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Interaction of ethynyloestradiol sulphate with plasma proteins
INTERACTION
OF ETHYNYLOESTRADIOL WITH PLASMA PROTEINS
SULPHATE
M. J. REED* and K. FOTHERBY Department of Steroid Biochemistry, Royal Postgraduate Medical School, Ducane Road, London W 12, England
(Reeeiued 6 April
1979)
SUMMARY Gel filtration on Sephadex G200 showed that ethynyloestradiol-3-suiphate, like oestrone sulphate, was bound to albumin. This was confirmed by equilibrium dialysis. Two groups of binding sites were present on albumin; high-affinity ones with values for the number of binding sites (n) and equilibrium association constant (K) of 1and 1.0 x IO6 M- ’ for ethynyloestradiol suiphate and 0.5 and 0.9 x 10’ M-’ for oestrone sulphate and lower-affinity ones with n of about 7 for both conjugates and with K of 1.8 x 10“ M-’ for ethynyl~stradioi sulphate and 1.0 x 104M- ’ for oestrone sulphate. EthynyloestradioI sulphate binding to albumin could be displaced by oestrogen sulphates but not by unconjugated oestrogens. INTRODUCTION Naturally occurring oestrogens circulate in the blood as sulphate conjugates [l-3] which are almost completely bound to albumin [4-6]. After administration of ethynyloestradiol to humans most of the steroid in the circulation is in the form of a conjugate [7] which also has been shown to be a sulphate [8,9]. No previous investigation of the ~teraction of ethy nyloestradiol sulphate with plasma proteins has been reported and the binding would be of importance in determining the level of free ethynyloestradiol in blood. Binding studies were carried out with ethynyloestradiol-3-sulphate rather than with the 17-sulphate of 3,i ‘I-disulphate since other studies [9, lo] had suggested that sulphation of this steroid occurred at C-3.
MATERIALS AND METHODS
Materials
[6,7-3HJ-Ethynyl~s~adiol (SA ~Ci/~ol) was obtained as a gift from Schering AG, Berlin and [6,7-3H]-oestrone (SA 4OCi/mmol) from the Radiochemical Centre, Amersham, U.K. Ethynyloestradiol, oestrone and human serum albumin were obtained from Sigma, London. and oestradiol-3-sulphate (F2-3-S), and oestradiol-3, 17~-disu~phate (E2-3, 17-diS) from Leo, Helsingborg, Sweden. [6,7-3H]ethynyloestradiol-3-suiphate (EE-3-S) and EE3-S) were synthesized as described previously [ 11, 121. Oestrogen conjugates were stored in 0.5 ml concentrated ammonia in 1 1 95% ethanol [S]. Gel fi Itr~t~5n [6.7-‘HI-EE-3-S
(0.1 &i) was incubated with 1 ml
* Present address: Department of Chemical Pathology, St. Mary’s Hospital Medical School, London W2 IPG, England. 495
plasma diluted 1:3 with 0.15 M phosphate buffer pH 7.4 at 37°C for 1h. The diluted plasma (2 ml) was applied to a Sephadex G2# column (50 x l.Ocm) and protein eluted with 0. i 5 M phosphate buffer [i 31. Fractions (2ml) were collected for measurement of the extinction at 28Onm and for radioactivity determination. Samples were counted in a Beckman liquid scintillation spectrometer (Model LS 200 B) in 10mi of ~Di~~urne~ (Packard Ltd) sinti~tion fluid. S&icient counts were recorded to give a counting error of less than 5%. Equilibrium dialysis. Equilibrium dialysis was carried out using either 0.2% human serum albumin in phosphate buffer or plasma diluted 1:x) with phosphate buffer to contain an equivalent mount of albumin [S]. Protein solutions were dialysed overnight against at least 40~01. phosphate buffer before every experiment. [6,7-3H]-EE-3-S (0.05 PCi) or [6,7-3H]-oestrone sulphate (El-S, 0.05 &i) and the unlabelied oestrogen sulphates in ~n~ntrations from O-133.3 &ml were added to 5 ml protein solution or plasma and the solutions were dialysed in cellulose tubing (Visking Corporation, 22mm flat width) at 37°C for 16 h against 10ml phosphate buffer. Duplicate samples (0.5 ml) were taken from inside and outside the dialysis bag for ~dioacti~ty dete~nation and the amount of steroid present in an unbound state was calculated [4]. RESULTS
Column chromatography on Sephadex G.200
Using Sephadex G200 plasma proteins were eiuted in the order: macrogiobulins (a and B globulins), globulins (mainly y globulins) and albumin. As shown in Fig. 1 [6,7-3HJ-EE-3-S was associated only with the albumin fraction and similarly El-S was also shown to be associated with this fraction.
