OESTRADIOL-17\G=B\AND OESTRIOL and of PREG- NANETRIOL in NORMAL MENSTRUATING WOMEN and in OOPHORECTOMIZED PATIENTS

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EFFECT OF 17\g=a\-ETHYNYL-19-NOR-TESTOSTERONE ON THE URINARY EXCRETION OF OESTRONE, OESTRADIOL-17\g=b\AND OESTRIOL AND OF PREG- NANETRIOL IN NORMAL MENSTRUATING WOMEN AND IN OOPHORECTOMIZED PATIENTS

J. WEIDENFELD, D. SERR and MICHAEL FINKELSTEIN Hormone Research Laboratory, The Hebrew University-Hadassah Medical School, Jerusalem, Israel [Received th February 1966)

Summary. Treatment of three normally menstruating women with 5 to 10 mg/day `Norlutin' (17\g=a\-ethynyl-19-nor-testosterone)for 16 to 20 days caused, in two ofthem, a decrease ofurinary oestrone, oestradiol\x=req-\ 17\g=b\and oestriol to undetectable levels. In the third case no change was observed in the excretion of the oestrogens, but this result was most probably due to an inadequate analytical procedure. In all three patients the urinary pregnanetriol decreased to about half of the mean level estimated during the respective control menstrual cycles. In three oophorectomized patients no oestrone, oestradiol-17\g=b\ or oestriol were detected and the level of pregnanetriol was significantly lower than in normally menstruating women. Treatment of these patients with `Norlutin' did not depress further the urinary excretion of pregnanetriol. The results suggest that `Norlutin' inhibits the biosynthesis of the ovarian steroids but has no effect on their biosynthesis in the adrenals. This selective action upon the ovaries may be useful for the evaluation of the ovarian function.

INTRODUCTION Administration of various 19-nor-steroid derivatives to normal women during the menstrual cycle results in a decrease in the urinary excretion of oestrogens, pregnanediol (5/?-pregnane-3a,20a-diol) and pregnanetriol (5/?-pregnane- 3a,17a,20a-triol) (Serr, Roth, Weidenfeld & Finkelstein, 1962; Fotherby, 1962; Brown, Fotherby & Loraine, 1962; Shearman, 1963; Loraine, Bell, Harkness, Mears & Jackson, 1963; Shearman, 1964). It is not certain, however, whether the decrease of these urinary steroids is due to the impairment of ovarian function alone, or whether biosynthesis of the adrenal steroids is also affected. 107 Downloaded from Bioscientifica.com at 10/01/2021 06:00:28AM via free access 108 J. Weidenfeld et al. Baulieu, Mauvais-Jarvis & Corpechot (1963) reported that in normal women 'Norethynodrel' (17a-ethynyl-17/?-hydroxy-5(10)-oestrene-3-one), per¬ sistently depressed the urinary excretion of 17-hydroxy-corticosteroids, of 11- oxygenated-17-ketosteroids and of dehydrowoandrosterone (3/?-hydroxy-5- androsten-17-one). This observation would imply that inhibition of both adrenal and ovarian function had resulted. The observation of Brooks & Prunty (1957), that the administration of 17a- ethyl-19-nor-testosterone to an oophorectomized woman caused a decrease in all fractions of urinary 17-ketosteroids which were investigated, gives further support to the concept of adrenal inhibition. However, Fotherby (1962) observed no depression of urinary dehydroûo- androsterone, of total 17-ketosteroids or 17-hydroxycorticosteroids following the administration of 'Norethisterone' (17a-ethynyl-19-nor-testosterone) or its acetate to normally menstruating women. Since treatment with either of these compounds prevented the rise of both pregnanediol and pregnanetriol in the second halfof the cycle, but did not affect their excretion following the adminis¬ tration of 'Norethisterone'-oenanthate to a post-menopausal woman, Fotherby (1962) suggested that the decrease observed in normal menstruating women was due to the inhibition of ovarian function alone. It seems, therefore, that estimation of neutral steroids does not give conclu¬ sive results with regard to the possible inhibitory effect of the anti-ovulatory steroids on the biosynthetic processes in the adrenals. The influence of various anti-ovulatory steroids on the urinary excretion of oestrone, oestradiol-17/? and oestriol in menstruating women was investigated by Brown et al. (1962), Shearman (1963), Loraine et al. (1963) and Shearman (1964). In most of the above cases the excretion of the above urinary oestrogens remained at the levels found during the follicular phase or decreased to levels normally seen in post-menopausal women but never disappeared completely. In one case, the administration of 'Norethisterone'-acetate to a menstruating woman, in a daily dose of 4 mg, caused an increase in the excretion of oestrone and oestriol far and above that of the control cycles (Shearman, 1963, 1964). Since these contradictory results could be, at least in part, due to factors involved in methodology, notably in the case ofurinary oestrogens, a study has been carried out with special attention to the rigorous control of the analytical procedures. METHODS Three normal menstruating women and three oophorectomized patients were selected for this study. 'Norlutin' (17a-ethynyl-19-nor-testosterone), supplied by Parke Davis Co., Detroit, U.S.A., was chosen as anti-ovulatory agent. Five milligrams of the compound contained less than 10 ^g, if any, of oestrone, oestradiol-17ß or ethynyl-oestradiol. Estimation of these compounds was carried out by methods described below. For the evaluation of the ovarian and the adrenal function respectively, urinary oestrone, oestradiol-17j?, oestriol and pregnanetriol were serially estimated during periods of treatment as well as during periods of no treatment.

