THE ESTROGENS OF THE PREGNANT MARE1 KENNETH SAVARD2 with the technical assistance of Downloaded from https://academic.oup.com/endo/article/68/3/411/2702391 by guest on 01 October 2021 BERNICE BURDULIS Endocrine Laboratory, University of Miami School of Medicine, Miami, Florida ABSTRACT The urinary ketonic estrogens were studied at monthly intervals in five pregnant mares. Estrone and equilin appear to be the principal components and equilenin a minor constituent of the mares' estrogens. The levels of equilin rise from the 4-5th months to equal and in some instances exceed those of estrone in the late months of pregnancy. A partition chromatographic method for the separation of the ketonic phenolic steroids of pregnant mares' urine is described. N THE early studies of mares' pregnancy urines, Girard and associates I (1, 2) observed that the Ring B unsaturated estrogens, equilin and equilenin, relative to the total estrogen titers, increased in the latter months of pregnancy, at a time when the total estrogen content of the urine diminished (3, 4). Prompted by the renewed interest in the biological origins and interrelationships of these unsaturated estrogens (5, 6, 7), and utilizing more refined analytical techniques (8), we have re-investigated this problem in several individual mares from the 5th month of pregnancy to its termination. METHODS Selection of Animals and Urine Collections. Thoroughbred, Quarterhorse and hybrid mares were initially studied in the 4th and 5th month after breeding in order to select those individuals secreting adequate amounts of urinary estrogens. Many mares ex- creted estrogen at very low levels, 20 mg. per day and less. Five mares were selected for the study; mares 1 and 2 were Quarterhorses, mares 3-5 were Thoroughbreds. Mares 1-4 were the property of a private stable and were kept in pasture land during the day- time in winter, and at night during the summer heat; otherwise the animals were brought to the barns where they were allowed water ad libitum at night in wintertime and during the day in summer. Urine was collected from the mares only while they were indoors in individual stalls. One such 8- to 12-hour urine collection was made from each mare at approximately the same time each month during the period of study. The estrogen titers, even on a 12-hour basis, were quite variable and in a few instances where com- plete 24-hour collections were made (mare 5), the estrogen excretion appeared to vary as Received June 24, 1960. 1 Supported in part by Grant No. C-4004, National Cancer Institute, United States Public Health Service. 2 Investigator, Howard Hughes Medical Institute. 411 412 SAVARD Volume 68 the volume of urine. The estrogen values on each urine collection, listed in Table 1, arc therefore given in mg. per liter of urine. Preparation of Estrogen Fraction. Treatment of the individual urines was as previ- ously described (7) and consisted of acidification to pH 1.0 with hydrochloric acid, ex- traction of the lipides with toluene during 0.5 and 2.5 hours of refluxing. The combined extracts were shaken with 10 per cent sodium carbonate and N sodium hydroxide; the sodium carbonate extracts (containing acidic material) and the final toluene layers (con- taining the neutral substances) were not studied. The sodium hydroxide extracts were adjusted to pH 8-9 and extracted several times with ether to provide the crude phenolic Downloaded from https://academic.oup.com/endo/article/68/3/411/2702391 by guest on 01 October 2021 fractions. The oily phenolic fractions were steam-distilled to remove non-estrogenic, volatile phenolic compounds, and the non-volatile portion from each urine specimen was separated into ketonic and non-ketonic fractions with Girard's reagent T. Chromatography of Ketonic Phenols. The chromatographic method of Haenni, Carol and Banes (8) for the non-ketonic estrogens of pregnant mares' urine, was modified to effect an adequate separation of estrone, equilin and equilenin. Celite (Johns-Manvillc TABLE 1. COMPOSITION OF KETONIC ESTROGEN FRACTION OF MARES' URINE DURING PREGNANCY Urinary Equilin Mare1 Month of Weeks before estrogen Estrone and Equilenin no. pregnancy foaling pe: liter % artefacts2 % mg.3 % 1 4 25 4.9 93 tr. tr. 5 21 31.7 85 9 tr. 6 17 34.2 73 16 6 7 13 43.7 49 38 8 8 9 13.2 — — .—. 