496
M. J. REEDand K.
3lt lx-3-s
binding by Sap&u
,o plccmc G-200
FOTHERBY
protwl wbmm chmm*~~apky
.3
1.2 0.0. 28Onm
.
I aa .
.2
Fig. I. Sephadex G200 coIumn chromatography of [3H]ethynyloestradioI sulphate bound to plasma proteins. Proteins ‘eluted are macroglobulins (fractions 15-23). globulins (fractions 25-42) and albumin (fractions 43-62).
Equilibrium
dialysis
The percentage binding of [%L]-EE-3-S and C3H]-E 1-S to 0.2% HSA is shown in Table I and a Sea&hard ,plot of this data is shown in Fig. 2. For both EE3-S and El-S there appeared to be at least two groups of binding sites on-&e albumin molecule with the concentration of high affinity binding sites for EE-3-S (approximately 29mol/l) being about twice that (15~mol/l) found for El-S. Assuming a molecular weight for albumin of 65,000, this gives a value for the number of binding sites (II) of about I and 0.5 for EE-3-S and for E I -S respectively per molecule of Plbumin. In addition to the hi& affinity binding sites there were a number of low affinity binding sites with n c 7 for both EE-3-S and El-S. Equilibrium association constants for the two binding sites were calculated to be 1.0 x 10” M- ’ and 1.8 x 104M-‘, for EE3-S and 0.9 x lo6 M-’ and 1.0 x lO*M-’ for El-S.
I :, 2o R ’ yF
lo
Fig. and : 2. Binding of [3H]-ethynyloestradiol-3-sulphate [“HI-oestrone sulphate to 0.27; human serum albumin studied by equilibrium dialysis. (M. ethynyloestradial-3-sulphate. A---A. oestrone sulphate).
Table I. Binding of oestrone sulphate (El-S) and ethynyloestradio.&3&phate (EE-3-S) to 0.2% human Serum albumin in 0.15M phosphate buffer. pH 7.4 and 37’C studied by equilibrium dialysis (Values are given for two separate experiments) Concentration of conjugate (&ml) 0
0.67 1.67 3.33 4.20 8.30 16.70 33.30
66.m 133.30
Ty0Binding El-S
EE-3-S
92.0.93.3 91.1.93.1 90.8.9 I.5 87.7.98.4 84.4,88.0 77.9.83.9 68.9, 71.0 60.0.66.0 49.0.55.2 36.5.39.8
96.6.97.0 96.4.97.0 952.96.6 95.6.95.8 94.9,95.7 93.6,95.4 90.9,92.9 80.2.83.9 65.4.70.8 48.4. 50.6
loI&_A 50
100 [Sl
( 150
200
Pmwl
Fig. 3. Binding of [‘HI-ethynyloestradiol-3-sulphate to 0.2% human -urn albumin and 5% plasma studied by equilibrium dialysis (-, 0.2% human serum albumin; A, 5% male plasma; A, 5% female plasma).
