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Journal of Clinical Endocrinology and Metabolism Copyright © 1978 by The Endocrine Society
Vol. 46, No. 6 Printed in U.S.A.
Longitudinal Studies of Plasma Aldosterone, Corticosterone, Deoxycorticosterone, Progesterone, 17Hydroxyprogesterone, Cortisol, and Cortisone Determined Simultaneously in Mother and Child at Birth and during the Early Neonatal Period. I. Spontaneous Delivery* WOLFGANG G. SIPPELL, HENNING BECKER, HANS T. VERSMOLD, FRANK BIDLINGMAIER, AND DIETRICH KNORR Division of Pediatric Endocrinology, Department of Pediatrics, Children's Hospital, and Second Department of Obstetrics and Gynecology, University of Munich, Medical School, Munich, Germany ABSTRACT. In order to obtain the still lacking reference data of individual plasma steroids in the immediate postnatal period needed for the assessment of adrenocortical function in various neonatal maladaptation syndromes, aldosterone (A), corticosterone, deoxycorticosterone (DOC), progesterone (P), 17-hydroxyprogesterone (17-OHP), cortisol, and cortisone were simultaneously followed in the same human newborn in a single 250-500 /il peripheral plasma sample obtained at constant times during the first week of life using a mechanized Sephadex LH-20 multicolumn chromatography and standardized RIAs. Mean concentrations in ng/ml MV UV 2h
4h 6h 12 h 24 h 4 days 7 days
A
ell, Division of Pediatric Endocrinology, Universitats- out a prior purification step (3-5). This applies Cinderklinik, Lindwurmstrasse 4, D-8000 Miinchen-2, even more to the "low level" but physiologiJermany. * This work was supported by grants from the cally highly important corticosteroids, aldosDeutsche Forschungsgemeinschaft (SFB 147 and 51). Pre- terone and deoxycorticosterone. iminary aspects of this study were reported in abstract Using a recently developed mechanized orm at the Pediatric Congress, Joint Meeting ESPR, SSPE, and WGMM, June 20-24, 1976, Rotterdam, The Sephadex LH-20 chromatography (6, 7) in Netherlands. combination with sensitive RIAs, all major 971
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 28 September 2015. at 03:17 For personal use only. No other uses without permission. . All rights reserved.
SIPPELL ET AL.
972
corticosteroids and their immediate biosynthetic precursor steroids can be determined specifically and simultaneously in a small plasma sample (8). Plasma aldosterone (A), corticosterone, deoxycorticosterone (DOC), progesterone (P), 17-hydroxyprogesterone (17-OHP), cortisol, and cortisone to date have not been studied simultaneously either in maternal and cord plasma at delivery or longitudinally in the same infant throughout the early neonatal period. In the present study, these seven steroids, therefore, were followed systematically in a group of spontaneously delivered full term infants during their phase of adaptation from intrauterine to extrauterine life. One major objective of this study was to establish reference data of individual plasma corticosteroid levels of major physiological importance for the immediate postnatal period which might help to assess the adrenocortical involvement in various maladaptation syndromes occurring in the human neonate, e.g. the idiopathic respiratory distress syndrome, neonatal convulsions, apnea attacks, or the postmaturity syndrome. Materials and Methods Subjects A total of 12 randomly selected, healthy mothers was investigated at the time of spontaneous vaginal delivery of uncomplicated pregnancy at term. Informed consent was obtained from all mothers after approval of the study by the hospitals' ethical comTABLE
No.
