BIOCHEMICAL
MEDICINE
Steroid
12, 22-31 (1975)
Effects
on Fibrinogen
Embryonic JOHANNA
Chicken
Synthesis
by Cultured
Hepatocytesl
PINDYCK, MICHAEL W. MOSESSON, AND RICHARD D. LEVERE
M. W. ROOMI,
The Department of Medicine, State University of New York, Downstate Medical Center, Brooklyn, New York 11203 Received
July
8, 1974
Studies in man and other animals suggest that steroid hormones increase fibrinogen synthesis. Increased levels of plasma fibrinogen have been observed in pregnant women ( l), animals (2), and in women taking oral contraceptive medication ( 3). Estradiol or testosterone treatment of rats after gonadectomy was associated with increased levels of circulating fibrinogen (4). Administration of hydrocortisone to rabbits (5) resulted in a measurable increase in fibrinogen production. Studies of the livers in normal dogs, using fluorescent antifibrinogen sera, have revealed few (5%) fib rmo * gen containing hepatic parenchymal cells, whereas more than 50%of these cells contained fibrinogen in livers from pregnant dogs (6). Perfusion of isolated rat liver with cortisol containing solutions resulted in increased fibrinogen synthesis ( 7). Reports have indicated that there is an increased incidence of stroke (8, 9) and thromboembolic disease (10, 11, 12) in women using oral contraceptive medication. The relationship between alterations in the levels of coagulation proteins, including fibrinogen, observed in women taking these drugs, and the apparent “hypercoagulable state” presumed to underlie these conditions, is uncertain. The role of oral contraceptive agents in contributing to the observed changes in the coagulation proteins has been difficult to define, a consideration which prompted the present studies. Fibrinogen has a central role in the coagulation process, is produced in relatively large amounts, and is readily identifiable by its biological capacity to form a clot or by specific antisera. Investigation of the effect of steroid hormones on fibrinogen production in tissue culture was therefore undertaken. For this purpose the technique of chicken embryo Iiver cell culture, developed by Granick ( 13), which favors the growth of
1 This
work
has appeared,
in part,
in abstract
22 Copyright All rights
@ 1975 by Academic Press, of reproduction in any form
Inc. reserved.
form.
J. Clin.
Invest.
51, 75a
( 1972).
FIBRINOGEN
SYNTHESIS
23
hepatic parenchymal cells, was adapted for the study of fibrinogen synthesis. The system was employed to test the hypothesis that some natural and synthetic sex steroid hormones exert a direct effect on the production of this protein. The results demonstrate that certain natural and synthetic steroids increase fibrinogen production. METHODS
Tissue Culture The tissue culture medium employed was prepared from the dry salts of Eagle’s Basal Medium (Grand Islands Biological Company, Berkeley, California). To each liter the following additions were made: 10 ml of 200 mM L-glutamine (Microbiological Associates), 100 ml Fetal Bovine Serum ( Microbiological Associates), 100 mg Streptomycin Sulfate ( Eli Lilly and Company, Indianapolis, Indiana), 100,000 units Penicillin G (Eli Lilly and Company), 25,000 units Mycostatin (E. R. Squibb & Son, Princeton, New Jersey), and 15.4 mg of Heparin (Connaught Medical Research Laboratories, Ontario, Canada). Chicken embryonic liver tissue was obtained from 16 day old White Leghorn chicken embryos (Shamrock Farms, N. J.). Sterile techniques were employed throughout all manipulations. Livers were removed from and Mg*+ free Earle’s solution. The 15 to 20 embryos and placed in Ca2+ livers were further washed in three successive changes of Earle’s solution, modified as above, and all visible fibrous strands were removed by dissection. The tissue was then finely minced with a razor blade, in a solution containing 200 mg of Trypsin (Worthington Biochemical Company, Freehold, N. J. ), and 30 mg of Pangestin (Difco Laboratories, Detroit, Michigan), dissolved in 21 ml of Ca2+ and Mg2+ free Earle’s solution. The minced tissue suspension was incubated in the enzyme solution at 37°C for 18-20 min. During the incubation period the suspension was passed into and out of a Pasteur pipette to assist separation into a single cell suspension. Following incubation the cell suspension was transferred to a conical centrifuge tube and permitted to stand for 5 min to allow large clumps of cells to settle. The supernatant cell suspension was then removed and added to a portion of the culture medium; the final volume of the suspension was adjusted by further addition of culture medium to give a final cell suspension containing 9 X lo5 cells/ml. Portions (2.5 ml) of the cell suspension were then transferred to 35 mm diam plastic culture dishes (Falcon Plastics, Bioquest, Los Angeles, California) and incubated at 37°C in 5% CO, in humid air. After 24 hr, the medium was removed by aspiration and the cell monolayer was washed with 1.0 ml of fresh culture medium to remove debris. Fresh culture medium (2.5 ml) which
24
PINDYCK
ET
AL.