9 44 11.4 47 36 11 2 6 21 50.8 60 28 7 7 18 71.6 46 41 8 8 14 29.1 49 31 15 9 10 24.3 36 51 9 10 6 13.5 29 53 13 11 2 7.9 — — — 3 5 25 44.8 85 4 6 6 21 17.4 68 22 5 7 17 32.9 55 33 8 8 13 45.5 45 42 8 9 9 25.8 39 48 8 10 5 17.6 55 33 7 11 1 5.8 92 tr. 2 4 5 27 75.5 88 6 1 6 23 111.3 64 26 5 7 19 53.7 41 43 11 8 15 41.7 37 46 12 9 10 42.4 40 48 7 10 5 53.2 — — -- 5 9 9 67.8 33 56 6 10 6 69.2 37 49 9 11 2 43.0 32 58 5 1 Mares 1-4 were the property of the Spring Hill Farm stables; mares Nos. 1 and 2 were Quarterhorse mares, Switchy-Mae and Cherryl Star, bred to Quiet Man, 1958; mares Nos. 3 and 4 were the Thoroughbreds, Menim and Plum Blossom, bred to Doctor Stanley, 1958. 2 Amounts of transformation products of equilin (see text) added. 3 Total estrogen determined by modified Kober test (for details, see text). * Mare foaled one month prematurely. March, 1961 ESTROGENS OF PREGNANT MARES 413 No. 545), washed according to Bauld (9), and IN sodium hydroxide (31.5 g. and 30 ml., respectively) were ground together in a mortar under ligroin-benzene (1:1 by volume). The impregnated Celite was transferred to a glass column (1.8 cm. i.d.) in small por- tions and packed as directed (9) to a height of 31-33 cm. Uniformity was determined by the use of Sudan Red; the flow-rate was usually approximately 1.5 ml. per minute for columns of these dimensions. The ketonic phenolic fraction was transferred to the column in ligroin: benzene (1:1) and developed in that solvent. Fractions (15 ml.) were collected on an automatic collector and alternate tubes were evaporated to dry ness. Freed of benzene, the residues Downloaded from https://academic.oup.com/endo/article/68/3/411/2702391 by guest on 01 October 2021 were dissolved in 5.0 ml. of freshly distilled methanol and examined in an ultraviolet- spectrophotometer in the range 350-220 m/u; weight of estrogen per tube was calculated from the intensity of the peak at 280 niju (10). When the zones of estrone and equilin had passed down the column, (tubes 75 and later) the solvent was changed and the develop- ment continued with benzene until the zone of equilenin had been eluted (tubes 120- 130). Little or no additional material was eluted with further volumes of benzene. The various steroids were characterized by their relative positions on the chromatograms, their ultraviolet absorption spectra (10) and by their absorption (700-220 mju) in sul- furic acid after two hours at room temperature (11). Variations in the concentration of sodium hydroxide, the nature of the developing solvent (hexane, cyclohexane, methyl cyclohexane, mixtures of the above with toluene), did not improve the operation of the column. It was found that temperatures above 25° C seriously impaired the resolution of the components; columns were therefore run in the range of 21-23° C. The weight of estrogenic material (determined by Brown modifica- tion of the Kober test (12)) applied to the column, ranged from 20-25 rag. of total estro- gen (expressed as estrone); the critical weight beyond which resolution was impaired appeared to be approximately 30 mg. and depended upon the content of equilin and equilenin, whose relative chromogenic values in the modified Kober test are 30 and 17%, respectively, relative to that of estrone (compare ref. 13). RESULTS AND DISCUSSION The results of the study of the components of the estrogen fraction of the monthly urine samples collected from Mare No. 3 are illustrated in Figure 1. It may be seen that an adequate separation of estrone and equilin took place on the chromatogram columns, the peaks being separated by approx- imately 25-30 tubes and the overlapping of the two zones amounting to 13- 18 per cent of the total weight of the two estrogens. Equilenin, on the other hand, was well separated, appearing only after the change in the mobile phase from 50 to 100% benzene; its mobility on these column corresponded approximately to that of estradiol-17/3. Two additional substances ap- peared in many of these chromatograms and were discernible by their ultraviolet absorption maxima. One compound was seen on occasion in the tubes immediately following the equilin zone or in the late (or trailing) tubes of that zone; the substance absorbed maximally at 274 m/x in meth- anol and in this respect resembled 8-dehydro-14-isoestrone (14). The second substance appeared in the tubes immediately preceding the equile- nin zone and is shown graphically (Fig. 1) as the "A9 E-l zone". This substance absorbed maximally at 263 m/x in methanol, resembling the spectrum of 9-dehydro-14-isoestrone (14). Both compounds are artefacts, 10 E-l 5th MONTH 6 th MONTH 7th MONTH 8th MONTH Pi 0.8 E- t E-l 0.6 I 0.4 1 0.2 68 ) 1 Z?E-IA E-| 0 20 40 60 80 100 120 20 40 60 80 100 120 20 40 60 80 100 120 140 20 40 60 80 100 120 TUBE NO.