Downloaded from Bioscientifica.com at 10/01/2021 06:00:28AM via free access Excretion ofsex steroids in women 109 Urine collection All patients collected complete 24-hr urine specimens. The urine was kept frozen until assayed. For daily estimation of oestrogens and pregnanetriol in the normally menstruating women, one-fifth or one-tenth of the 24-hr speci¬ men was used. In the oophorectomized patients half or all of the 24-hour urine specimen was used. Procedurefor estimation ofurinary oestrogens andpregnanetriol Urine was adjusted to pH 5-5 and was incubated with 10% volume buffer acetate 2 m and 300 units of /?-glucuronidase ('Glusulase', Endo Laboratories Inc.) per 1 ml urine at 37° C for 96 hr. The hydrolysed urine was extracted three times with an equal amount of ether-chloroform 4:1. After washing with bicarbonate and water, the phenolic fraction was separated from the neutral fraction by extracting the ether-chloro¬ form three times with half-volumes ofNaOH n/1, each. The NaOH extract was adjusted to pH 8-5 and was re-extracted with ether. Oestrone, oestradiol-17/? and oestriol were estimated in the phenolic fraction by the procedure ofLadany & Finkelstein (1963), with one exception, Patient l, M.E. In this patient, oestrone, oestradiol- ß and oestriol were estimated by a procedure previously used in our laboratory (Finkelstein, Jewelewicz, Klein, Pfeiffer & Sodmoria-Beck, 1960), but which has been found to overestimate the above oestrogens. The identification of ethynyl-oestradiol ( 17a-ethynyl-1,3,5(10)-oestratriene- 3,17/?-diol) was made by a modified procedure of Ercoli, Vitali & Gardi (1964). Following the initial separation of the compound on the thin layer plate prepared with AgN03 as described by the above authors, it was further purified by consecutive chromatography on silica gel plates in the following systems: (1) benzene-methanol 9:1; (2) benzene-ethylacetate 3:1; and (3) cyclohexane-ethylacetate-methanol 4-5 : 4-5 : 1. The compound was located by the fluorescence spot test of Ladany & Finkelstein (1963). In this test ethynyl-oestradiol exhibits a yellow fluorescence similar to that ofoestradiol- 17/?. One-half to 1 ^g of the compound may be easily detected. Pregnanetriol was estimated in the neutral fraction by the procedure of Finkelstein (1962), with the exception that the compound was isolated by sequen¬ tial chromatography in the following systems: (1) silica gel thin layer (tl), cyclohexane-ethylacetate-methanol 4-5 :4-5 : 1 ; (2) Whatman No. 1 paper, formamide-methanol 1 : 1-benzene; (3) tl, chloroform-ethanol 8:2; and (4) tl, benzene-methanol 8:2. Pregnanetriol was located on the respective thin layer chromatograms by the phosphoric acid reaction of Ladany & Finkelstein (1963), but the plates were heated for 5 min at 90° C. * For calculation of recovery tracer 4 4C-pregnanetriol* (6000 counts/min, specific activity 300,000 counts/min/mg) was added to several urine samples before extraction. The recovery was within the ranges of 75% to 90%.

