Endocrinol. Japon. 1988, 35 (2), 189-195

Age-Related Changes in Serum 17-Hydroxypregnenolone and 17-Hydroxypregnenolone Sulfate Concentrations in Human Infancy and Childhood

KAZUHIKO SHIMOZAWA, SUMITAKA SAISHO, JUN-ICHI YATA AND AKIRA KAMBEGAWA*

Department of Pediatrics, Faculty of Medicne, Tokyo Medical and Dental University, Tokyo 113, Japan Department of Obstetrics and Gynecology*, School of Medicine, Teikyo University, Tokyo 173, Japan

Abstract

In order to clarify some of the developmental processes of the human adrenal cortex or steroidogenesis in infancy and childhood, serum concentrations

of 17-hydroxypregnenolone, 17-hydroxypregnenolone sulfate and 17-hydroxy-

were measured by means of a combined radioimmunoassay method, and the age-related changes in these were also examined.

The actual ranges of serum concentrations of 17-hydroxypregnenolone, 17-

hydroxypregnenolone sulfate and 17-hydroxyprogesterone in umbilical cord blood were 27.1-80.5, 1,560-5,030 and 53.3-304nmol/l, respectively. These values subsequently decreased to nadirs of 0.95-2.09nmol/l of 17-hydroxy-

in subjects 1 to 2years old, 0.93-7.03nmool/l of 17-hydroxy- in subjects 3 to 6years old and 0.18-0.78nmol/l of 17- hydroxyprogesterone in subjects 1 to 2years old, respectively, and they

were followed by gradual increases to the adult levels. This study thus revealed the age-related changes in 17-hydroxypregnenolone

and its sulfate concentrations in infancy and childhood and indicated that, in the process in which the adrenal cortex was differentiated to the definitive form, the decrease in the activity of sulfotransferase in infancy and

childhood occurred more slowly than the increase in that of 3ƒÀ-hydroxy-

steroid dehydrogenase.

It is widely recognized that the activity Vihko, 1970; Laatikainen et al., 1973), while of 3 ƒÀ-hydroxysteroid dehydrogenase is lower that of is higher in