Interaction of ethynyloestradiol
The binding of [3H]-EE-3-S to 5% male and female plasma is compared with its binding to 0.2% HSA in Fig. 3. There was no significant difference in the binding of the conjugate in these three solutions confirming the results obtained by gel filtration that all the binding of EE-3-S in plasma is to albumin. Competitioe
binding of
other
oestrogens
The ability of unconjugated ethynyloestradiol and oestrone to compete with EE-3-S for the high affinity binding site was investigated. Addition of ethynyloestradiol or oestrone to the dialysis system to give a final concentration of 66.7 pg/ml did not displace C3H]-EE-3-S from the high affinity binding sites. In contrast when El-S, E2-3-S or E2-3,17-di-S were added at a similar concentration, C3H]-EE-3-S was displaced.
sulphate with plasma proteins
491
consistent with the presence of at least two groups of binding sites but other explanations for a non-linear Seatchard plot are possible [ 151. It has been postulated [6] from the binding of E2-3-S to 0.2% HSA that this conjugate might exist as a tetramer in addition ,to the monomer in solution. Preliminary attempts to purify the EE-3-S synthesized for the present study by Sephadex LH20 column chromatography using chloroform :methanol (1: 1, v/v) as solvents, suggested the presence of more than one form of EE-3-S as two distinct peaks were obtained, both having the same R, value on thin-layer chromatography as EE3-S. Acknowledgement-This work was supported by a grant from the Medical Research Council.
REFERENCES
1. Purdy R. H., Engel L. L. and Oncley J. L.: CharacterisDISCUSSION
As found for the sulphate conjugates of the naturally occurring oestrogens oestrone and oestradiol [S, 63. the present study has shown that EE-3-S circulates in blood bound to serum albumin. The concentration of ethynyloestradiol sulphate found in plasma 2 h after the administration of a contraceptive dose (50 pg) of ethynyloestradiol (1250 pg/ml) was much greater than that (< 100pg/ml) for the unconjugated steroid [7]. In the present study when a tracer amount of C3H]-EE-3-S was added to 5 ml undiluted plasma and submitted to equilibrium dialysis, 98.9% was present in a bound state. It is unlikely that this amount would be significantly reduced by a concentration of EE-3-S of about 1000 pg/ml since the results in Table 1 show that a concentration of EE-3-S of 670ng/ml did not significantly reduce the binding. Therefore in the human it would appear that even at the maximum concentrations of EE3-S that may occur in plasma after administration of EE, most will circulate in a form bound to serum albumin. Competition studies showed that oestrogen sulphates but not unconjugated oestrogens would displace C3H]-EE-3-S from albumin confirming previous observations [14] that the polar sulphate group is involved in the binding of oestrogen sulphates to serum albumin. Interestingly, when streptomyocin sulphate was included in the phosphate buffer in some initial experiments in order to inhibit bacterial growth, binding of C3H]-EE-3-S was greatly reduced showing that non-steroidal sulphates can also displace oestrogen sulphates from the albumin molecule. Although the association constants for the binding of EE-3-S to 0.2% HSA are similar to those reported for other oestrogen sulphates [S, 61 a difference in the concentration of high-affinity binding sites for EE3-S and El-S was apparent from the present study. Scatchard plot analysis of the binding data such as that illustrated in Fig. 2, is usually interpreted as being
