189
Ctinica Corsica Acta, 63 (1975) 189-192 0 Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
CCA 7256
EFFECT OF ESTROGEN-CONTAINING ORAL CONTRACEPTIVES ON URINARY CORTI~OSTEROID SULFATE EXCRETION
W.E. FAHL and D.P. ROSE Division of Clinical Oncology, (Received
University Hospitals, Madison, Wise. 53706
(U.S.A.)
April 21,1975)
Summary The excretion of corticosteroid sulfates and free cortisol in urine, and the total plasma cortisol, have been determined in 41 women receiving an estrogencontaining oral contraceptive and 53 age-matched female controls. The contraceptive steroids caused increases in the urinary corticosteroid sulfates and plasma cortisol similar to those observed in pregnancy; urinary free cortisol was unchanged.
Introduction The excretion of corticosteroid sulfates (CS) in urine [ 1,2] and the plasma cortisol [3] are elevated in the later stages of pregnancy. Recently it was found that these steroid conjugates are increased also in the urine of some breast cancer patients [4,5]. The suggestion was made that the finding of an elevated CS excretion might serve as a predictor of tumor response to hormone manipulation [4]. As an initial approach to defining more clearly the influence of other steroids on urinary CS levels we have determined the excretion of the conjugates and urinary free cortisol, and the plasma cortisol, in groups of women receiving estrogen-cont~ning oral contraceptives and age-matched controls. Subjects
and methods
Forty-one women (aged 20-47 years) were studied when they had been taking one of the estrogen-containing oral contraceptives listed in Table I for at least 6 months. The controls were 53 healthy women aged 19-45 years who were not taking any form of steroid therapy. Plasma samples and 24 h urine collections were obtained from the controls during the early luteal phase of the menstrual cycle, and from the oral contra-
I /.iO (Ortho)
Orthc+N-K~~\um Norln?l-J Oraci,n
(Svntey) 1 /x0
+ 80 (.llt,ad
ovult’n-21
(orth~l)
(Svnteu) .Johnson)
1 mg
(Srarlc)
Fnovld-F:
(SearIt’)
ceptive users when they had been taking their pill for 15. 21 days of a cycle. The blood samples were taken into heparinized tubes at 778 a.m. on the day of’ the urine collection. All specimens were stored at -2O’C: until analyzed. Plasma cortisol [ 61, urinary free cortisol [ 7 1, and CS 12 1 were assayed by competitive protein-binding techniques. Hydrolysis of the urinary sulfate conjugates was performed by the procedure of Burstein and Lieherman IS], and a petroleum ether treatment was included in the extraction step 121. The urinary CS as determined represented the sum of cortisol, c*ortisone. cxorticosterone. and 1 l-dehydrocorticosterone sulfates [ 2 1. Results The total plasma cortisol, urinary free cortisol, and urinary CS results are summarized in Table II. The oral contraceptive users have been divided into women taking a preparation containing 0.05 mg of estrogen and those receiving a higher dose of ethinyl estradiol or mestranol. At the same dose, the two CS-
TABL,E
II
PLASM.4 LEVELS Results
CORTJSOL. J:OR iire
URINARY
CONTROLS
given
as
mean
AND
FRE:E ORAL
S.1).
COR’J’JSOJ,
:\.?IKI
CONTRACEPTIVE
Numbrr
of
subjects
URINARY
COR’I‘JCOSTEROJJ)
SIIJ>FATb
CISERS m
rach
group
of
results
is glvrn
111 parenthwr~~.
Group
Controls
14.10
Oral contraceptive (0.05 Oral
mg
users
35.01
’
ll8.45 1 42.44 (5li) I;(;.19 (2(i)’
3.81i
(20)
52.36
7.02
(21)’
40.92
’ 25.20 (53) ’ 19.42 (26)
1.5x.92
47.21
’ 30.90 (151
1liO.03 ’ 77.42 (15)
estrogen)
contracept1cc
users
38.63
’ 10.82 (I I)’
(I-,0.05mg estrogrn) * Slgmficantly
dlfferrnt
from
control
group.
1’ I.