* 414C-Pregnanetriol was obtained from the urine of a patient with congenital adrenal hyperplasia following injection of 414'C-17a:-hyroxyprogesterone. The radioactive pregnanetriol was purified by chromatography in several systems until specific activity became constant.

Downloaded from Bioscientifica.com at 10/01/2021 06:00:28AM via free access 110 J. Weidenfeld et al. RESULTS Normally menstruating women Patient 1—M.E. age 24, para. 1. Regular menstrual periods, every 30 days. 'Norlutin' (5 mg/day) was administered from 7th day of the cycle until the 23rd day. The daily excretion of urinary oestrone, oestradiol-17/? and oestriol during a control cycle and during the period of medication were recorded. In this case the estimations were performed with the older method of Finkelstein et al. (1960). During the medicated cycle the excretion of oestriol significantly decreased (Text-fig. 1), but no significant changes were noted in the excretion of oestrone and oestradiol-17/? as compared with the preceding control cycle. Since menstrual bleeding did not occur following termination of the treat¬ ment, the urinary oestrogens were continuously estimated. The daily excretion of oestriol gradually increased from about 1 /tg/24 hr, 7 days after termination of the medication up to 45 /tg/24 hr on the 60th day (from the beginning of the medicated cycle). It was suspected that the marked rise in urinary oestriol

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.e SI o o o o in o in Text-fig. 1. Patient 1—M.E. Urinary excretion of oestriol estimated by the method of Finkelstein et al. ( 1960) before, during and after administration of 'Norlutin'. might have resulted from conception. To confirm pregnancy, rat hyperaemia tests (Zondek, Sulman & Black, 1945) were performed. This test became slight¬ ly positive on the 51st day (500 h.u./l) and on the 54th day it became definitely positive (5000 h.u./l). Apparently ovulation had occurred on or about the 34th day. In this case, urinary pregnanetriol was estimated in pooled urine collected between the 7th and the 23rd day of the control cycle and during the parallel days of the medicated cycle. The mean pregnanetriol level found in the pool of the control cycle was 0-4 mg/24 hr. In the pool of the medicated cycle the mean pregnanetriol level decreased to 0-24 mg/24 hr. In the post-medication period excretion of pregnanetriol increased from the mean level of 0T3 mg/24 hr estimated on Days 23 to 30 to the level of 0-55 mg/24 hr on the 50th to 55th day.

Downloaded from Bioscientifica.com at 10/01/2021 06:00:28AM via free access Excretion of sex steroids in women 111 Patient 2—D.Y. age 20, para. 1. Regular menstruation every 28 to 32 days. 'Norlutin' (5 mg/day) was administered from the 5th day of the cycle until the 25th day. Text-fig. 2 shows the excretion of oestrone, oestradiol-17/?, oestriol and pregnanetriol during the control and the medicated cycles. The oestrogens were estimated by the older method of Finkelstein et al. (1960). Since the levels of oestrone, oestradiol-17/? and oestriol found during medica¬ tion were similar to those in the control cycle, it was decided to check the re¬ liability of the results by a re-estimation of the respective oestrogens with the more exact procedure of Ladany & Finkelstein (1963). This was done on pooled urine, collected during 12 days of the control cycle (9th to 20th day), and on a

