The Metabolic Clearance Rate, the Brain Extraction and Distribution and the Uterine Extraction and Retention of Progesterone and R 5020 in Estrogen-Treated Ovariectomized Rabbits S. S. RAHMAN, R. B. BILLIAR, R. MIGUEL, W. JOHNSON,* AND B. LITTLE Department of Reproductive Biology, Case Western Reserve University, Cleveland, Ohio 44106 ± 3.9 (SE)%) was greater than that of progesterone (33.7 ± 7.7%) as was the uterine tissue:arterial blood ratio (28.1 ± 4 vs. 7.3). The brain and pituitary retention and distribution of R 5020 and progesterone were the same and provided no evidence for a selective accumulation of a progestin in the pituitary or hypothalamus. (Endocrinology 101: 464, 1977)
ABSTRACT. The metabolic clearance rate (MCR), uterine extraction, uterine retention and brain distribution of the synthetic progestin R 5020 and progesterone were studied in estrogen-treated ovariectomized rabbits. The MCR of R 5020 was 163 ± 15 (SE) I/day and was lower than that of progesterone. The uterine extraction of R 5020 (51.4
R
ECENTLY the existence of specific progesterone receptors in the uterine cytosol of various animals has been established (1). The concentration of the progesterone receptors in the uterus increases under the influence of estrogen and decreases in the presence of progesterone (1). Various synthetic steroids have been used to study and identify steroid receptors in the target tissues (2). The synthetic progestin R 5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione) is a useful compound for the study of progestin distribution and progestin tissue receptors as it is not bound by transcortin and binds to the uterine progesterone receptor(s) with a higher association constant than progesterone (1.5 x 109M-x vs. 0.36 x 109M-X) (2-5). R 5020 also is more active (wt/wt) than progesterone in the Clauberg bioassay (3). Although progesterone is known to affect central nervous system function(s) (6),
Received January 14, 1977. Supported by U.S.P.H.S. Program Project HD-07640 and HD-02378. These studies were initiated while W.AJ. was supported by NIH Training Grant HD00024 and Ford Foundation Training Grant 670-0135A, and R.B.B. was a recipient of Career Development Award, NICHHD Grant HD-42564. * Present address: Department of Psychology, Bowling Green State University, Bowling Green, Ohio.
definitive evidence for a selective distribution of progesterone or of a progesterone receptor(s) in the CNS or pituitary has not yet been demonstrated (2,6-13). The present study of estrogen-treated, ovariectomized rabbits was designed: 1) to determine the pharmacodynamics of R 5020 by comparing its metabolic clearance rate (MCR) with the MCR of progesterone, 2) to test for the presence of progesterone receptors in the brain and pituitary by determining the distribution and possible selective retention of R 5020, and 3) to compare the CNS and pituitary retention and extraction of R 5020 and progesterone with that of the uterus, a tissue in which the progesterone receptor has been identified and extensively studied. Materials and Methods Mature (3-4 kg) virgin, New Zealand rabbits were bilaterally ovariectomized. Three days later they received im injections of estradiol-17/3 (2 /xg/kg BW) for five consecutive days. On the 6th day the rabbits were anesthetized with ether and a femoral artery and femoral vein were catheterized with a teflon catheter. One group of rabbits received a priming dose of [14C]progesterone (1.6 /uCi) plus [3H]R 5020 (7 /xCi) in 2 ml of 10% ethanolic saline into the femoral vein followed by a continuous infusion of [14C]progesterone (25 /LtCi, 140 fxg) plus [3H]R
464
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IN VIVO METABOLISM OF PROGESTERONE AND R 5020 5020 (38 ju,Ci, 0.260 /-ig) in 30 ml 10% ethanolic saline solution (vol/vol). The second group received a priming dose of [3H]R 5020 (7 /xCi) in 2 ml of 10% ethanolic saline followed by a continuous infusion of [3H]R 5020 (38 /uCi) in 30 ml 10% ethanolic saline. The steroids were infused into the femoral vein at a constant rate of 0.1 ml/ min over a period of 4 h. At 120, 160, 200 and 240 min after the start of the continuous infusion, blood samples of 1.0 ml were taken from the femoral artery for the determination of the MCR of progesterone (6) and R 5020. At 240 min blood samples (1.0 ml) were obtained by direct puncture of the left and right uterine veins. The differences between femoral arterial and uterine venous concentrations of [14C]progesterone and [3H]R 5020 were determined and expressed as the uterine extraction of these steroids. At the end of the infusion, the brain, pituitary and the uterus were excised and stored at —20 C until analysis. The rabbits were anesthetized with ether during the entire procedure. To the 1.0 ml samples of blood were added 200 /u,g of progesterone and 200 fig of R 5020 in 0.1 ml of ethanol and 1.0 ml of water for the estimation of procedural losses. The blood samples were immediately frozen. The blood samples were extracted as previously reported (6). [3H]R 5020 and [14C]progesterone were isolated by thin layer chromatography (TLC): benzene:acetone (100:15, vol/vol), followed by benzene:ethyl acetate (7:3, vol/vol) and hexane: ethyl acetate (3:1, vol/vol). R 5020 and progesterone did not separate from each other in the TLC systems and were eluted together. Since progesterone absorbs at 240 nm and R 5020 at 300 nm, the Allen correction factor (14) was used for the determination of progesterone at 230, 240 and 250 nm and of R 5020 at 280, 300 and 320 nm for the estimation of procedural losses. Although R 5020 showed slight absorption at 230, 240 and 250 nm, this did not affect the absorbance of progesterone significantly, as the Allen correction [(A 240 nm x 2) - (A 230 nm -I- A 250 nm)] values for progesterone were the same in the presence or absence of R 5020. The recoveries for progesterone and R 5020 from the blood were 87 ± 7.5 (SD)% and 61.5 ± 9.0%, respectively. [14C]Progesterone and [3H]R 5020 were quantified by liquid scintillation spectrometry. The uteri were thawed at 5 C for 2 h. Weighed portions (2-5 g) were homogenized in a mechanical homogenizer in 60 ml of acetone which