0.001,
191
trogens had indistinguishable effects on the corticosteroid levels and so they have been considered together. The plasma cortisol was increased markedly by oral contraceptive administration; there was no significant difference between the elevations seen in the low and high estrogen dose groups. The urinary excretion of free cortisol was unaltered by oral contraceptives, but these steroids caused a significant elevation in CS excretion (p < 0.001). Again, there was no difference in the amount of CS excreted by the two oral contraceptive groups (Table II); when considered together they showed a strong correlat.ion between their plasma cortisol and urinary CS (I’ = 0.596; p < 0.001). Discussion In the non-pregnant state CS are formed from free corticosteroids by a sulfurylating enzyme system which is located predominantly in the liver 191. Klein et al. [l) showed that the urinary excretion of these steroid conjugates is elevated during the third trimester of pregnancy. The fetal adrenal gland is considered to secrete corticosterone, ll-deoxycortieosterone and cortisol as the sulfate esters [lo] and so it was postulated that CS are transferred to the maternal circulation and thence excreted, unchanged, in the urine. The present study suggests that there are alternative sources of elevated CS in pregnancy. Pregnant women [3] and those treated with estrogens [3,11] or estrogen-containing oral contraceptives 111,121 have increased plasma cortisol levels. Although most of this steroid is protein-bound Keller et al. [ 131 have produced evidence that increased circulating co~icosteroids arising from estrogen administration can enter hepatocytes by pinocytosis and there dissociate from the binding globulin to yield the free steroid. This may provide an increased amount of substrate for the hepatic sulfurylating enzyme systems in both pregnant and estrogen-treated women. The strong correlation between the plasma cortisol and urinary CS in our oral contraceptive-treated group supports this proposition. An alternative possibility arises from the observation by Nielsen et al. 1141 that the urinary excretion of 5cu-metabolites of cortisol and corticosterone is decreased in pregnancy and women receiving estrogen-progestogen preparations. Kornel et al. 1151 found that hypertensive patients have an elevated level of 17-hydroxycorticosteroid sulfates and a corresponding decrease in the glucuronides. This was the result of diminished enzymic 5a:-reduction of cortisol and corticosterone, a necessary preliminary to glucuronide synthesis, so that more substrate becomes available for sulfoconjugation. Finally, there is evidence that the sulfu~lating enzymes are responsive to changes in estrogen levels. Torday et al. 1161 reported recently that ovariectomy causes a marked decrease in the sulfoconjugation of corticosterone by rat liver, and an increase in enzyme activity has been observed in estradiol-treated male rats (Fahl, W.E. and Rose, D.P., unpublished). Dao and Libby [9,17] have demonstrated the presence of steroid-sulfurylating enzymes in some human breast cancers; the absence of activity in the advanced disease was associated with failure to respond to adrenalec~omy. Ghosh et al. j4] determined urinary CS in breast cancer patients, and found
192
that approximately half of them had elevated levels which they spec*uiated were a reflection of tumor sulfurylase activity. Our finding of increased urinary C% excretion in women taking estrogen-containing oral contraceptives, and the experimental evidence for estrogen dependence of hepatic sulfurylation is of particular interest in the light of these breast cancer studies. With the possible exception of the liver, the urinary free cortisol provides an index of the overall exposure of tissue to the unbound, physiologically active, steroid. Although Burke [ll] showed that treatment with large doses of estrogen will increase the urinary free cortisol he did not find any significant change in oral contraceptive users, and our results confirm this observation. Acknowledgement This work was supported by Grant CA 13302 from the National Institutes of Health, and by Cancer Center Grant CA 14520, National Cancer Institute. References 1
Klein,
G.P.,
Kertew,
J.P.,
Ghan,
S.K.
and
Giroud,
C.J.P.
(1971)
J.
Clin.
Endocrinol.
Metab.,
32.
333-340 2
Ghosh,
3
Slaunwhite,
P.C..
4 Ghosh. 5
Lockwood, Jr, W.R.
P.C.,
Fahl.
E. and
and
Lockwood,
W.E.,
Rose,
Pennington,
E. and
D.P.,
A.A.
Sandberg,
G.W.
Pennington,
Liskowski.