ation of estrone sulphate from human plasma. J. biol. Chem. 236 (1961) 104~1050. 2. Smith 0. W. and Hagerman D. D.: Quantitative estimation of oestrogen conjugates in late pregnancy plasma. J. clin. Endocr. Metab. 25 (1965) 732-741. 3. Nunez M., Aedo A. R., Landgren B. M., Cekan S. Z. and Diczfalusy E.: Studies on the pattern of circulating steroids in the normal menstrual cycle. Actu endocr., Cope& 86 (1977) 621-633. 4. Sandberg A. A., Slaunwhite W. R. and Antonides H. N.: Binding of steroids to human plasma proteins. Recenr Progr. Horm. Res. 13 (1957) 209-267. 5. Rosenthal H. E., Pietrzak E., Slaunwhite W. R. and Sandberg A. A.: Binding of oestrone sulphate in human plasma. J. clin. Endocr. Metab. 34 (1972) 805-813. 6. Rosenthal H. E., Ludwig G. A., Pietrzak E. and Sandberg A. A.: Binding of the sulphates of oestradiol-171 to hutian serum albumin and plasma. J. clin. Endocr. Met& 41 (1975) 1144-l 154. 7. Warren R. J. and Fotherby K.: Radioimmunoassay of ethynyloestradiol or mestranol to human subjects. J. Endocr. 63 (1974) 3&31. 8. Reed M. J., Fotherby K. and Steele S. J.: Metabolism of ethynyloestradiol in man. J. Endocr. 55 (1972) 351-361. 9. Bird C. E. and Clark A. F.: Metabolic clearance rates and metabolism of mestranol and ethynyloestradiol in normal young women. J. clin. Endocr. Metab. 36 (1973) 296-302. 10. Khan F. S. and Fotherby K.: (1976) Unpublished. 11. Fex H., Lundvall K. E. and Olsson A.: Hydrogen sulphates of natural oestrogens. Acra them. &and. 22 ( 1968) 254-264. 12. Reed M. J. and Fotherby K.: Intestinal absorption of synthetic steroids. J. steroid Biochem. II (1979) 1107-1112. 13. Rivera, J. V., Tore-Goyco, E. and Mates, M. L.: Melecular sieve in the study of plasma proteins. Amer. J. Med. Sci. 249 (1965) 371-377. 14. Sandberg A. A., Rosenthal H. E., Schneider S. L. and Slaunwhite W. R.: Protein-steroid interactions and their role in the transport and metabolism of steroids. In Steroid Dynamics (Edited by G. Pincus, T. Nakao and J. F. Tait). Academic Press, New York and London (1966) b. 1. 15. Buller R. E.. Schrader W. T. and O’Mallev B. W.: Steroids and’the practical aspects of performing binding studies. J. steroid Biochem. 7 (1976) 321-326.
OF ETHYNYLOESTRADIOL WITH PLASMA PROTEINS
SULPHATE
M. J. REED* and K. FOTHERBY Department of Steroid Biochemistry, Royal Postgraduate Medical School, Ducane Road, London W 12, England
(Reeeiued 6 April
1979)
SUMMARY Gel filtration on Sephadex G200 showed that ethynyloestradiol-3-suiphate, like oestrone sulphate, was bound to albumin. This was confirmed by equilibrium dialysis. Two groups of binding sites were present on albumin; high-affinity ones with values for the number of binding sites (n) and equilibrium association constant (K) of 1and 1.0 x IO6 M- ’ for ethynyloestradiol suiphate and 0.5 and 0.9 x 10’ M-’ for oestrone sulphate and lower-affinity ones with n of about 7 for both conjugates and with K of 1.8 x 10“ M-’ for ethynyl~stradioi sulphate and 1.0 x 104M- ’ for oestrone sulphate. EthynyloestradioI sulphate binding to albumin could be displaced by oestrogen sulphates but not by unconjugated oestrogens. INTRODUCTION Naturally occurring oestrogens circulate in the blood as sulphate conjugates [l-3] which are almost completely bound to albumin [4-6]. After administration of ethynyloestradiol to humans most of the steroid in the circulation is in the form of a conjugate [7] which also has been shown to be a sulphate [8,9]. No previous investigation of the ~teraction of ethy nyloestradiol sulphate with plasma proteins has been reported and the binding would be of importance in determining the level of free ethynyloestradiol in blood. Binding studies were carried out with ethynyloestradiol-3-sulphate rather than with the 17-sulphate of 3,i ‘I-disulphate since other studies [9, lo] had suggested that sulphation of this steroid occurred at C-3.