465
contained 200 fig of progesterone and 200 fig of R 5020 for the determination of procedural losses. The acetone solutions were centrifuged and the tissue was then reextracted with 15 ml of acetone-methylene dichloride (1:1, vol/vol) at 45 C for 2 h and the extracts were combined and taken to dryness. The residue was partitioned between ethyl acetate and water (20:5, vol/vol). The ethyl acetate extract was taken to dryness under nitrogen at 45. C and the residue was dissolved in 5 ml of heptane which was then warmed in a water bath at 45 C for 5 min. The steroids were extracted from the heptane by the addition of 10 ml of methanol: water (9:1, vol/vol). The methanol layer was separated, dried under nitrogen at 45 C and the residue dissolved in 2 ml of acetone. One-tenth ml of this extract was used for the determination of the total isotopic concentrations of 14C and 3 H. [I4C]Progesterone and [3H]R 5020 were then isolated from the remaining solution by the thin layer chromatography systems as described above. Procedural losses were estimated as described for the blood procedure and radioactivity was measured by liquid scintillation spectrometry (6). Recoveries of progesterone and R 5020 from the uteri were 84.0 ± 4.9 (SD)% and 71.6 ± 12.6%, respectively. [3H]R 5020 isolated from uteri in the procedure described above had the same specific activity after chromatography in the paper partition chromatographic system of petroleum ether:methanol: water (100: 96:4, vol ratios). During the continuous infusion, blood samples were also taken from the jugular vein at 120, 160, 200 and 240 min to calculate the extraction of the two steroids by the head. Head extractions were calculated from the difference in blood concentration of radioactive steroid in the removal artery and jugular vein divided by the femoral arterial blood concentration times 100. The brain was sectioned sagitally through the midline, selected brain areas were dissected, and steroids were extracted as described previously (15). [I4C]Progesterone and [3H]R 5020 were isolated from the brain samples and quantified as described above. Recoveries of progesterone and R 5020 from the brain samples were 91.5 ± 2.6 (SD)% and 66.0 ± 6.8%, respectively. All blood, uteri and brain samples were counted to an accumulation of at least 8000 counts of both 14C and 3 H. [14C] Progesterone was purchased from New
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466
TABLE 1. Blood metabolic clearance rates (MCR) of [HC]progesterone and [3H]R5020 in ovariectomized rabbits treated with estradiol Metabolic clearance rate (I/day) 14
[3H]R5020
Animal
[ C ] Progesterone
94* 96*
309 ± 8.7 409 ± 52.1 294 ± 29.6
192 203 142 123 155
337 ± 36
163:t 15
101* 102f 103t X ± SE
Endo i 1977 Vol 101 i No 2
RAHMAN ETAL.
dt 37.5$ dt 20.1 dt 19.5 d1:22.1 dt 8.9
Virgin rabbits were ovariectomized and 3 days later were injected daily with 2 fig estradiol per kg body weight for 5 days. On the sixth day the rabbits were anesthetized with ether and given a constant infusion of [l4C]progesterone and [3H]R5020* or [3H]R5020 alonef for 4 h. The MCR was determined by the rate of infusion of radioactive steroid and the concentration of radioactive steroid in the femoral artery. In a previous study (16) the MCR of [14C]progesterone in 7 ovariectomized rabbits treated with estradiol was 222 ± 23 (SE) I/day. | Mean ± standard deviation.
England Nuclear Corporation and purified by thin layer chromatography. [3H]R 5020 and R 5020 were a gift from Dr. J. P. Raynaud, and were used as supplied. Radiochemical purity of the [3H ]R 5020 was established by paper and thinlayer chromatography.
and distribution of R 5020 were not altered by the simultaneous infusion of ["Clprogesterone, so all MCR, distribution and extraction results for R 5020 are combined. The MCR of [14C]progesterone is in the same range as reported earlier (16) and is higher than the MCR of R 5020. The per cent of the total 3H retained in the uterus specifically as [3H]R 5020 is 59.9 ± 4.9 (SE)% and is similar to the per cent of the total 14C retained in the uterus specifically as [14C]progesterone (65 ± 7%, ref. 16). The uterine tissue/femoral arterial blood ratios indicate the capacity of the uterus to retain four times more R 5020 than [14C]progesterone (Table 2). The uterine extraction of [3H]R 5020 is 51.4%, which is greater than that of progesterone, 33.7% (Table 3). The head extraction of R 5020 of 36.1 ± 2.8 (SE)% is lower than the head extraction of progesterone, 53.7 ± 7.3%, but the brain distribution and retention of [3H]R 5020 and [14C]progesterone are similar (Table 4). The brain distribution and retention of [14C]progesterone in the present study are the same as that reported for a much larger series of animals (15).