mittees. Maternal age and parity, duration of labor, placental weight, fetal sex, gestational age, birth weight, and other pertinent clinical data are listed in Table 1. All pregnancies were uncomplicated, with no evidence of systemic or endocrine disease in either the mothers or the neonates. Postnatal course was uncomplicated in all infants. They were fed approximately 50 ml/kg on the first day, starting at 4-6 h after birth with 5% glucose, which was replaced at 8-12 h by a formula (Pomil) partially adapted to breast milk. Average daily feed volumes were 100 ml/kg BW on day 2 and 150 ml/kg from day 3 until the end of the first week of life. As the sodium and potassium contents of this formula were 1.9 and 2.15 meq/dl, respectively, the daily electrolyte intake averaged 1, 2, and 3 meq Na+ and K + /kg on days 1, 2, and 3-7, respectively. Maternal peripheral venous blood and umbilical venous blood was obtained by needle aspiration at delivery immediately after dissection of the umbilical cord, with the placenta still in situ. Neonatal blood samples of 1.0-1.5 ml were collected either by rapid puncture of a scalp or antecubital vein or by heel prick at 2, 4, 6,12, and 24 h as well as 4 and 7 days after birth in most instances. In infant 7, a 3-h sample was obtained instead of the 2- and 4-h samples. His 2- and 4-h data, therefore, were interpolated from the 0-, 3-, and 6-h values. Similarly, in infant 9, the 24-h sample was missed; in infant 12, the 12-h sample was missed. Instead, samples were taken at 32 and 13 h, respectively, the values of which were used to interpolate the 24- or 12-h data. There was no statistically significant change in the obtained mean values, whether the few interpolated data were included or omitted. Because of earlier discharge from the newborn nursery, 7-day samples could not be obtained in infants 5, 6, and 7. In order
1. Clinical data Maternal Duration of of Placental age, parity labor (h, Time birth wt(g) min) (yr)
1 2 3 4 5 6 7 8 9 10 11 12
29,1 26,11 21,11 22, III 21,11 27,11 23,11 21,1 27,11 26,11 34,1V 30,1
Mean
257/i2 4'/l2
± SD
JCE&M • 1978 Vol46 • No 6
„ , , sex Gestational „. ,, wt, v(g) .e>. Apgar score Fetal , , . Birth /,&._ min) .. age (weeks) (1/5
7,0 9, 10 4, 15 8,55 6,05 8,0 5, 12 12,0 4,25 2,20 8,0 7,35
1000 1100 1015 0855 1505 1100 1212 1400 1325 1020 1155 1035
700 600 680 590 640 720 810 680 600 850 800 730
F F M F F M M F F M M M
41 39 39 40 39.5 41 40 41 40 40 40 38.5
2900 2900 3050 3170 3180 3340 3350 3610 3700 3900 4100 3170
6,55 2,37
1133 150
700 87
6M, 6 F
39.9 0.82
3364
386
7/9 8/10 9/10 9/10 8/10 8/10 9/10 9/10
8/9 9/10
8/9 9/10 8.42/9.75 0.67/0.45
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MULTIPLE PLASMA STEROIDS IN NEONATES to prevent prolonged crying (9) of the infants, all neonatal blood samples were rapidly drawn by an experienced neonatologist. Thus, any significant stress to the neonate could be avoided in each instance. All blood samples were collected in sterile polystyrene tubes containing dried ammonium heparinate and were immediately centrifuged at 4 C. The plasma was then stored at — 20 C.
973
Results Maternofetal steroid gradients at vaginal delivery
P and 17-OHP. Individual and mean plasma levels of P and 17-OHP in the 12 mothers and in their newborn infants' umbilical veins are shown in Fig. 1. As could be expected from the placental and fetoplacental site of origin of P Multiple steroid RIAs and 17-OHP, respectively, both steroids After the addition of tracer amounts of each of showed considerably higher plasma concenthe seven steroids to each sample of 250-500 /til trations in the umbilical veins than in the plasma, unconjugated steroids were extracted twice maternal circulation, with the exception of with methylene chloride. The washed plasma expair 8 in which cord progesterone was slightly tract was subjected to a mechanized Sephadex LH20 multiple column chromatography, developed in lower (264 ng/ml) than in the mother, whose our laboratory (6), by which corticosterone, A, cor- plasma P level (298 ng/ml) was the highest of tisone, and cortisol were readily isolated, whereas the 12 mothers investigated and who also had P, DOC, and 17-OHP were collected in a combined the longest duration of labor (Table 1). Mean fraction and then separated from each other in a maternofetal gradients were 119.6 ± second chromatographic step on 40-cm LH-20 col24.7-270.8 ± 52.9 (SE) ng/ml (1.0:2.3) for P umns (7). After branching off a small aliquot for and 11.0 ± 2.4-32.9 ± 5.1 (SE) ng/ml (1:3) for internal standard recovery counting, each of the 17-OHP. Both gradients were highly signifiisolated steroid fractions was quantitated by RIAs, cant statistically (P < 0.01). Differentiation of which recently have been described in detail elsecord levels according to fetal sex revealed for where (8). The entire analytical procedure has been shown to be highly practicable and convenient, both