(Bermudez et al., 1972; Huhtaniemi and the fetal adrenal cortex and peripheral tissues than in those of adults (Korte et al., Received April 15, 1987 1982; Seron-Ferre et al., 1978). Address reprint requests to Dr. KAZUHIKO To better understand the developmental SHIMOZAWA at the Department of Pediatrics, Faculty of Medicine, Tokyo Medical and Dental process of the adrenal cortex and steroid- University. 1-5-45 Yushima, Bunkyo-ku, Tokyo ogenesis in human infancy and childhood, 113, Japan. it would help to study the age-related Endocrinol. Japon. 190 SHIMOZAWA et al. April 1988 changes in serum concentrations of adrenal one were measured by a combined RIA method unconjugated ƒ¢5-steroids and their 3-sulfates. previously reported (Shimozawa et al., 1985). The method consisted of the following pro- Although there have been many reports on cedures: (1) diethyl ether extraction and chro- the serum pregnenolone, dehydroepiandro- matographic separation of unconjugated steroids sterone (DHA) and their 3-sulfate con- (17-OH-pregnenolone and 17-OH-progesterone), centrations and their age-related changes (2) enzymatic hydrolysis of 17-OH-pregnenolone (Mathur et al., 1980; Peretti et al., 1976; sulfate using the residue of diethyl ether extrac- Reiter et al., 1977), little is known about 17- tion for a material, (3) diethyl ether extraction hydroxypregn enolone (17-OH-pregnenolone) and chromatographic purification of hydrolyzed 17-OH-pregnenolone sufate and (4) RIAs for 17- and its 3-sulfate (17-OH-pregnenolone OH-pregnenolone to estimate 17-OH-pregneno- sulfate), which are known as the inter- lone and its sulfate hydrolyzed, and for 17- mediate steroids in adrenal and gonadal OH-progesterone. steroid production. Extracted 17-OH-pregnenolone was well sepa- In this paper, we examined the age- rated from 17-OH-progesterone by Sephadex related changes in serum concentrations of LH-20 microcolumn chromatography, using a 17-OH-pregnenolone and its sulfate as well benzene/methanol=95/5 (v/v) solvent as a mobile as 17-hydroxyprogesterone (17-OH-progester- phase. The most suitable method for hydrolysis one), by means of a combined radio- of 17-OH-pregnenolone sulfate was an enzymatic hydrolysis with arylsulfatase from Helix immunoassay (RIA) method for a simul- Pomatia (obtained from Boehringer-Mannheim taneous determination of these steroids which BmbH, West Germany) in an appropriate we devised (Shimozawa et al., 1985). condition in which the percent hydrolysis was 92.9±2.7(mean±SD)%.Final recovery values Materials and Methods were 90.7±3.1% for 17-OH-pregnenolone,78.1 ±4.7% for 17-OH-pregnenolone sulfate and 88.7 Subjects ±2.7% for 17-OH-progesterone,respectively. We examined 11 umbilical cord blood 0.25ml of 1:12,000 diluted anti-17-OH-preg- specimens and sera from 82 children of various nenolone-3-succinate-BSA serum (obtained from ages and 20 adults with no endocrinological Teikoku Hormone Mfg, Kawasaki, Japan) was abnormalities. The 82 children were divided optimal for the reaction and 5,000 dpm of into the following groups: (1) 44 neonates and [7-3H(N)] 17-OH-pregnenolone (obtained from infants aged from 23days to 2months (23 boys Amersham International plc., Buckinghamshire, and 21 girls), (2) three infants aged from 3 to 4 England) was most suitable as a tracer for RIA months (two boys and one girl), (3) three in- of both 17-OH-pregnenolone and its sulfate fants aged from 5 to 11 months (one boy and hydrolyzed also. For RIA of 17-OH-progester- two girls), (4) three children aged from 1 to 2 one, 0.25ml of 1:32,000 diluted anti-17-OH- years (two boys and one girl), (5) seven progesterone-3-carboxymethyloxime-BSA serum children aged from 3 to 6years (three boys and (obtained from Teikoku Hormone Mfg.) and four girls), (6) eight school children aged from 10,000 dpm of [1, 2-3H(N)] 17-OH-progesterone 7 to 9years (four boys and four girls), (7) seven (obtained from New England Nuclear, Boston, preadolescents aged from 10 to 12years (five USA) were used. The specificities of both boys and two girls) and (8) seven adolescents antisera were discussed in previous papers (Saito, aged from 13 to 15years (two boys and five 1980; Shimozawa et al., 1985). In both RIAs, girls). The adults ranged from 21 to 32years we used a saturated ammonium sulfate solution old and included ten men and ten women, and for separation the bound from free steroid. The of the latter seven were in the follicular and intra- and interassay coefficients of variation three were in the luteal phase. for this method were 17.3•}2.1 (mean•}SD) % and 17.5•}2.5% for 17-OH-pregnenolone, 13.3•} Measurements 2.9% and 18.7±4.7% for its sulfate,and 12.2± Serum concentrations of 17-OH-pregnenolone, 2.1% and 15.0±2.4% for 17-OH-progesterone, 17-OH-pregnenolone sulfate and 17-OH-progester- respectively. Vol.35, No.2 SERUM 17-OH-PREGNENOLONE AND ITS SULFATE 191

Since the distribution of the data was and 17-OH-progesterone in the subjects of markedly skewed and their common logarithmic different ages are shown in Tables 1 and 2, values showed normal distribution, data are and the 95% toleran ce limits of 17-OH- expressed and statistically analyzed after common pregnenolone and its sulfate are shown in logarithmic transformation. Data are expressed Fig. 1. as the mean•}SD, and the median values, the actual ranges and their 95% tolerance limits are In umbilical cord blood, the serum con- also described. Statistical analysis was carried centrations of 17-OH-pregnenolone, 17-OH- out with Student's unpaired t-test. pregnenolone sulfate and 17-OH-progesterone were all significantly higher (P<0.001) than those in infancy, childhood and adulthood. Results These values all subsequently decreased to nadirs of 17-OH-pregnenolone in children 1 The serum concentrations of 17-OH- to 2years old, 17-OH-pregnenolone sulfate pregnenolone, 17-OH-pregnenolone sulfate in children 3 to 6yeras old and 17-OH-

