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An overview of four plus decades of research in estrogens: A personal history
An overview of four plus decades of research in estrogens: A personal history Mortimer Levitz Department of Obstetrics and Gynecology, New York University Medical Center, New York, New York 10016, USA
Introduction It was with considerable trepidation that I rashly agreed to the request of the editors of Steroids to share with the scientific community my recollections of 42 years in research on steroids with particular emphasis on estriol. Aside from an underlying concern of how to make these few pages interesting enough to attract a readership beyond my immediate family, I have rarely derived pleasure from turning the clock back. My underlying philosophy is that the record is there for anyone curious enough to delve into what I have done scientifically. Better my efforts should be expended on future pursuits. Moreover, even reflecting on a piece of work with pride, I find myself questioning why it took so long. Nevertheless, I accepted and am committed to the challenge. The strategy is to compare the state of the field in the early fifties with now, and then fill in the time gap, concentrating on my efforts. The former can be summarized succinctly. At mid-century steroid chemistry was dominated by sophisticated organic chemists completing work on the structure and synthesis of key steroids and their metabolites. They, myself included, knew virtually nothing about their mechanisms of action. On the other hand, today's steroid researcher at the forefront of the field knows little about the chemistry of the hormone, but is exquisitely perceptive of its action at the gene level. My piece of the jigsaw puzzle comprises the remainder of this remembrance.
conventional academic pursuits dominated the activities on several floors of the physics department laboratories. With the future in doubt and beyond individual control, I decided to interrupt my formal education when I interviewed successfully for a job in those hush-hush laboratories, under the aegis of the so-called Manhattan Project. The research, centering on the chemistry and corrosive properties of uranium hexafluoride, was not particularly exciting but it afforded the opportunity to learn valuable laboratory techniques including glassblowing and the construction and maintenance of high vacuum systems. Moreover, there was a pervasive aura that something crucial to the war effort was being contributed. But it was work down the hall that attracted my attention and directed my inclination to organic chemistry. Groups headed by experts in fluorine and polymer chemistry combined their talents to create
Prelude In a way my predoctoral experience dictated the course of my future professional career. In early 1942 with the world divided by forces of destruction, a lucky draft number afforded me the temporary luxury of enrolling for my doctorate at Columbia University. There I soon discovered that research activities unassociated with
Address reprint requests to Mortimer Levitz, Department of Obstetrics and Gynecology, New York University Medical Center, 550 First Avenue, New York, New York 10016, USA.
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Dr. Mortimer Levitz
© 1994 Butterworth-Heinernann
Four plus decades of research in estrogens: Levitz fascinating perfluorinated polymers. I often wonder whether teflon was developed there, a polymer that lent some interest to an otherwise dull 1984 presidential election. In late 1946, discharged from the army, it was back to graduate school under the tutelage of a remarkable teacher, Professor William Doering, who truly prepared one for a career in independent research. The first decade
After 9 years at Columbia University and environs on the Manhattan Project in both civilian and military capacities and as a graduate student in organic chemistry, the umbilical cord was not severed, merely stretched to the uptown campus of the College of Physicians and Surgeons. There began a long association with a superb gynecological surgeon, Gray H. Twombly, intensely interested in breast cancer research. In a brief interview, he expressed two concerns: whether I could synthesize radiolabeled estradiol and if I would be interested in biomedical research. I recall adroitly finessing the latter question, thinking that while working on the synthesis I could explore ideas concerning what I would do with the newly synthesized material. Although labeled estradiol was not available commercially in 1952, R. D. H. Heard of McGill University kindly provided me the details of a cumbersome, low-yield method he apparently never published. At least this would provide a source of material should my own efforts fail. The intermediary in this Montreal connection was a young graduate student, Sam Solomon, whose friendship and scientific interactions have been constant sources of pleasure and intellectual stimulation throughout my career. The synthesis of [16J4C]estradiol gave me great satisfaction professionally and involved me with interesting controversies with foremost scientists of the era, on which I will elaborate. The salient features of the synthesis are depicted in Figure 1. There are two crucial steps, the first being the introduction of t4C into 3-methoxymarrianolic acid, accomplished by treating II with triphenylmethylsodiumand 14CO2, and the second, the ring closure via an acyloin condensation to produce V. The former step was accomplished with dispatch since I discovered the powerful potential of the strong, ether-soluble base, triphenylmethylsodium during my thesis research. The condensation step posed a personal dilemma of sufficient interest to recount in detail. In 1952 I was fortunate to attend a Gordon Conference on Steroids. To a young investigator such a conference is a unique experience. Under idyllic
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surroundings information, frequently unpublished, and ideas are shared in a relaxed atmosphere, in sharp contrast to that pervading a typical national meeting. My excitement was unrestrained when in a session on newer reactions in steroid synthesis a discussion leader, John C. Sheehan of Massachusetts Institute of Technology (M.I.T.) showed that Na in liquid ammonia converted II into a ring D ketol (acyloin condensation). I presented him my problem and was delighted by his generous response. He invited me to Cambridge where he opened notebooks that contained the experimental details of the reaction. Parenthetically, the reaction is an esthetic experience. Sodium in liquid ammonia produces a brilliant deep blue solution and owing to the low boiling point of ammonia, a dry-ice condenser must be in place, a perfect grade B science fiction movie visual effect. The ensuing steps proceeded uneventfully except for an interesting twist. Dr. Sheehan informed me and later reported that the condensation produced a l%t-hydroxyketol, whereas I had obtained unequivocal evidence for the more desirable 17fl-hydroxy configuration, based primarily on the identification of the end-product as 17fl-estradiol. I quickly submitted a manuscript on the synthesis and at about the same time informed my benefactor in Cambridge of his error. The paper was accepted I but no communication was forthcoming from M.I.T. Instead a note appeared in the same journal several months later to the effect that upon independent investigation the authors would like to correct their original error. I thought it was a stretch of the definition of the word independent but let the matter rest. The beauty of the synthesis outlined in Figure 1 was that with minor modification the synthesis could be adapted to produce several labeled 16-oxygenated estrogens of interest. For example, the acyloin condensation applied to the 3-benzyl ether of dimethylmarrianolate afforded 16-ketoestradiol2 that on reduction with sodium amalgam produced estriol and 16-epiestriol in a rate of 7:3. a Our armamentarium of labeled compounds expanded at a time when erroneously estriol was being declared by some investigators as an impeded estrogen or even an antiestrogen, and 16-ketoestradiol - - in selected t u m o r cultures - appeared to impede cell proliferation. These labeled compounds were made available to the scientific community under a program sponsored by the American Cancer Society. The interest in 16-oxygenated estrogens prompted us to study the metabolism of estriol in women with emphasis on whether estriol was truly an end-product and whether 16-ketoestradiol was produced in vivo. a Finding the latter as well as 16-epiestriol in the urine of volunteers who received [14C]estriol settled these issues - - at least I thought so. Soon after we published this work, the American Cancer Society sponsored a symposium on estrogens and cancer in a mammoth auditorium of a New York City hotel. A featured speaker was the distinguished gentleman and renowned investigator, Guy Marrian. He presented an erudite address on the state of the field, but toward the end of his talk he
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Persona/History dropped a personal bombshell. To an audience of over 3000 he retracted his report that 16-ketoestradiol was an estrogen metabolite in women and that it arose from rearrangement caused by the conditions of urinary workup. No harm, until he proclaimed in a most delightful Scottish brogue that Levitz (heavy accent on the last syllable), Spitzer, and Twombly undoubtedly fell into the same trap and must also retract. At the conclusion, hardly waiting to be recognized by the chair, I sprang to my feet and declared that I was fully aware of the pitfalls of ring-D ketol rearrangements and that workup conditions were carefully designed to preclude this phenomenon and that we had no reason to retract. Nevertheless, in the ensuing 18 months Marrian papers on the subject echoed the same criticism of our work. Vindication came in August 1957 in a meeting at the Worcester Foundation, convened by the legendary Gregory Pincus, to which the foremost researchers in estrogens were invited. In his usual thorough style Egon Diczfalusy presented a paper confirming our 16ketoestradiol finding. I was sitting next to the redoubtable Scotsman, who leaning over, offered his sincere apology while commenting that if Diczfalusy reports it, it is true. Embarrasingly, in future papers, spanning some 5 years, Martian was profusely apologetic. That meeting cemented friendships with elite investigators on estrogens in several continents (Figure 2) and placed me in even better grace with Dr. Pincus; both did my career no harm. In the early part of the decade I entered into a collaboration - - that still endures - - with Joseph Dancis, a future professor and chairman of the department of pediatrics at our medical center with an insatiable thirst for knowledge that continues to reflect in a remarkable
Figure 2
Members of the conference on estrogens, Shrewsbury, Massachusetts, 1957. From left to r i g h t - front row: RI Dorfman, LL Engel, JW Goldzieher, S Kushinsky, E Bloch, WR Slaunwhite; second row: G Pincus, GF Marrian, E Diczfalusy, JK Preedy, TF Gallagher, K Savard; third row: R Jacobs, JB Brown, B Baggett, AE Kellie, M Levitz, Unidentified (according to the author's recollection he was a visiting scientist from Australia, named Brown); back row: CD West, RDH Heard, LT Samuels, J Fishman, E Schwartz.
