[4-14C]Estrone was injected intramuscularly into two mature laying Rhode Island Red hens. Radioactive steroids and steroid conjugates recovered from the urine on Amberlite XAD-2 columns were fractionated on columns (100 cm) of DEAE-Sephadex A-25 by NaCl gradients. The presences of the following were confirmed, the figures in brackets indicating average proportions as per cent of total radioactivity recovered after Sephadex column chromatography: -the 3-β-glucuronides of estrone (10. 9) and of estradiol-17α plus estradiol-17β(9.8); the 17-β-glucuronides of estradiol-17α plus estradiol-17β (2.1); the 3-sulfates of estrone (14. 5) and of estradiol-17α plus estradiol-17β (27. 4); and the disulfates of estradiol-17α plus estradiol-17β (2. 3). The following additional conjugates were identified:-a β-glucuronide of 16-epiestriol (0.2) and a β-glucuronide of 16-ketoestradiol-17β (0. 2); the 3-sulfates of 16-epiestriol (1. 4), of 17-epiestriol (0. 9), of 16, 17-epiestriol (0. 7), of 16-keto-estradiol-17β (1. 1), and of 2-methoxyestrone (0. 7). Some evidence was obtained for the presence of 16, 17-epoxy-estratrienol-3-sulfate (1.9).

[4- 14 C]Estrone was injected intramuscularly into two mature laying Rhode Island Red hens. Radioactive steroids and steroid conjugates recovered from the urine on Amberlite XAD-2 columns were fractionated on columns (100 cm) of DEAE-Sephadex A-25 by NaCl gradients. The presences of the following were confirmed, the figures in brackets indicating average proportions as per cent of total radioactivity recovered after Sephadex column chromatography: -the 3-β-glucuronides of estrone (10. 9) and of estradiol-17α plus estradiol-17β(9.8); the 17-β-glucuronides of estradiol-17α plus estradiol-17β (2.1); the 3-sulfates of estrone (14. 5) and of estradiol-17α plus estradiol-17β (27. 4); and the disulfates of estradiol-17α plus estradiol-17β (2. 3). The following additional conjugates were identified:-a β-glucuronide of 16-epiestriol (0.2) and a β-glucuronide of 16-ketoestradiol-17β (0. 2); the 3-sulfates of 16-epiestriol (1. 4), of 17-epiestriol (0. 9), of 16, 17-epiestriol (0. 7), of 16-keto-estradiol-17β (1. 1), and of 2-methoxyestrone (0. 7). Some evidence was obtained for the presence of 16, 17-epoxy-estratrienol-3-sulfate (1.9).

1. 1. [4- 14 C]oestrone was injected into the breast muscles of laying hens. An hour later the gall bladders were recovered and the contained bile was examined for radioactive phenolic steroids and conjugates thereof. 2. 2. The following conjugates were identified, the figures in brackets indicating average proportions as % of total radioactivity recovered after chromatographic fractionation: oestrone-β-glucuronide (13·2); oestradiol-17β-3-β-glucuronide (27·4); oestradiol-17β-17-β-glucuronide (3·1); oestrone sulphate (2·2); oestradiol-17β-3-sulphate (10·2); oestradiol-17α-3,17-disulphate (4·5). 3. 3. Free steroid (3·5), unidentified 3-monosulphate(s) (5·5) and other unidentified conjugated material (25·3), together with the conjugates listed above, accounted for 94·9% of the material fractionated on DEAE-Sephadex columns.

[4- 14 C] Estrone was injected intramuscularly into six laying hens. Fifty minutes later the hens were exsanguinated. The plasmas were examined for conjugates of radioactive phenolic steroids by recovery on columns of Amberlite XAD-2 or by extraction with tetrahydrofuran followed by chromatography on a column of DEAE-Sephadex A-25 in a gradient of NaC1. The biggest Sephadex Chromatographic fraction (50.4% of total) contained about 42% of its radioactivity as estradiol-17a-3-sulfate and 18% as estradiol-17β-3-sulfate and the remaining 40%-was identified tentatively as estradiol-17α-17-sulfate plus a small proportion of estradiol-17β-17-sulfate. The second biggest Sephadex Chromatographie fraction (12.7% of total) was a mixture of conjugates not further identified. Minor fractions identified comprised estrone-β-glucuronide (2.8%), estradiol-17α-3-β-glucuronide (2.8%), estradiol-17β-3-β-glucuronide (2.3%) and estrone sulfate (6.0%). Evidence was obtained for the presence of small proportions of estradiol-17α disulfate and estradiol-17β disulfate.

