatively small amount of the appropriate pure steroid labelled with c14. The mixture is chromatographed on a paper strip and then stained with a color reagent. The stained ohromatogram is photographed and then plaoed on X-ray ~ilm in a darkroom. After a certain period of exposure, the film is developed and the position of the dark spot oaused by the radioactive material is reoorded by photography. A positive identification hinges on a complete correspondance in detail between the position and shape of the spot as - 50 - indicated by the radioautograph with the position and shape indicated by the staining reaction.

The reverse of this fingerprint method has been used in the present work to identify radioactive estrogen metabolites isolated from avian excreta following the injection of estrogens labelled with c14. In this method, the radioactive spot (insufficient to give perceptible staining with DSA) was eluted from the chromatogram and mixed with 10-15 micrograms of the appropriate non-labelled estrogen. The mixture was then chromatographed on a paper strip and stained with DSA. The stained chromatogram was photographed and was then placed on X-ray film for exposure. The exposure time depended on the amount of radioactive metabolite isolated. The X-ray film was developed and the position of the dark spot was recorded by photography. A positive identification depends again on a complete correspondance in detail between the position and shape of the spot as indicated by the radioautograph and as indicated by the staining reaction. For the conclusion to be valid, the amount of labelled compound isolated should be insufficient to give perceptible staining with the color reagent, so that the perceptible staining reaction of the mixture can be considered attributable solely to the added non-active carrier. 3. EXPERIMENTAL PROCEDURES AND RESULTS - PART I The Isolation and Characterization of Estrogens from the Excrement of Laying Hans.

3. 1. Experiment I

3. 1. 1. Object.

The object of this experiment was to determine whether estrone, estradiol and estriol, or compounds similar to them, could be isolated and identified chromatographically from the excreta of laying hans.

3. 1 • 2. Me thod •

Rhode Island Red laying hans were housed in individual metabolism cages and fed a commercial laying ration. The excreta was collected in pyrex dishes which were fitted under the wire floor of the cage so as to encompass the entire floor area. In order to reduce bacterial action to a minimum, the excreta was collected several times daily, immediately dispersed in 70 per cent methanol and stored in a deep freeze (-20°C) pending analyais. Twenty-four hour samples from three laying hans were collected in this mannar and combined.

The methanolic suspension of faces was heated on a steam bath for 10 minutes and then filtered on a Buchner funnel. The residue was again extracted w~th 500 ml. of 70 per cent methanol and finally with 5oo ml. of 95 per cent ethanol. The combined extract was filtered on the Buchner - 52 - funnel using Wbatman No.l filter paper and the extract was left to stand overnight in a oold room (2°-5°c). The lipid-like material whioh settled out overnight was removed by filt~ation or centrifugation at 2°-5°c. The alooholic extract was evaporated to an aqueous sludge at reduoed pressu~e with mild heating in a rotary evaporato~.

The aqueous residue was diluted to 500 ml. with distilled water, to whioh was added 75 ml. of conoentrated HCl. The aoidified mixture was refluxed fo~ 40 minutes, oooled and thoroughly extraoted with ethyl ether (pe~oxide-free).

The procedure summarized in Figure 4 is essentially that devised for human plaoental tissue by Mitchell & Davies (1954), with the following modifications: a. The oombined extraots were not separated into "free", 11 oonjugated11 and "protein-bound" estrogen fractions.

b. Butanol was not used in the extraction of the excreta. This modification greatly reduced the time of heating required to evaporate the extracts to dryness. c. The partition of the final extract into an estriol (hydrophilio) and an estrone-estradiol (lipophilic) fraction was not oarried out, since a single chromatographie system was used to separate all three estrogens.

The evaporation of solvants at various stages of the extraction procedure shown in Fig. 4 was carried out under - 53 -

Fresh droppings (450 gm.) 1 Extracted with 70 percent methanol (2x500 ml.) Extracted with 95 percent ethanol (lx500 ml.)

Extra ct Residue 1 0 0 (discarded) Kept at 2 - 5 C overnight. Filtered. 1 Evaporated to aqueous sludge at reduced pressure with mild heating. Brought to 500 ml. with water, boiled, 75 ml. conc. HCl added, refluxed for 40 min. Cooled, extracted with ether (3x350 ml.)

Ether extract Aqueous phase (discarded)

Washed wit~ 10 percent (w/v) NaHco (2x0.1 vol.) 3 Ether phase 1 CC14 ,(0,..05 vol.) added, extracted wi th N Na OH Aqueous phase 1 Brought to pH 9.0 with H2so4 , using Napo3 as buffer. Extracted with ether (3x0.5 vol.) 1 Ether extract 1 Evaporated to dryness and transferred to paper strip for chromatography.

FIGURE 4. Procedure for extraction and purification of crude estrogen fractions. - 54 - nitrogen at the water pump. Mild heating on a water bath was sometimes necessary, but oare was taken to keep the heating time to a minimum.

The final extract was applied to two paper strips, each 6 cm. wide. After development for 8 hours in System I, pilot strips from the aides of the chromatogram were atained with DSA and DNPH. The upper two-third portion ot each chromatogram was eluted with dichlorometbane-methanol (1:1) and the combined eluates were applied to a single strip 6 cm. wide. The chromatogram was developed for 8 hours in System I. This proceas of rechromatographing was repeated a third time.

3. 1. 3. Resulta.

The original two chromatograms contained a large amount of brown and green contaminants which diffused over the entire length of the chromatogram, but which tended to concentrate more at the solvant front. Much of this contaminating material was removed by re-running the upper two-thirds of the cbromatograms as described above.

The final chromatogram, prepared from the eluates of the upper portions of the first chromatograms showed two distinct areas positive to DSA. The mobilities of these spots were similar to those of estrone and estriol on a - 55 - reference chromatogram. The zone corresponding in position to estrone gave a positive stain with DNPH while the zone corresponding to estriol was negative with this stain. The ultraviolet absorption spectra in ethanol of the eluates from these two areas showed definite absorption peaks at 280 mp.

A third DSA positive zone was observed on the chromatogram with a mobility corresponding to that of reference estradiol-17~. However, this zone could not be separated completely from contaminants which gave a positive reaction to DSA and which bad mobilities similar to that of estradiol-17~.

This experiment was repeated several times. In each case, similar evidence was obtained for the presence of estriol and estrone on the basis of chromatographie mobility, ultraviolet spectrophotometry and color reaction with DSA.

Again, estradiol-17~ could not be detected with any degree of certainty. Figure 5 shows typical ultraviolet absorption curves obtained with the eluates from the presumptive estriol and estrone zones.

Subsequently, the Kober reaction (Brown, 1952) was applied to the eluates from the preaumptive estriol and estrone zones. An extraneous brown color of considerable density was developed when these eluates from the filter - 56 -

0.15

0.10 Ul 0 z <{ [I) a: 0 Cf) co <( 0.05

240 250 260 270 280 290 300

WAVE LENGTH (Tf1f.L)

FIGURE 5. Ultraviolet absorption of presumptive estriol (curve 2) and estrone (curve 3) from 'SVian excreta. CUrve 1 shows the absorption spectrum of pure estrone. - 57 - paper were heated with sulphuric acid in the first stage of the reaction. Under these conditions there was a broad peak at 510 - 520 m~, but the extraneous absorption was so intense that it was practically impossible to make any reasonable estimate of the amount of estrogen present by this reaction.

While these resulta provide strong presumptive evidence for the presence of estrone, estradiol and estriol in avian excreta, it must be emphasized that even the final chromatograms of these extracts contained much contaminating material which gave staining reactions of various hues with DSA. Thus it became apparent that more rigorous purifica­ tion of the extracts was necessary before chromatography.

3. 2. Experiment II

3. 2. 1. Method.

Twenty-four hour samples of mixed excreta from three laying hens were collected, extracted and hydrolyzed as described in Experiment I. The ether extract was purified as outlined in Figure 6. This procedure is essentially that described by Bauld & Greenway (1957) for the extraction and purification of estrogen fractions from human urine, except that the ether extract was not partitioned between benzene and water to s eparate estriol from estrone and - 58 -

(I) Acid hydrolyzed extract of excreta

~xtracted with ether (2x200 ml. and lxlOO ml.) Ether extract 1 (2) Washed with conc. carbonate buffer, pH 10.5 (lOO ml.)

1 (3) Shaken with 2 N NaOH (25 ml.)

1 (4) M NaHco (lOO ml.) added, shaken, 3 combined aqueous extract discarded.

1 (5') Ether phase washed with 25 ml. M NaHco 3 and 15' ml. water.

1 (6) Éther evaporated

1 (7) Residue transferred to paper strip for chromatography.

FIGURE 6. Purification of crude estrogen fraction from avian excreta. - 59 - estradiol and the final saponification in N NaOH was omitted.

The wash with concentrated carbonate buffer (Step 2, Fig. 6) removed much brown colored contaminant from the ether extract. When the ether extract was shaken with 2 N NaOH (Step 3, Fig. 6), more brown pigments formed. When the pH was lowered to 9.5 by the addition ofM NaHC03 and shaken, the brown impurities remained in the aqueous phase. At this pH, the estrogens are re-extracted into the ether phase (Bauld & Greenway, 1957).

The final extraot was applied to ohromatograms and run in System I as deacribed in Experiment I.

3. 2. 2. Resulta.

Three rather broad areaa, positive to DSA, were looated on the original chromatograms, at positions indicative of estr1ol1 estradiol and estrone as judged by reference chromatograms. These three areas were eluted and rechromatographed aeparately in System I on strips 2 cm. wide. The DSA-positive areas on these ohromatograms corresponded in mobility to reference samples of estrone, estradiol-17~ and eatriol.

