I I. pp.
Journal OJ Steroid Biochemistry. Vol. 523 to 529 Pergamon Press Ltd 1979.Printed in GreatBritain
METABOLISM OF FETAL AND NEONATAL ADRENAL STEROIDS C.
H. L. SHACKLETON*, J. W. HoNouRt and N. F. TAYLOR with the technical assistance of RUBY PHILIP Division of Clinical Chemistry, Clinical Research Centre, Watford Road, Harrow, Middlesex HA1 3UJ, U.K. SUMMARY
There are marked differences between metabolism of steroids by the human fetus, neonatal infant and adult. These have been assessed by examination of the steroid excretion patterns in the fetus using analysis of pregnancy urine from patients with placental sulphatase deficiency and amniotic fluid from normal pregnancy, and in the neonate by analysis of urine collected during the first days of life. Metabolites of corticosterone and aldosterone have also been measured in infants with hypersecretion of these hormones. The major differences in the perinatal period in comparison with adulthood are: (1) 3fl-hydroxy-5-ene steroids are excreted in much greater absolute and relative amounts; (2) all the 3/3-hydroxy-5-ene steroids contain additional hydroxyl groups in the l6a, 18 or 21 positions; (3) of the metabolites of cortisol and corticosterone, 60% contain additional hydroxyl groups in the l/?- or 6a- position, compared with 2-S% in adults; (4) more than 90% of the corticosteroid metabolites contain an 11-0x0 group; (5) 21-dehydroxylation of corticosterone metabolites by gut bacteria is absent. Aldosterone metabolism does not differ greatly at different stages of life. The patterns of steroid conjugation are also similar with predominantly glucuronide conjugation of corticosteroids and sulphation of 3/?-hydroxy-5-ene steroids. However, a significant proportion of the more polar corticosteroid metabolites are excreted by infants as free compounds. Adrenal steroid output decreases dramatically post partum. Other changes include a relative increase of 15- and 18-hydroxylation of 3/?-hydroxy-5-ene steroids and an apparent decrease of 21-hydroxylation, the latter possibly due to utilisation for mineralocorticoid biosynthesis. Following progressive changes, the adult pattern of steroid metabolism is reached at about 1 year of age.
INTRODUCTION
In the perinatal period three populations of steroid metabolites may be considered to exist: (1) those arising from the metabolism of the large amounts of oestrogen and progesterone received from the placenta; after birth the excretion of these compounds by the infant decreases rapidly; (2) metabolites of 3fl-hydroxy-5-ene steroids produced by the fetal zone of the adrenal glands; postnatally the excretion of these compounds slowly decreases with regression of this zone; (3) metabolites of corticosteroids which remain for all of childhood the major urinary steroids. A few years ago we identified the major infant urinary steroid as 16{,18-dihydroxy DHA [l] although the stereochemistry of the hydroxyl group at position 16 has yet to be ascertained. The identification of this
* Present address: Biomedical Mass Spectrometry Resource, Space Sciences Laboratory, University of Cal;fornia, Berkelev. CA 94720. U.S.A. t Present adbress: Clinical Studies Center, San Francisco General Hospital, San Francisco, CA 94110, U.S.A. Non-standard steroid abbreviations: DHA, dehydroepiandrosterone, 3/l-hydroxy-5-androsten-17-one; androstenetriol, 5-androstene-3/I,16a,l7/?-triol; 16-oxo-androstenediol, 3/I,17b-dihydroxy-5-androsten-16-one; tetrahydrocortisone, THE, 3a,l7,21-trihydroxy-Sj3-pregnane-11,20dione; tetrahydro-Compound A, 3a,21-dihydroxy-5p-pregnane-ll,2l%dione. The prefix hydroxy is used to denote addition of a hydroxyl group.