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record of accomplishment in many areas of biomedical research. We did interesting studies on inborn errors of metabolism, in particular branched-chain ketoaciduria but that is peripheral to this biography. Dr. Dancis introduced me to the fascination of the placenta. In the steroid field, with emphasis on estrogens many metabolic and transfer studies followed exploiting both the human and guinea pig placenta. In all candor, though, one study turned out to be deeply embarrassing. First, a bit of background. In the 1950s the biochemical origin of estrogens was unknown. In fact, at the American Cancer Society Symposium alluded to above, a noted biochemist spun an intriguing fantasy, supported mechanistically, but not experimentally, purporting that estrogens are biosynthesized from aromatic amino acids. We decided to perfuse the human placenta with labeled acetate and were elated to find radioactive estrogen in the perfusate. When the result was confirmed we submitted an abstract for presentation at the ensuing Federation Meeting. On further exploration it became clear that our report was erroneous. I had thoughts on the origin of the contamination of our system with labeled estradiol, but I merely satisfied myself with spending three weekends perfusing placentas with absolutely fresh tubing and reporting our error. I gained an appreciation of the view of many senior biochemists of that era who maintained that results based on radioactive data were tentative at best. They had a point, but rigidity could produce premature scientific rigor mortis.
The second decade The same tide that carried me and numerous investigators into the areas of steroid metabolism and action drifted us further from pure organic chemistry. Two relatively minor contributions merit mention. [3H]Estriol was not readily available in the early sixties, restricting the kind of metabolic studies that could be done in man. The synthesis devised was essentially that described for that of [14C]estriol except that 3H204 was added in place of 14C in the appropriate step (see Figure 1 and reference 3 for further details). This era also witnessed a burst of activity in studies on the intermediary metabolism of estrogen conjugates. In order to study the metabolic fate of the sulfate moiety in estrone sulfate, 35S-labeled compound was desirable, preferably starting with the inexpensive H23sSO4. Treatment of [35S]-labeled sulfuric acid with pyridine in the presence of a 10% excess of acetic anhydride formed labeled pyridinium sulfate, which on reaction with estrone produced the desired conjugate in good yield. 5 All other conjugates used in many ensuing studies were produced biosynthetically. Thus, estriol is converted into estriol-3-glucosiduronate (E3-3G) in homogenate of guinea pig liver fortified with uridine diphosphate glucuronic acid 6 and to estriol-16-glucosiduronate (Ea-16G) with human liver under the same conditions. 7 Moreover, treatment of the latter with ATP and sulfate in a 100,000 x # supernate of guinea pig liver produced the double conjugate, estriol-3-sulfate-16-glucosiduronate (E3-3S-16G). Finally, fl-glucuronidase converts the
Four plus decades of research in estrogens: Levitz double conjugate into Ea-3S, making all the important estriol conjugates available for study. This point in my career is best described as the Swedish Connection. In 1961 I received the first of five invitations from Egon Diczfalusy to spend time in his laboratory at the Karolinska Hospital in Stockholm. Sweden had the most liberal abortion policy in the world and virtually all women undergoing the procedure consented to research on the fetus and placenta. The clinical material, the availability of labeled estriol and my experience in estriol metabolism coupled with Diczfalusy's biomedical and laboratory expertise fostered a productive partnership that lasted the decade. I spent two summers in Stockholm in the early sixties and made three other work-related visits later in the decade, all delightful and rewarding experiences. The laboratory attracted scientists from all over the world, but I particularly recall interacting with four young, budding investigators who have made significant contributions in reproductive endocrinology. I worked in closest association, literally day and night, with the late Uwe Goebelsmann, a scholar and a gentleman in the truest sense. The other three of note were Robert B. Jaffe, George Mikhail, and Philip Troen. Troen had the sense to ship his scintillation counter to Stockholm, permitting the prompt processing of samples. In 1965 1 was asked by the program committee of the Endocrine Society to present our work on estrogen transport and metabolism in the fetal-placental unit in a symposium at the annual meeting. The major stress was on my research at Karolinska. The salient features of the lecture were published s'9 and can be summarized briefly. Estrogen conjugates traverse the placenta poorly. Owing to sulfatase activity but insignificant glucuronidase in the placenta and fetal membranes, estrogen originally in the sulfate form reaches the fetus far more effectively than estrogen presented as the glucosiduronate. As pregnancy proceeds, the human fetus is exposed to increasing concentrations of free estrogens. Protection from hyperestrogenism is afforded by conjugation, each important portal to the fetus being an effective organ or tissue in that regard. This holds for the liver, the recipient of cord blood estrogens and fetal lung and skin which are exposed to the amniotic fluid. This kind of fetal-placental research came to an end with the introduction of prostaglandin as an aborting agent, virtually eliminating hysterotomy as a procedure for early second-trimester abortions. Research on the origin and intermediary metabolism of estrogens in the early sixties was intense, facilitated by the availability of isotopically-labeled compounds. The major contributions of our laboratory in this regard have been summarized. Of paramount importance, that era witnessed two other developments that would impact on the course of our research in the latter part of the decade. The first practical chemical method for the quantification of the classical estrogens; estrone, estradiol, and estriol in urine was reported by James B. Brown, of Edinburgh .1° Sequentially, he applied acid hydrolysis to a urine aliquot, solvent extraction of estrogens which were converted to their 3-methyl ethers, separation and
purification of the classical estrogens by column chromatography, and assay by the Kober reaction. The second important development was the elucidation of the origin of estriol in pregnancy, conversion by the placenta of 16~-hydroxydehydroisoandrosterone that is of fetal origin, tt'lz These considerations, coupled with the thinking on the part of many investigators that estriol levels in body fluids could provide a useful monitor of the status of pregnancy, prompted me to consider research in this area. Dr. Lila Nachtigall, fresh from her residency in medicine, requested a 2-year fellowship in my laboratory to work in an area Of estrogen research that would have direct clinical relevance. Literature was beginning to accumulate on the urinary excretion of estriol in pregnancy, but no convenient method for determining its concentration in plasma was described. There were many reasons to argue the superiority of a plasma method, the most compelling being that interruption of normal estriol synthesis would be reflected more promptly by a drop in blood levels than in urine concentrations. To our delight the method outlined on paper yielded cause for optimism in 6 weeks and was soon operative without significant change. 