(1972)
(1959)
L. and
G.W.
(1973)
Brown,
B.E.P.
(1967)
,I. Clin.
Endocrinol.
Metab.
27,
7
Murphy,
B.E.P.
(1968)
J. Clin.
Endocrinol.
Metab.
28,
8
Burstein,
S. and
9
Dao,
T.L.
200,
University
and
Libby,
10
Schweitzer,
M.,
Burke,
(1970)
12
Bulbrook,
R.D.,
13
Keller,
14
Nielsen.
N..
M.D.,
15
Kernel,
L..
16
Torday,
J.S..
17
Dno,
T.L.
P.R.
of Chicago
13
C.W.
(1972) C.
J. Clin. Hayward,
Stames, Klein, Libby,
and Giroud,
J.L.. and
C. and
W.R., G.P. P.R.
23,
Yates, S.R.
3. 75-81
J. 1. 328-330
Cancer
34.
16Sl-1695
973--990 343-348 233,
Target
Tissuesand
F.E.
331-335 Ncoplasia
(Dao,
T.L..
rd.),
PP. Ial--
(1969)
Hill.
Surgery
14.
Tong,
D.
Endocrinology Endocrinol.
(1969)
(1971) 66.
M.C.,
Acta A.
Steroids
519-532
11-18
Swain,
J. (1969) C.J.P.
(1969)
3.
M.,
and
Giroud,
(1969)
(1974)
Chem.
C..J.P.
Suppl.
Herian,
Starup.
Hill, and
Res.
Chicago
Pathot.
U.I.
Binder,
J. Biol.
in Estrogen
Press.
Branchaud,
Richardson,
and
S. (1958)
Lip.
BY. Med.
R.R.
G Murphy,
Lieberman,
Steroids
d. Clin. Invest. 38, 384-391
Can.
162-166
J. Clin.
and
84. 60.
D.Y.
(1973)
Lancet
49-62 473-485
Endocrinol.
J. Biochem.
Wang,
49,
Metab. 437-440
29.
1608m-1617
i,
Ctinica Corsica Acta, 63 (1975) 189-192 0 Elsevier Scientific Publishing Company,
Amsterdam
- Printed
in The Netherlands
CCA 7256
EFFECT OF ESTROGEN-CONTAINING ORAL CONTRACEPTIVES ON URINARY CORTI~OSTEROID SULFATE EXCRETION
W.E. FAHL and D.P. ROSE Division of Clinical Oncology, (Received
University Hospitals, Madison, Wise. 53706
(U.S.A.)
April 21,1975)
Summary The excretion of corticosteroid sulfates and free cortisol in urine, and the total plasma cortisol, have been determined in 41 women receiving an estrogencontaining oral contraceptive and 53 age-matched female controls. The contraceptive steroids caused increases in the urinary corticosteroid sulfates and plasma cortisol similar to those observed in pregnancy; urinary free cortisol was unchanged.
Introduction The excretion of corticosteroid sulfates (CS) in urine [ 1,2] and the plasma cortisol [3] are elevated in the later stages of pregnancy. Recently it was found that these steroid conjugates are increased also in the urine of some breast cancer patients [4,5]. The suggestion was made that the finding of an elevated CS excretion might serve as a predictor of tumor response to hormone manipulation [4]. As an initial approach to defining more clearly the influence of other steroids on urinary CS levels we have determined the excretion of the conjugates and urinary free cortisol, and the plasma cortisol, in groups of women receiving estrogen-cont~ning oral contraceptives and age-matched controls. Subjects
and methods
Forty-one women (aged 20-47 years) were studied when they had been taking one of the estrogen-containing oral contraceptives listed in Table I for at least 6 months. The controls were 53 healthy women aged 19-45 years who were not taking any form of steroid therapy. Plasma samples and 24 h urine collections were obtained from the controls during the early luteal phase of the menstrual cycle, and from the oral contra-
I /.iO (Ortho)
Orthc+N-K~~\um Norln?l-J Oraci,n
(Svntey) 1 /x0
+ 80 (.llt,ad
ovult’n-21
(orth~l)
(Svnteu) .Johnson)
1 mg
(Srarlc)
Fnovld-F:
(SearIt’)
ceptive users when they had been taking their pill for 15. 21 days of a cycle. The blood samples were taken into heparinized tubes at 778 a.m. on the day of’ the urine collection. All specimens were stored at -2O’C: until analyzed. Plasma cortisol [ 61, urinary free cortisol [ 7 1, and CS 12 1 were assayed by competitive protein-binding techniques. Hydrolysis of the urinary sulfate conjugates was performed by the procedure of Burstein and Lieherman IS], and a petroleum ether treatment was included in the extraction step 121. The urinary CS as determined represented the sum of cortisol, c*ortisone. cxorticosterone. and 1 l-dehydrocorticosterone sulfates [ 2 1. Results The total plasma cortisol, urinary free cortisol, and urinary CS results are summarized in Table II. The oral contraceptive users have been divided into women taking a preparation containing 0.05 mg of estrogen and those receiving a higher dose of ethinyl estradiol or mestranol. At the same dose, the two CS-
TABL,E
II
PLASM.4 LEVELS Results
CORTJSOL. J:OR iire
URINARY
CONTROLS
given
as
mean
AND
FRE:E ORAL
S.1).
