The Maternal-Fetal Cortisol Gradient During Pregnancy and at Delivery1 ALAIN L. CAMPBELL AND BEVERLY E. PEARSON MURPHY2 Reproductive Physiology Unit, The Montreal General Hospital, and the Faculty of Medicine, McGill University, Montreal, Canada ABSTRACT. Diffusible cortisol (DF) was estimated as total cortisol x per cent bound, determined by equilibrium dialysis at 37 C against an equal concentration of albumin. Maternal DF values in late pregnancy were about 2Vfc times those of non-pregnant women. In cord serum, DF levels correlated (r = 0.95) with the total cortisol levels and were higher at term than in early pregnancy. Amniotic fluid DF levels were similar to cord levels in early pregnancy but were only about V2 those of cord serum at term. A transcortin-like protein was demonstrable in amniotic fluid in early and late pregnancy. Maternal venous levels were higher than cord levels but if allowance was made for placental conversion of maternal cortisol to cortisone (estimated to be
C
ORTISOL is known to be bound to two proteins in human blood, albumin which has a high capacity but low affinity and transcortin (corticosteroid-binding globulin, CBG) which has a low capacity and high affinity. Binding to CBG is highly specific while that to albumin is relatively non-specific. Since the "unbound" moiety of cortisol is considered to be the physiologically active form in blood, it warrants careful scrutiny in the consideration of fetalmaternal relationships. Previous reports, recently reviewed (2), vary greatly regarding the extent of cortisol binding in umbilical cord plasma. Methods used have included equilibrium dialysis, ultrafiltration and gel filtration for determination of per cent unbound cortisol, andfluorometry,colorimetry and competitive protein-binding for cortisol determination. Most of the values obtained for total cord cortisol levels have been too high because of large amounts of steroids,
Received May 13, 1976. 1 Presented in part to the 57th Annual Meeting of tlie Endocrine Society, New York, June 1975, and to the International Study Group on Steroid Hormones, Rome, December 1975 (1). 2 Associate, Medical Research Council of Canada.
80%), the cord levels were higher. Cord arterial levels were higher than cord venous levels, the difference being significant in the spontaneous-onset group. Cord arterial levels in the group with spontaneous-labor were higher than those of the combined induced and cesarean section groups although the maternal levels were similar in all three. These data accord with the concept that suppression of the fetal pituitary-adrenal axis is prevented by placental conversion of maternal cortisol to cortisone, that the bulk of circulating cord cortisol is derived from the fetus, and that there is a surge of cortisol in the fetus in association with spontaneous-onset labor which occurs independently of the mother. (/ Clin Endocrinol Metab 45: 435, 1977)
particularly cortisone and progesterone, which interfered with the assays (3). Another factor which must be considered is the form in which maternal cortisol reaches the fetus. Thirty minutes after injecting 3H-cortisol into the mother in early pregnancy, we found 85% of the extractable labelled material in cord blood to be cortisone and only 15% to be cortisone (4). Recently, we demonstrated in vitro that under equilibrium conditions 80% of cortisol was converted to cortisone by the placental villi both in early (8-20 wk) and late (32 to 40 wk) pregnancy (6) and have suggested that this conversion provides a mechanism whereby the fetus can maintain some degree of independence of cortisol production from the mother (7). The placental activity is considerable—about 80 ng/g/min of cortisol (about 40 /xg/min for a 500 g placenta) is converted to cortisone with very little back reaction (Murphy, unpublished observations). Because of its low affinity, albuminbound cortisol diffuses more readily than does CBG-bound cortisol. Recently, it was shown (Murphy, unpublished observations) that the rate of entry of 3H-cortisol into