13 Three key considerations contributed to this success. First, recognizing that estriol circulates predominantly in the form of conjugates and that acid hydrolysis would produce varying degrees of estriol destruction, each assay tube was charged with representative tritiated estriol conjugates to serve for correction of methodological losses. Secondly, on benzene:hexane-water extraction, the bulk of interfering estrogens and other lipids distribute into the organic phase, while estriol remains in the water from which it is ether-extractable. Finally, estriol could be quantified by a highly specific, sensitive spectrofluorometric method. The method was applied to the study of diseases in pregnancy 14 as well as to specific research protocols. Numerous laboratories expressed interest in the technique to the extent of sending representatives to observe it firsthand. Over the past 15 years or so sophisticated techniques for monitoring the fetus in utero has, in the view of obstetricians, eliminated the need for estriol assays for assessing fetal status. Retrospectively, my view of the value of our research in this area is difficult to assess objectively. Interesting basic information was forthcoming. But in terms of patient care, no meaningfully statistical prospective study was run, or even conceivable in which estriol levels would be the only variable in the management of the pregnancy. Looking back, it was indeed a busy decade in our laboratory, virtually all the research focusing on estriol. The enterohepatic circulation and metabolism of estriol conjugates in the normal adult were investigated in depth; three studies are most worthy of mention. The first was an inquiry into the origin of E3-3G. A simply designed study yielded definitive results. The volunteer was a 29-year-old man with normal liver function who had undergone cholecystectomy for bile duct obstruction and whose bile was being drained via a T-tube. [14C]Estriol was injected into the duodenum via the T-tube while [3H]estriol was given i.v. E3-3G was found
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Persona/History only in the urine and labeled exclusively with ~C, demonstrating the intestinal origin of this conjugate, ts This study and two other studies showed: E3-3S-16G is the major estriol (E3) conjugate excreted with the bile, E3-16G is the major urinary metabolite, E3-3G is the true end-product of E 3 metabolism, and E3-3S is hydrolyzed extensively and the E 3 converted mainly to E3-16G before excretion in the urine. ~6'17 Other ring Dhydroxylated estrogens commanded interest in this period. Specifically, 3,15e,16e,17fl-tetrahydroxyestra1,3,5(10)-triene (esterol, E¢) levels were targeted as potentially superior to E 3 for assessing pregnancies at risk because E 4 was thought to be an estrogen metabolite unique to the fetus. We contradicted that claim by demonstrating the in vivo 15-hydroxylation of estrone in the nonpregnant adult woman. Is In March 1966, an event occurred that added spice to my research career for years to come. The legendary Morris Graft, Executive Secretary of the Endocrinology Study Section traced me to the bathtub of a hotel room in San Francisco at 6:30 AM. I was attending the Annual Meeting of the Society for Gynecologic Investigation. He invited me to become a member of the study section. I knew nothing of what was in store for me as I reflected that during the French Revolution Jean Paul Marat was assassinated by Charlotte Corday while taking a bath. The effort was enormous, invariably thankless, but the lasting friendships in endocrine circles and the learning experience more than compensated. Parenthetically, I must confess that prior to this experience I had no real concept of how the fate of proposals submitted to the National Institutes of Health (N.I.H.) was determined. I assume my contributions on study sections were appreciated since I was asked to serve as emergency fill-in for a second term, 1973-1975 and for a lengthy stint in a newly-forming Clinical Sciences Study Section in 1979-1981 (ad hoc) and -1985 (permanent), with the last two years as Chairman.
The third decade In the early seventies much effort was expended toward addressing unanswered questions on the intermediary metabolism of estriol in the normal and pregnant human. Fortunately we were able to recruit Helmut Jirku who trained with Donald S. Layne at the Worcester Foundation and Uma Raju who received the appropriate postdoctoral experience with Egon Diczfalusy. Moreover, Joseph Katz and Susan Kadner contributed both cerebrally and technically during their 25 years of research on many of the problems cited. Much of the research in this decade centered on the biosynthesis and quantification of estriol conjugates in pregnancy and diseased states. These studies were facilitated by the simplified methodology we developed for the separation of the four major conjugates of estriol and the availability of 3H-labeled compounds. One study centered on the renal clearances of estriol conjugates in normal human pregnancy at term. 19 It was found that more than 90% of the estriol in body fluids consists of four major conjugates: E3-3S, E3-3G, E3-16G and
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E3-3S-16G. The clearance of E3-16G was the highest, approaching the effective renal plasma flow. Then followed E3-3G which approximated the glomerutar filtration rate. The sulfurylated conjugates cleared the lowest, probably a reflection of their binding to serum albumin. The sensitivity of the method permitted the detection of minor estriol conjugates. A new conjugate that we identified, E 3-3,16-disulfate, comprised about 1% of total and was most concentrated in cord blood. The method, applied to the study of a patient with severe polycystic kidney disease, revealed the greatest aberration in the clearance of E3-3G. 2° The interest in 15-hydroxylated estrogens prompted us to study their metabolism and compare their intermediary metabolism with that of estriol. The prominent feature of these estrogens is the introduction of a novel mode of conjugation. Following administration of [3H]15ct-hydroxyestrone to pregnant women more than 50% of the radioactivity was excreted in the urine in the form of 15-N-acetylglucosaminides.21 This was the first report of this mode of conjugation of estrogens in the human, although N-acetylglucosaminides of estrogens in the rabbit 22 and progesterone metabolites in women 23 have been cited. Another surprising result in this study was the evidence for the urinary excretion of a glucosiduronate of 15c~-hydroxyestradiol-17~. Our in depth studies of the metabolism of estriol and 15ct-hydroxyestradiol in pregnancy indicated distinct differences in their conjugation profiles. Glucosiduronidation predominated for the former and Nacetylglucosaminidation for the latter. Estetrol may be viewed as the structural fusion of these two ring D estrogens, raising the question of its mode of conjugation. Our studies indicated that estriol exerted greater influence on the conjugation profile. Ring D glucosiduronates comprised about 90% of the urinary metabolites following administration of labeled estetrol to pregnant women, while only 1% was in the amino sugar form. 24 15~-Hydroxyestrogen N-acetylglucosaminyl transferase activity was demonstrated in vitro in human adult liver and in human adult and fetal kidney.25 Although these findings elucidated many interesting and novel features of the metabolism of 15~-hydroxyestrogens in pregnant and normal states, my chief regret on reflection is that the physiological impact remains obscure.