MATERIALS AND METHODS
Materials
[6,7-3HJ-Ethynyl~s~adiol (SA ~Ci/~ol) was obtained as a gift from Schering AG, Berlin and [6,7-3H]-oestrone (SA 4OCi/mmol) from the Radiochemical Centre, Amersham, U.K. Ethynyloestradiol, oestrone and human serum albumin were obtained from Sigma, London. and oestradiol-3-sulphate (F2-3-S), and oestradiol-3, 17~-disu~phate (E2-3, 17-diS) from Leo, Helsingborg, Sweden. [6,7-3H]ethynyloestradiol-3-suiphate (EE-3-S) and EE3-S) were synthesized as described previously [ 11, 121. Oestrogen conjugates were stored in 0.5 ml concentrated ammonia in 1 1 95% ethanol [S]. Gel fi Itr~t~5n [6.7-‘HI-EE-3-S
(0.1 &i) was incubated with 1 ml
* Present address: Department of Chemical Pathology, St. Mary’s Hospital Medical School, London W2 IPG, England. 495
plasma diluted 1:3 with 0.15 M phosphate buffer pH 7.4 at 37°C for 1h. The diluted plasma (2 ml) was applied to a Sephadex G2# column (50 x l.Ocm) and protein eluted with 0. i 5 M phosphate buffer [i 31. Fractions (2ml) were collected for measurement of the extinction at 28Onm and for radioactivity determination. Samples were counted in a Beckman liquid scintillation spectrometer (Model LS 200 B) in 10mi of ~Di~~urne~ (Packard Ltd) sinti~tion fluid. S&icient counts were recorded to give a counting error of less than 5%. Equilibrium dialysis. Equilibrium dialysis was carried out using either 0.2% human serum albumin in phosphate buffer or plasma diluted 1:x) with phosphate buffer to contain an equivalent mount of albumin [S]. Protein solutions were dialysed overnight against at least 40~01. phosphate buffer before every experiment. [6,7-3H]-EE-3-S (0.05 PCi) or [6,7-3H]-oestrone sulphate (El-S, 0.05 &i) and the unlabelied oestrogen sulphates in ~n~ntrations from O-133.3 &ml were added to 5 ml protein solution or plasma and the solutions were dialysed in cellulose tubing (Visking Corporation, 22mm flat width) at 37°C for 16 h against 10ml phosphate buffer. Duplicate samples (0.5 ml) were taken from inside and outside the dialysis bag for ~dioacti~ty dete~nation and the amount of steroid present in an unbound state was calculated [4]. RESULTS
Column chromatography on Sephadex G.200
Using Sephadex G200 plasma proteins were eiuted in the order: macrogiobulins (a and B globulins), globulins (mainly y globulins) and albumin. As shown in Fig. 1 [6,7-3HJ-EE-3-S was associated only with the albumin fraction and similarly El-S was also shown to be associated with this fraction.
496
M. J. REEDand K.
3lt lx-3-s
binding by Sap&u
,o plccmc G-200
FOTHERBY
protwl wbmm chmm*~~apky
.3
1.2 0.0. 28Onm
.
I aa .
.2
Fig. I. Sephadex G200 coIumn chromatography of [3H]ethynyloestradioI sulphate bound to plasma proteins. Proteins ‘eluted are macroglobulins (fractions 15-23). globulins (fractions 25-42) and albumin (fractions 43-62).
Equilibrium
dialysis
The percentage binding of [%L]-EE-3-S and C3H]-E 1-S to 0.2% HSA is shown in Table I and a Sea&hard ,plot of this data is shown in Fig. 2. For both EE3-S and El-S there appeared to be at least two groups of binding sites on-&e albumin molecule with the concentration of high affinity binding sites for EE-3-S (approximately 29mol/l) being about twice that (15~mol/l) found for El-S. Assuming a molecular weight for albumin of 65,000, this gives a value for the number of binding sites (II) of about I and 0.5 for EE-3-S and for E I -S respectively per molecule of Plbumin. In addition to the hi& affinity binding sites there were a number of low affinity binding sites with n c 7 for both EE-3-S and El-S. Equilibrium association constants for the two binding sites were calculated to be 1.0 x 10” M- ’ and 1.8 x 104M-‘, for EE3-S and 0.9 x lo6 M-’ and 1.0 x lO*M-’ for El-S.