Results The MCR of [3H]R 5020, when infused alone or in combination with ["Clprogesterone, in estrogen-treated ovariectomized rabbits is 163 I/day (Table 1). The MCR
Discussion The blood MCR of R 5020 is lower than the MCR of progesterone in the rabbit. Since R 5020 does not bind to transcortin
TABLE 2. Uterine retention of [HC]progesterone and [3H]R 5020 in ovariectomized rabbits treated with estradiol Uterine tissue/femoral arterial blood ratio [14C] Progesterone Animals
[3H]R5020
Right horn
Left horn
R.H[. + L.H.*
Right horn
Left horn
R.H. + L.H.*
96 101 102 103
6.7 7.7
7.3 7.5
7.0 7.6
X ± SE
7.2
7.4
7.3
35.3 26.1 31.0 16.2 27.2 ± 4.1
38.5 25.3 32.1 20.0 29.0 ± 4.0
36.9 25.7 31.5 18.1 28.1 ± 4.0
The study was the same as that described in the legend to Table 1. After uterine venous blood was drawn, the uterus was removed, the right and left uterine horns were cleaned of fat and connective tissue, weighed and frozen until analysis for [14C]progesterone and [3H]R 5020. In a previous study (16) the uterine tissue/femoral blood arterial ratio of [14C]progesterone in 7 ovariectomized rabbits treated with estradiol was 5.1 ± 0.7 (SE). * Average of right and left horns.
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IN VIVO METABOLISM OF PROGESTERONE AND R 5020
467
TABLE 3. Uterine extraction of [l4C]progesterone and [3H]R 5020 in ovariectomized rabbits treated with estradiol Uterine extraction (%) M
[3H]R5020
1' C] Progesterone Animals 94 96 101 102 103 X ± SE
Right horn
Left hom
R.H . + L.H.*
Right horn
Left horn
R.H. + L.H.*
30.0 57.5 20.8
26.6 40.5 27.2
28.3 49.0 24.0
36.1 ± 11.0
31.4 ± 4.5
33.7 ± 7.7
59.5 39.3 44.5 54.1 50.0 49.5 ± 3.5
64.0 37.3 52.4 57.8 54.6 53.2 ± 4.4
61.8 38.3 48.5 56.0 52.3 51.4 ± 4.0
The study was the same as that described in the legend to Table 1. At the end of the 4th h of infusion of radioactive steroids venous blood was obtained from veins draining the right and left uterine horns at the same time as blood was drawn from the femoral artery. The uterine extraction equals femoral arterial blood concentration - uterine venous blood concentration/femoral arterial blood concentration) x 100. In a previous study (16) the uterine extraction of [I4C]progesterone in 7 ovariectomized rabbits treated with estradiol was 27 ± 4 (SE)%.
* Average of right and left horns.
(2-3) and progesterone does, this result was terone. At the isotopic steady state, the exsomewhat unexpected since 5a-preg- traction of a steroid by the uterus would be nane-3,20-dione also does not bind to trans- due to metabolism. However, if the uterine cortin but its MCR is about twice that of tissue is not at an isotopic steady state, progesterone (16). However, the MCR of then the uterine extraction would be due to 20a-hydroxy-4-pregnen-3-one, which also distribution and metabolism of the steroid. binds only weakly to transcortin, is the same Since the uterine venous blood and tissue as the MCR of progesterone in the rabbit samples were obtained only at the end of a (Rahman et al., unpublished observations). 4 h infusion, we do not know if the uterine These results suggest that the MCR of progesterone and progestins may be more de- TABLE 4. Brain and pituitary distribution of [14C]propendent upon their metabolism in various gesterone and [ H]R 5020 in ovariectomized rabbits tissues than their binding to transcortin treated with estradiol (17). In addition, plasma proteins other than Ratio of tissue (DPM/g wet weight) to femoral artery transcortin {e.g., oroscomucoid) may in(DPM/ml) ± SE fluence the MCR of progesterone and R [ C] Pro5020. However, this possibility was not ingesterone [ H]R5020 [ ll]R5020/ vestigated. (n = 3) Tissue ["C] Progesterone (n = 5) In the Clauberg test 2.0 fig of R 5020 Cortex 3.32 ± 1.05 3.87 ± 0.27 1.17 3.17 ± 1.30 2.83 ±0.41 0.89 was reported to be as effective as 200 fxg of POA* 2.88 ± 0.99 2.84 ± 0.29 Hypothalamus 0.99 progesterone in producing the endometrial Thalanms 1.09 2.89 ± 1.95 3.16 ± 0.43 0.87 ± 2.09 4.32 ± 0.44 response in the rabbit (3). The greater Midbrain retieulum 4.95 4.17 ± 1.74 0.91 Central grey 3.81 ±0.19 biological activity of R 5020 in the Clau- Pons reticulum 4.67 ± 0.66 5.08 ± L.99 0.92 5.32 + 2.55 4.33 ± 0.76 0.81 berg test cannot be explained by differences Pons 3.80 ± 0.58 3.35 ± 1.15 Cerebellum 1.13 in the rate of clearance of progesterone Hippocampus 2.76 ± 0.97 0.89 2.47 ± 0.23 0.97 3.05 ± 1.29 2.97 ± 0.37 and R 5020 from the blood alone since Amygdala 1.77 Anterior pituitary 2.25 ± 0.58 3.99 ± 1.41 the MCR of progesterone is only 1.5 times Posterior pituitary 4.01 ± 1.58 3.91 ± 0.75 0.98 greater than the MCR of R 5020. * Preoptie-anterior hypothalamus. Although the MCR of R 5020 was lower The study was the same as that described in the legend of Table 1. brain and pituitary were removed and frozen until the brains were than that of progesterone, the uterine ex- The dissected and the samples weighed and analyzed for [ C]progesterone traction of R 5020 was twice that of proges- and [ H]R5020. 3
IJ
3
3
14
3
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Endo • 1977 Vol 101 • No 2
RAHMAN ETAL.