COR’J’JSOJ,
:\.?IKI
CONTRACEPTIVE
Numbrr
of
subjects
URINARY
COR’I‘JCOSTEROJJ)
SIIJ>FATb
CISERS m
rach
group
of
results
is glvrn
111 parenthwr~~.
Group
Controls
14.10
Oral contraceptive (0.05 Oral
mg
users
35.01
’
ll8.45 1 42.44 (5li) I;(;.19 (2(i)’
3.81i
(20)
52.36
7.02
(21)’
40.92
’ 25.20 (53) ’ 19.42 (26)
1.5x.92
47.21
’ 30.90 (151
1liO.03 ’ 77.42 (15)
estrogen)
contracept1cc
users
38.63
’ 10.82 (I I)’
(I-,0.05mg estrogrn) * Slgmficantly
dlfferrnt
from
control
group.
1’ I.
0.001,
191
trogens had indistinguishable effects on the corticosteroid levels and so they have been considered together. The plasma cortisol was increased markedly by oral contraceptive administration; there was no significant difference between the elevations seen in the low and high estrogen dose groups. The urinary excretion of free cortisol was unaltered by oral contraceptives, but these steroids caused a significant elevation in CS excretion (p < 0.001). Again, there was no difference in the amount of CS excreted by the two oral contraceptive groups (Table II); when considered together they showed a strong correlat.ion between their plasma cortisol and urinary CS (I’ = 0.596; p < 0.001). Discussion In the non-pregnant state CS are formed from free corticosteroids by a sulfurylating enzyme system which is located predominantly in the liver 191. Klein et al. [l) showed that the urinary excretion of these steroid conjugates is elevated during the third trimester of pregnancy. The fetal adrenal gland is considered to secrete corticosterone, ll-deoxycortieosterone and cortisol as the sulfate esters [lo] and so it was postulated that CS are transferred to the maternal circulation and thence excreted, unchanged, in the urine. The present study suggests that there are alternative sources of elevated CS in pregnancy. Pregnant women [3] and those treated with estrogens [3,11] or estrogen-containing oral contraceptives 111,121 have increased plasma cortisol levels. Although most of this steroid is protein-bound Keller et al. [ 131 have produced evidence that increased circulating co~icosteroids arising from estrogen administration can enter hepatocytes by pinocytosis and there dissociate from the binding globulin to yield the free steroid. This may provide an increased amount of substrate for the hepatic sulfurylating enzyme systems in both pregnant and estrogen-treated women. The strong correlation between the plasma cortisol and urinary CS in our oral contraceptive-treated group supports this proposition. An alternative possibility arises from the observation by Nielsen et al. 1141 that the urinary excretion of 5cu-metabolites of cortisol and corticosterone is decreased in pregnancy and women receiving estrogen-progestogen preparations. Kornel et al. 1151 found that hypertensive patients have an elevated level of 17-hydroxycorticosteroid sulfates and a corresponding decrease in the glucuronides. This was the result of diminished enzymic 5a:-reduction of cortisol and corticosterone, a necessary preliminary to glucuronide synthesis, so that more