The chromatograma of theae extracts were considerably - 60 - cleaner than thoae described in ExperLment I. The zone corresponding to estradiol-17~ did not contain as much interfering contaminant as that obtained in Experiment I. However, there were several diffuse bands with mobilities similar to that of estradio1-17~.

The ultraviolet absorption spectra of eluates of the zones corresponding in mob111ty to estriol, estradiol-17~ and estrone were similar to those shown in Figure 5. All three eluates and their corresponding blanks were evaporated to dryness and the Kober reaction was applied to the residue. A bread absorption was obtained for each e1uate between 510 - 520 mJ.l, but again, the extraneous absorpti.on was quite intense.

Ex:periment III

3. 3. 1. Object.

The experimenta described previously, provide strong presumptive evidence for the presence of estrone, estradiol and estriol in avian excreta. These findings are in agreement with those of Hurst, Kukis & Bendel1 (1958) who reported chromatographie evidence for the presence of these three estrogens in avian excreta.

The quantitative observations made by Marlow & Richert - 61 -

(1940), Riddle (1942), Fraps & Sykes (1956) and Layne (1956) have all pointed to a relatively large production of estrogen by the laying hen. With this in mind, the present experiment was designed to attempt the isolation, in crystalline form, of estrone, estradiol and estriol from avian excreta.

3. 3. 2. Method

Rhode Island Red laying hens were kept in a laying battery am the droppings w:ere collected and extracted as described in Experimenta I and II. This procedure was continued until approximately 0.5 kgm. fresh droppings had been treated. The combined extracts were evaporated as previously described. The residue was taken up in water, hydrolyzed in the usual manner and thoroughly extracted with ethyl ether •

The further purification of the estrogen fractions was performed as described in Figure 6. In all, sixteen batches each of approximately o.5 kgm. fresh droppings were brought to this stage. The extracts were then combined, dissolved in a small volume of ethyl alcohol and transferred to a separatory funnel with four 25 ml. portions of benzene. One hundred ml. water was then added to the benzene solution. The separatory funnel was gently shaken, but sufficiently vigorous to ensure equilibration of the two - 62 - phases. This partition separates estriol (aqueous phase) from estradiol and estrone (benzene phase). Considerable emulsion was formed at this stage and it was necessary to allow the mixture to stand for a considerable time before the emulsion broke.

A small amount of solid, fluffy material was observed to separate at the benzene-water interface. This fluffy material was collected separately and will be discussed below.

The benzene phase was evaporated to dryness at reduced pressure under nitrogen. The residue was treated with Girard's Reagent T to separate estrone (ketonic) from estradiol (non-ketonic) by the method described by Talbot,

Butler. & Ma cLachlan ( 1940). Portions of the aq_ueous phase, ketonic and non-ketonic fractions were chromatographed separately in System I and the remaining main portions of each fraction were reserved for attempts at crystallization of the estrogens.

3. 3. 3. Resulta.

Aqueous Phase - The aqueous phase from the benzene­ water partition was relatively free from contaminating material. The chromatogram obtained from the small portion of this phase, yielded a zone which had the mobility of - 63 - reference estriol, stained strongly with DSA and negatively with DNPH, and gave an ultraviolet absorption spectrum agreeing with its identification as estriol.

Repeated attempts to obtain crystalline estriol from the main portion of the aqueous phase, uaing various solvants and mixtures of solvants, proved unsuccessful.

Ketonic Fraction - The chromatogram obtained from a small portion of the ketonic fraction yielded a zone that corresponded in mobility with reference estrone and gave strongly positive stains with DNBH and with DSA. The ultraviolet absorption spectrum agreed with that of reference estrone. Attempts to crystallize estrone from the main portion of the ketonic fraction were unsuccessful.

Material Obtained from Benzene-Water Interface - This material was dissolved in a small volume of absolute ethanol. Water was added until the mixture became just perceptibly cloudy. The mixture was left overnight at -10°C. The small amount of semi-crystalline material that separated out was again taken up in absolute ethanol and crystallized as before. This recrystallization was then repeated twice to affect further purification. The final yield of air dried material weighed approximately 0.8 mgm. and consisted of fine needles. The melting point of this material,determined with a microscope hot-stage and - 64 - uncorrected for emergent stem, was 1710 -177 0 C (MP of 0 0 reference estradiol-17~, 173 -179 C). Unfortunately, there was insuffioient materiel to permit of a mixed melting point determination. Its chromatographie behaviour, staining reactions with DSA and DNPH agreed with those of reference estradiol-17~. The ultraviolet absorption spectrum agreed with that of the reference compound (Fig. 7).

The infrared absorption spectrum of the materiel was determined in KBr in a single bearn Perkin Elmer Spectrometer by Dr. W. Zaharia. The spectrum, Fig. 8, showed extremely close agreement witb that of reference estradiol"l7~ and with published spectra (Dobriner et al, 1953) for this substance.

This experiment was repeated twice. Eaoh time, approximately 8 kgm. fresh droppings were processed as desoribed above. In each of tbese two experimenta, a small amount of solid material separated at the benzene-water interface. This materiel was collected separately, but attempts to orystallize estradiol were unsuccessful. When this solid materiel was dissolved in ethanol and subjeoted to ultraviolet absorption analysis, the spectrum displayed a small but definite peak at 2BO ~ with four distinct peaks in the range 240 - 265 ~· The solvant was evaporated to dryness at reduced pressure and under nitrogen. The residus - 65-

0.6

0.5

0.4- w 0 z ..( ro 0.3 cr 0 Ci) CD <( 0.2

0.1

240 250 260 270 280 290 300

WAVE LENGTH('mfl)

FIGURE 7. Ultraviolet absorption of presumptive crystalline estradiol-17 ~ isolated from avian excreta (curve l)o Curve 2 shows ultraviolet absorption spectrum of pure estradiol-17 ~ • -66-

80 z 0- 60 ~ a... }\ J~\ B a:: 40 0 cf) 8 0 CD ~ 1-- 60 A z LJ.J u a:: 4 0 w CL

20~~-L~~~--~~~~--~~~--~~~~~ 1700 1500 1400 1300 12 (; 0 1100_,1000 900 800 WAVf_ NUI'12ER CM

FIGURE 8. Infrared absorption of crysta11ine estradio1-17 ~ • A. Infrared absorption curve of reference estradio1-17 ~ • B. Infrared absorption curve of presumpti ve estradiol-17 ~isola ted from droppings of 1aying hens. - 67- was chromatographed in System I. A zone with an Rf value of 0.07 gave a faint but distinct positive reaction with DSA. When the main portion of this zone eluted from the chromatogram and subjeoted to UV analysis, the spectrum was found to be identical with the original material. Later it was observed that the spectrum of this material, in the range 240 - 265 mp, was identical with that of pure benzene. The extremely tenacious mannar in which benzene was bound to this solid material is difficult to explain. In spite of several evaporations of solvant in vacuo, and a chromatographie run in System I, the material retained an absorption spectrum in the range 240 - 265 mp identical with that of pure benzene.

Experiment IV

3. 4. l.Method

Droppings from laying hens were collected, extracted and hydrolyzed as described in section 3. 1. 2. In this experiment, the total amount of excreta processed was increased from 8 kgm. to approximately 16 kgm. (equivalent to lOO bird-days of excrement). The ether extracts were purified as outlined in Fig. 6. The partition between benzene and water to separate estriol from estrone and - 68 - estradiol waa omitted. Inatead, the purified ether extracts were combined and partitioned as followa:

Ether extract 1 Extracted with 0.1 N NaOH

Ether Phase Aqueous Phase (weak phenols-eatrone, (strong phenola-estriol) estradiol and neutrals)

1 Extracted with 2 N NaOH 1

Ether Phase Aqueous phase (neutrals) (weak phenola-estrone and estradiol)

Girard r s T Reagent

Non-ketonic Ketonic Fraction Fraction

3. 4. 2. Resulta.

Attempts to crystallize estriol, estrone and estradiol by conventional methods were unsuccessf ul.

In a further attempt to isolate estrone, the ketonic fraction was subjected to high vacuum sublimation at a pressure of 0.08 mm. Hg. When the bath temperature was - 69 - increased from 100 0 C to 150 0 c, a small amount of crystalline material deposited on the sublimation tube. This crystalline m.aterial was contaminated with a light yellow oil. After several re-crystallizations from petroleum ether, it yielded a srnall amount of crystalline material resembling small plates. A further small amount of the same material was isolated in a similar experiment and this compound will henceforward be designated''HK11 for purpose of reference.

The ultraviolet absorption spectrum of HK displayed a major peak at 271 m~ with a distinct shoulder at 278 ~. This curve resembled the ultraviolet absorption spectrum ofA5,7,9-estratrienol-3-one-17, a ketonic steroid isolated by Heard & Hofûman (1940) from marets pregnancy urine and later isolated from the same source by Glen, Barber & Papineau-Couture (1958). A sample of this compound (kindly presented by Dr. W.L. Glen) was subjected to ultraviolet absorption analysis and the resulta showed that the isolated crystalline compound HK was not identical witha5,7,9-estratrienol -3-one-17, (Fig. 9).

The infrared absorption spectrum of compound HK was determined by Dr. w. Zaharia (Fig. 10). The IR absorption spectrum of this compound was not, in general, similar to that given by pureA5,7,9-estratrienol-3-one .. l7. The spectrum of compound HK was compared with the spectrum of - 70 -

0.3

0.2 LU u z < co Ir 0 (/) co <( 0.1

240 250 260 270 280 290 300 WAVE LENGTH(rrTl;U.)