Table 1. Serum concentrations of 17-OH-pregnenolone, 17-OH-pregnenolone sulfate and 17-OH-progesterone in normal human umbilical cords, infants and children

The median value, actual range and the 95% tolerance limit: nmol/l Endocrinol. Japon. 192 SHIMOZAWA et al. April 1988

Table 2. Serum concentrations of 17-OH-pregnenolone, 17-OH-pregnenolone sulfate and 17-OH-progesterone in normal adults

The median value, actual range and the 95% tolerance limit: nmol/1 LP: luteal phase, FP: follicular phase

17-OH-pregnenolone 17-OH-pregnenolone sulphate

Fig. 1. The 95% tolerance limits of serum concentra- tions of 17-OH- pregnenolone and 17-OH-pregneno- lone sulfate in nor- mal control sub- jects. UB: umbili- cal cord blood, A: adult, M: male, F: female.

progesterone in children 1 to 2years old, including pregnenolone, 17-OH-pregnenolone and they were followed by gradual increases and DHA, which are mainly secreted as up to the adult levels. their sulfate conjugates. Although the role of the high activity of sulfotransferase in the fetal adrenal cortex has not been Discussion revealed, a possible explanation is that the fetal adrenal cells are protected by this In fetal adrenal steroidogenesis, the phenomenon from the accumulation of free major products are 3ƒÀ-ƒ¢5-hydroxysteroids, or unconjugated steroids which are much Vol.35, No.2 SERUM 17-OH-PREGNENOLONE AND ITS SULFATE 193

more potent inhibitors of 3ƒÀ-hydroxysteroid maternal plasma. However, there have been

dehydrogenase than their sulfate conjugates no reports on the age-related changes in

(Winter 1985). Besides this, to account for serum 17-OH-pregnenolone sulfate concen- the increased adrenal secretion in tration in man. In this study, therefore, adrenarche, various changes in adrenal in order to obtain more detailed information enzyme activity from childhood to adult- on the maturation of the adrenal cortex, hood have been demonstrated. It is we examined the serum concentrations of

generally recognized that the adrenarche is 17-OH-pregnenolone, 17-OH-pregnenolone associated with reduced 3ƒÀ-hydroxysteroid sulfate and 17-OH-progesterone in umbilical dehydrogenase activity and increased 17, cord blood, infancy, childhood and adult- 20-desmolase and 17ƒ¿-hydroxylase activity hood.

(Kelnar and Brook, 1983; Rich et al., 1981; The age-related changes in plasma 17- Schiebinger et al., 1981), although the OH-pregnenolone concentrations were ex- mechanisms of change in adrenal enzyme amined by Abraham et al., (1973), together activity remain controversial. Therefore, with pregnenolone and DHA. They reported 17-OH-pregnenolone, 17-OH-pregnenolone that the plasma concentration of 17-OH-

sulfate and 17-OH-progesterone are con- pregnenolone was significantly higher in sidered to be key substrates for adrenal neonates (18.9•}11.1nmol/l) than in pre- steroidogenesis, since these are intermediate adolescents (boys: 2.86•}2.91nmol/l, girls: steroids having the potentiality to be 2.71•}3.61nmol/l), adult males (2.20•}1.20 converted to both and adrenal nmol/‚Œ) and nonpregnant women. Besides . this, our more detailed examinations demon- Many papers describing the serum con- strated that the serum 17-OH-pregnenolone centrations and the age-related changes in concentration decreased once to a nadir at