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(/Jg/24hr) — 5 m 10 20 0 10 20 Days Text-fig. 2. Patient 2—D.Y. Urinary excretion of pregnanetriol and of oestrone, oestra¬ diol- 17/Î, oestriol before and during administration of 'Norlutin'. The oestrogens were estimated by the method of Finkelstein et al. (1960). * No oestrogen was detected when estimated with the method of Ladany & Finkelstein (1963) (see Table 1). parallel pool collected during the medicated cycle. Comparative results ob¬ tained by the two methods are shown in Table 1. It may be seen that in the urine pool of the control cycle the quantities of oestrone, oestradiol-17/? and oestriol recorded by the method of Ladany & Finkelstein (1963) were roughly half of those estimated by the older method (Finkelstein et al., 1960). However, in the 12-day pool of the medicated cycle neither oestrone, oestradiol-17/? nor oestriol were detected with the sensitivity of the test adjusted to 2 /ig/12-days ofurine collection. The excretion of pregnanetriol during the control cycle was typical of a normal menstrual cycle (Pickett, Kyriakides, Stern & Sommerville, 1959;

Downloaded from Bioscientifica.com at 10/01/2021 06:00:28AM via free access 112 J. Weidenfeld et al. Fotherby, 1960), ranging from 0-4 to 1-25 mg/24 hr. During treatment with 'Norlutin', excretion of pregnanetriol decreased to a level ranging from 0-3 to 0-55 mg/24 hr. Patient 3—Z·^· a§e 22, para. 1. Menstrual periods usually regular every 30 to 32 days but the control cycle was unexpectedly 6 days longer than usual. 'Norlutin' (10 mg/day) was administered from the 5th day of the cycle until the 25th day. Text-fig. 3 shows the excretion of oestrone, oestradiol-17)?, oestriol and pregnanetriol which were estimated every 2 to 3 days during the control and the medicated cycles. In the control cycle the excretion of oestrone, of oestradiol-17/? and of oestriol was within the limits of 1 to 15 /tg/24 hr, 1 to 8-5 /ig and 1 to 16 ^g/24 hr, respectively. The identity of each of the three oestrogens was confirmed on several samples by the spot test of Ladany & Finkelstein (1963). The intensity of the fluores¬ cent colour on the respective chromatograms was in agreement with the result Table 1 comparison of results obtained by methods of finkelstein et al. (1960) (method I) and of ladany & finkelstein (1963) (method II) on the urinary excretion of oestrone, oestradiol- 17/? and oestriol of patient 2 (d.y.) during control and norlutin medicated cycles

Oestrone Oestradiol-17ß Oestriol Period of («/24 hr) (W/24 hr) te/24 hr) study Method Method Method Method Method Method I II I II I II Control cycle 9th to 20th day 47-9 28-1 24-6 9 83 43 Medicated cycle 9th to 20th day 47-5 <2 51-5 <2 103 <2 of the quantitative estimation. Neither oestrone, oestradiol-17)? or oestriol could be detected during the medicated cycle. The excretion ofpregnanetriol during the control cycle was typical ofa normal menstrual cycle (Pickett et al., 1959; Fotherby, 1960) ranging from 0-6 to 1·7 mg/24 hr. In the medicated cycle pregnanetriol excretion was within the range 0-52 to 0-6 mg/24 hr. In this patient the detection of urinary ethynyl-oestradiol as a possible meta¬ bolite of 'Norlutin' (Breuer, Dardenne & Nocke, 1960) was attempted. This was done on a pool of 500 ml urine collected on the 8th, 10th, 12th, 15th and 17th day of the medicated cycle, equal aliquots being taken from each collection. To check the reliability of the procedure, which is described in the experi¬ mental part of this paper, 0-5 ^g of crystalline ethynyl-oestradiol was added to 500 ml urine, collected during the control cycle. Following the above procedure and on viewing the chromatogram under the U.V. lamp, a fluorescent spot was detected with the mobility, intensity and shade of colour identical with 0-5 µg of crystalline ethynyl-oestradiol which was chromatographed in parallel.