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JCE & M . 1977 Vol 45 • No 3
CAMPBELL AND MURPHY
placental tissue in vitro, as measured by its rate of conversion to cortisone, was similar from protein-free media (75 ± 3 ng/g/min, n = 11) or maternal serum heated to 60 C for 20 min to denature CBG (79 ± 4 ng/g/min, n = 6) but was decreased in the presence of unheated maternal serum (51 ± 4 ng/g/ min, n = 6, P < 0.01). Incubation in albumin solutions (5-50 mg/ml) had little effect (see Addendum). It thus appears that in considering the fetal-maternal gradient across the placenta we are chiefly concerned with CBG binding. Albumin is present in much the same concentration in both circulations while the level of CBG is much higher in maternal plasma (1.2 x 10~6M) than in cord plasma (0.25 x 10-6M) (8). The present study describes the use of equilibrium dialysis of undiluted serum at 37 C against an equal volume of saline containing a similar content of albumin to assess the diffusible cortisol (DF, i.e., that which is not bound to CBG and therefore able to diffuse rapidly into the placenta) in fetal cord and maternal serum during pregnancy and at delivery. Some preliminary studies of cortisol binding in amniotic fluid are also reported. Materials and Methods Serum was obtained from an antecubital vein in non-pregnant women and in pregnant women at the time of vaginal delivery, elective cesarean section (C-section) without labor or hysterotomy. At term, a 30 cm segment of cord was clamped at both ends before delivery of the placenta and blood immediately taken from the two umbilical arteries and then the vein. From 10 to 20 weeks' gestation, blood was taken from the placental and fetal ends of the cord as described previously (9). Amniotic fluid was obtained at hysterotomy and at cesarean section. Non-radioactive steroids were obtained from the Sigma Chemical Co., St. Louis, Mo., and cortisol—1,2,6,7-3H (N) (SA 98 Ci/mMol) and cortisone—1,2-3H (N) (SA 48 Ci/mMol) from the New England Nuclear Corp., Boston, Mass. All were stored in redistilled ethanol at - 1 0 C. When required, radioactive cortisol was repurified by Sephadex LH-20 column chromatography
(methylene chloride: methanol 98:2) (10). Human albumin 25 g%, lot 832-1) was obtained through the Canadian Red Cross from Connaught Medical Research Laboratories, Toronto, Canada. Albumin concentrations were determined by electrophoresis on cellulose acetate in barbital buffer (pH 8.6). Unbound cortisol levels were measured in duplicate by equilibrium dialysis at 37 C using microdialysis cells (Model A, capacity 0.1 cc No. 9035/408, Chemical Rubber Co., Cleveland, Ohio). Each cell was prepared by inserting a moistened piece of Viskose casing between the two halves of the dialysis chamber and tightening the bolts. Using a Hamilton syringe, 0.1 ml serum was pipetted into one side, and 0.1 ml 0.9% saline solution containing tracer cortisol (15 pg) and albumin (in a concentration equal to that of the serum) into the other. The cells were then closed and placed in a gently shaking Dubnoff bath at 37 C for 2 h. The material on each side was transferred individually using a syringe with a flexible Teflon needle to a piece of parafilm where it formed a discrete drop. A Hamilton syringe was then used to transfer 50 fi\ of each drop to a counting vial, to which was added 10 ml scintillator solution. Each vial was counted for 10 min in a Phillips liquid scintillation counter (maximum efficiency for tritium 60%). The per cent free cortisol was calculated as cpmsaline aibumin/cpmplasma x 100. Total cortisol was determined by a specific radiotransinassay recently described (3). The CBG of horse serum was used as the binding protein, 3H-corticosterone as the tracer and Florisil as the adsorbent. Diffusible cortisol (DF) was calculated as the total cortisol x the fraction of cortisol not bound to transcortin.
