Variations in urinary steroid profiles after birth

CLINICA CHIMICA ACTA

VARIATIONS

MARJORIE EVAN

AND

261

IN URINARY

G. HORNING*, C. HORNING*

STEROID

AMELIA

HUNG*,

Institute for Lipid Research+ and Department Houston, Texas 77025 (U.S.A.)

PROFILES

REBA

AFTER

BIRTH

M. HILL**

of Pediatrics+ l, Baylor College of Medicine,

SUMMARY

Quantitative steroid profiles were obtained in fifty-three newborn infants. The excretion of steroids (mg/z4 h) did not correlate with birth weight, sex or gestational age. The steroid profiles of individual infants were followed for four months. During this period the profiles changed completely from a profile in which neonatal steroids predominate to a profile composed of metabolites of adrenocortical steroids.

INTRODUCTION

Urinary steroids excreted by the newborn infant differ markedly in structure from the urinary steroids of the adult human and should be considered as a new group of mammalian steroids (neonatal steroids). The major neonatal steroids are derivatives of 3/?-hydroxy-A%ndrostenes and As-pregnenes; 5-androsten-3p,r6adiol-r7-one; 5-androsten-3,9-17/?-diol-r6-one; 5-androsten-3fi,x6a,r7/?-triol; 5-pregnen-3/I,2r-diol-zo-one are present in all samples. Small amounts of the isomeric androsten-triols are also present. In addition, several isomeric androsten-triolones and androsten-tetrols are normally present as major componentW. In preliminary studies, wide variations were observed in the amount (mg) of urinary steroids excreted in 24 h, and in the amounts of individual steroids. Quantitative analyses were carried out on 24-h urine samples collected from both male and female premature and term infants using gas-chromatographic procedures described previously’. The identity of the individual steroid peaks in the gas-chromatographic record was established by mass spectrometry (MS). It was found that the excretion of steroids declined during the first month from an average of 7.2 mg/z4 h to 1-2 mg/z4 h and then remained at the lower level for at least six months. During this period, the neonatal steroids disappeared from the urine, and metabolites of the adrenocortical steroids became the major urinary steroids. The time required for this change in steroid profile appears to be different for each infant and varies from 30 to 75 days. This changeover from neonatal steroids to corticoids probably is related to biochemical changes associated with the disappearance of the fetal adrenal zone. Clin. Chim. Acta, 34

(1971)

261-268

262

HORNING

et al.

EXPERIMENTAL

Materials Twenty-four hour urine samples were obtained from both male and female newborn infants using a pediatric urine collector. The urines were kept at - 14’ until analyzed. Methods The

steroid

hydrolysis

with Glusulase

with ether

fraction

and ethyl

was isolated

as described

(Endo Laboratories,

acetate.

After washing

saturated 5% sodium bicarbonate vents were evaporated (Rotovap). stored in ethyl acetate-methanol ative formation.

previously’.

After

enzymic

Inc.), the free steroids were extracted the combined

extracts

with cold salt-

solution and drying (magnesium sulfate), the solThe residue containing the steroid fraction was (I : I) at - 14~ and aliqupts

Derivative formation Both methoxime-trimethylsilyl silyl ether (BO-TM%) derivatives

were removed

for deriv-

ether (MO-TMSi) and benzyloxime-trimethylwere prepared. After removal of the solvent

(nitrogen) from an aliquot (115) of the steroid extract, the dry residue was dissolved in 0.5 ml of pyridine and IO mg of methoxylamine hydrochloride was added. The solution was allowed to stand at room temperature overnight. evaporated (nitrogen) and 30 ,ul of N-trimethylsilyl-imidazole