Downloaded from Bioscientifica.com at 10/01/2021 06:00:28AM via free access Excretion ofsex steroids in women 113 On the other hand, no ethynyl-oestradiol was seen on the chromatogram of the urine extracts collected during the treatment with 'Norlutin'. Even when 1 µg of ethynyl-oestradiol is being taken as the limit of sensitivity of the spot test reaction, it is evident that 1 litre of urine contained less than 2 /ig of this oestrogen. Since the patient has taken 10 mg 'Norlutin' daily it follows that less than 0-02% could have been converted to urinary ethynyl-oestradiol. Thus it seems improbable that the high readings in the oestradiol- ß fraction obtained on Patients 1 and 2 when the method of Finkelstein et al. (1960) was used, could have been due to ethynyl-oestradiol contaminating oestradiol-17/?.

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OestrioL 10- :1pg/24hr (¿jg/24hr) 5- 0- "WwJlinn || J.U.UJJ.U 20 30 0 10 20 0 Days Text-fig. 3. Patient 3—Z.S. Urinary excretion of pregnanetriol and of oestrone, oestra¬ diol- 17/?, oestriol before and during administration of 'Norlutin'. * In these samples the oestrogens were identified by the spot test of Ladany & Finkelstein (1963).

Patient 4—R.G. age 44, para. 4. Bilateral oophorectomy and hysterectomy were performed 4 months before this study for a necrotic uterine myoma. Ten milligrams of 'Norlutin' /day were administered for 20 days. The results obtained in this case are shown in Table 2. Urinary oestrogens were estimated during the control period and on the 20th day of treatment. No oestrone, oestradiol- ß or oestriol were detected. The excretion of pregnanetriol estimated during the control and medicated periods varied within the range of 0 85 to 0-32 mg/24 hr. However, no sig¬ nificant differences were noted between the mean excretion level estimated in the control and the medicated periods.

Patient 5—B.A. age 57, para. 2. Bilateral oophorectomy and hysterectomy were performed 12 months before this study because of adenocarcinoma of the

Downloaded from Bioscientifica.com at 10/01/2021 06:00:28AM via free access 114 J. Weidenfeld et al. corpus uteri. 'Norlutin', 10 mg daily, was given for 18 days. The results obtained are shown in Table 2. No oestrone, oestradiol-17/? or oestriol were detected either during the control period or on the 18th day of treatment. In this patient, very low values of urinary pregnanetriol were estimated, ranging from 0 03 to 0-01 mg/24 hr. No significant changes were noted in the mean pregnanetriol excretion levels between the control and the medicated period.

Patient 6—I.S. age 52. Bilateral oophorectomy and hysterectomy were per¬ formed 18 months before this study because of uterine myoma. 'Norlutin', 10 mg/day, was administered for 21 days. Table 2 shows the values of urinary pregnanetriol estimated before and during treatment with 'Norlutin'. Because

Table 2 urinary excretion of oestrone (e-1), oestradiol- 17ß (e-2), oestriol (e-3) and pregnanetriol (p'triol) in oophorectomized patients receiving 'norlutin' 10 mg/day

Patient and treatment E-\ E-2 E-3 P'triol (W/24 hr) (Ag/25 hr) («/24 hr) (mg¡24- hr) Patient 4, Mrs R.G. Control period (mean four estimations) <10 <1·0 <1·0 0-200 6th day of medication 0-320 15th day of medication 0-290 17th day of medication 0-160 20th day of medication

of insufficient data in this case, due to inadequate urine collections, it is difficult to say whether the decrease of pregnanetriol seen on the 21st day of medication was indeed influenced by 'Norlutin', or whether it was due to daily variation. DISCUSSION The administration of 'Norlutin' to menstruating women in a daily dose of 5 or 10 mg caused an almost total disappearance of urinary oestrone, oestradiol- 17/? and oestriol. This was shown in two normally menstruating women (Patients 2 and 3) when the oestrogenic fractions were rigorously purified before the quantitative estimation. In the treated cycles of these patients the urinary oestrogens decreased to levels which were below 1 /ig/24 hr. On the other hand