contained steroids to be tested, or cycloheximide, was then added. All agents for testing were dissolved in 1 PI of propylene glycol per ml of medium. Control cultures were treated with propylene glycol alone (1 J/ml). For purposes of timed sampling of the culture, the time of the change of medium was taken as “starting time” (t = 0). The culture dishes were then incubated for 24 hr at 37°C in 5% CO, in humid air. At the end of this period the medium was removed from the cells by aspiration, centrifuged at 10,000 rpm at 4°C for 5 min, and the supernatant solution analyzed for fibrinogen content. The residual material in the culture dishes was washed three times with 2.0 ml cold saline. The cells were scraped from the dish with a rubber spatula and the total cell protein per dish determined ( 14). Crystalline bovine serum albumin was the standard. Ebctroimmunoassay Antiserum against adult chicken fibrinogen was produced in rabbits by three injections of about 1 mg of fibrinogen (Fraction I obtained from titrated plasma of adult White Leghorn chickens) in Freund’s complete adjuvant. The resulting antiserum was made specific for chicken fibrinogen by absorption with lyophilized chicken serum. The monospecific nature of the absorbed antiserum for fibrinogen was demonstrated by the formation of a single line after performance of immunodiffusion, immunoelectrophoresis ( 15) and electroimmunoassay ( 16) against chicken plasma. The specificity for fibrinogen was verified by the disappearance of the line after clotting the plasma with thrombin. Quantitation of the fibrinogen elaborated by the chick embryo hepatocytes into the surrounding media was accomplished by a modification of the Laurel1 technique of electroimmunoassay ( 16). The procedure was carried out on film strips (Cronar, 70 mm unperforated movie film, E. I. du Pont de Nemours and Co., Inc., Wilmington, Delaware) layered with 6 ml of agarose (E. Behring, Marburg-Lahn, Germany) in barbital buffer (ionic strength 0.05, 0.005 M EDTA, pH = 8.6), containing a dilution of antiserum previously determined to be optimal. A 2.4 ,J sample of standard plasma or medium for testing was placed in each of eight 2.6 PI wells, and electrophoresis was performed at room temperature for 150-180 min at a constant current of 16 mA per slide ( 14O-150 V). After electrophoresis the agarose covered film strips were immersed in a solution of tannic acid (0.5%) and acetic acid ( 1%) to develop the precipitin pattern. A standard curve was constructed for each slide, using dilutions of a reference chicken plasma which had been calibrated against a human fibrinogen standard. The sensitivity of the system, under the conditions of use, allowed quantitation of chicken fibrinogen at concentrations as low as 1 pg/ml.