P and 17-OHP significantly (P < 0.05) particularly for pediatric studies, and, in addition, higher plasma levels in the male [P, 363.2 ± 82.1 (SE) ng/ml; 17-OHP, 43.8 ± 7.4 (SE) to be at least as specific, sensitive, precise, and ng/ml] than in the female newborns [P, 178.4 accurate as most single steroid assays published to ± 46.6 (SE) ng/ml; 17-OHP, 21.9 ± 3.5 (SE) date. Coefficients of variation of the complete determination (including extraction, chromatography, ng/ml]. The mothers with male infants had a and RIA) were between 6.9 and 14.5% within assays higher mean 17-OHP level of 14.4 ± 4.0 (SE) and between 11.9 and 16.3% between assays, Tor all ng/ml, as compared to 7.7 ± 2.0 (SE) ng/ml in of the seven steroids. Because of the limited amount the mothers with female infants, although this of plasma available, about 50% of the DOC meassex difference was statistically not sufficiently urements from day 4 and 7 after birth fell below significant (P < 0.10). No such difference was the sensitivity of the assay, thus, relatively high SE seen within the maternal P levels [126.1 ± 32.0 values were obtained. Plasma sodium and potas(SE) ng/ml in the male, 113.1 ± 40.5 (SE) sium were determined by routine procedures (flame ng/ml in the female pregnancies]. Moreover, photometry). a significant (P < 0.05) positive correlation For statistical evaluations of differences between the means of two samples, either parametric (e.g. was found between placental weights and umbilical vein P concentrations, whereas no cort test or paired t test) or nonparametric tests of significance [e.g. Wilcoxon's signed rank test (10), relations were obtained either between plaMann-Whitney's U test (11)] were used. Statistical cental weights and maternal P or between comparisons of more than two means of paired maternal and fetal P levels. There was a fairly samples, i.e. analyzing the postnatal course of the high and significant correlation (r = 0.744, P mean plasma levels of a given steroid, were made < 0.01) between individual maternal and cord utilizing the ranked analysis of variance according venous levels of 17-OHP. No relationship, to Friedman (12), in combination with Wilcoxon and Wilcox's multiple comparison of paired samples however, was found between placental weights (13). Correlations between two different parameters and either maternal or cord 17-OHP concentrations. Differences in maternal parity were were checked by linear regression analysis.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 28 September 2015. at 03:17 For personal use only. No other uses without permission. . All rights reserved.
SIPPELL ET AL.
974 PROGESTERONE ng/ml
-•
JCE&M • 1978 V0U6 • No 6
17-HYDR0XYPR0GESTER0NE nmol/l r 200
p
Journal of Clinical Endocrinology and Metabolism Copyright © 1978 by The Endocrine Society
Vol. 46, No. 6 Printed in U.S.A.
Longitudinal Studies of Plasma Aldosterone, Corticosterone, Deoxycorticosterone, Progesterone, 17Hydroxyprogesterone, Cortisol, and Cortisone Determined Simultaneously in Mother and Child at Birth and during the Early Neonatal Period. I. Spontaneous Delivery* WOLFGANG G. SIPPELL, HENNING BECKER, HANS T. VERSMOLD, FRANK BIDLINGMAIER, AND DIETRICH KNORR Division of Pediatric Endocrinology, Department of Pediatrics, Children's Hospital, and Second Department of Obstetrics and Gynecology, University of Munich, Medical School, Munich, Germany ABSTRACT. In order to obtain the still lacking reference data of individual plasma steroids in the immediate postnatal period needed for the assessment of adrenocortical function in various neonatal maladaptation syndromes, aldosterone (A), corticosterone, deoxycorticosterone (DOC), progesterone (P), 17-hydroxyprogesterone (17-OHP), cortisol, and cortisone were simultaneously followed in the same human newborn in a single 250-500 /il peripheral plasma sample obtained at constant times during the first week of life using a mechanized Sephadex LH-20 multicolumn chromatography and standardized RIAs. Mean concentrations in ng/ml MV UV 2h
4h 6h 12 h 24 h 4 days 7 days
A
ell, Division of Pediatric Endocrinology, Universitats- out a prior purification step (3-5). This applies Cinderklinik, Lindwurmstrasse 4, D-8000 Miinchen-2, even more to the "low level" but physiologiJermany. * This work was supported by grants from the cally highly important corticosteroids, aldosDeutsche Forschungsgemeinschaft (SFB 147 and 51). Pre- terone and deoxycorticosterone. iminary aspects of this study were reported in abstract Using a recently developed mechanized orm at the Pediatric Congress, Joint Meeting ESPR, SSPE, and WGMM, June 20-24, 1976, Rotterdam, The Sephadex LH-20 chromatography (6, 7) in Netherlands. combination with sensitive RIAs, all major 971
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 28 September 2015. at 03:17 For personal use only. No other uses without permission. . All rights reserved.
SIPPELL ET AL.