FIGURS 9. Ultraviolet absorption of compound HK (curve 2). CUrve 1 shows ultraviolet spectrum of pure ~ 5,7,9-estratrienol-3-one-17. - 71 -

100 z 0 .... ~ 80 a: 0 fi) al < 60 ...... z ~ u 40 a: ~ ~ 20 1800 1600 1400 1300 12 00 Il 00 1000 900 WAVE N UM BER CM-1

FIGURE 10. Infrared absorption of compound HK. - 72 - several other compounds. This comparison revealed that compound HK was aimilar to but not identical with 2,3- dihydroxybenzoic acid. It is possible that derivatives of benzoic acid might be present in extracts of avian excreta. This compound was not further investigated.

The aqueous phase (estriol fraction) was also subjected to high vacuum sublimation. When the temperature was increased from 130°C to 150°C at a pressure of 0.08 mm.Hg., a minute trace of white material appeared on the sublimation tube. This material was dissolved in ethanol and subjected to ultraviolet spectroscopy. The UVspectrum was identical with that of reference estriol (Fig. 11). Its extinction in alcohol indicated an amount approximately equivalent to 5 - 10 pg. of estriol. Its chromatographie behavior in System I was identical with that of reference eatriol. It may be concluded that this observation constituted an isolation of either estriol or 16-epiestriol.

Repeated attempts to crystallize estradiol from the non-ketonic fraction were uniformly unsuccessful. However, chromatographie evidence was obtained for the presence of estradiol in this fraction. - 73 -

0.3

0.2 w 0 z <{ co CL 0 (f) ((} <( 0.1

240 250 260 270 280 290 300 WAVE LENGTH (mf-l')

FIGURE 11. Ultraviolet absorption of presumptive estriol isolated by high vacuum sublimation from avian excreta (curve 2). CUrve 1 shows ultraviolet absorption spectrum of pure es triol. - 74 -

3. 5. Experiment V

3. 5. 1. Object.

In view of the failures in the previous experimenta to isolate estrone and estriol in orystalline form, it was deoided that further work on mixed excreta presented too many difficulties and that recourse would have to be had to separation of urine and faces. The amount of contaminants, even in the final estrogen fractions from mixed excreta, was considerable. Moreover, much material of plant origin undoubtedly was carried through the extraction procedures and caused considerable interference in attempts to isolate the phenolic estrogens. The use of urine separated from feces would, to a large extent, eliminate this latter problem.

3. 5. 2. Method.

The urine and feces of birds are voided into the cloaca. In order to obtain urine free from fecal matter, it is necessary to cannulate the ureters or surgically to exteriorize the openings of the ureters or of the rectum.

Fortunately, a new surgical technique for the exteriorization of the ureters of birds was developed by Dixon & Wilkinson (1957) about this time. The operation can be performed quite rapidly and, once healing bas - 75 - occurred, the bird may be used indefinitely. This operative technique was used in the present work. The main surgical problem encountered was that the birds developed fistulas, usually about one week after the operation. The fistulas developed in the region where the mucosa containing the uretral openings was separated from the mucosa of the cloaca. Two Rhode Island Red laying hens were eventually successfully modified by this technique so as to allow of separate collection of urine and feces. The surgery was performed by Dr. D.G. Dale, Animal Pathology Department, Macdonald Collage.

The operated birds were kept in specially constructed metabolism cages for several days following the operation to allow of healing to occur. The birds were fitted with a canvas harness which was used to hold the urine collection tubes in place. The urine was collected through latex rubber tubes (artificiel vagina reliners) which were sutured to the upper lip of the vent. Later, it was found that the birds thrived better in ordinary large individuel cages where they were free to move about, and in this environment they soon came into lay. With this arrangement, it was merely necessary to tie the lower end of the collection tube over the back of the bird. Urine collections could be made readily at frequent intervals throughout the day. Urine volumes for each laying hen were recorded for each - 76 - twenty-four hour period and egg-laying records were kept. Soma interesting subsidiary observations on the affect of surgery and egg-laying on urine volumes will be presented in the Appendix.

Birds provided with exteriorized ureters required great care each day. The urine was usually quite con­ centrated. It contained thick mucoid material and was abundant in urates. The urates tended to collect in the region of the exteriorized ureters and when the urates dried, they formed a hard crust over the area. The tiny uretral openings may become blocked and once this has occurred, the bird will die usually within twenty-four hours. It was essential, therefore, to wash this area daily so as to prevent any appreciable collection of urates. The washings were always added to the urine sample. The operation caused quite a severe diuresis which persisted for several days. Under these conditions the urine was quite dilute and urates did not tend to collectas described above.

Urine was collected from the two laying hans for fifty days (100 bird-days of urine} and was stored in polythene bottles in the deep-freeze. The urine was processed in batches of two litera. Each batch was brought to the boil under reflux condenser and concentrated HCl - 77 -

(230 ml.) was added. Boiling was continued for one hour. The hydrolysate was cooled and thoroughly extracted with ethyl ether. The ether extracts were purified as outlined in Figure 6. The ether extracts were then combined and partitioned as described in section 3. 4. 1.

3. 5. 3. Resulta.

The residue from the aqueous phase (estriol fraction) was dissolved in a minimum volume of ethyl acetate and kept at -10°C overnight. The small amount of semi­ crystalline material which settled out was recovered by centrifugation and washed with a few drops of cold ethyl acetate. The residue was dissolved in a small volume of hot methanol. Water was then added until the mixture became perceptibly turbid. On cooling slowly, a minute amount of crystalline material separated out. This material was recrystallized from aqueous methanol, applied to a paper strip and run in chromatographie System I. A pilot strip from this chromatogram was stained with DSA. A barely perceptible DSA positive zone was observed in the expected position of estriol. The spectrum was similar to that of reference estriol. The amount of material isolated was insufflaient to permit further identification.

Repeated attempts to i solate estrone f rom the ketonic fraction or estradiol from the non-ketonic fraction were again unsuccessf ul. - 78-

3. 6. Discussion

The resulta presented in the foregoing experimenta suggeat that the amounta of eatrogen produced by the laying hen may have been grossly overestimated in the paat. Repeated failure to isolate estriol or estrone from relatively large amounts of mixed excreta or from urine may have been due to the amall amounta present. In addition, chromatographie evidence, as judged by intenaity of staining reaction, suggested that the amounts of theae eatrogens in avian excreta waa very small. ·

It is somewhat astonishing that the successful isolation of estradiol-17B from extracta of mixed excreta waa not reproduced in the two subsequent attempta to repeat this isolation nor was it realized in the experiment with urine. The successful isolation, therefore, appears to have been rather lucky and the ressons for the divergent resulta of the four attempted isolations is not at all clear. The fact that the successful isolation was from materiel which had separated out at the benzene-water interface suggesta that the solubility of estradiol may also have been greatly affected by the large amount of contaminant which was dissolved in the .benzene phase.

Estrogens excreted !!! the alimentary tract are likely to be subjected to bacterial action. The pregnant - 79 - cow excretes more estrogen in the faces than in the urine (Levin, 1945; Pearlman, Rakoff, Cantarow & Paschkis, 1947; El-Attar & Turner, 1957) and most of the estrogenic activity appears to be due to estradiol. Levin (1945) originally suggested that this was a consequence of bacterial conversion of biliary estrone to estradiol. The situation in the fowl may be analogous and this may explain, in part, the isolation of estradiol from avian excreta.

The most serious handicap in working with mixed excreta is probably the presence of relatively large amounts of materials of plant origin which may be carried into estrogen fractions. While the primary purpose of the previous experimenta was the isolation and identifica­ tion of the steroid estrogens, several unidentified compounds were isolated in crystalline form with relative ease (e.g.,compound HK). It is not improbable that these materials were of plant origin, as they were not found when urine extracts were examined by methods similar to those employed in the examination of mixed excreta.

The presence of estriol or its isomers in avian excreta is of particular interest because of the beliet, in the past, that estriol is exclusive to the human species. The minute amounts of crystalline material isolated from estriol fractions in the present work (vide sections 3. 4. 2. - 80 - and 3. 5. 3.) were insufficient to identify this material conclusively. However, the resulta obtained strongly suggest that this material was in fact estriol or an isomer of estriol. Further studies on estriol in avian excreta will be presented in Part II of this thesis.

Finally, the foregoing experimenta point to the enormous technical difficulties encountered in attempts to isolate, from avian excreta, small amounts of material in the presence of relatively large amounts of contaminants. This problem is greatly reduced when urine is used, but the importance of the fecal excretion of estrogen in the bird means that the problems of isolation from the feces cannot be evaded in quantitative studies. SUMMARY - PART I

1. Methods were developed for the extraction and purification of estrogen fractions from avian excreta. These methods were based on the methods of F.L. Mitchell & R.F. Davies and of w.s. Bauld.

2. Estrone and estriol were detected on chromatograms of extracts of avian excreta. The chromatographie behaviour of these compounds in an absolute methanol-light petroleum ether system was similar to that of reference samples of estrone and estriol. The ultraviolet absorption spectrum of the materials after elution from the chromatograms showed absorption peaks at 280 - 282 mp. Estradiol could not be detected by similar methods with any degree of certainty because of interfering contaminants.

3. Estradiol-17~ was isolated in crystalline torm from extracts of large amounts of avian excreta. Its identity was established by infrared spectrophotometry.

4. Laying hans were provided with exteriorized ureters by surgical means so as to permit of separate collection of urine and faces. Birds modified in this mannar began to lay once healing bad occuiTed and could be used indefinitely.

5. Repeated attempts to isolate estrone in crystalline fo~ from both urine and mixed excreta were unsuccessful. - 82 -

However, a small amount of crystalline material {designated HK) was isolated from the ketonic estrogen fraction by high vacuum sublimation. The ultraviolet and infrared absorption spectra of this materiel did not provide positive information as to its chemical nature. Attempts to obtain this material from urine were unsuccessful.