FIBRINOGEN
25
SYNTHESIS
RESULTS Fibrinogen
Production
by Chicken
Hepatocytes
Fibrinogen was consistently demonstrable in the hepatocyte culture medium after 24 hr of incubation, and the level was great enough to permit direct quantitation, without concentration of the medium. The amount of fibrinogen produced in 24 hr in control dishes of different experiments varied widely (0.7 pg-2.0 rg of fibrinogen per 100 pg of cell protein). However, within any given experiment the variability between control culture dishes was small (SEM ~fr 0.03-0.09 pg fibrinogen/ 100 pg cell protein) ( 17). The production of fibrinogen under any given experimental condition was determined from the means of the fibrinogen produced by 3-5 culture dishes. No visible clots were observed in unconcentrated samples of media after addition of thrombin, presumably because of the low concentration of fibrinogen. However, addition of thrombin to media from control or steroid containing cultures resulted in loss of the fibrinogen precipitin pattern detected by electroimmunoassay when compared with saline treated samples. The absence of fibrinogen by electroimmunoassay after thrombin treatment is interpreted as due to the formation of fibrin which failed to enter the agarose gel. These observations strongly suggest that the cells produce fibrinogen molecules which are functionally, as well as antigenically identifiable as fibrinogen. The Time Course of Fibrinogen of Cycloheximide
Production
and the Effect
Production of fibrinogen was linear during the first 24 hr after the medium change. It was important to establish whether the fibrinogen elaborated into the culture medium during the period of observation was the result of de no00 synthesis and release rather than due to release of preformed fibrinogen. To investigate this question, cycloheximide, an inhibitor of protein synthesis by virtue of its ability to interfere with the function of the ribosomal-mRNA complex (18), was added to unstimulated and steroid treated hepatocyte cultures at final concentrations of 0.05, 0.1, 0.25, 0.40, 1.0, and 2.0 pg/ml. At cycloheximide concentrations of 0.25 pg/ml or higher, fibrinogen was not detected in the culture medium of control or steroid treated cultures during a 24-hr sampling period (Fig. 1). At the lesser cycloheximide concentrations a relative decrease in the amount of fibrinogen produced was noted. These results support the conclusion that the fibrinogen produced during any given observation period results from the release of newly synthesized fibrinogen. Furthermore, there was no evidence to suggest the existence of a
26
PINDYCK
0
3
6
ET
8
AL.
12
I6
20
24
Hours
FIG. 1. Fibrinogen synthesis by cultured embryonic chicken hepatocytes during 24 hr. The medium contained 10” M mestranol (O---o), lOa M mestranol and 0.1 gg cycloheximide (A -A), or lo4 M mestranol and 0.25 ag cycloheximide (&--A). Each point represents the mean of triplicate determinations, differing by not more than 5%. Similar curves were observed in nonsteroid treated cultures.
significant storage pool of fibrinogen in the cultured hepatic parenchymal C&S.
Effect of Certain Natural and Synthetic Steroid Hormones on Fibrinogen Production Several steroids induced increased fibrinogen production in the tissue culture system after 24 hr (Table 1). The adrenal steroid, cortisol, increased fibrinogen production 21% above control values at HP8 M (p < 0.03). Three substituted estrogenic steroids also increased fibrinogen synthesis as follows: 17-0x0-1,3,5( lO)-estratrien3-yloxy acetic acid, 40% increase at 1O-sM (p < 0.05) (Fig. 2), ethinyl estradiol, 27%increase at
g q
1
,
,
Control IO“’ lo-‘~Io-~
,
,
10-a IO-’ 10-b
Concentration.
,
t
10-q
10-z
M
FIG. 2. Fibrinogen production by cultured embryonic chicken hepatocytes, in the presence of various concentration of 17-0x0-1,3,5( lO)-estratrien-3-yloxy acetic acid during 24 hr. Each point represents the mean of the fibrinogen produced by five dishes within the culture. The vertical lines through each point represent SEM.
17-acetate)
a Embryonic chicken hepatocytes were cultured from lo+ M to lo-” M. The fibrinogen produced * SEM.
pregnen-17Lu-ol-3,20-dione
M M
0.77 1.10
0.91
1.54 0.83
* 0.09 +_ 0.07
_+ 0.06
OF CHICK
1.08 1.77
1.16
1.87 1.18
+ 0.06 10.19
+ 0.07
++ 0.11 0.13
Stimulated
per 100 rg cell protein
CULTURES
*+ 0.076 0.06
Control
bg fibrinogen
1 IN PRIMARY
-
EMBRYO
in Eagle’s medium for 24 hr in the presence of the steroids indicated, by five dishes was related to 5 control dishes within each experiment.