Results
The maternal level of albumin (3.4%) was slightly lower (P < 0.025) than the cord level (4.3%) at term but higher (P< 0.001) than the cord level at 10-20 wk (1.5%) (Table 1). Since levels in amnioticfluidwere very low, albumin was omitted on the saline side during dialysis. Equilibrium time was determined by adding tritiated cortisol on the cord serum side and dialyzing against 4.0% albumin in saline, and also by adding the radioactive ma-
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437
MATERNAL-FETAL CORTISOL GRADIENT terial to the albumin side and dialyzing against cord serum until a constant value for unbound cortisol was obtained. No further change occurred from IY2 hrs to 72 h and 2 h was adopted for routine use. The precision (calculated from analysis of duplicates using the formula SD = VX(xi - X2)2/2n where Xi and X2 were the duplicate values and n the number of duplicate pairs) was ±3.2%. Values for total cortisol, per cent unbound cortisol and DF are given in Table 2. Total cortisol levels, per cent unbound and DF were all higher in all types of sample at delivery than at 10 to 20 wks. At delivery in maternal serum there were no differences in total cortisol among the various groups. In the mothers the per cent unbound cortisol was higher (P < 0.01) in the spontaneous group than in the elective C-section group but there was no significant difference in DF. Cord venous per cent unbound was higher (P
C
ORTISOL is known to be bound to two proteins in human blood, albumin which has a high capacity but low affinity and transcortin (corticosteroid-binding globulin, CBG) which has a low capacity and high affinity. Binding to CBG is highly specific while that to albumin is relatively non-specific. Since the "unbound" moiety of cortisol is considered to be the physiologically active form in blood, it warrants careful scrutiny in the consideration of fetalmaternal relationships. Previous reports, recently reviewed (2), vary greatly regarding the extent of cortisol binding in umbilical cord plasma. Methods used have included equilibrium dialysis, ultrafiltration and gel filtration for determination of per cent unbound cortisol, andfluorometry,colorimetry and competitive protein-binding for cortisol determination. Most of the values obtained for total cord cortisol levels have been too high because of large amounts of steroids,
Received May 13, 1976. 1 Presented in part to the 57th Annual Meeting of tlie Endocrine Society, New York, June 1975, and to the International Study Group on Steroid Hormones, Rome, December 1975 (1). 2 Associate, Medical Research Council of Canada.
80%), the cord levels were higher. Cord arterial levels were higher than cord venous levels, the difference being significant in the spontaneous-onset group. Cord arterial levels in the group with spontaneous-labor were higher than those of the combined induced and cesarean section groups although the maternal levels were similar in all three. These data accord with the concept that suppression of the fetal pituitary-adrenal axis is prevented by placental conversion of maternal cortisol to cortisone, that the bulk of circulating cord cortisol is derived from the fetus, and that there is a surge of cortisol in the fetus in association with spontaneous-onset labor which occurs independently of the mother. (/ Clin Endocrinol Metab 45: 435, 1977)
particularly cortisone and progesterone, which interfered with the assays (3). Another factor which must be considered is the form in which maternal cortisol reaches the fetus. Thirty minutes after injecting 3H-cortisol into the mother in early pregnancy, we found 85% of the extractable labelled material in cord blood to be cortisone and only 15% to be cortisone (4). Recently, we demonstrated in vitro that under equilibrium conditions 80% of cortisol was converted to cortisone by the placental villi both in early (8-20 wk) and late (32 to 40 wk) pregnancy (6) and have suggested that this conversion provides a mechanism whereby the fetus can maintain some degree of independence of cortisol production from the mother (7). The placental activity is considerable—about 80 ng/g/min of cortisol (about 40 /xg/min for a 500 g placenta) is converted to cortisone with very little back reaction (Murphy, unpublished observations). Because of its low affinity, albuminbound cortisol diffuses more readily than does CBG-bound cortisol. Recently, it was shown (Murphy, unpublished observations) that the rate of entry of 3H-cortisol into
435
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436
JCE & M . 1977 Vol 45 • No 3
CAMPBELL AND MURPHY