Downloaded from Bioscientifica.com at 10/01/2021 06:00:28AM via free access Excretion of sex steroids in women 115 in the respective control cycles, oestrone, oestradiol-17/? and oestriol were iso¬ lated and identified and the values, estimated either by daily estimation or in pooled urine, were within the normal ranges previously reported (Ladany & Finkelstein, 1963). The observation, that in normally menstruating patients none of the three oestrogens was detectable during 'Norlutin' medication, suggests that the bio¬ synthesis of ovarian oestrogens is completely impaired under the influence of 'Norlutin'. However, in other investigations (Brown et al., 1962; Shearman, 1963; Loraine et al., 1963; Shearman, 1964) when Brown's colorimetrie method was employed (Brown, 1955) a continuous excretion of oestrogens was observed during the medicated cycles and, in some cases, the oestrogen levels were found to be as high (Brown et al., 1962) or even higher than (Shearman, 1963; Shearman, 1964) in the respective control cycles. Similar results were obtained in the present study when the urinary oestrogens were estimated on the partially purified fractions. It has been shown by Ladany & Finkelstein (1963) that without rigorous purification of the oestrogenic fractions before the quantitative estimation the true oestrogen values may be markedly overestimated. It seems, therefore, that the relatively high readings, obtained when Brown's method was used or when the estimations in the present study were carried out on the partially purified oestrone, oestradiol-17)? and oestriol, were due to unidentified material which interfered with the estimation of the above oestrogens. The possibility that the apparent increase in the oestradiol-17)? fraction could be due to ethynyl-oestradiol being converted in vivo from the administered 17ot-ethynyl-19-nor-testosterone or its derivatives has been expressed by Breuer et al. (1960), Besch, Watson, Vorys & Barry (1963) and others. About 0-1% of the administered dose was recovered as urinary ethynyl-oestradiol (Breuer et al., 1960; Besch et al., 1963). If such a conversion had occurred in the present study of Patient 3 (Z.S.), it was less than 0-02%. Whether 'Norlutin' had any inhibitory effect on the function of the adrenal cortex, could not have been ascertained from the study of the urinary excretion of the oestrogens because the normal contribution of the adrenals to the total pool of urinary oestrogens is so low that it could not have been estimated by the present method (Ladany & Finkelstein, 1963). To follow the possible effect of 'Norlutin' on the adrenal function, the urinary excretion of pregnanetriol was studied. Pregnanetriol is an urinary metabolite of both adrenal and ovarian precursors. It has been observed that administration of'Norlutin' or its acetate to normal menstruating women decreases the urinary excretion of pregnanetriol (Serr et al., 1962; Fotherby, 1962; Brown et al., 1962; and the present study). This decrease apparently reflects the inhibition of ovarian steroid hormone bio¬ synthesis. The possibility that 'Norlutin' inhibits also the biosynthesis of the adrenal precursors of pregnanetriol can be ruled out, because there was no decrease of its urinary excretion in three oophorectomized patients receiving comparable doses of'Norlutin'.

Downloaded from Bioscientifica.com at 10/01/2021 06:00:28AM via free access 116 J. Weidenfeld et al. Thus, the differential between the excretion of pregnanetriol in the basal condition and during treatment with 'Norlutin' (or its analogues) may be used as a measure to distinguish between the adrenal and the ovarian sources of pregnanetriol. This differentiation may be of diagnostic value for the evaluation of ovarian function. For instance, in several cases of secondary amenorrhoea studied by us, the excretion of pregnanetriol remained unchanged when the patients were treated with 'Norlutin' or 'Enovid' (norethynodrel + ethynyl-oestradiol-3-methyl- ether) ; however, there was a definite decrease in a few others also classified as 'secondary amenorrhoea' (Finkelstein, Serr & Weidenfeld, 1966). It can be assumed that the patients in whom there was no change in the excretion of pregnanetriol had non-functioning ovaries, but in those patients who re¬ sponded to the treatment with a decrease of pregnanetriol the ovaries were functioning, at least to the stage of the biosynthesis of the precursors of preg¬ nanetriol (17a-hydroxypregnenoloneor 17a-hydroxyprogesterone). Thus estimation of pregnanetriol in patients receiving 'Norlutin' or similar agents may be of value in locating the possible biochemical defects in the chain of reactions involved in the biosynthesis of ovarian hormones.