compound emphasized the efficiency of the fetus and neonate at producing highly polar metabolites of steroids. Hepatic hydroxylating mechanisms are also important in the metabolism of corticosteroids. Early studies demonstrated the increased excretion of polar metabolites [2] and it was more recently noted that only half of the cortisol metabolites excreted were of the adult-type[3]. Many of these have now been identified [4,5] and it the purpose of this communication to report their quantitative significance in the neonate and relate the results to the excretion of the neonatal 3/I-hydroxy-5-ene steroids. METHODS
Urine processing. Urine (and amniotic fluid) samples were extracted using Amberlite XAD-2 resin and conjugates were separated by LH-20 chromatography according to the method of Jiinne et a/$6]. Columns contained 4g of LH-20 swollen in the solvent system methanol-chloroform, 1: 1 V/V, salt saturated. The samples were applied in 2 x 2.5 ml of the solvent and the first 30ml were collected (free and glucuronide conjugates). Steroid monosulphates and disulphates were recovered by elution with 40ml methanol. Both fractions were dried, dissolved in buffer (pH 4.6) and hydrolysed with Helix pomatia digestive juice (48 h). Hydrolysates were extracted by XAD-2 and filtered through 1 g Sephadex LH-20
523
c‘. H. L. SHACKLETUN. J. W. HONOUR and N. F. TAYI.OR
524
columns (cyclohexaneeethanol, 4: 1 V/V), 40 ml being collected. Derirdsution. Following addition of internal standards (5a-androstane-3a,l7cc-diol, stigmasterol and methyloxime-trimethylsilyl cholesteryl butyrate), ethers were prepared according to methods previously described [7, 81. GUS clrromu~ograpk~. Gas chromatography was carried out using a Becker 409 instrument containing 20m OV-1 or OV-101 wall coated open-tubular columns. The temperature was programmed linearly between 160 and 260°C. Detection was by FID. GLISchromcrtography-muss spectrometry. Gas chromatography-mass spectrometry was carried out on a Varian MAT-731 instrument housing a packed OV-1 column. Samples were analysed by repetitive scanning at 10 s intervals over the range 5NNO amu during temperature programmed runs. Data was stored on tape in mass converted form for further analysis. RESULTS AND
Sepurution
qf inditG4d
SULPHATE
-4
FREE+G
DISCUSSION
components
Metabolites of cortisol and corticosterone cannot be measured satisfactorily by gas chromatography of unfractionated extracts because of interference by the predominant 3P-hydroxy-5-ene compounds. However, these are mostly excreted as sulphates while cortisol corticosterone metabolites are principally and excreted as glucoronides or free compounds, and this enables both groups to be determined if conjugate separation or selective hydrolysis are employed. We chose to use an adaptation of the simple Sephadex LH-20 procedure of Janne et tr/.[6] which gives two fractions: (1) free + glucoronide; and (2) monosulphates + disulphates. Fractionation of conjugates does not give a completely clear-cut separation between metabolites of corticosteroids and 3/?-hydroxy-5-ene steroids and results must be interpreted with caution since Giroud[9] has shown that significant amounts of cortisol and corticosterone are excreted as sulphates in the neonatal period and it is impossible to determine these with accuracy in the presence of the 3b-hydroxy-5-ene steroids. In addition. certain infants excrete a disproportionate amount of 3/?-hydroxy-5-ene steroids in the free or glucuronide conjugated forms. Finally, the 16-glucuronide of oestriol is predominantly found in the sulphate fraction and this is particularly significant where large amounts are present (e.g. in amniotic fluid). Separation of the free + glucuronide and sulphate fractions from a &day-old infant and pooled amniotic fluid are illustrated in Figs 1 and 2. The amount of extract taken for the sulphate fraction of baby urine was 2/5 that for the free + glucuronide. Some of the major steroids have been indicated but the stereochemistry of the hydroxyl groups has not been resolved in all cases. The chromatograms of the baby
Fig. 1. Newborn infant (2 days): Gas chromatographic separation of sulphate (S) and free + glucuronide (F + G) steroids as methyloxime-trimethylsilyl ethers. The amount of extract analysed for the S fraction represents 215 that for the F + G fraction. 16
Journal OJ Steroid Biochemistry. Vol. 523 to 529 Pergamon Press Ltd 1979.Printed in GreatBritain
METABOLISM OF FETAL AND NEONATAL ADRENAL STEROIDS C.