972
corticosteroids and their immediate biosynthetic precursor steroids can be determined specifically and simultaneously in a small plasma sample (8). Plasma aldosterone (A), corticosterone, deoxycorticosterone (DOC), progesterone (P), 17-hydroxyprogesterone (17-OHP), cortisol, and cortisone to date have not been studied simultaneously either in maternal and cord plasma at delivery or longitudinally in the same infant throughout the early neonatal period. In the present study, these seven steroids, therefore, were followed systematically in a group of spontaneously delivered full term infants during their phase of adaptation from intrauterine to extrauterine life. One major objective of this study was to establish reference data of individual plasma corticosteroid levels of major physiological importance for the immediate postnatal period which might help to assess the adrenocortical involvement in various maladaptation syndromes occurring in the human neonate, e.g. the idiopathic respiratory distress syndrome, neonatal convulsions, apnea attacks, or the postmaturity syndrome. Materials and Methods Subjects A total of 12 randomly selected, healthy mothers was investigated at the time of spontaneous vaginal delivery of uncomplicated pregnancy at term. Informed consent was obtained from all mothers after approval of the study by the hospitals' ethical comTABLE
No.
mittees. Maternal age and parity, duration of labor, placental weight, fetal sex, gestational age, birth weight, and other pertinent clinical data are listed in Table 1. All pregnancies were uncomplicated, with no evidence of systemic or endocrine disease in either the mothers or the neonates. Postnatal course was uncomplicated in all infants. They were fed approximately 50 ml/kg on the first day, starting at 4-6 h after birth with 5% glucose, which was replaced at 8-12 h by a formula (Pomil) partially adapted to breast milk. Average daily feed volumes were 100 ml/kg BW on day 2 and 150 ml/kg from day 3 until the end of the first week of life. As the sodium and potassium contents of this formula were 1.9 and 2.15 meq/dl, respectively, the daily electrolyte intake averaged 1, 2, and 3 meq Na+ and K + /kg on days 1, 2, and 3-7, respectively. Maternal peripheral venous blood and umbilical venous blood was obtained by needle aspiration at delivery immediately after dissection of the umbilical cord, with the placenta still in situ. Neonatal blood samples of 1.0-1.5 ml were collected either by rapid puncture of a scalp or antecubital vein or by heel prick at 2, 4, 6,12, and 24 h as well as 4 and 7 days after birth in most instances. In infant 7, a 3-h sample was obtained instead of the 2- and 4-h samples. His 2- and 4-h data, therefore, were interpolated from the 0-, 3-, and 6-h values. Similarly, in infant 9, the 24-h sample was missed; in infant 12, the 12-h sample was missed. Instead, samples were taken at 32 and 13 h, respectively, the values of which were used to interpolate the 24- or 12-h data. There was no statistically significant change in the obtained mean values, whether the few interpolated data were included or omitted. Because of earlier discharge from the newborn nursery, 7-day samples could not be obtained in infants 5, 6, and 7. In order
1. Clinical data Maternal Duration of of Placental age, parity labor (h, Time birth wt(g) min) (yr)
1 2 3 4 5 6 7 8 9 10 11 12
29,1 26,11 21,11 22, III 21,11 27,11 23,11 21,1 27,11 26,11 34,1V 30,1
Mean
257/i2 4'/l2
± SD
JCE&M • 1978 Vol46 • No 6
„ , , sex Gestational „. ,, wt, v(g) .e>. Apgar score Fetal , , . Birth /,&._ min) .. age (weeks) (1/5
7,0 9, 10 4, 15 8,55 6,05 8,0 5, 12 12,0 4,25 2,20 8,0 7,35
1000 1100 1015 0855 1505 1100 1212 1400 1325 1020 1155 1035
700 600 680 590 640 720 810 680 600 850 800 730
F F M F F M M F F M M M
41 39 39 40 39.5 41 40 41 40 40 40 38.5
2900 2900 3050 3170 3180 3340 3350 3610 3700 3900 4100 3170
6,55 2,37
1133 150
700 87
6M, 6 F
39.9 0.82
3364
386
7/9 8/10 9/10 9/10 8/10 8/10 9/10 9/10
8/9 9/10
8/9 9/10 8.42/9.75 0.67/0.45
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 28 September 2015. at 03:17 For personal use only. No other uses without permission. . All rights reserved.
MULTIPLE PLASMA STEROIDS IN NEONATES to prevent prolonged crying (9) of the infants, all neonatal blood samples were rapidly drawn by an experienced neonatologist. Thus, any significant stress to the neonate could be avoided in each instance. All blood samples were collected in sterile polystyrene tubes containing dried ammonium heparinate and were immediately centrifuged at 4 C. The plasma was then stored at — 20 C.