lo* lo+
M
M M
Concentrations at which maximal fibrinogen production observed
TABLE PRODUCTION
lo-’ acid
FIBRINOGEN
Ethinyl estradiol(l,3,5(10)-estratrien-17=ethinyl-3,17fi-diol) 17-oxo-1,3,5(lO)estratrien-3-yloxy acetic Medroxyprogesterone acetate(6a-met,hyl-4-
TO STIMULATE
10” lo-*
Compound
OBSERVED
Cortisol(4-pregnen-ll&17q2l-triol-3,2O-dione) Mestranol(l,3,5(10)-estratrien-17cu-ethinyl3,178-diol 3-methyl ether)
STEROIDS
4070 60%
27yo
21% 40%
at concentrations
p < 0.05 p < 0.001
p < 0.02
pp
MEDICINE
Steroid
12, 22-31 (1975)
Effects
on Fibrinogen
Embryonic JOHANNA
Chicken
Synthesis
by Cultured
Hepatocytesl
PINDYCK, MICHAEL W. MOSESSON, AND RICHARD D. LEVERE
M. W. ROOMI,
The Department of Medicine, State University of New York, Downstate Medical Center, Brooklyn, New York 11203 Received
July
8, 1974
Studies in man and other animals suggest that steroid hormones increase fibrinogen synthesis. Increased levels of plasma fibrinogen have been observed in pregnant women ( l), animals (2), and in women taking oral contraceptive medication ( 3). Estradiol or testosterone treatment of rats after gonadectomy was associated with increased levels of circulating fibrinogen (4). Administration of hydrocortisone to rabbits (5) resulted in a measurable increase in fibrinogen production. Studies of the livers in normal dogs, using fluorescent antifibrinogen sera, have revealed few (5%) fib rmo * gen containing hepatic parenchymal cells, whereas more than 50%of these cells contained fibrinogen in livers from pregnant dogs (6). Perfusion of isolated rat liver with cortisol containing solutions resulted in increased fibrinogen synthesis ( 7). Reports have indicated that there is an increased incidence of stroke (8, 9) and thromboembolic disease (10, 11, 12) in women using oral contraceptive medication. The relationship between alterations in the levels of coagulation proteins, including fibrinogen, observed in women taking these drugs, and the apparent “hypercoagulable state” presumed to underlie these conditions, is uncertain. The role of oral contraceptive agents in contributing to the observed changes in the coagulation proteins has been difficult to define, a consideration which prompted the present studies. Fibrinogen has a central role in the coagulation process, is produced in relatively large amounts, and is readily identifiable by its biological capacity to form a clot or by specific antisera. Investigation of the effect of steroid hormones on fibrinogen production in tissue culture was therefore undertaken. For this purpose the technique of chicken embryo Iiver cell culture, developed by Granick ( 13), which favors the growth of
1 This
work
has appeared,
in part,
in abstract
22 Copyright All rights
@ 1975 by Academic Press, of reproduction in any form
Inc. reserved.
form.
J. Clin.
Invest.
51, 75a
( 1972).
FIBRINOGEN
SYNTHESIS
23
hepatic parenchymal cells, was adapted for the study of fibrinogen synthesis. The system was employed to test the hypothesis that some natural and synthetic sex steroid hormones exert a direct effect on the production of this protein. The results demonstrate that certain natural and synthetic steroids increase fibrinogen production. METHODS
Tissue Culture The tissue culture medium employed was prepared from the dry salts of Eagle’s Basal Medium (Grand Islands Biological Company, Berkeley, California). To each liter the following additions were made: 10 ml of 200 mM L-glutamine (Microbiological Associates), 100 ml Fetal Bovine Serum ( Microbiological Associates), 100 mg Streptomycin Sulfate ( Eli Lilly and Company, Indianapolis, Indiana), 100,000 units Penicillin G (Eli Lilly and Company), 25,000 units Mycostatin (E. R. Squibb & Son, Princeton, New Jersey), and 15.4 mg of Heparin (Connaught Medical Research Laboratories, Ontario, Canada). Chicken embryonic liver tissue was obtained from 16 day old White Leghorn chicken embryos (Shamrock Farms, N. J.). Sterile techniques were employed throughout all manipulations. Livers were removed from and Mg*+ free Earle’s solution. The 15 to 20 embryos and placed in Ca2+ livers were further washed in three successive changes of Earle’s solution, modified as above, and all visible fibrous strands were removed by dissection. The tissue was then finely minced with a razor blade, in a solution containing 200 mg of Trypsin (Worthington Biochemical Company, Freehold, N. J. ), and 30 mg of Pangestin (Difco Laboratories, Detroit, Michigan), dissolved in 21 ml of Ca2+ and Mg2+ free Earle’s solution. The minced tissue suspension was incubated in the enzyme solution at 37°C for 18-20 min. During the incubation period the suspension was passed into and out of a Pasteur pipette to assist separation into a single cell suspension. Following incubation the cell suspension was transferred to a conical centrifuge tube and permitted to stand for 5 min to allow large clumps of cells to settle. The supernatant cell suspension was then removed and added to a portion of the culture medium; the final volume of the suspension was adjusted by further addition of culture medium to give a final cell suspension containing 9 X lo5 cells/ml. Portions (2.5 ml) of the cell suspension were then transferred to 35 mm diam plastic culture dishes (Falcon Plastics, Bioquest, Los Angeles, California) and incubated at 37°C in 5% CO, in humid air. After 24 hr, the medium was removed by aspiration and the cell monolayer was washed with 1.0 ml of fresh culture medium to remove debris. Fresh culture medium (2.5 ml) which
24
PINDYCK
ET
AL.