6. Minute amounts of crystalline material similar to estriol or an isomer of estriol were isolated from mixed excreta and from urine. The amounts isolated were insufficient to permit of a conclusive identification of this material.

7. Rea sons are advanced in support of the view tha t the amount of estrogen excreted by the laying hen is quite small. 4. EXPERIMENTAL PROCEDURES A~ID RESULTS - PART II Studies on the Metabolism of the Estrogens in the Laying Hen.

4- 1. Experiment I

4. 1. 1. Objeot.

The metabolic inter-relationships of the estrogens have been reviewed in Section 1. 2. of this thesis. While many of the relationships of the more recently discovered estrogens are far from being fully elucidated (seeFig.I), the conversion of estradiol estrone estriol has been wall established for various mammalian species. It seemed desirable to confirm whether or not the fowl also can produce estrone and estriol from estradiol.

4. 1. 2. Method.

One mg. estradiol-17~-16-c 14 oontaining 2.7 microcuries of radioactivity par mg. {Chas.Frosst and Co., Montreal) was dissolved in propylene glycol and injected into the wing vein of a laying han. One hour later the bird was killed by bleeding from the jugular vain. The blood was collected in excess methanol, which was at once heated to inactivate enzymes. The blood was examined for estrogens as described by Layne and Common (1956) and the extract was chromatographed in System I. A radioautogram of this - 84 - chromatogram was prepared as described in Section 2. 2. 6.

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 bappened that the bird did not void any excrement during the hour after injection~ The intestinal tract and excreta was examined for estrogens as described in Section 3• 1. 2., chromatographed in System I and a radioautogram of this chromatogram was prepared.

The gall bladder was punctured and the bladder bile was taken up in a small volume of methanol. The bile was examined for estrogens by the same methods as were used for the excreta.

4. 1. 3. Resulta and Discussion.

The radioautogram of the chromatogram prepared from the extract of the blood yielded one radioactive zone which corresponded with the expected position of estradiol-17~ in System I. No other radioactive zone was observed on the chromatogram.

The radioautogram of the chromatogram of the bile showed the presence of estradiol and of a faint trace of zones corresponding in position to reference estriol and estrone, but no other radioactive zones were detected. - 85 -

The first chromatogram of the extract of the intestinal tract and excreta yielded two broad radioactive zones, A and B. These were eut out, eluted and chromato­ graphed separately. The more polar zone (A) now gave one narrow radioactive zone (A1 ). The less polar zone (B) gave three radioactive zones (B , B , B ). 1 2 3

Narrow stripswere eut from the papers and stained with DSA and with DNPH. The resulta of staining and mobilities in System I are summarized as follows: Tentative Zone DSA DNPH identification Al most polar + es triol Bl lesa polar than + es trone Al B2 lesa than A1 but + unknown more than B2 . B3 least polar estrone

Since the amounts present were minute, the DSA staining where perceptible was too raint to permit or photography. However, the faintness or absence of percept­ ible staining was of advantage in so far as it offered the possibility of applying fingerprinting methodology in reverse (see Section 2. 2. 6) in order to confirm the tentative identifications. - 86 -

Accordingly, the four radioactive zones were eut out from the main unstained portions of the strips and eluted with dichloromethan~ethanol. Twenty-five micrograms of non-radioactive reference steroid ~as added as carrier to the four eluates as follows:

El ua te Non-radioactive steroid added

Al es triol

~ estradiol B2 es trone B3 es trone

The four eluates then were evaporated, chromatographed again on separate strips in System I, stained with DSA, photographed, and radioautographed. The amounts of radio­ activity ~ere auch that two months exposure was necessary to get sufficient blackening of the film. The resulta are presented in Figure 12.

Strip A1 oontained radioactive estriol and this identification was confirmed by the close correspondance in both shape and position of the radioautogram witb the stained zone, the latter staining being due to the reference estriol added. Strip A1 also contained a radioactive zone in the presumptive position of estradiol but was insufficient to give any staining reaction. - 8f,l -

SR SR S R 5 R Al BI B2 B3

FIGURE 12. Staining reactions with diazbtized sulphanilic acid (S) and radioautograms (R) of chroma tograms of stsroids fr·om excreta of laying hen. Ar - Presumptive estriol fraction with added carrier estriol. ~ote ~lso presence of broad radioactive ~one in presumptive position of estradiol (amount too small to e:ive perceptible stain with t)SA).­ The, double nature of the estriol zone ( s·een also in Br) probRbly arose from application of much contaminatine: materiel and relatively slight movement of estriol from point of application in Rystem T.

~- Presumptive estradiol fraction with added carrier estradiol. Note presence also of some estriol es shown by radioautoe:ram.

B2 - Unidentified z·one wi th added carrier estrone·. The staining at the point of application was due to sorne unknown constituent; it could not have been estriol since it was not radioactive. B3 - Presumptive estrone with added carrier estrone. - 88 -

Strip B contained radioactive estradiol and this 1 identification was confirmed by correspondance in both shape and position of the radioautogram with the staining of the reference estradiol added. It also showed some estriol as shown by the radioautogram. The perceptible staining reaction in this region was probably due to contaminants.

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 the estradiol and

lesa polar than estriol. Strip B2 also yielded a minute trace of radioactive estrone, identifiable by correspondance in position with the stained zone of carrier estrone. The

identity of B2 was not established in this experiment.

Strip B3 contained radioactive estrone, confirmed by close correspondance of the radioautogram with the staining. It will be recalled that the estrone was in auch minute amounts that it gave no stain with DSA. No other band was apparent either by staining or radioautography.

The foregoing experiment bas shown, therefore, that the laying hen is able to convert estradiol into eatrone and estriol. One hour after the intravenous injection of radioactive estradiol, these metabolites could be detected by chromatographie and radioautographic methods, even though - 89 - the amounts were minute.

The presence of a fourth radioactive metabolite other than estradiol, estrone or estriol was of considerable interest as it contained more radioactivity than either of the other three zones. Although it gave a positive DNPH stain,)llo it did not yield a positive DSA stain. Its relative position on the chromatogram corresponded with that reported for the 16-hydroxyestrones in chromatographie System I. However, the instability of the 16-hydroxyestrones in alkali made it highly improbable that they would survive the extraction procedures used in this experiment. The possibility that this material was formed secondarily by bacterial action in the intestine was not excluded, but equally, the possibility remained that it represented a major metabolite of estrogens in the fowl. This fourth radioactive metabolite of estradiol will be discussed further in the following experiment.

The extract of the blood yielded only estradiol. This was probably a residue of the injected estradiol. The presence of estradiol and trace amountsaf estrone and estriol in the bile is probably not surprising as only the very small amount of bile, which was present in the gall

•This positive DNPH stain was almost certainly due to a contaminant as will appear more clearly in the light of Section 4. 2. 2. - 90 - bladder at the time or killing, was collected and examined ror eatrogena.

Experiment II

4. 2. 1. Object.

It was considered desirable to repeat the previous experiment (Section 4. 1.) with a view to a repeated isolation and possible identification of the unknown radioactive metabolite or estradiol-17~-16-cl4 in the lay­ ing ben.

4. 2. 2. Method.

One mg. estradiol-17~-16-cl4 (Chas.Frosst and Co., Montreal) containing 2.7 microcuries of radioactivity, was dissolved in 1 ml. propylene glycol and injected into the wing vain of a laying hen. Two hours later the bird was killed by decapitation. The bile was recovered ror separate analysis. The intestinal tract, from the gizzard to the cloaca including contents, was removed. The excreta voided since the injection or estradiol was collected and dispersed along with the intestinal tract in methanol by means of a Waring Blendor.

The mixture was then worked up for estrogens by the modification of Bauld's method described in Section 3. 2. 1., - 91 - but included the final partition between benzene (lipophilic fraction) and water (hydrophilic fraction) as described in Section 3. 3. 2.

The bile was taken up in methanol, extracted and partitioned in the same fashion.

The extracts were chromatographed in System I and radioautographed as described previously.

4. 2. 3. Resulta and Discussion.

Lipophilic fraction from intestinal tract and excreta:- The chromatogram of this fraction was examined by radioautography (two weeks' exposure). The resulta indicated the necessity .for further chromatography. After two further chromatographie runa in System I, the material was sufficiently purified to yield two distinct radioactive zones with a trace of a third radioactive zone. This third zone corresponded in position with the unknown radioactive metabolite described in Section 4. 1. 3.

The two major zones were then eluted separately. An aliquot of the eluate from the presumptive estrone zone, auch that it was insufflaient to give a perceptive stain with DSA, was mixed with 20 micrograms reference estrone and re-chromatographed. Congruence of atain and radio­ autogram established the identity of the zone as estrone - 92 -

(Fig. 13, As & Ar). The presumptive estradiol zone was identified as estradiol by the same technique using reference estradiol-17~ (Fig. 13, Bs & Br). These observations confirmed the previous findings (Section 4. 1. 3.) in which a different procedure bad been used and the benzene-water partition had been omitted •.

Hydrophilic fraction from intestinal tract and excreta:- The radioautogram of the chromatogram of the hydrophilic fraction (estriol) yielded a broad zone that suggested the presence of two components. This zone was eluted and chromatographed again. The radioautogram now showed two well-marked zones (Fig. 14). These two zones were eluted separately. An aliquot of the presumptive estriol zone, known by a pilot to be insufficient to give perceptible staining with DSA, was mixed with 20 micro­ grams reference estriol, re-chromatographed, stained with DSA and radioautographed. Congruence of stain and radio­ autogram conf irmed the identification as estriol (Fig . 15, S & R).