The pyridine was (TSIM) and 20 ~1

of bis trimethylsilylacetamide (BSA) were added and the solution was heated for 3 to 4 h at 150’. An aliquot was used directly for gas chromatography (GC) and GCMS analysis. Benzyloxime-trimethylsilyl ether (BO-TMSi) derivatives were prepared by adding 20 mg of 0-benzylhydroxylamine hydrochloride to an aliquot (I/S) of the steroid extract dissolved in 0.5 ml of pyridine. After heating overnight at 60 to 7o”, the pyridine was evaporated (nitrogen) and 40 to 50 ,ul of TSIM added and the solution was heated at 150’ for 3 to 4 h394. An aliquot was used directly for GC and GC-MS analysis. Gas chromatography Gas-chromatographic analyses were carried out with Barber-Colman model 5000 instruments equipped with Keithley model 417 picoammeters and Texas Instruments recorders. The columns were 12 ft x 4 mm glass W-columns. The column packings were 1% SE-30 on 100-120 mesh Gas Chrom P prepared according to the usual procedure of this laboratory5. The injector temperature was 260”; the detector bath temperature was 330’. Typical gas pressures were nitrogen, 26 psi; air, 40 psi; hydrogen, 14 psi. The nitrogen flow rate was 40-50 ml per min at 200’. Methylene unit (MU) values were determined in temperature-programmed separations as described previouslya. Mass

spectrometry Mass spectra were obtained with an LKB model 9000 gas chromatograph-mass spectrometer. The column was a g ft ~4 mm glass coil with a 1% SE-30 column Clin. Chim. Acta,34(1971) 261-268

STEROIDS AFTER BIRTH

263

packing. The ionizing current was 60 ,uA; the voltage was 70 eV; the ion source was at 270”; the usual scan time was 3 to 6 sec.

Most of the neonatal steroids are not available as pure compounds and it is necessary to express the quantities of neonatal steroids in terms of area relationships to cholesteryl butyrate, the reference standard, assuming a response factor of unity. RESULTS AND DISCUSSION

Quantitative analyses were carried out on urines (24-h samples) collected from 53 infants. This group included zz female and 31 male infants whose birth weight varied from 3.4 to 9.6 pounds; there were 17premature infants in this group. There was no significant difference in the quantity of urinary steroids excreted by male and female infants. The excretion of steroids by premature infants averaged 5.5 mg/z4 h compared to 8.1 mg/24 h for term infants for urines collected during days I to 4. This difference may not be significant because of the small number of premature infants and the wide range in values. In Fig. I, the excretionof steroids (mg/z4 h) is plotted against the age (days) of the infant when the urine was collected. It is apparent that the excretion of steroids (mg/z4 h) is greatest during the first 4 days of life. The average excretion during this period was 7.2 mg/z4 h with a range in values from 0.5 to 27 mg124 h. When analyses

53 INFRNTS BIRTH

WEIGHT

34-96

LBS

Fig. I. Urinary steroid excretion (mgjq h) plotted against the age in days for 53 infants. The analysis of the data and the plotting of the graph was done by an IBM 3601150 computer. Clin. Cham. Ada,

34 (rgy1)

261-268

HORNING

264

et d.

on consecutive urine samples from individual infants were carried out, it was found that the highest excretion of steroids occurred during the first week followed by a gradual decline to an average excretion of I to 2 mg/24 h after 15 to 30 days. The wide variation in urinary steroid levels suggests that factors other than sex, birth weight and gestational age are significant in regulating the steroid metabolism and excretion in the newborn. The gas chromatographic

analyses

provided

qualitative

as well as quantitative

data on each of the 53 infants. By comparing steroid profiles, it was possible to see how infants differed in the types of steroids excreted. Fig. z and 3 demonstrate some of the

URINARY STEROIDS MO-TMSI TP I’/MIN BABY FR DAY 2

565.

I % SE-30

I~

610

80

60

40

k309

MIN URINARY

STEROIDS

1

MO-TMSI TP I”/MIN

I%SE-30 BABY

FR

DAY

45

(175’)

II-KEI B-CORT

THE

b

\ I!