468
tissue was at the isotopic steady state with respect to R 5020. The higher extraction of R 5020 may be due, in part, to a greater accumulation of R 5020 than progesterone in the uterine tissue and/or an increased metabolism. The accumulation of progesterone and R 5020 in the uterus is most likely due to the presence of progesterone receptors induced by the estrogen treatment of the rabbit (16, 18, 19). Also, the four times greater retention of R 5020 than progesterone in the uterus compares with the reported 3-4 times greater affinity of R 5020 than progesterone for the rabbit uterine progesterone receptor (3). The head extraction of R 5020 was lower
than the head extraction of progesterone which suggests that the head can metabolize progesterone more readily than R 5020. The retention and distribution of R 5020 and progesterone were the same in the brain and pituitary. As in previous studies (6-9, 15) no evidence for a selective accumulation of a progestin in the pituitary or hypothalamus was observed. This would suggest that progesterone and R 5020 accumulation in the brain are not dependent on the same mechanism(s) as that of the uterus, or if there is a progesterone receptor present in CNS, then its quantity is not sufficient to show selective retention of progesterone or R 5020 in brain areas studied, or its relative binding affinity for progesterone and R 5020 is different than the uterine progesterone receptors). If there is a low concentration of progesterone receptor(s) in the brain, they may have become saturated with the progesterone secreted by the adrenals of the ovariectomized rabbits (20). Acknowledgments The authors wish to acknowledge the technical assistance of Mr. Augusts Heinsons and Mrs. Helen
Chen during the course of this work. We thank Dr. J. P. Raynaud, Roussel-UCLAF, Romainville, France for his generous gift of [3H]R 5020 and R 5020.
References 1. Thi, M. T. L., E. E. Baulieu, and E. Milgrom, J Endocrinol 66: 349, 1975. 2. Raynaud, J.-P., M.-M. Bouton, E. Philibert, and B. Vannier, In Labrie, F., J. Meites, and G. Pelletier (eds.), Hypothalamus and Endocrine Function, Plenum Press, New York, 1976, p. 171. 3. Philibert, D., and J.-P. Raynaud, Endocrinology 94: 627, 1974. 4. Philibert, D., and J.-P. Raynaud, Steroids 22: 89, 1973. 5. Philibert, D., and J.-P. Raynaud, Contraception 10: 457, 1974. 6. Billiar, R. B., B. Little, I. Kline, P. Reier, Y. Takaoka, and R. J. White, Brain Res 94: 99, 1975. 7. Wade, G. N., and H. H. Feder, Brain Res 45: 525, 1972. 8. Luttge, W. G., C. J. Wallis, and N. R. Hall, Brain Res 71: 105, 1974. 9. Karavolas, H. J., D. Hodges, and D. O'Brien, Endocrinology 98: 164, 1976. 10. Seiki, K., and M. Hattori, Endocrinol Jap 20: 111, 1973. 11. Sar, M., and W. E. Stumpf, Science 182: 1266, 1973. 12. Atger, J., E.-E. Baulieu, and E. Milgrom, Endocrinology 94: 161, 1974. 13. Kato, J. J Steroid Biochem 6: 979, 1975. 14. Allen, W. M.J Clin Endocrinol Metab 10: 71,1950. 15. Johnson, W. A., R. B. Billiar, S. S. Rahman, and B. Little, Brain Res 111: 147, 1976. 16. Little, B., R. B. Billiar, S. S. Rahman, W. A. Johnson, Y. Takaoda, and R. J. White, Am J Obstet Gynecol 123: 527,'1975. 17. Billiar, R. B., M. Jassani, and B. Little, Am] Obstet Gynecol 121: 877, 1974. 18. Faber, L. E., M. L. Sandmann, and H. E. Stavely, 7 Biol Chem 247: 5648, 1972. 19. Rao; B. R., W. G. Wiest, and W. M. Allen, Endocrinology 92: 1229, 1973. 20. Rahman, S. S., R. B. Billiar, and B. Little, Biol Reprod 12: 305, 1975.