The other and less polar zone corresponded clearly to the unknown radioactive metabolite observed in the previous experiment. In particular, its separation in this experiment in the aqueous phase along with estriol suggested that it might be 16-epiestriol. To determine whether ar not System I was capable of separating estriol from -93-

As Ar Bs Br

FIGURE 13. Chroma to·graphic .fingerprinting of presumpti ve estrone (A) and estradio1 (B) from 1ipophific fraction. As. Chromatogram of presumptive estrone to which 20 micrograms reference estrone had been added. Stained with DSA. Ar. Radioautogram of As.

Bs. Chromatogram of presumptive estradio1-17~ to which 20 micrograms reference estradio1-17 ~ bad been added. Sta ined with DSA. Br. Radioautogram of Bs. 94 -

FIGURE 14. Radioautogram of chromatogram of hydrophilic estrogen fraction from extract of droppings from a laying hen which had been injected with estradiol-~ -16-c14. The uppermost zone corresponded in position with estriol. The lower zone corresponded with the presumptive position of 16-epiestriol. Note that the two dark zones did not differ greatly in intensity. -95 -

s R

FtGURE 15 •. Chromatographi'c fingerprinting of presumptive estriol from hydrophilic fraction. s •. Chromatogram of presumpti ve es triol to which 20 micragrams reference estriol had been added. Stained with DSA.

R• . Radioautogram of S~ - 96 -

16-~estriol, 20 micrograms of 16-epiestriol (kindly presented by Dr. T.J. Gallagher) waa spotted on a paper strip along with a spot containing 20 micrograms each o~ estriol and 16-epiestriol. The paper strip was then chromatographed in System I and stained with DSA (Fig. 16). This result confirmed that the system waa capable of separating estriol from 16-~estriol.

Accordingly, the lesa polar zone (see above) ~rom the hydrophilic fraction was divided into two halvas. One bal~ waa eluted, chromatographed in System I, stained witb DSA and then radioautographed (Fig. 17, Cs and Cr). The radioautogram (Fig. 17, Cr) displayed a strong zone that corresponded in position with that of 16-epiestriol, together with a faint trace o~ a lesa polar radioactive zone. The other half was similarily eluted and mixed with 20 miorograms of non-radioactive reference 16-epiestriol (comparison of Cs and Ds, Fig. 17). The congruence of staining and radioautogram (Fig. 16, Ds and Dr) confirmed the identification of the radioactive zone as 16-!E!estriol. The identity of the very faint, less polar radioactive zone remains uncertain. It occupied a position approximately the same as would have estradiol-17~, so that it may have been due to occluded traces of estradiol that bad separated with the aqueous phase on partition between benzene and water. - · 97 - ·

-

ES TRIOL 16-epiESTR!OL 1.6-epiESTRIOL

A B

FIGURE 16. Chromatographie separation of estrio1 and 16-~estrio1 in System I.

Spot A. 20 micrograms 16-mestrio1 •.

Spot B. 20 micrograms each of 16-.m_estrio1 and estrio1. Running time: 6 hours. Stained with OSA. NOTE: The estrio1 gave a pinkish ye1low col our. The 16-~est riol gave a distinct1y brownish yellow colour. - 98 -

..

Cs Cr Ds Dr

FIGURE 17. ChroiJ18tographic fingerprinting of presumptive 16-,W_estriol •.

Cs. Chromatogram of one half of presumptive 16-~estriol, stained with DSA. Note absence of perceptible staining •. Cr. Radioautogram of Cs. Ds·• . Chromatogram of other half of presumptive 16-ill_estriol plus· 20 micrograms reference 16-mestriol, stained with DSA •. Dr. Radioautogram of Ds. Note congruence of staining of ca·rrier 16-mestriol and radioautogram of presumpti ve radioactive 16-mestriol •. - 99 -

In the previous experiment, it was noted that the radioactive zone, now shown to be 16-~estriol, gave a stain with DNPH though not with DSA. The present experi­ ment indicated that this positive reaction with DNPH1 which was confusing at the time, must have been due to contaminating material. The negative DSA stain, of course, was a consequence of the minute amounts present.

The chromatograms of the extracts of the bile showed the presence of radioactive estrone and estriol but did not provide any indication of the presence of 16-!E!estrio1. This confirmed the observation reported in Section 4. 1. 3.

The foregoing experiment showed that 16-!E!estriol is a prominent metabolite of estradiol in the fowl, along with estrone and estrio1. Its presence in the aqueous phase of the benzene-water partition, a piace of evidence that was lacking in the previous experiment, provided the first clue to its identity. The fact t hat a trace of 16-epiestrio1 showed up in the radioautograms of the benzene phase is in agreement with the finding that 16-

~estriol appears in both aqueous and benzene phases in this partition (Givner, 1959).

While the identification of 16-~estrio1 as a metabolite of estradiol-17~ in the 1aying hen is of interest in itself, the present experiment raised a further point - lOO - of interest in relation to the pattern of estrogen metabolism in the laying hen as compared to that of the pregnant woman.

The ratio estriol: 16-!E!estriol in pregnancy urine bas been reported as about 60:1 {Marrian, Watson & Panattoni, 1957). The estriol is excreted in mgm. quantities and is easily crystallized from extracts of pregnancy urine.

The following evidence, therefore, supports the view that the ratio estriol: 16-!E!estriol in the excreta of the laying hen is much lower than in pregnancy urine:

(a) The 16-~estriol zones on the radioautogram of the extracts of excreta of the laying hen were darker than any other radioactive zone and comparable to the estriol zones; the resulta, in faot, indicate that 16-~estriol is a major metabolite of estradiol in the laying hen.

(b) Repeated and consistent failure to obtain any appreciable amount of crystalline estriol from extracts of droppings or from the urine (amounts equivalent to 100 bird­ days) of laying hens, although chromatographie, spectrometrie and chemical evidence showed that estriol was present. These difficulties suggest that the estriol content of laying hens• droppings or urine is relatively small.

(c) If the ratio estriol: 16-epiestriol were as high - 101 - in the excreta of laying hens as in pregnancy urine, it is unlikely that it would have been possible to separate estriol from 16-~estriol so readily, in the chromatographie system used, as has proved possible in these experimenta.

(d) Three twenty-four hour urine samples were collected from a laying hen and quantitatively analyzed for estrogens aooording to the method outlined by Bauld and Greenway (1957). The aqueous phase from the benzene­ water partition was ohromatographed on Celite oolumns (desoribed by Bauld & Greenway, 1957), using 70% methanol as the stationary phase and ethylene dichloride as the mobile phase. Two Kober-positive fractions were taken from the oolumn corresponding to presumptive 16-~estriol and estriol fractions. The average resulta (using the Allen correction) for the three determinations yielded 3 micrograms for the presumptive 16-epiestriol fraction and 1 microgram for the presumptive estriol fraction. While no claim is made that these amounts give an accurate measurement of the daily excretion of these estrogens by the laying hen, the r atio of the amounts found may be considered to be significant.

It appears, therefore, that the ratio estriol: 16-

~estriol in the excreta of the ~ying hen must be very much lower than the ratio in human pregnancy urine, and - 102 -

that this constitutes a difference in the pattern of estrogen excretion as between the pregnant woman and the laying hen.

4. 3. Experiment III

4. 3. 1. Object.

If the metabolic pathway of estrogens in the laying hen is generally similar to that in humans (see Fig. 1), then injection of radioactive estrio1 wou1d be expected to give rise to radioactive estriol, radioactive 16-ketoestradiol

and radioactive 16-~estriol in vivo, but to no other radioactive estrogen, provided that the extraction procedure involved acid hydrolysis which would destroy the unstable hydroxyestrones. It is well established that estriol can give rise to 16-!E!estriol in the human subject. There is uncertainty, however, as to whether estrio1 is transformed

in the human subject directly to 16-~estriol or indirectly

!!! 16-ketoestradiol-17~. Levitz, Spitzer & Twombly (1958) have furnished evidence in support of the latter view.

This experiment was designed to determine the metabolic products of injected radioactive estriol in the laying hen. Since studies on the droppings are complicated by the possibility of secondary changes due to bacterial action, it was considered desirable to examine urine and feces separa tely. - 103 -

4. 3. 2. Method.

A Rhode Island Red l,aying hen, provided with exteriorized ureters by the operative procedure described in Section 3. 5. 2., was used in this experiment. On the morning of the first day of experiment, 5.9 microcuries (0.27 mg.) estriol-16-c14 i~ propylene glycol (1 ml.) was injected into the wing vein. The urine and feces were collected separately for each of the next two succeeding twenty-four hour periods.

All four samples were worked up separately for estrogens by the following method:- The urine (or suspension of feces in water) was diluted to 500 ml. with distilled water. To this was added 75 ml. concentrated HCl. This solution was then boiled under reflux for an hour, cooled and extracted thrice with diethyl ether (peroxide-free) so that the final volume of the ethereal extract was approxim­ ately 500 ml. One-hundred ml. concentrated carbonate buffer (pH 10.5) was shaken with the ethereal extract. The aqueous phase was discarded and the ethereal extract was washed once Wlth 15 ml. M NaHco3 and subaequently with two successive 15 ml. portions of water. The ethereal layer was dried by the addition of Na so . The ether was removed 2 4 under reduced pressure and the residue was taken up in 2 ml. ethanol. The partition into hydrophilic and lipophilic fractions was done as described previously. - 104 -

The benzene phase containing the lipophilic estrogens, was evaporated under reduced pressure and obromatographed and radioautographed.

The aqueous phase was brought to pH 9.0 by careful addition of NaOH solution and with the aid of a pH meter. The resultant solution was extracted at once with diethyl ether. The ethereal solution of bydrophilio estrogens was dried over anhydrous Na so , cbromatographed and radio­ 2 4 autographed. Both chromatographie Systems I and II were used.

4. 3. 3. Resulta and Discussion.