Two previously uncharacterized radioactive estrogen conjugates, 17β-estradiol-17-β-D-glucuronide (3-hydroxyestra-1,3,5(10)-trien-17β-yl-β-D-glucopyranosiduronate) and 17α-estradiol-17-β-D-glucuronide (3-hydroxyestra-1,3,5(10)-trien-17α-yl-β-D-glucopyranosiduronate), have been identified in small but significant amounts in avian urine and in a ratio of approximately 2:1 after intramuscular injection of [4-14C]estrone.

[4--14C] Estrone was injected intramuscularly into six laying hens. Fifty minutes later the hens were exsanguinated. The plasmas were examined for conjugates of radioactive phenolic steroids by recovery on columns of Amberlite XAD-2 or by extraction with tetrahydrofuran followed by chromatography on a column of DEAE-Sephadex A-25 in a gradient of NaCl. The biggest Sephadex chromatographic fraction (50,4% of total) contained about 42% of its radioactivity as estradiol-17alpha-3-sulfate and 18% as estradiol-17beta-3-sulfate and the remaining 40% was identified tentatively as estradiol-17alpha-17-sulfate plus a small proportion of estradiol-17beta-17-sulfate. The second biggest Sephadex chromatographic fraction (12.7% of total) was a mixture of conjugates not further identified. Minor fractions identified comprised estrone-beta-glucuronide (2.8%), estradiol-17alpha-3-beta-glucuronide (2.8%), estradiol-17beta-3-beta-glucuronide (2.3%) and estrone sulfate (6.0%). Evidence was obtained for the presence of small proportions of estradiol-17alpha disulfate and estradiol-17beta disulfate.

If equol is subjected to acidic hydrolysis by refluxing with 1.5 n HCl, a large proportion of the equol is altered to other substances. Subjection to enzymic hydrolysis by aryl sulfatase does not bring about any such alterations.

The concentrations of total androgen, determined by radioimmunoassay and expressed as testosterone equivalents, were measured in the peripheral plasma of laying hens. Total androgen attained peak values at from 8 to 2 h before ovulation. Peak values were not observed either on days when the terminal egg of a sequence was laid or when oviposition failed to occur during the experimental period.For 12 ovulatory cycles each observed on a different hen, the total androgen concentrations, in testosterone equivalents, ranged from 125 to 1100 pg/ml. For six birds in which ovulation did not accompany oviposition, the total androgen concentrations ranged from 150 to 900 pg/ml. For six birds, which were not in lay during the experimental period, the concentrations of total androgen ranged from 200 to 800 pg/ml.

[4-14C]Estrone was injected into three mature Rhode Island hens. Radioactive steroids and steroid conjugates were recovered from the urine on columns of Amberlite XAD-2 and fractionated on columns of DEAE-Sephadex A-25 in a NaCl gradient. Two minor less polar radioactive fractions (I and II) were due to free steroids. Six radioactive conjugate fractions were separated and designated III to VIII in order of increasing polarity. Fraction III was identified as estrone-β-glucuronide; Fraction IV as estradiol-17β-3-β-glucuronide plus estradiol-17α-3-β-glucuronide; Fraction V as estrone sulphate; and Fraction VII as estradiol-17β-3-sulphate plus estradiol-17α-3-sulphate. Fraction VI was identified as a monosulphate. Fraction VIII was not identified definitively.

An application of radioimmunoassay to measurement of concentrations of estradiol and estrone in peripheral plasma of hens is described. Evidence is presented that the estradiol measured by the procedure was essentially estradiol-17β.For 13 ovulatory cycles the average concentration of estradiol was 123 pg/ml with a range from 36 pg/ml to 284 pg/ml. The corresponding average concentration for estrone was 64 pg/ml with a range from 14 pg/ml to 138 pg/ml. For 12 days of no ovulation the average concentration of estradiol was 142 pg/ml with a range from 42 pg/ml to 364 pg/ml. The corresponding average value for estrone was 91 pg/ml with a range from 18 pg/ml to 242 pg/ml.The results are interpreted as demonstrating the existence of peak levels of plasma estradiol at 22–18 h and again at 6–2 h before ovulation, and a minimal level at 14–10 h before ovulation. The corresponding plasma estrone concentrations displayed a similar cycle but the earlier peak and the minimum occurred 2–4 h later than the corresponding values for estradiol. Such peak values were not demonstrable on days when ovulation did not occur.The results are discussed briefly in relation to variation in the levels of blood progesterone during the ovulatory cycle.