The fourth decade A hallmark of the eighties in obstetrics was the introduction of sophisticated ultrasound and electronic equipment for assessing the status of the fetus at risk. Paralleling the expansive use of these innovations was the phasing out of the reliance on estriol concentrations in maternal blood for monitoring the status of the pregnancy. In the research arena the estriol molecule was not moribund; the emphasis shifted to nonpregnant states. Our laboratory was involved in two studies worthy of comment. The first concerned a research inquiry conducted in connection with a clinical study. In a collaboration with Isaac Schiff at The Boston
Four plus decades of research in estrogens: Levitz Hospital for Women, the experimental design was to give estriol either orally or intravaginally to menopausal women and to compare the physiologic responses with mode of administration. The measure of biologic response was the decline in blood FSH and LH levels at 6 h. Significant declines were observed after intravaginal administration only. Analysis of the sera for estriol and its conjugates indicated a much higher rate of absorption following oral administration; however, significant levels of free estriol were seen only with the vaginal route. We concluded that only unconjugated estriol suppresses gonadotropins. 26 It is apparent that with the correct preparation and mode of administration estriol can be effective in estrogen replacement therapy, but it must be borne in mind that estriol is an active estrogen when administered continually, and not an impeded estrogen as was suggested over 30 years ago. My last major thrust into estriol research, culminating in a most surprising finding can be described as the epitome of serendipity. In terms of nostalgia and human interest this section dates back to the first decade of the narrative. At that time a carpool formed which included biochemists from Sloan Kettering and myself. Parenthetically, my participation lasted 25 years until my move to New York. Lively discussions usually on politics and steroid biochemistry made us oblivious to the endless stream of traffic between Queens and Manhattan. These discussions spawned collaborations, one of which was with a distinguished steroid biochemist, H. Leon Bradlow. The focus was on gross cystic disease (GCD) of the breast, a benign condition requiring simple needle aspiration of the breast cyst fluid (BCF) usually to relieve pain and anxiety. The interest was two-fold: epidemiologically, women with GCD were at greater risk for developing breast cancer and according to fragmentary reports many BCFs contain high concentrations of steroids. Is there a connection? The question is not resolved, but meritorious of investigation. We decided that Dr. Bradlow would focus on estradiol, estrone, and protein hormones, while our laboratory would investigate estriol and later, bile acids and some enzymes of particular interest to us. There was no compelling reason to expect to find E3 in BCF. We had shown that the levels of unconjugated E 3 in plasma was less than 0.02 nmol/L and for its conjugates it was barely twice that value. BCFs were analyzed for E 3 and its four major conjugates. Surprisingly, concentrations of E3 several hundred-fold greater than in blood were observed, specifically relegated to E3-3S. Many cysts also manifest unusually high levels of potassium ion, a phenomenon thought by many investigators to be associated with risk for breast cancer. Accordingly, we examined our E3-3S data in terms of the K+/Na ÷ ratios, classifying Type I as > 1.0; Type II, < 0.25, and Type III, 0.25--0.1.0. By Student's t-test, the concentrations of E3-3S differed between each BCF Type (p < 0.002), with Type I cysts showing the highest levels and Type II the lowest. Moreover, by linear regression analysis, there was a significant correlation between the concentrations of E3-3S and the sulfates of estrone and dehydroisoandrosterone. 27
The origin of the extraordinarily high concentrations of E3-3S in BCF has yet to be elucidated. One thought was that the sulfate transferred to the cyst from blood against a huge concentration gradient, as we have shown for bile acids. 2s This was considered a possibility since steroid sulfates generally have blood half-lives in excess of 6 h. Unexpectedly though, we found that E3-3S had a half-life of only 0.5 h, precluding the use of radioactive E3-3S to investigate this phenomenon. 29 In connection with our studies on the biochemistry of BCF, we found that this fluid contained formidable levels of long-chain fatty acid esters of steroids. Since these biologically inactive substances can be rendered active by hydrolysis to the parent steroid, we initiated a search for esterases in BCF. An unusual esterase was identified and characterized. 3° Thomas H, Finlay and Sila Banerjee, close collaborators, purified the esterase to near chromatographic homogeneity. It is a 23 kDa peptide and appears to be of breast epithelial origin since its properties differ from any blood esterase. The esterase is present in about 40% of BCFs and statistically, its concentrations associate positively with E3-3S, other steroid sulfates and K+/Na + ratios) 1 The take-home message of the decade is that the steroid biochemist is not a dinosaur. Unpredictable metabolic pathways impacting on disease, particularly cancer, continued to surface. The number of players has dwindled sharply, but those who have stayed the course, still too numerous to cite, clearly have justified their efforts. F o u r and a quarter - - Epilogue
By rough calculation, at the appearance of this memoir, my research career will have approached the second quarter of the fifth decade. What about the future? This is hard to predict. If this were the script for an old radio soap opera, I could hear the announcer droning, "This is a saga seeking the answers to two related questions: does life begin at 40, and can quality research continue without N.I.H. support?" It is superfluous to state that research support is harder than ever to obtain. The grant supporting the bulk of the research described herein, the seventh highest in terms of longevity at the National Cancer Institute at the time of inquiry in 1992, 39 years, failed renewal by the proverbial cat's whisker. On resubmission the result was the same but the margin was by a bigger bunch of hairs. I have no plans for retirement. Before that day, I would like to 1) have contributed substantially to the solution of the problem of the source and physiological implications of the extraordinarily high concentration of estriol, bile acids and esterase in BCF; 2) have witnessed unequivocal verification of our observation that bioavailable estradiol constitutes a risk for cancer (paper submitted for publication); and 3) have increased our understanding of how nutrients and deleterious drugs cross the placenta - - at present we are doing cocaine. The latter two studies are considered peripheral to the main thrust of this narrative, but continue to be major contributors to the joy and excitement of just going to work in the morning.
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Persona/History Acknowledgments The research cited was supported principally by a grant from the National Cancer Institute, National Institutes of Health, CA2071 (01-39). I wish to express my thanks and deepest appreciation to Dr. Gordon W. Douglas, Professor and Chairman of the Department of Obstetrics and Gynecology at our Medical Center through three decades of my research career. His appreciation of the value and importance of basic research to a clinical department was reflected in his personal support and commitment of resources, particularly in times of departmental budgetary stress. I am also grateful to our present Acting Chairman, Robert F. Porges, M.D., who has made it clear that continued productivity and vitality, and not retirement rules, would be the criteria for continued support.
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Levitz M (1953), The synthesis of 17fl-estradiol-16-C 14. J Am Chem Soc 75:5352-5355. Levitz M, Spitzer JR (1956). The synthesis of 16-ketoestradiol17fl-16-C 14. J Biol Chem 222:979-980. Levitz M, Spitzer JR, Twombly GH (1958). Interconversions of 16-oxygenated estrogens. 1. The synthesis of estriol-16-C14 and its metabolism in man. J Biol Chem 231:787-797. Levitz M, Katz J (1965). The synthesis and some properties of estriol- 15-3H. Steroids 5:11-20. Levitz M (1963). Synthesis of estrone- 6,7-3H sulfate-aSS. Steroids 1:117-120. Gobelsmann U, Diczfalusy E, Katz J, Levitz M (1965). Biosynthesis of radioactive estriol-3-glucosiduronate by guinea pig liver homogenate. Steroids 6:859-864. Slaunwhite WR Jr, Lichtman MA, Sandberg AA (1964). Studies of phenolic steroids in human subjects: VI. Biosynthesis of estriolglucosiduronic acid-16-14C by human liver. J Clin Endocrinol Metab 24:638-643. Levitz M (1966). Conjugation and transfer of fetal-placental steroid hormones. J Clin Endocrinol Metab 26:773-777. Levitz M, Condon GP, Dancis J, Goebelsmann U, Eriksson G, Diczfalusy E (1967). Transfer of estriol and estriol conjugates across the human placenta perfused in situ at midpregnancy. J Clin Endocrinol Metab 27:1723-1729. Brown JB (1959). The metabolism of estrogens and the measurement of the excretory products in the urine. Br J Obstet Gynaecol 66: 795-803. Colas A, Heinrichs WL, Tatum HJ (1964). Pettenkofer chromogens in the maternal and fetal circulations: Detection of 3fl, 16ct-dihydroxyandrost-5-en-17-one in umbilical cord blood. Steroids 3:417-434. Magendantz HG, Ryan KJ (1964). Isolation of an estriol precursor, 16ct-hydroxydehydroepiandrosterone from human umbilical sera. J Clin Endocrinol Metab 24:1155-1162. Nachtigall L, Bassett M, Hogsander U, Slagle S, Levitz M (1966).
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28. 29.
30. 31.