Lipophilio fraction from urine:- The lipophilio fraction from the urine sample for the first twenty-four hours was ohromatographed in System I. It yielded a single broad radioactive zone. The strip was eut in half longitudinally. The radioactive zone was out from the first half and eluted. Reference 16-ketoestradiol-17~ (15 micrograms) was added to the eluate, which was then ohromatographed in System II. Staining with DSA yielded two strongly atained zones (Fig. 18, AS).

Of the two strongly stained zones, that with the greater Rf value was strongly radioactive and displayed congruence in shape and position of the staining with the - 105 -

FIGURE 18. - 106 -

AS AR BS _BR --- -

EQUOL

EQUOL 16-ept.-EST RIOL

16-KETOESfRAOIOL 16-~pi-ES T Rl OL

16-KETOESTRAOIOL - 107 - blackening of the X-ray film (Fig. l~AR). As will be shawn below, the staining could be attributed entirely to the added reference 16-ketoestradiol, so that this result constituted a chromatographie fingerprinting of the radioactive 16-ketoestradiol. The strongly staining zone of lower Rf value was devoid of radioactivity; it represented part of the urinary equol, a point which will be explained later. The radioautogram also showed the presence of a strongly radioactive but non-staining zone witb an Rf that corresponded with that of 16-!E!estriol. This tentative identification was confirmed by fingerprint­ ing (see below) so that this zone represented the part of the radioactive 16-~estriol which had separated with the lipophilic fraction. It should be noted that 16-~estriol,

16-ketoestradiol-17~ and equo1 bad simi1ar mobilities in System I, but that they could be separated clean1y in System II.

The radioactive zone of the second half of the chromatogram of the lipophilic fraction in System I was now eluted and chromatographed in System II without addition of any reference estrogen. A strip 1 cm. wide was eut longitudinally from the chromatogram, stained with DSA and ràdioautographed. The staining yielded a strongly stained equol zone and four minor faintly stained zones (Fig. 18, BS). Comparison with the radioautogram (Fig. 18,BR) - 108 - showed that none of these stained zones was radioactive, and that neither the radioactive 16-~estriol nor the radioactive 16-ketoestradiol was present in sufficient amount to give perceptible staining with DSA, and hence the observations shown in Fig. 18, AS & AR constituted a chromatographie fingerprinting of the 16-ketoestradio1-17~. It remained to confirm the identity of the radioactive 16-!E!estrio1. This was done by cutting out the appropriate zone from the remaining unstained portion of the chromatogram, e1uting it, adding reference 16-~estrio1 (20 micrograms) and chromatographing the resultant solution in System II. Congruence of shape and position of the staining with DSA with the blackening of the radioautogram confirmed the identity of the 16-!E!estrio1 (Fig. 19, S & R).

Since no other radioactive zone was detected on the chromatograms of the lipophilic fraction of the urine, it was evident that the injected radioactive estriol had yie1ded1 in this fraction, 16-~estriol and 16-ketoestrad­ iol-17~, but no other radioactive estrogen.

Attention may now be given to the evidence for the identification of the equol zone. The intensity with which this zone stained, in circumstances where the amounts of the estrogens present were too minute to stain with DSA, of itself suggested that this material was unlikely to be an estrogen. Furthermore, the mobility of the zone in System II - 109 -

s R

FIGURE 19. Chromatographie fingerprinting of presumptive 16-~estriol from lipoph11ic fraction of urine. s. Chromatogram of presumptive 16-~estrio1 to which 20 micrograms reference 16-~estrio1 had been added. Stained with DSA. R. Radioautogram of s. - llO - did not correspond with that of any of the following:­ estradiol-17~, estrone, estriol, 16-epiestriol, or 2- methoxyestrone. Aeeordingly, the presumptive equol zone was eut out from the remaining unstained portion of the chromatogram of the lipophilic fraction and was eluted. The solution was evaporated and the residue was dissolved in 3 ml. of ethanol. The UV absorption spectrum was determined in a reeording speetrophotometer (Warren Spectroehord) and compared with the UV absorption speetrum of a sample of reference equol that bad been isolated from goat•s urine. (The reference equol had been prepared by Dr. w. Klyne and was presented by Professor G.F. Marrian). Both speetra (Fig. 20) displayed a smooth absorption curve with a peak at 280 - 282 m~ and devoid of shoulders. The UV spectrometrie evidence, therefore, was consistent with the identification of the material as equol. The alcoholic solution was evaporated to drynesa, the residue was taken up in methanol - dichloromethane, and portions of this solution were chromatographed in System II as follows:- Spot A presumptive equol Spot B presumptive equol plus reference equol (20 micrograms) Spot C reference equol (20 mierograms)

Staining with DSA showed that the presumptive equol and the reference equol had the same mobility in System II (Fig. 21) and that they gave the same hue with the stain. These - 111 -

0.6

2 0.5

0.4 u.J 0 z <( m 0.3 cr 0 U) (l) <( 0.2

0.1

240 250 260 270 280 290 300

WAVE LENGTH ('mfL)

FIGURE 20. Ultraviolet absorption of presumptive equol after elution from chromatograms of extracts of avian excreta (curve 2)~ Curve 1 shows ultraviolet absorption spectrum of pure equol. - ]!2

A B c

FIGURE 21 •. Chromstography of equol obtained from avian excreta and reference equol in System II. Stained with DSA •. Spot A. Presumptive equol. Spot H. Presumptive equol plus reference equol ( 20 mi crograms) •. Spot c .. Reference equol ( 20 micrograms) •. - 113 - observations left little doubt that the strongly staining, non-radioactive zone on the chromatograms of the lipophilic fraction was due to equol.

Equol was first discovered by Marrian & Haslewood (1932b), who isolated it from marets pregnancy urine. It has been round in the urine of the goat (Klyne & Wright, 1957) and of the cow (Klyne & Wright, 1959). Equol is thought to be a metabolite of plant !!2flavones; and its occurrence in the excreta of the fowl would be consistent with the fact that the diet contained dried alfalfa meal. Its possible presence calls for close attention in studies on estrogens in the fowl.

Hydrophilic fraction from urine:- The hydrophilic fraction from the urine for the first 24-hours was chromato­ graphed in System I. Like the lipophilic fraction, it yielded one broad radioactive zone. This zone was eluted and chromatographed again in System I. This time, the single broad zone was separated into two distinct radioactive zones (Fig. 22, CR). These zones corresponded in position with the expeoted positions of eatrio1 and 16-epieatriol (aee Fig. 14). The part of the strip that oontained both radio­ active zones was eut out, e1uted and chromatographed in System II. (The remaining parts of the strip from System I were stained with DSA, which revea1ed strong ataining at the edge immediately behind the estriol zone but none in the - 1!14 - CR os DR ~ ·

} ESTRIOL ESTRtOL 16-epl·ESTRI OL EQUOL

16-epi·ESTRIOL

16-KETOESTRADtOL

FIGURE 2"2 •. Chromato·graphy of hydrophil!ic· urinary astrogens of larang hen a~ter injection of estr1o1-16-C .. CR •. Radioautogram of chromatogram (System I) of hydrophilic urinary estrogens. DS. Chromatogram (System II) of radioactive material eluted from strip CR. Stained with DSA. Note strongly-staining equol zone and other stained contaminants of lower Rf. DR. Radioautogram of DS. Note (a) the appearance of two estriol zones due to interference of contaminants; (b) the absence of perceptible staining of the radioactive 16-epiestrio1 zone; and (c) the presence of traces of 16-ketoestradio1. - 115 - region immediate1y in advance of the 16-~estrio1 zone. This staining could be attributed to the trailing edge of the equol zone, for equo1 appears to have a slightly lasser mobility than estriol in System I).

The chromatogram of the radioactive zone in System II was stained with DSA and radioautographed, with the resulta shown in Fig. 22, DS & DR. There were five distinct stained zones. Of these, one was practically on the lina of application and was not radioactive. Two others of low Rf value were also not radioactive. Presumab1y all three represent contaminating materials. A fourth faintly stained zone carried soma radioactivity. A fifth major, stained, non-radioactive zone was due to equol, which showed that this substance was present in relatively large amounts in the hydrophilic fraction as wall as in the lipophi1ic fraction.

Four radioactive zones were present. The most strongly radioactive, corresponded in Rf value with 16-

~estriol (Fig. 22, DS & DR) and this observation confirmed the identification from mobility of the radioactive zones in System I (Fig. 22 1 CR).

The next most strongly radioactive zone, which was not associated with staining, had an Rf corresponding with that of estriol and represented most of the radioactive estriol - 116 present. The third most radioactive zone, corresponded with the rourth faint1y staining zone. This re1ative1y weak, narrow radioactive zone representa a fraction of the estrio1 which did not move with the main fraction of the estrio1 but with a contaminant of 1ower Rf than estrio1. This effect, whereby estrio1 may give rise to two zones on chromatograpby or extracts of hen's excreta, has been observed in a previous experiment (see Section 4. 1. }., Fig. 12). Chromatography of the hydrophi1ic fraction of an extract or races to which soma radioactive estrio1 bad been added confirmed that auch extracts may contain contaminants that cause estrio1 to give two zones on chromatography.

There was a1so a very faint radioactive zone in the position corresponding to 16-ketoestradio1. This may have represented ketoestradio1 that separated with the aqueous phase on partition, or it may have been due to entrainment of drop1ets or the benzene phase in the aqueous phase. In any event, the amount was extremely small.

Finally, attention is directed to the fact that estrio1 may be separated c1eanly from equol in System II.