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ABSTRACT. The metabolic clearance rate (MCR), uterine extraction, uterine retention and brain distribution of the synthetic progestin R 5020 and progesterone were studied in estrogen-treated ovariectomized rabbits. The MCR of R 5020 was 163 ± 15 (SE) I/day and was lower than that of progesterone. The uterine extraction of R 5020 (51.4
R
ECENTLY the existence of specific progesterone receptors in the uterine cytosol of various animals has been established (1). The concentration of the progesterone receptors in the uterus increases under the influence of estrogen and decreases in the presence of progesterone (1). Various synthetic steroids have been used to study and identify steroid receptors in the target tissues (2). The synthetic progestin R 5020 (17,21-dimethyl-19-nor-4,9-pregnadiene-3,20-dione) is a useful compound for the study of progestin distribution and progestin tissue receptors as it is not bound by transcortin and binds to the uterine progesterone receptor(s) with a higher association constant than progesterone (1.5 x 109M-x vs. 0.36 x 109M-X) (2-5). R 5020 also is more active (wt/wt) than progesterone in the Clauberg bioassay (3). Although progesterone is known to affect central nervous system function(s) (6),
Received January 14, 1977. Supported by U.S.P.H.S. Program Project HD-07640 and HD-02378. These studies were initiated while W.AJ. was supported by NIH Training Grant HD00024 and Ford Foundation Training Grant 670-0135A, and R.B.B. was a recipient of Career Development Award, NICHHD Grant HD-42564. * Present address: Department of Psychology, Bowling Green State University, Bowling Green, Ohio.
definitive evidence for a selective distribution of progesterone or of a progesterone receptor(s) in the CNS or pituitary has not yet been demonstrated (2,6-13). The present study of estrogen-treated, ovariectomized rabbits was designed: 1) to determine the pharmacodynamics of R 5020 by comparing its metabolic clearance rate (MCR) with the MCR of progesterone, 2) to test for the presence of progesterone receptors in the brain and pituitary by determining the distribution and possible selective retention of R 5020, and 3) to compare the CNS and pituitary retention and extraction of R 5020 and progesterone with that of the uterus, a tissue in which the progesterone receptor has been identified and extensively studied. Materials and Methods Mature (3-4 kg) virgin, New Zealand rabbits were bilaterally ovariectomized. Three days later they received im injections of estradiol-17/3 (2 /xg/kg BW) for five consecutive days. On the 6th day the rabbits were anesthetized with ether and a femoral artery and femoral vein were catheterized with a teflon catheter. One group of rabbits received a priming dose of [14C]progesterone (1.6 /uCi) plus [3H]R 5020 (7 /xCi) in 2 ml of 10% ethanolic saline into the femoral vein followed by a continuous infusion of [14C]progesterone (25 /LtCi, 140 fxg) plus [3H]R
464
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IN VIVO METABOLISM OF PROGESTERONE AND R 5020 5020 (38 ju,Ci, 0.260 /-ig) in 30 ml 10% ethanolic saline solution (vol/vol). The second group received a priming dose of [3H]R 5020 (7 /xCi) in 2 ml of 10% ethanolic saline followed by a continuous infusion of [3H]R 5020 (38 /uCi) in 30 ml 10% ethanolic saline. The steroids were infused into the femoral vein at a constant rate of 0.1 ml/ min over a period of 4 h. At 120, 160, 200 and 240 min after the start of the continuous infusion, blood samples of 1.0 ml were taken from the femoral artery for the determination of the MCR of progesterone (6) and R 5020. At 240 min blood samples (1.0 ml) were obtained by direct puncture of the left and right uterine veins. The differences between femoral arterial and uterine venous concentrations of [14C]progesterone and [3H]R 5020 were determined and expressed as the uterine extraction of these steroids. At the end of the infusion, the brain, pituitary and the uterus were excised and stored at —20 C until analysis. The rabbits were anesthetized with ether during the entire procedure. To the 1.0 ml samples of blood were added 200 /u,g of progesterone and 200 fig of R 5020 in 0.1 ml of ethanol and 1.0 ml of water for the estimation of procedural losses. The blood samples were immediately frozen. The blood samples were extracted as previously reported (6). [3H]R 5020 and [14C]progesterone were isolated by thin layer chromatography (TLC): benzene:acetone (100:15, vol/vol), followed by benzene:ethyl acetate (7:3, vol/vol) and hexane: ethyl acetate (3:1, vol/vol). R 5020 and progesterone did not separate from each other in the TLC systems and were eluted together. Since progesterone absorbs at 240 nm and R 5020 at 300 nm, the Allen correction factor (14) was used for the determination of progesterone at 230, 240 and 250 nm and of R 5020 at 280, 300 and 320 nm for the estimation of procedural losses. Although R 5020 showed slight absorption at 230, 240 and 250 nm, this did not affect the absorbance of progesterone significantly, as the Allen correction [(A 240 nm x 2) - (A 230 nm -I- A 250 nm)] values for progesterone were the same in the presence or absence of R 5020. The recoveries for progesterone and R 5020 from the blood were 87 ± 7.5 (SD)% and 61.5 ± 9.0%, respectively. [14C]Progesterone and [3H]R 5020 were quantified by liquid scintillation spectrometry. The uteri were thawed at 5 C for 2 h. Weighed portions (2-5 g) were homogenized in a mechanical homogenizer in 60 ml of acetone which