The concentration of progesterone in the peripheral blood plasma of laying hens attained a peak value 4–6 h before ovulation. A peak value was not observed on days where ovulation did not occur, i.e., on days when the terminal egg of a sequence was laid.For nine ovulatory cycles the average level was 2.45 ng/ml with a range of 0.5 to 8.8 ng/ml. For five periods covering a day of no ovulation the average was 2.95 ng/ml with a range of 0.5 to 12.5 ng/ml.

Estrone concentrations in whole peripheral blood of seven pregnant cows that calved normally were measured daily from at least three but not more than six days prepartum to four days postpartum. The highest average daily value was 8.2±1.2ng per milliliter at five days prepartum. Blood estrone declined nearly continuously up to one day prepartum, but onset of this decline varied from one to five days before parturition. There was a relatively steep and significant decline between one day prepartum and one day postpartum. The lowest average blood estrone was four days postpartum, beyond which no samples were collected. A heifer that calved abnormally displayed an abnormally high blood estrone at the time of calving.

Blood estrone concentrations of 65 pregnant dairy cows were measured by an Ittrich-Kober fluorometric procedure. Average estrone increased slowly from 1.2ng/ml at 16 to 14 weeks prepartum to 2.5ng/ml at 8 to 6 weeks prepartum, and then more rapidly to 4.8ng/ml at 4 to 2 weeks before parturition. The highest average, 8.3ng/ml, was 5 days preparturn; no sanlples were taken closer to parturition. The lowest average (0.7ng/ml) mas 5 days postpartum.

Progesterone-4-14C was administered intramuscularly to hens. The urine subsequently excreted contained radioactive 5β-pregnane-3α,20β-diol and 5β-pregnan-3α-ol-20-one, the identities being established by thin-layer chromatographic mobilities of the isolated materials and derivatives thereof, and by crystallization of the alcohols and their acetates to constant specific activity with reference carrier materials. Radioactive progesterone was not detected in the urine, neither did the results provide any evidence for the presence of radioactive 5α-pregnane derivatives.

Estradiol-17β-4-14C was injected into a mature hen when it was not in lay and when it was in lay. In each instance the major radioactive conversion products in the urine were estrone and estradiol-17α. The percentage of the total radioactive phenolic steroids present in the urine as estradiol-17β was higher, and the percentages present as estradiol-17α and as estrone were lower when the hen was not in lay than when the hen was in lay. Estrone-4-14C was injected into a mature hen in a second similar experiment, and the major radioactive conversion products in the urine were estradiol-17β and estradiol-17α. As in the first experiment, the percentage of the total radioactive phenolic steroids present in the urine as estradiol-17β was higher and the percentages present as estradiol-17α and as estrone were lower when the hen was not in lay than when she was in lay.Irrespective of whether estrone-4-14C or estradiol-17β-4-14C were injected, the major radioactive phenolic steroids in the urine were estradiol-17β, estrone, and estradiol-17α, in that order, when the hen was not in lay, and estrone and estradiol-17β or estradiol-17α, in that order, when the hen was in lay.

A conversion product of injected 14 C-labelled estrone in hen's urine which was formerly identified tentatively as 16-ketoestrone was shown to contain approximately 90% nonketonic material that corresponded in chromatographic mobility with estradiol-17α, a known urinary conversion product of injected estradiol-17β or estrone. Contrary to the previous suggestion, therefore, 16-ketoestrone is not a major urinary conversion product of injected estrone.

The reliability of a chemical analytical method is a composite property of the method. This composite property may be resolved into four more elementary properties which may be designated as (1) specificity, (2) accuracy, (3) precision, and (4) sensitivity. It is desirable to define these terms technically, to elucidate some of their interrelationships and, wherever possible, to assign an appropriate statistical parameter to each property.

A mixture of estradiol-17α-4-14C and estradiol-17α-17β-3H was injected into a laying hen. The ratios dpm 3H:dpm 14C of the urinary radioactive estradiol-17α and 17-epiestriol were practically identical whereas the corresponding ratio for the urinary radioactive 16, 17-epiestriol was considerably higher and approached the value for the injected estradiol-17α. These results show (a) that the 17-epiestriol and 16, 17-epiestriol were derived directly from the estradiol-17α and that estrone was not an intermediate product; and (b) that the formation of 16, 17-epiestriol was a relatively fast process. A general scheme for the pathways of in vivo derivation of the four estriol epimers in the hen is proposed on the basis of these and previous findings.