contained steroids to be tested, or cycloheximide, was then added. All agents for testing were dissolved in 1 PI of propylene glycol per ml of medium. Control cultures were treated with propylene glycol alone (1 J/ml). For purposes of timed sampling of the culture, the time of the change of medium was taken as “starting time” (t = 0). The culture dishes were then incubated for 24 hr at 37°C in 5% CO, in humid air. At the end of this period the medium was removed from the cells by aspiration, centrifuged at 10,000 rpm at 4°C for 5 min, and the supernatant solution analyzed for fibrinogen content. The residual material in the culture dishes was washed three times with 2.0 ml cold saline. The cells were scraped from the dish with a rubber spatula and the total cell protein per dish determined ( 14). Crystalline bovine serum albumin was the standard. Ebctroimmunoassay Antiserum against adult chicken fibrinogen was produced in rabbits by three injections of about 1 mg of fibrinogen (Fraction I obtained from titrated plasma of adult White Leghorn chickens) in Freund’s complete adjuvant. The resulting antiserum was made specific for chicken fibrinogen by absorption with lyophilized chicken serum. The monospecific nature of the absorbed antiserum for fibrinogen was demonstrated by the formation of a single line after performance of immunodiffusion, immunoelectrophoresis ( 15) and electroimmunoassay ( 16) against chicken plasma. The specificity for fibrinogen was verified by the disappearance of the line after clotting the plasma with thrombin. Quantitation of the fibrinogen elaborated by the chick embryo hepatocytes into the surrounding media was accomplished by a modification of the Laurel1 technique of electroimmunoassay ( 16). The procedure was carried out on film strips (Cronar, 70 mm unperforated movie film, E. I. du Pont de Nemours and Co., Inc., Wilmington, Delaware) layered with 6 ml of agarose (E. Behring, Marburg-Lahn, Germany) in barbital buffer (ionic strength 0.05, 0.005 M EDTA, pH = 8.6), containing a dilution of antiserum previously determined to be optimal. A 2.4 ,J sample of standard plasma or medium for testing was placed in each of eight 2.6 PI wells, and electrophoresis was performed at room temperature for 150-180 min at a constant current of 16 mA per slide ( 14O-150 V). After electrophoresis the agarose covered film strips were immersed in a solution of tannic acid (0.5%) and acetic acid ( 1%) to develop the precipitin pattern. A standard curve was constructed for each slide, using dilutions of a reference chicken plasma which had been calibrated against a human fibrinogen standard. The sensitivity of the system, under the conditions of use, allowed quantitation of chicken fibrinogen at concentrations as low as 1 pg/ml.