H. L. SHACKLETON*, J. W. HoNouRt and N. F. TAYLOR with the technical assistance of RUBY PHILIP Division of Clinical Chemistry, Clinical Research Centre, Watford Road, Harrow, Middlesex HA1 3UJ, U.K. SUMMARY
There are marked differences between metabolism of steroids by the human fetus, neonatal infant and adult. These have been assessed by examination of the steroid excretion patterns in the fetus using analysis of pregnancy urine from patients with placental sulphatase deficiency and amniotic fluid from normal pregnancy, and in the neonate by analysis of urine collected during the first days of life. Metabolites of corticosterone and aldosterone have also been measured in infants with hypersecretion of these hormones. The major differences in the perinatal period in comparison with adulthood are: (1) 3fl-hydroxy-5-ene steroids are excreted in much greater absolute and relative amounts; (2) all the 3/3-hydroxy-5-ene steroids contain additional hydroxyl groups in the l6a, 18 or 21 positions; (3) of the metabolites of cortisol and corticosterone, 60% contain additional hydroxyl groups in the l/?- or 6a- position, compared with 2-S% in adults; (4) more than 90% of the corticosteroid metabolites contain an 11-0x0 group; (5) 21-dehydroxylation of corticosterone metabolites by gut bacteria is absent. Aldosterone metabolism does not differ greatly at different stages of life. The patterns of steroid conjugation are also similar with predominantly glucuronide conjugation of corticosteroids and sulphation of 3/?-hydroxy-5-ene steroids. However, a significant proportion of the more polar corticosteroid metabolites are excreted by infants as free compounds. Adrenal steroid output decreases dramatically post partum. Other changes include a relative increase of 15- and 18-hydroxylation of 3/?-hydroxy-5-ene steroids and an apparent decrease of 21-hydroxylation, the latter possibly due to utilisation for mineralocorticoid biosynthesis. Following progressive changes, the adult pattern of steroid metabolism is reached at about 1 year of age.
INTRODUCTION
In the perinatal period three populations of steroid metabolites may be considered to exist: (1) those arising from the metabolism of the large amounts of oestrogen and progesterone received from the placenta; after birth the excretion of these compounds by the infant decreases rapidly; (2) metabolites of 3fl-hydroxy-5-ene steroids produced by the fetal zone of the adrenal glands; postnatally the excretion of these compounds slowly decreases with regression of this zone; (3) metabolites of corticosteroids which remain for all of childhood the major urinary steroids. A few years ago we identified the major infant urinary steroid as 16{,18-dihydroxy DHA [l] although the stereochemistry of the hydroxyl group at position 16 has yet to be ascertained. The identification of this
* Present address: Biomedical Mass Spectrometry Resource, Space Sciences Laboratory, University of Cal;fornia, Berkelev. CA 94720. U.S.A. t Present adbress: Clinical Studies Center, San Francisco General Hospital, San Francisco, CA 94110, U.S.A. Non-standard steroid abbreviations: DHA, dehydroepiandrosterone, 3/l-hydroxy-5-androsten-17-one; androstenetriol, 5-androstene-3/I,16a,l7/?-triol; 16-oxo-androstenediol, 3/I,17b-dihydroxy-5-androsten-16-one; tetrahydrocortisone, THE, 3a,l7,21-trihydroxy-Sj3-pregnane-11,20dione; tetrahydro-Compound A, 3a,21-dihydroxy-5p-pregnane-ll,2l%dione. The prefix hydroxy is used to denote addition of a hydroxyl group.