substrate becomes available for sulfoconjugation. Finally, there is evidence that the sulfu~lating enzymes are responsive to changes in estrogen levels. Torday et al. 1161 reported recently that ovariectomy causes a marked decrease in the sulfoconjugation of corticosterone by rat liver, and an increase in enzyme activity has been observed in estradiol-treated male rats (Fahl, W.E. and Rose, D.P., unpublished). Dao and Libby [9,17] have demonstrated the presence of steroid-sulfurylating enzymes in some human breast cancers; the absence of activity in the advanced disease was associated with failure to respond to adrenalec~omy. Ghosh et al. j4] determined urinary CS in breast cancer patients, and found
192
that approximately half of them had elevated levels which they spec*uiated were a reflection of tumor sulfurylase activity. Our finding of increased urinary C% excretion in women taking estrogen-containing oral contraceptives, and the experimental evidence for estrogen dependence of hepatic sulfurylation is of particular interest in the light of these breast cancer studies. With the possible exception of the liver, the urinary free cortisol provides an index of the overall exposure of tissue to the unbound, physiologically active, steroid. Although Burke [ll] showed that treatment with large doses of estrogen will increase the urinary free cortisol he did not find any significant change in oral contraceptive users, and our results confirm this observation. Acknowledgement This work was supported by Grant CA 13302 from the National Institutes of Health, and by Cancer Center Grant CA 14520, National Cancer Institute. References 1
Klein,
G.P.,
Kertew,
J.P.,
Ghan,
S.K.
and
Giroud,
C.J.P.
(1971)
J.
Clin.
Endocrinol.
Metab.,
32.
333-340 2
Ghosh,
3
Slaunwhite,
P.C..
4 Ghosh. 5
Lockwood, Jr, W.R.
P.C.,
Fahl.
E. and
and
Lockwood,
W.E.,
Rose,
Pennington,
E. and
D.P.,
A.A.
Sandberg,
G.W.
Pennington,
Liskowski.
(1972)
(1959)
L. and
G.W.
(1973)
Brown,
B.E.P.
(1967)
,I. Clin.
Endocrinol.
Metab.
27,
7
Murphy,
B.E.P.
(1968)
J. Clin.
Endocrinol.
Metab.
28,
8
Burstein,
S. and
9
Dao,
T.L.
200,
University
and
Libby,
10
Schweitzer,
M.,
Burke,
(1970)
12
Bulbrook,
R.D.,
13
Keller,
14
Nielsen.
N..
M.D.,
15
Kernel,
L..
16
Torday,
J.S..
17
Dno,
T.L.
P.R.
of Chicago
13
C.W.
(1972) C.
J. Clin. Hayward,
Stames, Klein, Libby,
and Giroud,
J.L.. and
C. and
W.R., G.P. P.R.
23,
Yates, S.R.
3. 75-81
J. 1. 328-330
Cancer
34.
16Sl-1695
973--990 343-348 233,
Target
Tissuesand
F.E.
331-335 Ncoplasia
(Dao,
T.L..
rd.),
PP. Ial--
(1969)
Hill.
Surgery
14.
Tong,
D.
Endocrinology Endocrinol.
(1969)
(1971) 66.
M.C.,
Acta A.
Steroids
519-532
11-18
Swain,
J. (1969) C.J.P.
(1969)
3.
M.,
and
Giroud,
(1969)
(1974)
Chem.
C..J.P.
Suppl.
Herian,
Starup.
Hill, and
Res.
Chicago
Pathot.
U.I.
Binder,
J. Biol.
in Estrogen
Press.
Branchaud,
Richardson,
and
S. (1958)
Lip.
BY. Med.
R.R.
G Murphy,
Lieberman,
Steroids
d. Clin. Invest. 38, 384-391
Can.
162-166
J. Clin.
and
84. 60.
D.Y.
(1973)
Lancet
49-62 473-485
Endocrinol.
J. Biochem.
Wang,
49,
Metab. 437-440
29.
1608m-1617
i,