A rapid method for the assay of plasma estriol in pregnancy. J Clin Endocrinol Metab 26:941-948. Levitz M, Young BK (1977). Estrogens in pregnancy. Vitam Horm 35:109-147. Stoa KF, Levitz M (1968). Comparison of the conjugated metabolites of intravenously and intraduodenally administered oestriol. Acta Endocrinol (Copenh) 57:657-668. Emerman S, Twombly GH, Levitz M (1967). Biliary and urinary metabolites of estriol-15-3H-3-sulfate-35S in women. J Clin Endocrinol Metab 27:539 548. Levitz M, Katz J (1968). Enterohepatic metabolism of estriol-3-sulfate-16-glucosiduronate in women. J Clin Endocrinol Metab 28:862-868. Jirku H, Hogsander U, Levitz M (1967). 15:¢-Hydroxyestrone sulfate: a biliary metabolite of estrone sulfate in the non-pregnant female. Biochim Biophys Acta 137:588-591. Young BK, Jirku H, Kadner S, Levitz M (1976). Renal clearances of estriol conjugates in normal human pregnancy at term. Am J Obstet Gynecol 126:3842. Levitz M, Kadner S, Young BK (1983). Profile of serum estriol and its conjugates at delivery and in the immediate postpartum period in a patient with severe polycystic kidney disease: a comparison with normal pregnancy. Am J Obstet Gynecol 145:465-468. Jirku H, Kadner S, Levitz M (1972). Metabolism of 15ct-hydroxyestradiol in human pregnancy. J Clin Endocrinol Metab 35:522-534. Jirku H, Layne DS (1965). The formation of estriol-3glucuronoside-17ct-N-acetylglucosaminide by rabbit liver homogenate. Biochemistry 4:2126-2131. Arcos M, Lieberman S (1967). 5-Pregnene-3fl,20~-diol-3-sulfate20-(2'-acettamido-2'-deoxy-ct-D-glucoside) and 5-pregnene3fl,20ct-diol-3,20-disulfate. Two novel urinary conjugates. Biochemistry 6:2032-2039. Jirku H, Kadner S, Levitz M (1972). Pattern of estetriol conjugation in the human. Steroids 19:519-534. Jirku H, Levitz M (1972). 15~-Hydroxyestrogen N-acetylglucosaminyl transferase activity in human adult liver. J Clin Endocrinol Metab 35: 322-325. Schiff I, Tulchinsky D, Ryan KJ, Kadner S, Levitz M (1980). Plasma estriol and its conjugates following oral and vaginal administration of estriol to postmenopausal women: correlations with gonadotropin levels. ,4m J Obstet Gyneco1138:1137-1141. Levitz M, Raju U, Arcuri F, Brind JL, Vogelman JH, Orentreich N, Granata OM, Castagnena L (1992). Relationship between the concentrations of estriol sulfate and estrone sulfate in human breast cyst fluid. J Clin Endocrinol Metab 75:726-729. Javitt NB, Budai K, Miller DG, Cahan AC, Raju U, Levitz M (1994). Breast-gut connection: origin of chenodeoxycholic acid in breast cyst fluid. The Lancet 343:633-635. Raju U, Noumoff J, Levitz M, Bradlow HL, Breed CN (1981). On the occurrence and transport of estriol-3-sulfate in human breast cyst fluid: the metabolic disposition of blood estriol-3sulfate in normal women. J Clin Endocrinol Metab 53:847-851. Banerjee S, Katz J, Levitz M, Finlay TH (1991). Purification and properties of an esterase from human breast cyst fluid. Cancer Res 51:1092-1098. Levitz M, Raju U, Katz J, Finlay TH, Brind JL, Arcuri F, Castagnetta L (1992). Esterase activity in human breast cyst fluid: associations with steroid sulfates and cations. Steroids 57:485-487.
Introduction It was with considerable trepidation that I rashly agreed to the request of the editors of Steroids to share with the scientific community my recollections of 42 years in research on steroids with particular emphasis on estriol. Aside from an underlying concern of how to make these few pages interesting enough to attract a readership beyond my immediate family, I have rarely derived pleasure from turning the clock back. My underlying philosophy is that the record is there for anyone curious enough to delve into what I have done scientifically. Better my efforts should be expended on future pursuits. Moreover, even reflecting on a piece of work with pride, I find myself questioning why it took so long. Nevertheless, I accepted and am committed to the challenge. The strategy is to compare the state of the field in the early fifties with now, and then fill in the time gap, concentrating on my efforts. The former can be summarized succinctly. At mid-century steroid chemistry was dominated by sophisticated organic chemists completing work on the structure and synthesis of key steroids and their metabolites. They, myself included, knew virtually nothing about their mechanisms of action. On the other hand, today's steroid researcher at the forefront of the field knows little about the chemistry of the hormone, but is exquisitely perceptive of its action at the gene level. My piece of the jigsaw puzzle comprises the remainder of this remembrance.
conventional academic pursuits dominated the activities on several floors of the physics department laboratories. With the future in doubt and beyond individual control, I decided to interrupt my formal education when I interviewed successfully for a job in those hush-hush laboratories, under the aegis of the so-called Manhattan Project. The research, centering on the chemistry and corrosive properties of uranium hexafluoride, was not particularly exciting but it afforded the opportunity to learn valuable laboratory techniques including glassblowing and the construction and maintenance of high vacuum systems. Moreover, there was a pervasive aura that something crucial to the war effort was being contributed. But it was work down the hall that attracted my attention and directed my inclination to organic chemistry. Groups headed by experts in fluorine and polymer chemistry combined their talents to create
Prelude In a way my predoctoral experience dictated the course of my future professional career. In early 1942 with the world divided by forces of destruction, a lucky draft number afforded me the temporary luxury of enrolling for my doctorate at Columbia University. There I soon discovered that research activities unassociated with
Address reprint requests to Mortimer Levitz, Department of Obstetrics and Gynecology, New York University Medical Center, 550 First Avenue, New York, New York 10016, USA.
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Dr. Mortimer Levitz
© 1994 Butterworth-Heinernann
Four plus decades of research in estrogens: Levitz fascinating perfluorinated polymers. I often wonder whether teflon was developed there, a polymer that lent some interest to an otherwise dull 1984 presidential election. In late 1946, discharged from the army, it was back to graduate school under the tutelage of a remarkable teacher, Professor William Doering, who truly prepared one for a career in independent research. The first decade
After 9 years at Columbia University and environs on the Manhattan Project in both civilian and military capacities and as a graduate student in organic chemistry, the umbilical cord was not severed, merely stretched to the uptown campus of the College of Physicians and Surgeons. There began a long association with a superb gynecological surgeon, Gray H. Twombly, intensely interested in breast cancer research. In a brief interview, he expressed two concerns: whether I could synthesize radiolabeled estradiol and if I would be interested in biomedical research. I recall adroitly finessing the latter question, thinking that while working on the synthesis I could explore ideas concerning what I would do with the newly synthesized material. Although labeled estradiol was not available commercially in 1952, R. D. H. Heard of McGill University kindly provided me the details of a cumbersome, low-yield method he apparently never published. At least this would provide a source of material should my own efforts fail. The intermediary in this Montreal connection was a young graduate student, Sam Solomon, whose friendship and scientific interactions have been constant sources of pleasure and intellectual stimulation throughout my career. The synthesis of [16J4C]estradiol gave me great satisfaction professionally and involved me with interesting controversies with foremost scientists of the era, on which I will elaborate. The salient features of the synthesis are depicted in Figure 1. There are two crucial steps, the first being the introduction of t4C into 3-methoxymarrianolic acid, accomplished by treating II with triphenylmethylsodiumand 14CO2, and the second, the ring closure via an acyloin condensation to produce V. The former step was accomplished with dispatch since I discovered the powerful potential of the strong, ether-soluble base, triphenylmethylsodium during my thesis research. The condensation step posed a personal dilemma of sufficient interest to recount in detail. In 1952 I was fortunate to attend a Gordon Conference on Steroids. To a young investigator such a conference is a unique experience. Under idyllic