Lipophilic fraction from faces:- The lipophilic fraction from the faces was given a preliminary chromato­ graphy in System I and the chromatogram was radioautographed. It yielded one broad radioactive zone. This zone was e1uted - 117 - and the eluate was chromatographed in System II. A narrow strip was eut from the chromatogram, stained with DSA and radioautographed. It yielded a faintly stained zone in the position corresponding to equol (Rf•O.l8) and four other very faintly stained zones (Fig. 23, ES}. None of these five stained zones was radioactive (Fig. 23 1 ER). There was, however, {a) a strongly radioactive zone that corresponded in position to 16-epiestriol; (b) a very faintly radioactive zone corresponding in position to 16~ketoestradiol-17~; and (c) a faint, diffuse zone of radioactivity immediately behind the solvant front (Fig. 23, ER-X)~ This latter radio­ active zone was not observed on chromatograms of either lipophilic or hydrophilic fractions from urine or of the hydrophilic fraction from faces, and its possible identity was, therefore, of considerable interest.

The presumptive 16-~estriol zone (non-staining) was eut from the main, unstained portion of the chromatogram, eluted, and reference 16-~estriol was added to the eluate. The solution was chromatographed in System II. Congruence of staining and radioautogram of the resultant chromatogram confirmed the identification of the radioactive zone as 16-!œ!estriol ..

The faint diffuse radioactive zone just behind the solvant front was eluted from the appropriate part of the main unstained portion of the chromatogram and reference - 118 - ES ER FS FR

1-- EQUOL -

16-eptESTR\OL ESTRADIOt 17~ x

x

FIGURE 23. Chromatography of lipophilic fecal estrogens of laying hen after injection of estriol-16-C14 • ES. Chromatogrsm (System II) of lipophilic fraction. Note strongly stained equol zone. ER . Radioautogram of stained chromatogram, ES. Note presence of radioactive 16 - ~estriol and of a faintly radioactive zone near the solvent front and marked X. (A trace of radioactive 16- ketoestradiol was too fa int for reproduction) •. FS . Chromatogram (System I) of eluate corresponding to zone X, to which had been added reference estradiol - 17 ~ (10 micrograms). FR . Radioautogram of chromatogram FS . Note that zone X was mainly due to a radioactive constituent less polar than estradiol- 17 (l . - 119 - estradio1-17~ was added. The solution was then chromato­ graphed in System I, stained with DSA, and radioautographed. A comparison of the staining and radioautogram (Fig. 23, FS & FR) showed that the major component of the radioactive material in question was not estradiol-17~ but a less polar material, possibly estrone. It was not further identified.

Hydrophilic fraction from feces:- The hydrophilic fraction yielded a single broad radioactive zone on prelim­ inary chromatography in System I. This radioactive zone was eluted and the eluate was chromatographed in System II. The strip was stained with DSA and radioautographed. The radioautogram showed main and subsidiary estriol zones of low Rf, as had been observed for the hydrophilic fraction from the urine. There was also a strongly radioactive 16-!E!estriol zone that did not stain, together with an extremely faint radioactive 16-ketoestradiol zone (Fig. 24, GS & GR). A striking result was that thel6-epiestriol zone on the radioautogram was much blacker than the corresponding zone for the hydrophilic fraction of the urine. A strongly staining non-radioactive zone due to equol was also present.

Distribution of radioactivity between urine and feces:­ Radioautograms were prepared from the total hydrophilic and lipophilic fractions of the estrogen fractions of both urine and feces. The resulta, shown in Fig. 25, indicate that the -120-

- - ESTRIOL

EQUOL

16-epiESTRIOL

GS GR

FIGURE 24. Chromatography of hydrophilic fecal estrogens of laying hen after injection of estr-ioi-16-cll.f. ·- crs •. Chromatogram (System II) of hydrophilic feca 1 estrogens •. Stained w1 th DSA •. Note strongly staining equol zone and ether stained contaminants at point of application. GR. Radioautogram of GS. Note strongly radioactive 16-~estriol zone that did not stain. -121-

URINE FE CES

Lipophilic Hydrophilic Lipophilic Hydrophilic

FIGURE 27. Distribution of radioactivity between lipophilic and hydrophilic estrogen fractions from both urine and f~~es after injection of estriol-16-C~ • - 122 - amount of radioactivity in the feces was of the same order of magnitude as that present in the urine. This observation points to the importance of the fecal route in the excretion of estrogens by the laying hen in that roughly half of the total radioactive metabolites of estriol could be accounted for in fecal estrogen extracts.

Urinary and fecal samples for the second 24-hour period:- The samples of urine and feces for the second 24-hour period yielded resulta which were in general similar to those described for the samples of the first 24-hour period, but the levels of radioactivity on the chromatogra~ were extremely low. The amounts of equol, however, were comparable with thoae found in the samples for the first 24-hours. By far the greater part of the radioactive urinary and fecal estrogens bad obviously been excreted by the end of the first 24 hours.

The faregoing resulta have demonstrated that estriol is metabolized by the laying ben in vivo to 16-ketoestradiol-

17~ and 16-epiestriol. If the resulta far urine alone are considered, then it would appear that no other estrogen was formed in vivo from the injected estriol. This result is in agreement with the resulta and conclusions of Levitz, Spitzer & Twombly (1958) regarding the metabolism of estriol in t he human subject. The r esulta showed that the hen•s f eces contained considerable amounts of radioactive 16-~estriol - 123 - and little, if any, radioactive 16-ketoes~radiol-17~. However, the faces also contained a detectable amount of a radioactive constituent lesa polar than estradiol-17~.

The latter observation does not necessarily invalidate the conclusion that the injected radioactive estriol gave rise in vivo to 16-~estriol and 16-ketoestradiol-17~ but to no other radioactive estrogen, apart from auch as may have been destroyed by acid hydrolysis. Estrogens secreted

~ the alimentary tract are likely to be subject to bacterial action. It is possible and, indeed, likely that the appearance of an additional radioactive constituent in the feces was a consequence of the action of bacterie (see discussion, Section 3. 6.).

The demonstration that equol ia a possible major constituent of extracts of estrogens from either feces or urine points to the necessity of taking into account the possible presence of this substance in studies on the estrogens in avian excreta. It is fortunate tbat equol may be separated cleanly from estriol in System II (chloroform­ formamide system).

Finally, the effects of contaminants on the chromato­ graphie behaviour of estriol is of technical importance in studies of thi s kind. SUMMARY - PART II

1. When estradio1-17~-16-c1 4 bad been administered intravenous1y to a laying ben, radioactive estradiol-17~, estrone, estrio1 and 16-~estrio1 could be detected in the gut plus excreta by chromatographie and radioautographic methods. Examination of the bile revealed the presence of radioactive estradiol-17~ and traces of radioactive estriol and estrone. Radioactive 16-~estriol was not detected in the bile. Radioactive estradiol, but no other radioactive steroid, was detected in the blood.

2. Evidence is preaented in support of the view that the ratio estriol: 16-!E!estrio1 in the excreta of the 1aying ben is considerab1y lower than the corrésponding ratio in human pregnancy urine, and that 16-!E!estriol is quantitatively, an important metabolite of estradio1-17~ in the laying ben.

3. Estrio1-16-cl4 was administered intravenously. to a laying ben furniahed with exteriorized ureters. Urine and feces were col1ected separately and examined for estrogens by a method that included acid hydrolyais but avoided expoaure of the extract to alka1inity in excesa of pH 10.5. The urine secreted in the firat 24-hour period -·125- after injection contained radioactive estriol, radioactive

16-ketoestradiol-17(3 and radioactive 16-~estriol. No other radioactive estrogen was detected in the urine. The feces excreted in the first 24-hour period after injection contained radioactive estriol and radio­ active 16-!l?.!.estriol, but little, if any radioactive 16-ketoestradiol-17(3. The feces yielded also small amounts of a radioactive constituent less polar than estradiol-17(3. Reasons are advanced in support of the .view that this constituent was present in consequence of bacterial action in the gut or feces.

4. A high proportion of the metabolites of injected radioactive estriol was excreted by the fecal route, as shown by comparisons of blackening of radioautograms. The greatest single fraction of the radioactivity in the excreta, as gauged by blackening of X-ray film, was present as 16-.epiestriol.

5. Examination of the urine and feces for the second 24-hour period after injection showed that the amounts of radioactive estrogens were insignificant in comparison with those excreted in the first 24-hour period.

6. The lipophilic and hydrophilic fraction of estrogen extracts both from urine and feces contained amourits of equol that were large relative to the amounts of the various estrogens excreted, as judged by staining of the chromato- - 126 - grams. Extracts for the first and second 24-hour periods contained equol in amounts of about the same order as gauged by staining. CLAIMS TO ORIGINAL RESFARCH

1. The isolation of crystalline estradiol-17~ from the droppings of the laying hen and its characterization by IR spectrophotometry. This work invo1ved the elaboration of extraction methods applicable to avian droppings and based on the methods of w.s. Bauld. The resu1t is be1ieved to represent the first isolation of a crystalline estrogen from avian material.

2. The demonstration that injected radioactive

estradiol-17~ gives rise to the following radioactive

estrogens in the droppings: estradio1-17~, estrone, estriol and 16-!E!estriol. This work constituted the first study on the metabolism of estrogens in vivo in an avian species, so far as the author is aware.

3. The first demonstration of the presence of 16-

~estriol in avian excreta, so far as the author is aware.

4. The demonstration that injected radioactive estriol

givea rise to radioactive estrio1, 16-~estrio1, and radio­

active 16-ketoestradiol-17~ but to no other radioactive estrogen (within the limita of sensitivity of the methods

used) in the urine; and to radioactive estrio1 1 16-epiestriol,

traces of 16-ketoestradiol-17~ (which may have been a contaminant from the urine) and detectab1e amounts of another unidentified estrogen in the feces. This work is believed to represent the first study on the metabolism of estrogens - 128 - in an avian species in which urine and feces have been examined separately by use of suitably operated birds.