pregnenolone, DHA and these sulfates in 1 to 2years and then gradually increased man have been published (Hopper and Yen, to the adult levels. 1975; Korth-Schutz et al., 1976; Laatikainen As to the 17-OH-pregnenolone sulfate et al., 1973; Mathur et al., 1980; Peretti concentration, our data indicated that, and Forest, 1976; Reiter et al.. 1977). As similar to 17-OH-pregnenolone, the serum for 17-OH-pregnenolone and its sulfate con- concentration was highest in umbilical cord centrations in man, there have been blood and decreased once to a nadir at 3 several papers on 17-OH-pregnenolone to 6years, followed by a gradual increase sulfate concentrations in umbilical cord to adult levels. blood, amniotic fluid, maternal blood and These age-related changes are also ob- adult females (Belisle et al.. 1978; Laatkainen served in the serum concentrations of 17- et al., 1973), and on the age-related changes OH-progesterone (Hughs and Winter, 1976; in the plasma concentrations of 17-OH- Shimozawa et al., 1985), DHA and its

pregnenolone (Abraham et al., 1973). As sulfate (Korth-Schutz et al., 1976; Peretti for the 17-OH-pregnenolone concentration and Forest, 1976; Reiter et al., 1977). They in the early neonatal period, Wiener et al., are thought to result from the maturation

(1987) reported in a paper on several process of adrenal and gonadal steroid adrenal steroids levels in healthy full-term production. However, there was a small 3-day-old infants, together with DHA- difference between 17-OH-pregnenolone and sulfate, 17-OH-progesterone, 11-deoxycortisol its sulfate at the ages the nadirs were and cortisol. Magyar et al., (1981) also recorded, presumably secondary to the reported a study on 17-OH-pregnenolone difference between the change in the activity and its sulfate levels in ovine fetal and of 3ƒÀ-hydroxysteroid dehydrogenase and Endocrinol. Japon. 194 SHIMOZAWA et al. April 1988 steroid sulfotransferase. Hughes, I. A. and J. S. D. Winter (1976). The This study therefore reveals the age- application of a serum 17OH-progesterone radioimmunoassay to the diagnosis and manage- related changes in the serum concentrations ment of congenital hyperplasia. J. Pediatr. of these steroids in human infancy and 88, 766-773. childhood, and demonstrates that, in the pro- Huhtaniemi, I. and R. Vihko (1970). Deter- cess in which the adrenal cortex differentiates mination of unconjugated and sulfated neutral from the fetal to the adult form, the de- steroids in human fetal blood of early and crease in the activity of steroid sulfotrans- mid-pregnancy. Steroids 16, 197-206. ferase in infancy and childhood occurs Kernar, C. J. H. and C. G. D. Brook (1983). more slowly than the increase in that of A mixed longitudinal study of adrenal steroid excretion in childhood and the mechanism of 3ƒÀ-hydroxysteroid dehydrogenase, although adrenarche. Clin. Endocrinol. 19, 117-129. changes in the activity of these enzymes in Korte, K., P. G. Hemsell and J. I. Mason adolescence remain to be determined. (1982). Steroid sulfate metabolism in the adrenals of the human fetus, anencephalic newborn, and adult. J. Clin. Endocrinol. Acknowledgements Metab. 55, 671-675. Korth-Schutz, S., L. S. Levine and M. I. New The authors wish to thank to Misses Y. (1976). sulfate (DS) Chiba and M. Seki for their painstaking technical levels, a rapid test for abnormal adrenal assistance. androgen secretion. J. Clin. Endocrinol. Metab. This work was supported in part by a Grant- 42, 1005-1013. in-Aid for scientific research from the Ministry Laatikainen, T., J. Peltonen and D. Nylandar of Education, Science and Culture (58570416) (1973). Determination of , estriol sulfate, and by those for research on "Prevention of progesterone and neutral steroid mono- and Physical and Mental Disabilities" from the disulfates in umbilical cord blood plasma. Ministry of Health and Welfare, Japan. Steroids 21, 347-359. Magyar, D. M., C. W. Elsner, J. Eliot, T. Glatz, P. W. Nathanielsz and J. E. Buster References (1981). A combined radioimmunoassay for the measurement of unconjugated and sulfo- conjugated pregnenolone, 17-hydroxypregne- Abraham, G. E., J. E. Buster, F. W. Kyle, P. nolone, dehydroepiandrosterone, and C. Corrales and R. C. Teller (1973). Radio- applied to fetal and maternal ovine plasma. immunoassay of plasma pregnenolone, 17- Steroids 37, 423-443. hydroxypregnenolone and dehydroepiandro- sterone under various physical conditions. Mathur, R. S., S. Landgrebe, L. O. Moody, S.