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surroundings information, frequently unpublished, and ideas are shared in a relaxed atmosphere, in sharp contrast to that pervading a typical national meeting. My excitement was unrestrained when in a session on newer reactions in steroid synthesis a discussion leader, John C. Sheehan of Massachusetts Institute of Technology (M.I.T.) showed that Na in liquid ammonia converted II into a ring D ketol (acyloin condensation). I presented him my problem and was delighted by his generous response. He invited me to Cambridge where he opened notebooks that contained the experimental details of the reaction. Parenthetically, the reaction is an esthetic experience. Sodium in liquid ammonia produces a brilliant deep blue solution and owing to the low boiling point of ammonia, a dry-ice condenser must be in place, a perfect grade B science fiction movie visual effect. The ensuing steps proceeded uneventfully except for an interesting twist. Dr. Sheehan informed me and later reported that the condensation produced a l%t-hydroxyketol, whereas I had obtained unequivocal evidence for the more desirable 17fl-hydroxy configuration, based primarily on the identification of the end-product as 17fl-estradiol. I quickly submitted a manuscript on the synthesis and at about the same time informed my benefactor in Cambridge of his error. The paper was accepted I but no communication was forthcoming from M.I.T. Instead a note appeared in the same journal several months later to the effect that upon independent investigation the authors would like to correct their original error. I thought it was a stretch of the definition of the word independent but let the matter rest. The beauty of the synthesis outlined in Figure 1 was that with minor modification the synthesis could be adapted to produce several labeled 16-oxygenated estrogens of interest. For example, the acyloin condensation applied to the 3-benzyl ether of dimethylmarrianolate afforded 16-ketoestradiol2 that on reduction with sodium amalgam produced estriol and 16-epiestriol in a rate of 7:3. a Our armamentarium of labeled compounds expanded at a time when erroneously estriol was being declared by some investigators as an impeded estrogen or even an antiestrogen, and 16-ketoestradiol - - in selected t u m o r cultures - appeared to impede cell proliferation. These labeled compounds were made available to the scientific community under a program sponsored by the American Cancer Society. The interest in 16-oxygenated estrogens prompted us to study the metabolism of estriol in women with emphasis on whether estriol was truly an end-product and whether 16-ketoestradiol was produced in vivo. a Finding the latter as well as 16-epiestriol in the urine of volunteers who received [14C]estriol settled these issues - - at least I thought so. Soon after we published this work, the American Cancer Society sponsored a symposium on estrogens and cancer in a mammoth auditorium of a New York City hotel. A featured speaker was the distinguished gentleman and renowned investigator, Guy Marrian. He presented an erudite address on the state of the field, but toward the end of his talk he
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Persona/History dropped a personal bombshell. To an audience of over 3000 he retracted his report that 16-ketoestradiol was an estrogen metabolite in women and that it arose from rearrangement caused by the conditions of urinary workup. No harm, until he proclaimed in a most delightful Scottish brogue that Levitz (heavy accent on the last syllable), Spitzer, and Twombly undoubtedly fell into the same trap and must also retract. At the conclusion, hardly waiting to be recognized by the chair, I sprang to my feet and declared that I was fully aware of the pitfalls of ring-D ketol rearrangements and that workup conditions were carefully designed to preclude this phenomenon and that we had no reason to retract. Nevertheless, in the ensuing 18 months Marrian papers on the subject echoed the same criticism of our work. Vindication came in August 1957 in a meeting at the Worcester Foundation, convened by the legendary Gregory Pincus, to which the foremost researchers in estrogens were invited. In his usual thorough style Egon Diczfalusy presented a paper confirming our 16ketoestradiol finding. I was sitting next to the redoubtable Scotsman, who leaning over, offered his sincere apology while commenting that if Diczfalusy reports it, it is true. Embarrasingly, in future papers, spanning some 5 years, Martian was profusely apologetic. That meeting cemented friendships with elite investigators on estrogens in several continents (Figure 2) and placed me in even better grace with Dr. Pincus; both did my career no harm. In the early part of the decade I entered into a collaboration - - that still endures - - with Joseph Dancis, a future professor and chairman of the department of pediatrics at our medical center with an insatiable thirst for knowledge that continues to reflect in a remarkable
Figure 2
Members of the conference on estrogens, Shrewsbury, Massachusetts, 1957. From left to r i g h t - front row: RI Dorfman, LL Engel, JW Goldzieher, S Kushinsky, E Bloch, WR Slaunwhite; second row: G Pincus, GF Marrian, E Diczfalusy, JK Preedy, TF Gallagher, K Savard; third row: R Jacobs, JB Brown, B Baggett, AE Kellie, M Levitz, Unidentified (according to the author's recollection he was a visiting scientist from Australia, named Brown); back row: CD West, RDH Heard, LT Samuels, J Fishman, E Schwartz.
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record of accomplishment in many areas of biomedical research. We did interesting studies on inborn errors of metabolism, in particular branched-chain ketoaciduria but that is peripheral to this biography. Dr. Dancis introduced me to the fascination of the placenta. In the steroid field, with emphasis on estrogens many metabolic and transfer studies followed exploiting both the human and guinea pig placenta. In all candor, though, one study turned out to be deeply embarrassing. First, a bit of background. In the 1950s the biochemical origin of estrogens was unknown. In fact, at the American Cancer Society Symposium alluded to above, a noted biochemist spun an intriguing fantasy, supported mechanistically, but not experimentally, purporting that estrogens are biosynthesized from aromatic amino acids. We decided to perfuse the human placenta with labeled acetate and were elated to find radioactive estrogen in the perfusate. When the result was confirmed we submitted an abstract for presentation at the ensuing Federation Meeting. On further exploration it became clear that our report was erroneous. I had thoughts on the origin of the contamination of our system with labeled estradiol, but I merely satisfied myself with spending three weekends perfusing placentas with absolutely fresh tubing and reporting our error. I gained an appreciation of the view of many senior biochemists of that era who maintained that results based on radioactive data were tentative at best. They had a point, but rigidity could produce premature scientific rigor mortis.