I :, 2o R ’ yF
lo
Fig. and : 2. Binding of [3H]-ethynyloestradiol-3-sulphate [“HI-oestrone sulphate to 0.27; human serum albumin studied by equilibrium dialysis. (M. ethynyloestradial-3-sulphate. A---A. oestrone sulphate).
Table I. Binding of oestrone sulphate (El-S) and ethynyloestradio.&3&phate (EE-3-S) to 0.2% human Serum albumin in 0.15M phosphate buffer. pH 7.4 and 37’C studied by equilibrium dialysis (Values are given for two separate experiments) Concentration of conjugate (&ml) 0
0.67 1.67 3.33 4.20 8.30 16.70 33.30
66.m 133.30
Ty0Binding El-S
EE-3-S
92.0.93.3 91.1.93.1 90.8.9 I.5 87.7.98.4 84.4,88.0 77.9.83.9 68.9, 71.0 60.0.66.0 49.0.55.2 36.5.39.8
96.6.97.0 96.4.97.0 952.96.6 95.6.95.8 94.9,95.7 93.6,95.4 90.9,92.9 80.2.83.9 65.4.70.8 48.4. 50.6
loI&_A 50
100 [Sl
( 150
200
Pmwl
Fig. 3. Binding of [‘HI-ethynyloestradiol-3-sulphate to 0.2% human -urn albumin and 5% plasma studied by equilibrium dialysis (-, 0.2% human serum albumin; A, 5% male plasma; A, 5% female plasma).
Interaction of ethynyloestradiol
The binding of [3H]-EE-3-S to 5% male and female plasma is compared with its binding to 0.2% HSA in Fig. 3. There was no significant difference in the binding of the conjugate in these three solutions confirming the results obtained by gel filtration that all the binding of EE-3-S in plasma is to albumin. Competitioe
binding of
other
oestrogens
The ability of unconjugated ethynyloestradiol and oestrone to compete with EE-3-S for the high affinity binding site was investigated. Addition of ethynyloestradiol or oestrone to the dialysis system to give a final concentration of 66.7 pg/ml did not displace C3H]-EE-3-S from the high affinity binding sites. In contrast when El-S, E2-3-S or E2-3,17-di-S were added at a similar concentration, C3H]-EE-3-S was displaced.
sulphate with plasma proteins
491
consistent with the presence of at least two groups of binding sites but other explanations for a non-linear Seatchard plot are possible [ 151. It has been postulated [6] from the binding of E2-3-S to 0.2% HSA that this conjugate might exist as a tetramer in addition ,to the monomer in solution. Preliminary attempts to purify the EE-3-S synthesized for the present study by Sephadex LH20 column chromatography using chloroform :methanol (1: 1, v/v) as solvents, suggested the presence of more than one form of EE-3-S as two distinct peaks were obtained, both having the same R, value on thin-layer chromatography as EE3-S. Acknowledgement-This work was supported by a grant from the Medical Research Council.