465
contained 200 fig of progesterone and 200 fig of R 5020 for the determination of procedural losses. The acetone solutions were centrifuged and the tissue was then reextracted with 15 ml of acetone-methylene dichloride (1:1, vol/vol) at 45 C for 2 h and the extracts were combined and taken to dryness. The residue was partitioned between ethyl acetate and water (20:5, vol/vol). The ethyl acetate extract was taken to dryness under nitrogen at 45. C and the residue was dissolved in 5 ml of heptane which was then warmed in a water bath at 45 C for 5 min. The steroids were extracted from the heptane by the addition of 10 ml of methanol: water (9:1, vol/vol). The methanol layer was separated, dried under nitrogen at 45 C and the residue dissolved in 2 ml of acetone. One-tenth ml of this extract was used for the determination of the total isotopic concentrations of 14C and 3 H. [I4C]Progesterone and [3H]R 5020 were then isolated from the remaining solution by the thin layer chromatography systems as described above. Procedural losses were estimated as described for the blood procedure and radioactivity was measured by liquid scintillation spectrometry (6). Recoveries of progesterone and R 5020 from the uteri were 84.0 ± 4.9 (SD)% and 71.6 ± 12.6%, respectively. [3H]R 5020 isolated from uteri in the procedure described above had the same specific activity after chromatography in the paper partition chromatographic system of petroleum ether:methanol: water (100: 96:4, vol ratios). During the continuous infusion, blood samples were also taken from the jugular vein at 120, 160, 200 and 240 min to calculate the extraction of the two steroids by the head. Head extractions were calculated from the difference in blood concentration of radioactive steroid in the removal artery and jugular vein divided by the femoral arterial blood concentration times 100. The brain was sectioned sagitally through the midline, selected brain areas were dissected, and steroids were extracted as described previously (15). [I4C]Progesterone and [3H]R 5020 were isolated from the brain samples and quantified as described above. Recoveries of progesterone and R 5020 from the brain samples were 91.5 ± 2.6 (SD)% and 66.0 ± 6.8%, respectively. All blood, uteri and brain samples were counted to an accumulation of at least 8000 counts of both 14C and 3 H. [14C] Progesterone was purchased from New
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466
TABLE 1. Blood metabolic clearance rates (MCR) of [HC]progesterone and [3H]R5020 in ovariectomized rabbits treated with estradiol Metabolic clearance rate (I/day) 14
[3H]R5020
Animal
[ C ] Progesterone
94* 96*
309 ± 8.7 409 ± 52.1 294 ± 29.6
192 203 142 123 155
337 ± 36
163:t 15
101* 102f 103t X ± SE
Endo i 1977 Vol 101 i No 2
RAHMAN ETAL.
dt 37.5$ dt 20.1 dt 19.5 d1:22.1 dt 8.9
Virgin rabbits were ovariectomized and 3 days later were injected daily with 2 fig estradiol per kg body weight for 5 days. On the sixth day the rabbits were anesthetized with ether and given a constant infusion of [l4C]progesterone and [3H]R5020* or [3H]R5020 alonef for 4 h. The MCR was determined by the rate of infusion of radioactive steroid and the concentration of radioactive steroid in the femoral artery. In a previous study (16) the MCR of [14C]progesterone in 7 ovariectomized rabbits treated with estradiol was 222 ± 23 (SE) I/day. | Mean ± standard deviation.
England Nuclear Corporation and purified by thin layer chromatography. [3H]R 5020 and R 5020 were a gift from Dr. J. P. Raynaud, and were used as supplied. Radiochemical purity of the [3H ]R 5020 was established by paper and thinlayer chromatography.
and distribution of R 5020 were not altered by the simultaneous infusion of ["Clprogesterone, so all MCR, distribution and extraction results for R 5020 are combined. The MCR of [14C]progesterone is in the same range as reported earlier (16) and is higher than the MCR of R 5020. The per cent of the total 3H retained in the uterus specifically as [3H]R 5020 is 59.9 ± 4.9 (SE)% and is similar to the per cent of the total 14C retained in the uterus specifically as [14C]progesterone (65 ± 7%, ref. 16). The uterine tissue/femoral arterial blood ratios indicate the capacity of the uterus to retain four times more R 5020 than [14C]progesterone (Table 2). The uterine extraction of [3H]R 5020 is 51.4%, which is greater than that of progesterone, 33.7% (Table 3). The head extraction of R 5020 of 36.1 ± 2.8 (SE)% is lower than the head extraction of progesterone, 53.7 ± 7.3%, but the brain distribution and retention of [3H]R 5020 and [14C]progesterone are similar (Table 4). The brain distribution and retention of [14C]progesterone in the present study are the same as that reported for a much larger series of animals (15).