The second decade The same tide that carried me and numerous investigators into the areas of steroid metabolism and action drifted us further from pure organic chemistry. Two relatively minor contributions merit mention. [3H]Estriol was not readily available in the early sixties, restricting the kind of metabolic studies that could be done in man. The synthesis devised was essentially that described for that of [14C]estriol except that 3H204 was added in place of 14C in the appropriate step (see Figure 1 and reference 3 for further details). This era also witnessed a burst of activity in studies on the intermediary metabolism of estrogen conjugates. In order to study the metabolic fate of the sulfate moiety in estrone sulfate, 35S-labeled compound was desirable, preferably starting with the inexpensive H23sSO4. Treatment of [35S]-labeled sulfuric acid with pyridine in the presence of a 10% excess of acetic anhydride formed labeled pyridinium sulfate, which on reaction with estrone produced the desired conjugate in good yield. 5 All other conjugates used in many ensuing studies were produced biosynthetically. Thus, estriol is converted into estriol-3-glucosiduronate (E3-3G) in homogenate of guinea pig liver fortified with uridine diphosphate glucuronic acid 6 and to estriol-16-glucosiduronate (Ea-16G) with human liver under the same conditions. 7 Moreover, treatment of the latter with ATP and sulfate in a 100,000 x # supernate of guinea pig liver produced the double conjugate, estriol-3-sulfate-16-glucosiduronate (E3-3S-16G). Finally, fl-glucuronidase converts the
Four plus decades of research in estrogens: Levitz double conjugate into Ea-3S, making all the important estriol conjugates available for study. This point in my career is best described as the Swedish Connection. In 1961 I received the first of five invitations from Egon Diczfalusy to spend time in his laboratory at the Karolinska Hospital in Stockholm. Sweden had the most liberal abortion policy in the world and virtually all women undergoing the procedure consented to research on the fetus and placenta. The clinical material, the availability of labeled estriol and my experience in estriol metabolism coupled with Diczfalusy's biomedical and laboratory expertise fostered a productive partnership that lasted the decade. I spent two summers in Stockholm in the early sixties and made three other work-related visits later in the decade, all delightful and rewarding experiences. The laboratory attracted scientists from all over the world, but I particularly recall interacting with four young, budding investigators who have made significant contributions in reproductive endocrinology. I worked in closest association, literally day and night, with the late Uwe Goebelsmann, a scholar and a gentleman in the truest sense. The other three of note were Robert B. Jaffe, George Mikhail, and Philip Troen. Troen had the sense to ship his scintillation counter to Stockholm, permitting the prompt processing of samples. In 1965 1 was asked by the program committee of the Endocrine Society to present our work on estrogen transport and metabolism in the fetal-placental unit in a symposium at the annual meeting. The major stress was on my research at Karolinska. The salient features of the lecture were published s'9 and can be summarized briefly. Estrogen conjugates traverse the placenta poorly. Owing to sulfatase activity but insignificant glucuronidase in the placenta and fetal membranes, estrogen originally in the sulfate form reaches the fetus far more effectively than estrogen presented as the glucosiduronate. As pregnancy proceeds, the human fetus is exposed to increasing concentrations of free estrogens. Protection from hyperestrogenism is afforded by conjugation, each important portal to the fetus being an effective organ or tissue in that regard. This holds for the liver, the recipient of cord blood estrogens and fetal lung and skin which are exposed to the amniotic fluid. This kind of fetal-placental research came to an end with the introduction of prostaglandin as an aborting agent, virtually eliminating hysterotomy as a procedure for early second-trimester abortions. Research on the origin and intermediary metabolism of estrogens in the early sixties was intense, facilitated by the availability of isotopically-labeled compounds. The major contributions of our laboratory in this regard have been summarized. Of paramount importance, that era witnessed two other developments that would impact on the course of our research in the latter part of the decade. The first practical chemical method for the quantification of the classical estrogens; estrone, estradiol, and estriol in urine was reported by James B. Brown, of Edinburgh .1° Sequentially, he applied acid hydrolysis to a urine aliquot, solvent extraction of estrogens which were converted to their 3-methyl ethers, separation and
purification of the classical estrogens by column chromatography, and assay by the Kober reaction. The second important development was the elucidation of the origin of estriol in pregnancy, conversion by the placenta of 16~-hydroxydehydroisoandrosterone that is of fetal origin, tt'lz These considerations, coupled with the thinking on the part of many investigators that estriol levels in body fluids could provide a useful monitor of the status of pregnancy, prompted me to consider research in this area. Dr. Lila Nachtigall, fresh from her residency in medicine, requested a 2-year fellowship in my laboratory to work in an area Of estrogen research that would have direct clinical relevance. Literature was beginning to accumulate on the urinary excretion of estriol in pregnancy, but no convenient method for determining its concentration in plasma was described. There were many reasons to argue the superiority of a plasma method, the most compelling being that interruption of normal estriol synthesis would be reflected more promptly by a drop in blood levels than in urine concentrations. To our delight the method outlined on paper yielded cause for optimism in 6 weeks and was soon operative without significant change. 13 Three key considerations contributed to this success. First, recognizing that estriol circulates predominantly in the form of conjugates and that acid hydrolysis would produce varying degrees of estriol destruction, each assay tube was charged with representative tritiated estriol conjugates to serve for correction of methodological losses. Secondly, on benzene:hexane-water extraction, the bulk of interfering estrogens and other lipids distribute into the organic phase, while estriol remains in the water from which it is ether-extractable. Finally, estriol could be quantified by a highly specific, sensitive spectrofluorometric method. The method was applied to the study of diseases in pregnancy 14 as well as to specific research protocols. Numerous laboratories expressed interest in the technique to the extent of sending representatives to observe it firsthand. Over the past 15 years or so sophisticated techniques for monitoring the fetus in utero has, in the view of obstetricians, eliminated the need for estriol assays for assessing fetal status. Retrospectively, my view of the value of our research in this area is difficult to assess objectively. Interesting basic information was forthcoming. But in terms of patient care, no meaningfully statistical prospective study was run, or even conceivable in which estriol levels would be the only variable in the management of the pregnancy. Looking back, it was indeed a busy decade in our laboratory, virtually all the research focusing on estriol. The enterohepatic circulation and metabolism of estriol conjugates in the normal adult were investigated in depth; three studies are most worthy of mention. The first was an inquiry into the origin of E3-3G. A simply designed study yielded definitive results. The volunteer was a 29-year-old man with normal liver function who had undergone cholecystectomy for bile duct obstruction and whose bile was being drained via a T-tube. [14C]Estriol was injected into the duodenum via the T-tube while [3H]estriol was given i.v. E3-3G was found
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Persona/History only in the urine and labeled exclusively with ~C, demonstrating the intestinal origin of this conjugate, ts This study and two other studies showed: E3-3S-16G is the major estriol (E3) conjugate excreted with the bile, E3-16G is the major urinary metabolite, E3-3G is the true end-product of E 3 metabolism, and E3-3S is hydrolyzed extensively and the E 3 converted mainly to E3-16G before excretion in the urine. ~6'17 Other ring Dhydroxylated estrogens commanded interest in this period. Specifically, 3,15e,16e,17fl-tetrahydroxyestra1,3,5(10)-triene (esterol, E¢) levels were targeted as potentially superior to E 3 for assessing pregnancies at risk because E 4 was thought to be an estrogen metabolite unique to the fetus. We contradicted that claim by demonstrating the in vivo 15-hydroxylation of estrone in the nonpregnant adult woman. Is In March 1966, an event occurred that added spice to my research career for years to come. The legendary Morris Graft, Executive Secretary of the Endocrinology Study Section traced me to the bathtub of a hotel room in San Francisco at 6:30 AM. I was attending the Annual Meeting of the Society for Gynecologic Investigation. He invited me to become a member of the study section. I knew nothing of what was in store for me as I reflected that during the French Revolution Jean Paul Marat was assassinated by Charlotte Corday while taking a bath. The effort was enormous, invariably thankless, but the lasting friendships in endocrine circles and the learning experience more than compensated. Parenthetically, I must confess that prior to this experience I had no real concept of how the fate of proposals submitted to the National Institutes of Health (N.I.H.) was determined. I assume my contributions on study sections were appreciated since I was asked to serve as emergency fill-in for a second term, 1973-1975 and for a lengthy stint in a newly-forming Clinical Sciences Study Section in 1979-1981 (ad hoc) and -1985 (permanent), with the last two years as Chairman.
The third decade In the early seventies much effort was expended toward addressing unanswered questions on the intermediary metabolism of estriol in the normal and pregnant human. Fortunately we were able to recruit Helmut Jirku who trained with Donald S. Layne at the Worcester Foundation and Uma Raju who received the appropriate postdoctoral experience with Egon Diczfalusy. Moreover, Joseph Katz and Susan Kadner contributed both cerebrally and technically during their 25 years of research on many of the problems cited. Much of the research in this decade centered on the biosynthesis and quantification of estriol conjugates in pregnancy and diseased states. These studies were facilitated by the simplified methodology we developed for the separation of the four major conjugates of estriol and the availability of 3H-labeled compounds. One study centered on the renal clearances of estriol conjugates in normal human pregnancy at term. 19 It was found that more than 90% of the estriol in body fluids consists of four major conjugates: E3-3S, E3-3G, E3-16G and
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E3-3S-16G. The clearance of E3-16G was the highest, approaching the effective renal plasma flow. Then followed E3-3G which approximated the glomerutar filtration rate. The sulfurylated conjugates cleared the lowest, probably a reflection of their binding to serum albumin. The sensitivity of the method permitted the detection of minor estriol conjugates. A new conjugate that we identified, E 3-3,16-disulfate, comprised about 1% of total and was most concentrated in cord blood. The method, applied to the study of a patient with severe polycystic kidney disease, revealed the greatest aberration in the clearance of E3-3G. 2° The interest in 15-hydroxylated estrogens prompted us to study their metabolism and compare their intermediary metabolism with that of estriol. The prominent feature of these estrogens is the introduction of a novel mode of conjugation. Following administration of [3H]15ct-hydroxyestrone to pregnant women more than 50% of the radioactivity was excreted in the urine in the form of 15-N-acetylglucosaminides.21 This was the first report of this mode of conjugation of estrogens in the human, although N-acetylglucosaminides of estrogens in the rabbit 22 and progesterone metabolites in women 23 have been cited. Another surprising result in this study was the evidence for the urinary excretion of a glucosiduronate of 15c~-hydroxyestradiol-17~. Our in depth studies of the metabolism of estriol and 15ct-hydroxyestradiol in pregnancy indicated distinct differences in their conjugation profiles. Glucosiduronidation predominated for the former and Nacetylglucosaminidation for the latter. Estetrol may be viewed as the structural fusion of these two ring D estrogens, raising the question of its mode of conjugation. Our studies indicated that estriol exerted greater influence on the conjugation profile. Ring D glucosiduronates comprised about 90% of the urinary metabolites following administration of labeled estetrol to pregnant women, while only 1% was in the amino sugar form. 24 15~-Hydroxyestrogen N-acetylglucosaminyl transferase activity was demonstrated in vitro in human adult liver and in human adult and fetal kidney.25 Although these findings elucidated many interesting and novel features of the metabolism of 15~-hydroxyestrogens in pregnant and normal states, my chief regret on reflection is that the physiological impact remains obscure.