ACKNOWLEDGMENT This investigation was supported by a grant from the Population Council, New York.

REFERENCES Baulieu, E.-E., Mauvais-Jarvis, P. & Corpeohot, C. (1963) Steroid studies in a case of Stein- Leventhal syndrome with hirsutism. J. clin. Endocr. Metab. 23, 374. Besch, P. K., Watson, D. J., Vorys, N. & Barry, R. D. (1963) Aromatization in vivo of 3ß,\7ß- bisacetoxy-17a¡-ethynyl-19-nor-androst-4-ene (SC-11 800). Biochim. biophys. Ada, 69, 417. Breuer, H., Dardenne, U. & Nocke, W. (1960) Ausscheidung von 17-ketosteroiden, 17-ketogenen steroiden und oestrogenen beim menschen nach gaben von 17a-athynyl-19-nor-testosteron- estern. Ada endocr., Copenh. 33, 10. Brooks, R. V. & Prunty, F. T. G. (1957) The suppression of adrenocortical secretion with 17a-ethyl- 19-nortestosterone. J. Endocr. 15, 385. Brown, J. B. (1955) Urinary excretion of oestrogens during the menstrual cycle. Lancet, i, 320. Brown, J. B., Fotherby, K. & Loraine, J. A. (1962) The effect of norethisterone and its acetate on ovarian and pituitary function during the menstrual cycle. J. Endocr. 25, 331. Ercoli, ., Vitali, R. & Gardi, R. (1964) Absorbents for detection, isolation and evaluation of ethynyl steroids. Steroids, 3, 479. Finkelstein, . (1962) Pregnanetriolone, an abnormal urinary steroid. Methods in Hormone Research, Vol. 1. Ed. R. I. Dorfman. Academic Press, New York. Finkelstein, M., Jewelewicz, D. R., Klein, O., Pfeiffer, V. & Sodmoriah-Beck, S. (1960) Fluoro- metric analysis of urinary oestrogens and its clinical application. Prenatal Care, p. 12. P. Noordhoff, Groningen. Finkelstein, M., Serr, D. & Weidenfeld, J. (1966) Evaluation of ovarian function in women by the estimation of urinary pregnanetriol during treatment with 19-nor-steroids. Proc. 2nd Meeting of International Study Group for Steroid Hormones. Research on Steroids, Vol. 2. Il Pensiero "Scien¬ tifico", Rome. Fotherby, K. (1960) Excretion of pregnanetriol during the normal menstrual cycle. Br. med. J. i, 1545 Fotherby, K. (1962) The ovarian production of a pregnanetriol precursor. J'. Endocr. 25, 19. Ladany, S. & Finkelstein, M. (1963) Isolation of oestrone, oestradiol-17ß and oestriol from female human urine. Steroids, 2, 297. Loraine, J. ., Bell, E. T., Harkness, R. ., Mears, E. & Jackson, M. C. N. (1963) Effect of long- term administration of hormone excretion in normally menstruating women. Lancet, ii, 902. Pickett, M. T., Kyriakides, E. C, Stern, M. I. & Sommerville, I. F. (1959) Urinary pregnanetriol throughout the menstrual cycle. Lancet, ii, 829.

Downloaded from Bioscientifica.com at 10/01/2021 06:00:28AM via free access Excretion ofsex steroids in women 117 Serr, D. M., Roth, R., Weidenfeld, J. & Finkelstein, M. (1962) Effect of Norlutin on urinary excre¬ tion of steroids. Excerpta Med., int. Cong. Series N, 51, Abstract No. 174. Shearman, R. P. (1963) Excretion of ovarian steroids in patients treated with an 'ovulation inhibitor'. Lancet, i, 197. Shearman, R. P. (1964) Ovarian function during and after long-term treatment with ovulation inhi¬ bitors. Lancet, ii, 557. Zondek, B., Sulman, F. & Black, R. (1945) The hyperemia effect of gonadotropins in the ovary. J. Am. med. Ass. 128, 939.

Downloaded from Bioscientifica.com at 10/01/2021 06:00:28AM via free access