J. Clin. Endocrinol. Metab. 37, 140-144. Powell and H. O. Williamson (1980). Plasma Belisle, S., M. Fend., R. Osthanondh and D. steroid concentrations in maternal and um- Tulchinsky (1978). Sources of 17ƒ¿-hydroxy- bilical circulation after onset of labor. J. Clin. Endocrinol. Metab. 51, 1235-1238. pregnenolone and its sulfate in human pregnan- cy. J. Clin. Endocrinol. Metab. 46, 721-728. Peretti, E. and M. G. Forest (1976). Uncon- Bermudez, J. A. and M. B. Lippsett (1972). jugated dehydroepiandrosterone plasma levels

Early adrenal response to ACTH: plasma- in normal subjects from birth to adolescence concentrations of pregnenolone, 17-hydroxy- in human: the use of a sensitive radio- immunoassay. J. Clin. Endocrinol. Metab. 43, pregnenolone, progesterone and 17-hydroxy- progesterone. J. Clin. Endocrinol. Metab. 34, 982-991. 241-243. Reiter, E. O., V. G. Fuldauer and A. W. Root Hopper, B. R. and S. S. C. Yen (1975). Cir- (1977). Secretion of the adrenal androgen, culating concentrations of dehydroepiandro- dehydroepiandrosterone sulfate, during normal sterone and dehydroepiandrosterone sulfate infancy, childhood, adolescence, in sick infants

during puberty. J. Clin. Endocrinol. Metab. and children with endocrinologic abnormalities. 40, 458-461. J. Pediatr. 90, 776-770. Vol.35, No.2 SERUM 17-OH-PREGNENOLONE AND ITS SULFATE 195

Rich, B. H., R. L. Rosenfeld, A. W. Lucky, J. androgens during infancy and adrenarche are C. Helke and P. Otto (1981). Adrenarche: associated with changing activities of adrenal changing adrenal response to adrenocortico- microsomal 17-hydroxylase and 17, 20- tropin. J. Clin. Endocrinol. Metab. 52, 1129- desmolase. J. Clin. Invest. 67, 1177-1182. 1136. Shimozawa, K., S. Saisho, J. Yata and A. Saito, N.(1980). Serum 17-hydroxyprogesterone, Kambegawa (1985). A combined radioim- 21-deoxycortisol and cortisol levels in patients munoassay method for the determination of 17- and heterozygotes with congenital adrenal hydroxypregnenolone, 17-hydroxypregnenolone hyperplasia. Ochanomizu Igakuzasshi 28, 117- sulfate and 17-hydroxyprogesterone in human 125.(in Japanese) blood. Folia Endocrinol. Jpn. 61, 631-641. Seron-Ferre, M., C. C. Lawrence, P. K. Sitteri (in Japanese) and R. B. Jaffe (1978). Steroid production by Wiener, D., J. Smith, S. Dahlem, G. Berg and definitive and fetal zones of the human fetal T. Moshang (1987). Serum adrenal steroid adrenal gland. J. Clin. Endocrinol. Metab. levels in healthy full-term 3-day-old infants. 47, 603-609. J. Pediatr. 110, 122-124. Shiebinger, R. J., B. D. Albertson, F. G. Cas- Winter, J. S. D.(1985). The adrenal cortex in sorla, D. W. Bowyer, G. W. Geelhoed, G. B. the fetus and neonate. In: Anderson, D. C. Cutler, Jr, D. L. Loriaux (1981). The de- and J. S. D. Winter (eds.) Adrenal Cortex, velopmental changes in plasma adrenal 32-56, Butterworth, London.