REFERENCES
1. Purdy R. H., Engel L. L. and Oncley J. L.: CharacterisDISCUSSION
As found for the sulphate conjugates of the naturally occurring oestrogens oestrone and oestradiol [S, 63. the present study has shown that EE-3-S circulates in blood bound to serum albumin. The concentration of ethynyloestradiol sulphate found in plasma 2 h after the administration of a contraceptive dose (50 pg) of ethynyloestradiol (1250 pg/ml) was much greater than that (< 100pg/ml) for the unconjugated steroid [7]. In the present study when a tracer amount of C3H]-EE-3-S was added to 5 ml undiluted plasma and submitted to equilibrium dialysis, 98.9% was present in a bound state. It is unlikely that this amount would be significantly reduced by a concentration of EE-3-S of about 1000 pg/ml since the results in Table 1 show that a concentration of EE-3-S of 670ng/ml did not significantly reduce the binding. Therefore in the human it would appear that even at the maximum concentrations of EE3-S that may occur in plasma after administration of EE, most will circulate in a form bound to serum albumin. Competition studies showed that oestrogen sulphates but not unconjugated oestrogens would displace C3H]-EE-3-S from albumin confirming previous observations [14] that the polar sulphate group is involved in the binding of oestrogen sulphates to serum albumin. Interestingly, when streptomyocin sulphate was included in the phosphate buffer in some initial experiments in order to inhibit bacterial growth, binding of C3H]-EE-3-S was greatly reduced showing that non-steroidal sulphates can also displace oestrogen sulphates from the albumin molecule. Although the association constants for the binding of EE-3-S to 0.2% HSA are similar to those reported for other oestrogen sulphates [S, 61 a difference in the concentration of high-affinity binding sites for EE3-S and El-S was apparent from the present study. Scatchard plot analysis of the binding data such as that illustrated in Fig. 2, is usually interpreted as being
ation of estrone sulphate from human plasma. J. biol. Chem. 236 (1961) 104~1050. 2. Smith 0. W. and Hagerman D. D.: Quantitative estimation of oestrogen conjugates in late pregnancy plasma. J. clin. Endocr. Metab. 25 (1965) 732-741. 3. Nunez M., Aedo A. R., Landgren B. M., Cekan S. Z. and Diczfalusy E.: Studies on the pattern of circulating steroids in the normal menstrual cycle. Actu endocr., Cope& 86 (1977) 621-633. 4. Sandberg A. A., Slaunwhite W. R. and Antonides H. N.: Binding of steroids to human plasma proteins. Recenr Progr. Horm. Res. 13 (1957) 209-267. 5. Rosenthal H. E., Pietrzak E., Slaunwhite W. R. and Sandberg A. A.: Binding of oestrone sulphate in human plasma. J. clin. Endocr. Metab. 34 (1972) 805-813. 6. Rosenthal H. E., Ludwig G. A., Pietrzak E. and Sandberg A. A.: Binding of the sulphates of oestradiol-171 to hutian serum albumin and plasma. J. clin. Endocr. Met& 41 (1975) 1144-l 154. 7. Warren R. J. and Fotherby K.: Radioimmunoassay of ethynyloestradiol or mestranol to human subjects. J. Endocr. 63 (1974) 3&31. 8. Reed M. J., Fotherby K. and Steele S. J.: Metabolism of ethynyloestradiol in man. J. Endocr. 55 (1972) 351-361. 9. Bird C. E. and Clark A. F.: Metabolic clearance rates and metabolism of mestranol and ethynyloestradiol in normal young women. J. clin. Endocr. Metab. 36 (1973) 296-302. 10. Khan F. S. and Fotherby K.: (1976) Unpublished. 11. Fex H., Lundvall K. E. and Olsson A.: Hydrogen sulphates of natural oestrogens. Acra them. &and. 22 ( 1968) 254-264. 12. Reed M. J. and Fotherby K.: Intestinal absorption of synthetic steroids. J. steroid Biochem. II (1979) 1107-1112. 13. Rivera, J. V., Tore-Goyco, E. and Mates, M. L.: Melecular sieve in the study of plasma proteins. Amer. J. Med. Sci. 249 (1965) 371-377. 14. Sandberg A. A., Rosenthal H. E., Schneider S. L. and Slaunwhite W. R.: Protein-steroid interactions and their role in the transport and metabolism of steroids. In Steroid Dynamics (Edited by G. Pincus, T. Nakao and J. F. Tait). Academic Press, New York and London (1966) b. 1. 15. Buller R. E.. Schrader W. T. and O’Mallev B. W.: Steroids and’the practical aspects of performing binding studies. J. steroid Biochem. 7 (1976) 321-326.