FIBRINOGEN
25
SYNTHESIS
RESULTS Fibrinogen
Production
by Chicken
Hepatocytes
Fibrinogen was consistently demonstrable in the hepatocyte culture medium after 24 hr of incubation, and the level was great enough to permit direct quantitation, without concentration of the medium. The amount of fibrinogen produced in 24 hr in control dishes of different experiments varied widely (0.7 pg-2.0 rg of fibrinogen per 100 pg of cell protein). However, within any given experiment the variability between control culture dishes was small (SEM ~fr 0.03-0.09 pg fibrinogen/ 100 pg cell protein) ( 17). The production of fibrinogen under any given experimental condition was determined from the means of the fibrinogen produced by 3-5 culture dishes. No visible clots were observed in unconcentrated samples of media after addition of thrombin, presumably because of the low concentration of fibrinogen. However, addition of thrombin to media from control or steroid containing cultures resulted in loss of the fibrinogen precipitin pattern detected by electroimmunoassay when compared with saline treated samples. The absence of fibrinogen by electroimmunoassay after thrombin treatment is interpreted as due to the formation of fibrin which failed to enter the agarose gel. These observations strongly suggest that the cells produce fibrinogen molecules which are functionally, as well as antigenically identifiable as fibrinogen. The Time Course of Fibrinogen of Cycloheximide
Production
and the Effect
Production of fibrinogen was linear during the first 24 hr after the medium change. It was important to establish whether the fibrinogen elaborated into the culture medium during the period of observation was the result of de no00 synthesis and release rather than due to release of preformed fibrinogen. To investigate this question, cycloheximide, an inhibitor of protein synthesis by virtue of its ability to interfere with the function of the ribosomal-mRNA complex (18), was added to unstimulated and steroid treated hepatocyte cultures at final concentrations of 0.05, 0.1, 0.25, 0.40, 1.0, and 2.0 pg/ml. At cycloheximide concentrations of 0.25 pg/ml or higher, fibrinogen was not detected in the culture medium of control or steroid treated cultures during a 24-hr sampling period (Fig. 1). At the lesser cycloheximide concentrations a relative decrease in the amount of fibrinogen produced was noted. These results support the conclusion that the fibrinogen produced during any given observation period results from the release of newly synthesized fibrinogen. Furthermore, there was no evidence to suggest the existence of a
26
PINDYCK
0
3
6
ET
8
AL.
12
I6
20
24
Hours
FIG. 1. Fibrinogen synthesis by cultured embryonic chicken hepatocytes during 24 hr. The medium contained 10” M mestranol (O---o), lOa M mestranol and 0.1 gg cycloheximide (A -A), or lo4 M mestranol and 0.25 ag cycloheximide (&--A). Each point represents the mean of triplicate determinations, differing by not more than 5%. Similar curves were observed in nonsteroid treated cultures.
significant storage pool of fibrinogen in the cultured hepatic parenchymal C&S.
Effect of Certain Natural and Synthetic Steroid Hormones on Fibrinogen Production Several steroids induced increased fibrinogen production in the tissue culture system after 24 hr (Table 1). The adrenal steroid, cortisol, increased fibrinogen production 21% above control values at HP8 M (p < 0.03). Three substituted estrogenic steroids also increased fibrinogen synthesis as follows: 17-0x0-1,3,5( lO)-estratrien3-yloxy acetic acid, 40% increase at 1O-sM (p < 0.05) (Fig. 2), ethinyl estradiol, 27%increase at
g q
1
,
,
Control IO“’ lo-‘~Io-~
,
,
10-a IO-’ 10-b
Concentration.
,
t
10-q
10-z
M
FIG. 2. Fibrinogen production by cultured embryonic chicken hepatocytes, in the presence of various concentration of 17-0x0-1,3,5( lO)-estratrien-3-yloxy acetic acid during 24 hr. Each point represents the mean of the fibrinogen produced by five dishes within the culture. The vertical lines through each point represent SEM.
17-acetate)
a Embryonic chicken hepatocytes were cultured from lo+ M to lo-” M. The fibrinogen produced * SEM.
pregnen-17Lu-ol-3,20-dione
M M
0.77 1.10
0.91
1.54 0.83
* 0.09 +_ 0.07
_+ 0.06
OF CHICK
1.08 1.77
1.16
1.87 1.18
+ 0.06 10.19
+ 0.07
++ 0.11 0.13
Stimulated
per 100 rg cell protein
CULTURES
*+ 0.076 0.06
Control
bg fibrinogen
1 IN PRIMARY
-
EMBRYO
in Eagle’s medium for 24 hr in the presence of the steroids indicated, by five dishes was related to 5 control dishes within each experiment.
lo* lo+
M
M M
Concentrations at which maximal fibrinogen production observed
TABLE PRODUCTION
lo-’ acid
FIBRINOGEN
Ethinyl estradiol(l,3,5(10)-estratrien-17=ethinyl-3,17fi-diol) 17-oxo-1,3,5(lO)estratrien-3-yloxy acetic Medroxyprogesterone acetate(6a-met,hyl-4-
TO STIMULATE
10” lo-*
Compound
OBSERVED
Cortisol(4-pregnen-ll&17q2l-triol-3,2O-dione) Mestranol(l,3,5(10)-estratrien-17cu-ethinyl3,178-diol 3-methyl ether)
STEROIDS
4070 60%
27yo
21% 40%
at concentrations
p < 0.05 p < 0.001
p < 0.02
pp