Results The MCR of [3H]R 5020, when infused alone or in combination with ["Clprogesterone, in estrogen-treated ovariectomized rabbits is 163 I/day (Table 1). The MCR
Discussion The blood MCR of R 5020 is lower than the MCR of progesterone in the rabbit. Since R 5020 does not bind to transcortin
TABLE 2. Uterine retention of [HC]progesterone and [3H]R 5020 in ovariectomized rabbits treated with estradiol Uterine tissue/femoral arterial blood ratio [14C] Progesterone Animals
[3H]R5020
Right horn
Left horn
R.H[. + L.H.*
Right horn
Left horn
R.H. + L.H.*
96 101 102 103
6.7 7.7
7.3 7.5
7.0 7.6
X ± SE
7.2
7.4
7.3
35.3 26.1 31.0 16.2 27.2 ± 4.1
38.5 25.3 32.1 20.0 29.0 ± 4.0
36.9 25.7 31.5 18.1 28.1 ± 4.0
The study was the same as that described in the legend to Table 1. After uterine venous blood was drawn, the uterus was removed, the right and left uterine horns were cleaned of fat and connective tissue, weighed and frozen until analysis for [14C]progesterone and [3H]R 5020. In a previous study (16) the uterine tissue/femoral blood arterial ratio of [14C]progesterone in 7 ovariectomized rabbits treated with estradiol was 5.1 ± 0.7 (SE). * Average of right and left horns.
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IN VIVO METABOLISM OF PROGESTERONE AND R 5020
467
TABLE 3. Uterine extraction of [l4C]progesterone and [3H]R 5020 in ovariectomized rabbits treated with estradiol Uterine extraction (%) M
[3H]R5020
1' C] Progesterone Animals 94 96 101 102 103 X ± SE
Right horn
Left hom
R.H . + L.H.*
Right horn
Left horn
R.H. + L.H.*
30.0 57.5 20.8
26.6 40.5 27.2
28.3 49.0 24.0
36.1 ± 11.0
31.4 ± 4.5
33.7 ± 7.7
59.5 39.3 44.5 54.1 50.0 49.5 ± 3.5
64.0 37.3 52.4 57.8 54.6 53.2 ± 4.4
61.8 38.3 48.5 56.0 52.3 51.4 ± 4.0
The study was the same as that described in the legend to Table 1. At the end of the 4th h of infusion of radioactive steroids venous blood was obtained from veins draining the right and left uterine horns at the same time as blood was drawn from the femoral artery. The uterine extraction equals femoral arterial blood concentration - uterine venous blood concentration/femoral arterial blood concentration) x 100. In a previous study (16) the uterine extraction of [I4C]progesterone in 7 ovariectomized rabbits treated with estradiol was 27 ± 4 (SE)%.
* Average of right and left horns.
(2-3) and progesterone does, this result was terone. At the isotopic steady state, the exsomewhat unexpected since 5a-preg- traction of a steroid by the uterus would be nane-3,20-dione also does not bind to trans- due to metabolism. However, if the uterine cortin but its MCR is about twice that of tissue is not at an isotopic steady state, progesterone (16). However, the MCR of then the uterine extraction would be due to 20a-hydroxy-4-pregnen-3-one, which also distribution and metabolism of the steroid. binds only weakly to transcortin, is the same Since the uterine venous blood and tissue as the MCR of progesterone in the rabbit samples were obtained only at the end of a (Rahman et al., unpublished observations). 4 h infusion, we do not know if the uterine These results suggest that the MCR of progesterone and progestins may be more de- TABLE 4. Brain and pituitary distribution of [14C]propendent upon their metabolism in various gesterone and [ H]R 5020 in ovariectomized rabbits tissues than their binding to transcortin treated with estradiol (17). In addition, plasma proteins other than Ratio of tissue (DPM/g wet weight) to femoral artery transcortin {e.g., oroscomucoid) may in(DPM/ml) ± SE fluence the MCR of progesterone and R [ C] Pro5020. However, this possibility was not ingesterone [ H]R5020 [ ll]R5020/ vestigated. (n = 3) Tissue ["C] Progesterone (n = 5) In the Clauberg test 2.0 fig of R 5020 Cortex 3.32 ± 1.05 3.87 ± 0.27 1.17 3.17 ± 1.30 2.83 ±0.41 0.89 was reported to be as effective as 200 fxg of POA* 2.88 ± 0.99 2.84 ± 0.29 Hypothalamus 0.99 progesterone in producing the endometrial Thalanms 1.09 2.89 ± 1.95 3.16 ± 0.43 0.87 ± 2.09 4.32 ± 0.44 response in the rabbit (3). The greater Midbrain retieulum 4.95 4.17 ± 1.74 0.91 Central grey 3.81 ±0.19 biological activity of R 5020 in the Clau- Pons reticulum 4.67 ± 0.66 5.08 ± L.99 0.92 5.32 + 2.55 4.33 ± 0.76 0.81 berg test cannot be explained by differences Pons 3.80 ± 0.58 3.35 ± 1.15 Cerebellum 1.13 in the rate of clearance of progesterone Hippocampus 2.76 ± 0.97 0.89 2.47 ± 0.23 0.97 3.05 ± 1.29 2.97 ± 0.37 and R 5020 from the blood alone since Amygdala 1.77 Anterior pituitary 2.25 ± 0.58 3.99 ± 1.41 the MCR of progesterone is only 1.5 times Posterior pituitary 4.01 ± 1.58 3.91 ± 0.75 0.98 greater than the MCR of R 5020. * Preoptie-anterior hypothalamus. Although the MCR of R 5020 was lower The study was the same as that described in the legend of Table 1. brain and pituitary were removed and frozen until the brains were than that of progesterone, the uterine ex- The dissected and the samples weighed and analyzed for [ C]progesterone traction of R 5020 was twice that of proges- and [ H]R5020. 3
IJ
3
3
14
3
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Endo • 1977 Vol 101 • No 2
RAHMAN ETAL.