The fourth decade A hallmark of the eighties in obstetrics was the introduction of sophisticated ultrasound and electronic equipment for assessing the status of the fetus at risk. Paralleling the expansive use of these innovations was the phasing out of the reliance on estriol concentrations in maternal blood for monitoring the status of the pregnancy. In the research arena the estriol molecule was not moribund; the emphasis shifted to nonpregnant states. Our laboratory was involved in two studies worthy of comment. The first concerned a research inquiry conducted in connection with a clinical study. In a collaboration with Isaac Schiff at The Boston
Four plus decades of research in estrogens: Levitz Hospital for Women, the experimental design was to give estriol either orally or intravaginally to menopausal women and to compare the physiologic responses with mode of administration. The measure of biologic response was the decline in blood FSH and LH levels at 6 h. Significant declines were observed after intravaginal administration only. Analysis of the sera for estriol and its conjugates indicated a much higher rate of absorption following oral administration; however, significant levels of free estriol were seen only with the vaginal route. We concluded that only unconjugated estriol suppresses gonadotropins. 26 It is apparent that with the correct preparation and mode of administration estriol can be effective in estrogen replacement therapy, but it must be borne in mind that estriol is an active estrogen when administered continually, and not an impeded estrogen as was suggested over 30 years ago. My last major thrust into estriol research, culminating in a most surprising finding can be described as the epitome of serendipity. In terms of nostalgia and human interest this section dates back to the first decade of the narrative. At that time a carpool formed which included biochemists from Sloan Kettering and myself. Parenthetically, my participation lasted 25 years until my move to New York. Lively discussions usually on politics and steroid biochemistry made us oblivious to the endless stream of traffic between Queens and Manhattan. These discussions spawned collaborations, one of which was with a distinguished steroid biochemist, H. Leon Bradlow. The focus was on gross cystic disease (GCD) of the breast, a benign condition requiring simple needle aspiration of the breast cyst fluid (BCF) usually to relieve pain and anxiety. The interest was two-fold: epidemiologically, women with GCD were at greater risk for developing breast cancer and according to fragmentary reports many BCFs contain high concentrations of steroids. Is there a connection? The question is not resolved, but meritorious of investigation. We decided that Dr. Bradlow would focus on estradiol, estrone, and protein hormones, while our laboratory would investigate estriol and later, bile acids and some enzymes of particular interest to us. There was no compelling reason to expect to find E3 in BCF. We had shown that the levels of unconjugated E 3 in plasma was less than 0.02 nmol/L and for its conjugates it was barely twice that value. BCFs were analyzed for E 3 and its four major conjugates. Surprisingly, concentrations of E3 several hundred-fold greater than in blood were observed, specifically relegated to E3-3S. Many cysts also manifest unusually high levels of potassium ion, a phenomenon thought by many investigators to be associated with risk for breast cancer. Accordingly, we examined our E3-3S data in terms of the K+/Na ÷ ratios, classifying Type I as > 1.0; Type II, < 0.25, and Type III, 0.25--0.1.0. By Student's t-test, the concentrations of E3-3S differed between each BCF Type (p < 0.002), with Type I cysts showing the highest levels and Type II the lowest. Moreover, by linear regression analysis, there was a significant correlation between the concentrations of E3-3S and the sulfates of estrone and dehydroisoandrosterone. 27
The origin of the extraordinarily high concentrations of E3-3S in BCF has yet to be elucidated. One thought was that the sulfate transferred to the cyst from blood against a huge concentration gradient, as we have shown for bile acids. 2s This was considered a possibility since steroid sulfates generally have blood half-lives in excess of 6 h. Unexpectedly though, we found that E3-3S had a half-life of only 0.5 h, precluding the use of radioactive E3-3S to investigate this phenomenon. 29 In connection with our studies on the biochemistry of BCF, we found that this fluid contained formidable levels of long-chain fatty acid esters of steroids. Since these biologically inactive substances can be rendered active by hydrolysis to the parent steroid, we initiated a search for esterases in BCF. An unusual esterase was identified and characterized. 3° Thomas H, Finlay and Sila Banerjee, close collaborators, purified the esterase to near chromatographic homogeneity. It is a 23 kDa peptide and appears to be of breast epithelial origin since its properties differ from any blood esterase. The esterase is present in about 40% of BCFs and statistically, its concentrations associate positively with E3-3S, other steroid sulfates and K+/Na + ratios) 1 The take-home message of the decade is that the steroid biochemist is not a dinosaur. Unpredictable metabolic pathways impacting on disease, particularly cancer, continued to surface. The number of players has dwindled sharply, but those who have stayed the course, still too numerous to cite, clearly have justified their efforts. F o u r and a quarter - - Epilogue
By rough calculation, at the appearance of this memoir, my research career will have approached the second quarter of the fifth decade. What about the future? This is hard to predict. If this were the script for an old radio soap opera, I could hear the announcer droning, "This is a saga seeking the answers to two related questions: does life begin at 40, and can quality research continue without N.I.H. support?" It is superfluous to state that research support is harder than ever to obtain. The grant supporting the bulk of the research described herein, the seventh highest in terms of longevity at the National Cancer Institute at the time of inquiry in 1992, 39 years, failed renewal by the proverbial cat's whisker. On resubmission the result was the same but the margin was by a bigger bunch of hairs. I have no plans for retirement. Before that day, I would like to 1) have contributed substantially to the solution of the problem of the source and physiological implications of the extraordinarily high concentration of estriol, bile acids and esterase in BCF; 2) have witnessed unequivocal verification of our observation that bioavailable estradiol constitutes a risk for cancer (paper submitted for publication); and 3) have increased our understanding of how nutrients and deleterious drugs cross the placenta - - at present we are doing cocaine. The latter two studies are considered peripheral to the main thrust of this narrative, but continue to be major contributors to the joy and excitement of just going to work in the morning.
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Persona/History Acknowledgments The research cited was supported principally by a grant from the National Cancer Institute, National Institutes of Health, CA2071 (01-39). I wish to express my thanks and deepest appreciation to Dr. Gordon W. Douglas, Professor and Chairman of the Department of Obstetrics and Gynecology at our Medical Center through three decades of my research career. His appreciation of the value and importance of basic research to a clinical department was reflected in his personal support and commitment of resources, particularly in times of departmental budgetary stress. I am also grateful to our present Acting Chairman, Robert F. Porges, M.D., who has made it clear that continued productivity and vitality, and not retirement rules, would be the criteria for continued support.
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