compound emphasized the efficiency of the fetus and neonate at producing highly polar metabolites of steroids. Hepatic hydroxylating mechanisms are also important in the metabolism of corticosteroids. Early studies demonstrated the increased excretion of polar metabolites [2] and it was more recently noted that only half of the cortisol metabolites excreted were of the adult-type[3]. Many of these have now been identified [4,5] and it the purpose of this communication to report their quantitative significance in the neonate and relate the results to the excretion of the neonatal 3/I-hydroxy-5-ene steroids. METHODS
Urine processing. Urine (and amniotic fluid) samples were extracted using Amberlite XAD-2 resin and conjugates were separated by LH-20 chromatography according to the method of Jiinne et a/$6]. Columns contained 4g of LH-20 swollen in the solvent system methanol-chloroform, 1: 1 V/V, salt saturated. The samples were applied in 2 x 2.5 ml of the solvent and the first 30ml were collected (free and glucuronide conjugates). Steroid monosulphates and disulphates were recovered by elution with 40ml methanol. Both fractions were dried, dissolved in buffer (pH 4.6) and hydrolysed with Helix pomatia digestive juice (48 h). Hydrolysates were extracted by XAD-2 and filtered through 1 g Sephadex LH-20
523
c‘. H. L. SHACKLETUN. J. W. HONOUR and N. F. TAYI.OR
524
columns (cyclohexaneeethanol, 4: 1 V/V), 40 ml being collected. Derirdsution. Following addition of internal standards (5a-androstane-3a,l7cc-diol, stigmasterol and methyloxime-trimethylsilyl cholesteryl butyrate), ethers were prepared according to methods previously described [7, 81. GUS clrromu~ograpk~. Gas chromatography was carried out using a Becker 409 instrument containing 20m OV-1 or OV-101 wall coated open-tubular columns. The temperature was programmed linearly between 160 and 260°C. Detection was by FID. GLISchromcrtography-muss spectrometry. Gas chromatography-mass spectrometry was carried out on a Varian MAT-731 instrument housing a packed OV-1 column. Samples were analysed by repetitive scanning at 10 s intervals over the range 5NNO amu during temperature programmed runs. Data was stored on tape in mass converted form for further analysis. RESULTS AND
Sepurution
qf inditG4d
SULPHATE
-4
FREE+G
DISCUSSION
components
Metabolites of cortisol and corticosterone cannot be measured satisfactorily by gas chromatography of unfractionated extracts because of interference by the predominant 3P-hydroxy-5-ene compounds. However, these are mostly excreted as sulphates while cortisol corticosterone metabolites are principally and excreted as glucoronides or free compounds, and this enables both groups to be determined if conjugate separation or selective hydrolysis are employed. We chose to use an adaptation of the simple Sephadex LH-20 procedure of Janne et tr/.[6] which gives two fractions: (1) free + glucoronide; and (2) monosulphates + disulphates. Fractionation of conjugates does not give a completely clear-cut separation between metabolites of corticosteroids and 3/?-hydroxy-5-ene steroids and results must be interpreted with caution since Giroud[9] has shown that significant amounts of cortisol and corticosterone are excreted as sulphates in the neonatal period and it is impossible to determine these with accuracy in the presence of the 3b-hydroxy-5-ene steroids. In addition. certain infants excrete a disproportionate amount of 3/?-hydroxy-5-ene steroids in the free or glucuronide conjugated forms. Finally, the 16-glucuronide of oestriol is predominantly found in the sulphate fraction and this is particularly significant where large amounts are present (e.g. in amniotic fluid). Separation of the free + glucuronide and sulphate fractions from a &day-old infant and pooled amniotic fluid are illustrated in Figs 1 and 2. The amount of extract taken for the sulphate fraction of baby urine was 2/5 that for the free + glucuronide. Some of the major steroids have been indicated but the stereochemistry of the hydroxyl groups has not been resolved in all cases. The chromatograms of the baby
Fig. 1. Newborn infant (2 days): Gas chromatographic separation of sulphate (S) and free + glucuronide (F + G) steroids as methyloxime-trimethylsilyl ethers. The amount of extract analysed for the S fraction represents 215 that for the F + G fraction. 16