973
Results Maternofetal steroid gradients at vaginal delivery
P and 17-OHP. Individual and mean plasma levels of P and 17-OHP in the 12 mothers and in their newborn infants' umbilical veins are shown in Fig. 1. As could be expected from the placental and fetoplacental site of origin of P Multiple steroid RIAs and 17-OHP, respectively, both steroids After the addition of tracer amounts of each of showed considerably higher plasma concenthe seven steroids to each sample of 250-500 /til trations in the umbilical veins than in the plasma, unconjugated steroids were extracted twice maternal circulation, with the exception of with methylene chloride. The washed plasma expair 8 in which cord progesterone was slightly tract was subjected to a mechanized Sephadex LH20 multiple column chromatography, developed in lower (264 ng/ml) than in the mother, whose our laboratory (6), by which corticosterone, A, cor- plasma P level (298 ng/ml) was the highest of tisone, and cortisol were readily isolated, whereas the 12 mothers investigated and who also had P, DOC, and 17-OHP were collected in a combined the longest duration of labor (Table 1). Mean fraction and then separated from each other in a maternofetal gradients were 119.6 ± second chromatographic step on 40-cm LH-20 col24.7-270.8 ± 52.9 (SE) ng/ml (1.0:2.3) for P umns (7). After branching off a small aliquot for and 11.0 ± 2.4-32.9 ± 5.1 (SE) ng/ml (1:3) for internal standard recovery counting, each of the 17-OHP. Both gradients were highly signifiisolated steroid fractions was quantitated by RIAs, cant statistically (P < 0.01). Differentiation of which recently have been described in detail elsecord levels according to fetal sex revealed for where (8). The entire analytical procedure has been shown to be highly practicable and convenient, both P and 17-OHP significantly (P < 0.05) particularly for pediatric studies, and, in addition, higher plasma levels in the male [P, 363.2 ± 82.1 (SE) ng/ml; 17-OHP, 43.8 ± 7.4 (SE) to be at least as specific, sensitive, precise, and ng/ml] than in the female newborns [P, 178.4 accurate as most single steroid assays published to ± 46.6 (SE) ng/ml; 17-OHP, 21.9 ± 3.5 (SE) date. Coefficients of variation of the complete determination (including extraction, chromatography, ng/ml]. The mothers with male infants had a and RIA) were between 6.9 and 14.5% within assays higher mean 17-OHP level of 14.4 ± 4.0 (SE) and between 11.9 and 16.3% between assays, Tor all ng/ml, as compared to 7.7 ± 2.0 (SE) ng/ml in of the seven steroids. Because of the limited amount the mothers with female infants, although this of plasma available, about 50% of the DOC meassex difference was statistically not sufficiently urements from day 4 and 7 after birth fell below significant (P < 0.10). No such difference was the sensitivity of the assay, thus, relatively high SE seen within the maternal P levels [126.1 ± 32.0 values were obtained. Plasma sodium and potas(SE) ng/ml in the male, 113.1 ± 40.5 (SE) sium were determined by routine procedures (flame ng/ml in the female pregnancies]. Moreover, photometry). a significant (P < 0.05) positive correlation For statistical evaluations of differences between the means of two samples, either parametric (e.g. was found between placental weights and umbilical vein P concentrations, whereas no cort test or paired t test) or nonparametric tests of significance [e.g. Wilcoxon's signed rank test (10), relations were obtained either between plaMann-Whitney's U test (11)] were used. Statistical cental weights and maternal P or between comparisons of more than two means of paired maternal and fetal P levels. There was a fairly samples, i.e. analyzing the postnatal course of the high and significant correlation (r = 0.744, P mean plasma levels of a given steroid, were made < 0.01) between individual maternal and cord utilizing the ranked analysis of variance according venous levels of 17-OHP. No relationship, to Friedman (12), in combination with Wilcoxon and Wilcox's multiple comparison of paired samples however, was found between placental weights (13). Correlations between two different parameters and either maternal or cord 17-OHP concentrations. Differences in maternal parity were were checked by linear regression analysis.
The Endocrine Society. Downloaded from press.endocrine.org by [${individualUser.displayName}] on 28 September 2015. at 03:17 For personal use only. No other uses without permission. . All rights reserved.
SIPPELL ET AL.
974 PROGESTERONE ng/ml
-•
JCE&M • 1978 V0U6 • No 6
17-HYDR0XYPR0GESTER0NE nmol/l r 200
p