468
tissue was at the isotopic steady state with respect to R 5020. The higher extraction of R 5020 may be due, in part, to a greater accumulation of R 5020 than progesterone in the uterine tissue and/or an increased metabolism. The accumulation of progesterone and R 5020 in the uterus is most likely due to the presence of progesterone receptors induced by the estrogen treatment of the rabbit (16, 18, 19). Also, the four times greater retention of R 5020 than progesterone in the uterus compares with the reported 3-4 times greater affinity of R 5020 than progesterone for the rabbit uterine progesterone receptor (3). The head extraction of R 5020 was lower
than the head extraction of progesterone which suggests that the head can metabolize progesterone more readily than R 5020. The retention and distribution of R 5020 and progesterone were the same in the brain and pituitary. As in previous studies (6-9, 15) no evidence for a selective accumulation of a progestin in the pituitary or hypothalamus was observed. This would suggest that progesterone and R 5020 accumulation in the brain are not dependent on the same mechanism(s) as that of the uterus, or if there is a progesterone receptor present in CNS, then its quantity is not sufficient to show selective retention of progesterone or R 5020 in brain areas studied, or its relative binding affinity for progesterone and R 5020 is different than the uterine progesterone receptors). If there is a low concentration of progesterone receptor(s) in the brain, they may have become saturated with the progesterone secreted by the adrenals of the ovariectomized rabbits (20). Acknowledgments The authors wish to acknowledge the technical assistance of Mr. Augusts Heinsons and Mrs. Helen
Chen during the course of this work. We thank Dr. J. P. Raynaud, Roussel-UCLAF, Romainville, France for his generous gift of [3H]R 5020 and R 5020.
References 1. Thi, M. T. L., E. E. Baulieu, and E. Milgrom, J Endocrinol 66: 349, 1975. 2. Raynaud, J.-P., M.-M. Bouton, E. Philibert, and B. Vannier, In Labrie, F., J. Meites, and G. Pelletier (eds.), Hypothalamus and Endocrine Function, Plenum Press, New York, 1976, p. 171. 3. Philibert, D., and J.-P. Raynaud, Endocrinology 94: 627, 1974. 4. Philibert, D., and J.-P. Raynaud, Steroids 22: 89, 1973. 5. Philibert, D., and J.-P. Raynaud, Contraception 10: 457, 1974. 6. Billiar, R. B., B. Little, I. Kline, P. Reier, Y. Takaoka, and R. J. White, Brain Res 94: 99, 1975. 7. Wade, G. N., and H. H. Feder, Brain Res 45: 525, 1972. 8. Luttge, W. G., C. J. Wallis, and N. R. Hall, Brain Res 71: 105, 1974. 9. Karavolas, H. J., D. Hodges, and D. O'Brien, Endocrinology 98: 164, 1976. 10. Seiki, K., and M. Hattori, Endocrinol Jap 20: 111, 1973. 11. Sar, M., and W. E. Stumpf, Science 182: 1266, 1973. 12. Atger, J., E.-E. Baulieu, and E. Milgrom, Endocrinology 94: 161, 1974. 13. Kato, J. J Steroid Biochem 6: 979, 1975. 14. Allen, W. M.J Clin Endocrinol Metab 10: 71,1950. 15. Johnson, W. A., R. B. Billiar, S. S. Rahman, and B. Little, Brain Res 111: 147, 1976. 16. Little, B., R. B. Billiar, S. S. Rahman, W. A. Johnson, Y. Takaoda, and R. J. White, Am J Obstet Gynecol 123: 527,'1975. 17. Billiar, R. B., M. Jassani, and B. Little, Am] Obstet Gynecol 121: 877, 1974. 18. Faber, L. E., M. L. Sandmann, and H. E. Stavely, 7 Biol Chem 247: 5648, 1972. 19. Rao; B. R., W. G. Wiest, and W. M. Allen, Endocrinology 92: 1229, 1973. 20. Rahman, S. S., R. B. Billiar, and B. Little, Biol Reprod 12: 305, 1975.
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