placental tissue in vitro, as measured by its rate of conversion to cortisone, was similar from protein-free media (75 ± 3 ng/g/min, n = 11) or maternal serum heated to 60 C for 20 min to denature CBG (79 ± 4 ng/g/min, n = 6) but was decreased in the presence of unheated maternal serum (51 ± 4 ng/g/ min, n = 6, P < 0.01). Incubation in albumin solutions (5-50 mg/ml) had little effect (see Addendum). It thus appears that in considering the fetal-maternal gradient across the placenta we are chiefly concerned with CBG binding. Albumin is present in much the same concentration in both circulations while the level of CBG is much higher in maternal plasma (1.2 x 10~6M) than in cord plasma (0.25 x 10-6M) (8). The present study describes the use of equilibrium dialysis of undiluted serum at 37 C against an equal volume of saline containing a similar content of albumin to assess the diffusible cortisol (DF, i.e., that which is not bound to CBG and therefore able to diffuse rapidly into the placenta) in fetal cord and maternal serum during pregnancy and at delivery. Some preliminary studies of cortisol binding in amniotic fluid are also reported. Materials and Methods Serum was obtained from an antecubital vein in non-pregnant women and in pregnant women at the time of vaginal delivery, elective cesarean section (C-section) without labor or hysterotomy. At term, a 30 cm segment of cord was clamped at both ends before delivery of the placenta and blood immediately taken from the two umbilical arteries and then the vein. From 10 to 20 weeks' gestation, blood was taken from the placental and fetal ends of the cord as described previously (9). Amniotic fluid was obtained at hysterotomy and at cesarean section. Non-radioactive steroids were obtained from the Sigma Chemical Co., St. Louis, Mo., and cortisol—1,2,6,7-3H (N) (SA 98 Ci/mMol) and cortisone—1,2-3H (N) (SA 48 Ci/mMol) from the New England Nuclear Corp., Boston, Mass. All were stored in redistilled ethanol at - 1 0 C. When required, radioactive cortisol was repurified by Sephadex LH-20 column chromatography
(methylene chloride: methanol 98:2) (10). Human albumin 25 g%, lot 832-1) was obtained through the Canadian Red Cross from Connaught Medical Research Laboratories, Toronto, Canada. Albumin concentrations were determined by electrophoresis on cellulose acetate in barbital buffer (pH 8.6). Unbound cortisol levels were measured in duplicate by equilibrium dialysis at 37 C using microdialysis cells (Model A, capacity 0.1 cc No. 9035/408, Chemical Rubber Co., Cleveland, Ohio). Each cell was prepared by inserting a moistened piece of Viskose casing between the two halves of the dialysis chamber and tightening the bolts. Using a Hamilton syringe, 0.1 ml serum was pipetted into one side, and 0.1 ml 0.9% saline solution containing tracer cortisol (15 pg) and albumin (in a concentration equal to that of the serum) into the other. The cells were then closed and placed in a gently shaking Dubnoff bath at 37 C for 2 h. The material on each side was transferred individually using a syringe with a flexible Teflon needle to a piece of parafilm where it formed a discrete drop. A Hamilton syringe was then used to transfer 50 fi\ of each drop to a counting vial, to which was added 10 ml scintillator solution. Each vial was counted for 10 min in a Phillips liquid scintillation counter (maximum efficiency for tritium 60%). The per cent free cortisol was calculated as cpmsaline aibumin/cpmplasma x 100. Total cortisol was determined by a specific radiotransinassay recently described (3). The CBG of horse serum was used as the binding protein, 3H-corticosterone as the tracer and Florisil as the adsorbent. Diffusible cortisol (DF) was calculated as the total cortisol x the fraction of cortisol not bound to transcortin.
Results
The maternal level of albumin (3.4%) was slightly lower (P < 0.025) than the cord level (4.3%) at term but higher (P< 0.001) than the cord level at 10-20 wk (1.5%) (Table 1). Since levels in amnioticfluidwere very low, albumin was omitted on the saline side during dialysis. Equilibrium time was determined by adding tritiated cortisol on the cord serum side and dialyzing against 4.0% albumin in saline, and also by adding the radioactive ma-
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 26 January 2015. at 05:12 For personal use only. No other uses without permission. . All rights reserved.
437
MATERNAL-FETAL CORTISOL GRADIENT terial to the albumin side and dialyzing against cord serum until a constant value for unbound cortisol was obtained. No further change occurred from IY2 hrs to 72 h and 2 h was adopted for routine use. The precision (calculated from analysis of duplicates using the formula SD = VX(xi - X2)2/2n where Xi and X2 were the duplicate values and n the number of duplicate pairs) was ±3.2%. Values for total cortisol, per cent unbound cortisol and DF are given in Table 2. Total cortisol levels, per cent unbound and DF were all higher in all types of sample at delivery than at 10 to 20 wks. At delivery in maternal serum there were no differences in total cortisol among the various groups. In the mothers the per cent unbound cortisol was higher (P < 0.01) in the spontaneous group than in the elective C-section group but there was no significant difference in DF. Cord venous per cent unbound was higher (P