5. The first demonstration, so~r as the author is aware, of the presence of 16-ketoestradiol-17~ in the excreta of the fowl.

6. The detection of equol as a contaminant of both hydrophilic and lipophilic estrogen extracts from both urine and feces of the domestic fowl. This is beiieved to constitute the first identification of equol in avian excreta.

7. The observation that a large part of the metabolites of estriol, probably about half of the total, is excreted by the fecal route, with the inference that account must be taken of fecal estrogens in any future quantitative studies of total estrogen excretion.

8. The first examination, so far as the author is aware, of the bile of the domestic fowl for the presence of estrogen. Estradiol, estrone and estriol were shown to be present. REFERENCES

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Nowaczynski, W.J. and Steyermark, P.R. The absorption spectra of steroids in "lOO per cent" phosphoric acid. Arch. Biochem. and Biophys. 58:453-~-60. 1955. Parkes, A.S. and Emmens, c.w. Effect of androgens and estrogens on birds. Vitamins and Hormones 2:361-408. 1944. Pearlman, W.H., Rakoff, A.E., Cantarow, A. and Paschkis, K.E. The isolation of estrone from the bile of pregnant cows •. J. Biol. Chem. 170:173-179. 1947. Pincus, G. and Pearlman, W.H. Metabo1ism of estrogen in men and nonpregnant women. Endocrinology 31:507-514. 1942. Reineke, L.M .. Paper chromatography in steroid determination. Anal. Chem. 28:1853-1858. 1956. Riddle, o. Cyclic changes in blood calcium, phosphorus and fat in relation to egg laying and estrogen production. Endocrinology 31:498-506. 1942. Roberts, R.B., Abelson, P.H., Cowie, D.B., Bolton, E.T. and Britten, R.J. Studies of biosynthesis in Escherichia coli. Carnegie Inst. of Washington, Pub. No. 607. 1955. Roberts, G., Gallagher, B.S. and Jones, R.N. Infrared absorption spectra of steroids -An atlas. Vol. II, Interscience Publishers Inc., New Yo!'k. 1958. Roberts, S. and Szego, c.M. Steroid interactions in the metabolism of reproductive target organs. Physiol. Reviews 33:593-629. 1953. Rosenheim, o. and King, H. Ring system of sterols and bile a cids. Chem. and Indust!'y 51:464-466. 1932a. Rosenheim, o. and King, H. Ring systems of sterols and bile acids. Chem. and Industry 51:954-956. 1932b.

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Ryan, K. J. Metabolism or C-16-oxygenated steroids by human placenta: the for.mation of estriol. J. Biol. Chem. 234:2006-2008. 1959. Salhanick, H.A. and Berliner, D.L. Isolation of steroids from a feminizing adrenal carcinoma. J. Biol. Chem. 227:583-590. 1957. Savard, K. Sorne theoretical and seme practical aspects of partition chromatography of keto steroids. Recent Prog. Hormone Res;l85-211. 1954. Serchi, G. The separation of a estratrienic urinary metabolite in crystalline form, by extraction from paper chromatograms. Chimica (Milan) 8:9-11. 1953. Cited from Chem. Abstr. 47:12496c. 1953. Slaunwhite, W.R. Jr. and Sandberg, A.A. Phenolic steroid~ in human subjects. I. Tpe conversion of estrone-16-cl4 to 16-ketoestrone-16-cl4. Arch.Biochem. and Biophys. 63!478-480. 1956. Stevenson, M.F. and Marrian, G.F. The determination of oestrogens in human pregnancy urine. A new method of correcting for the brown colour developed in the Kober reaction by non-estrogenic substances, Biochem. J. 41:507-511. 1947.

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Watson, E.J.D. and Marrian, G.F. The isolation of 16-oxo-oestradio1-17~ in the urine of pregnant women. Biochem. J. 61:xxiv. 1955. Zaffaroni, A. Absorption spectra of sulphuric acid chromogens obtained from adrenal steroids and related compounds. J. Am. Chem. Soc. 72:3828. 1950. Zaffaroni, A., Burton, R.B. and Keutmann, E.H. The application of paper partition chromatography to steroid analysis. I. Ketosteroids. J. Biol. Chem. 177:109-116. 1949. APPENDIX A NOTE ON THE AMOUNI' OF URINE VOIDED

BY THE LAYING HEN

The urine and ~eces o~ the bird are voided into the cloaca. In order to obtain urine ~ree from ~ecal matter, it is necessary either to cannulate the ureters or surgically to exteriorize the opening o~ the ureters or rectum. Most of the early work relating to quantities of urine voided by the bird was based on collections made over short periods. The data were then extrapolated to represent excretion over a 24-hour period. The values tbus obtained led various workers to conclude: (1) that the volume of uretral urine entering the cloaca and rectum was as much as 700 to 1 1 000 ml. in 24 hours; and (2) that 240 to 270 ml. of water was reabsorbed from this urine in the cloaca and rectum in 24 hours (Sharpe, 1923; Davis, 1927;

Rester, Essex&: Mann, 1940). The estimated amount o~ urine voided daily was very large and greatly exoeeded the daily water consumption. Water balance was believed to be main­ tained by reabsorption of water from urine in the cloaca and rectum.

Hester, Essex &: Mann (1940) and Hart &: Essex (1942) considered that auch estimates were in error because they bad found uretral urine volumes of 5o to 180 ml. per day, based on 24-hour quantitative collection periods. They also concluded that cannulation and handli ng of the bird caused a diuresis which persisted for at least 30 minutes. - 142 -

Dixon (1958) reinvestigated the question of re­ absorption of water from the urine in the cloaca and rectum by comparison of the water balances of normal hens with those of hens with exteriorized ureters or with the rectum exteriorized. The data presented by Dixon indicated that little or no water was reabsorbed from the uretral urine in the rectum and cloaca of the hen. Once the bird had recovered from the operation, the surgical modification used in his experiment had no affect on water excretion. Dixon allowed about 10 days for recovery in order to permit of thorough healing and presumably also to avoid complica­ tions from post-operative diuresis, although Dixon does not specifically mention this latter point.

In the present work records of daily urinary volumes were kept for two laying hens over a period of 80 days. Collections were begun immediately after operation and were based on the period 10 a.m. until 10 a.m. the following morning.

The data for Hen A (liveweight, 2.32 kg.) are presented in Fig. 26. The urinary volume rose steadily to a maximum of 725 ml. for the eighth day after operation. Thereafter it declined steeply and steadied off at a leval of 57 ml. per day from day 11 to day 22. The volume then rose sharply to a level around 190 ml. from day 23 to day 29. The first egg was laid on day 30 and it is suggested - 143 -

1000 ...1 ·1:

UJ 800 ~ :::J _J 60 0 > w 400 z

0:: 200 ::::>

EGGS îO 20 3 0 40 50 60 70 80 DA YS

FIGURE 26. Daily urine volumes ~or Hen A.

Arrow indicates day o~ operation. - 144-

that the data indicates an increase in urine volume in relation to the approach of laying. The general ·level was maintained for a further eight days, but then fell off gradually and for 60 - 80 days the daily volume averaged 60 ml.

While the data are admittedly limited, they do suggest (a) that there is a marked post-operative diuresis that continued for at least 10 days, (b) that there is an increase of urinary volume with the onset of reproductive actlvity and (c) that this increase is not necessarily maintained throughout laying. It may be remarked in passing that poultrymen are familiar with the fact that the droppings in a laying house are, in general, wetter than in a fattening house or in a house with non-laying pullets.

The data for Hen B (liveweight, 3.38 kg.) are presented in Fig. 27. The data are complicated (a) by the necessity for reoperation on day 29 and (b) by the oircum­ stance that a solitary egg was laid on day 21, with no further eggs until day 37. Post-operative diuresis was marked during the first 10 days (average 575 ml. per day) and then declined to a leval of 105 ml. from day 11 to 23. From day 24 there may have been a tendency toward increased urinary volume in association with the onset of laying, but this was complicated by the diuresis consequent on the second operation. For the first ten days after this - 145 -

I w 800 L :::> _j 600 0 > w 400 2

0:: 200 :::>

EGGS 1 1 Il Il 1 1 Il 111111111 Il Il 1111 Ill 10 2 0 30 40 50 60 70 80 0 A YS

FIGURE 27. Daily urine volumes for Han B. Arrows indicate days of operation. - 146 - operation, the urinary volume averaged 767 ml. per day. (The urinary volume for the firat day after the operation actually attained the astoniahing value of 960 ml.). From day 44 to day 55 the volume declined somewhat (average, 252 ml.). It then increased again, poasibly in association with a period of intensive egg production, to an average of 417 ml. per day.

The resulta, though admittedly ltmited, show that post-operative diuresia persista for at least 10 daya after operation, and that the volume of urine may average around 350 ml. per day over this period. The resulta also auggest that the urinary volumeincreaaes in the 7 to 10 days immediately before the beginning of laying. The resulta also suggeat that birds may display considerable individual variation in their pattera of urinary excretion.

Finally, it is remarkable that a hen should excrete almost 1 litre of urine in 24 hours. REFERENCES

Davis, R.E •. The nitrogenous constituants of hen•s urine. J. Biol. Chem. 74:509-513. 1927. Dixon, J.M. Investigation of urinary water reabsorption in the cloaca and rectum of the ben. Poultry Sei. 37:410-414. 1958. Hart, W.M. and Essex, H.E. Water metabolism in the chicken with special reference to the ro1e of the cloaca. Am. J. Physiol. 136:657-688.1942. Hester, H.R., Essex, H.E. and Mann, F.C. Secretion of the urine in the chicken. Am. J. Physio1. 128:592-602. 1940. Sharpe, N.c. On absorption from the cloaca in birds. Am. J. Physiol. 66:209-213. 1923.