STD

:-60

80 MIN

100

Fig. 2. Urinary steroid profile for a newborn infant (baby FR) for days 2 and 45 of life. The steroids were separated as TMSi and MO-TMSi derivatives with a rz-ft I o/0SE-30 column with temperature programming at I’/min from 175 or rgo”. The compounds identified with Roman numerals are I, 5-androsten-3p,r6cc-diol-r7-one; II, 5-androsten-3B,r7/&diol-r6-one; III, 5-androsten-3B,16cc, 17p-triol. The compounds identified with numbers (molecular weight) are derivatives of steroids for which a definite structural assignment can not be made at this time; androsten-triol, M = 522; androsten-triolone, M = 565; androsten-tetrol, M = 610; II-KEt, II-ketoetiocholanolone; I I/?HOAn, I r,%hydroxyandrosterone; ,!?-tort, ,G-cortolone; THE, tetrahydrocortisone; THA, 5/3pregnan-3a,21-diol-r I, zo-dione; CHOL, cholesterol; C pB,n-hexacosane; STD, cholesteryl butyrate. Clin. Chim. Acta, 34 (1971) 261-268

265

STEROIDS AFTER BIRTH

URINARY

sTEmlDS

MO-TMS! TP PlMlN IIs

IX SE-30

BABY GU DAY I

URINARY I

STEROIDS

MO-TM9 TPI*/MIN

IXSE-30 BABY

W.57

GU

DAY 44

40

80

60 MIN

Fig. 3. Urinary steroid profiles for a newborn infant (baby GU) for days I and 44. The GC conditions were the same as those for Fig. 2. The retention of neonatal steroids indicates persistence of fetal adrenal tissue.

differences between infants. The major FR (Fig. 2) was an androsten-triolone

neonatal steroid excreted on day 2 by baby (M = 565). The major steroids excreted by

baby GU (Fig. 3) on day I were androsten-3/3,r6ct-diol-r7-one (I), an androsten-tetrol (M = 610) and (M = 595). The steroid profiles of the two infants were quite different qualitatively, but the total amounts of steroids excreted were comparable (5 to 6 mg/ 24 h).

The corticoids are found as minor components in steroid profiles of the neonate. However, several corticoids including THE, THF and THB have been identified in urines collected during the first week of life. THB was identified in the urine of baby. FR on day 2. Pregnenetriol, estriol, isomeric estriols and estetrol have also been identified by GC-MS in urines collected during days I to 4. Steroid profiles were obtained for several infants at intervals up to the age of six months. It was found that the neonatal steroids gradually disappeared from the urine but the time required was variable. For example, no neonatal steroids were identified Cl&a. Chim. Acta, 84 (1971) 261-268

266

HORNING

URINARY

et d.

d-CORT

STEROIDS

CHOL

MO - TMSi TP I”/MIN.(1800)

I% SE-30

BABY FR DAY 77

60 URINARY

STEROIDS

0

MO-TMSi

THE TP I’/MIN

I% SE-30

80

MIN

(175’)

4 -CORT

BABY FR DAY II5

’

~-CORTOL

$ CHOL

’ STD

697

C-26

Il4-HOAn

60

MIN

IO0

80

Fig. 4. Urinary steroid profile for a newborn conditions were the same as those for Fig. 2.

infant (baby

FR)

for days 77 and 115. The GC

in the steroid profile of baby RF on day 45 (Fig. 2). However, baby GU was still excreting large amounts of 5-androsten-3B,r6a-diol-r7-one, (I); 5-androsten-3p,r7/?diol-r6-one, (II); 5-androsten-3,!?,16a,r7/?-triol, (III); several androsten-triolones (M = 565) and 5-pregnen-3b, r6a-diol-zo-one (IV) on day 44. By day 45, the major steroids in the profile of baby FR were THE, THA, ,!?-cortolone, II-ketoetiocholanolone and II/?-hydroxyandrosterone. Urinary steroids profiles weer obtained for baby FR for days 77 and 115. Qualitatively the profiles for days 35,77 and 115 were similar but the quantitative relationships were quite different (Fig. 4). The steroid profile, however, appeared to be reaching a fairly regular pattern. Similar analyses have been carried out for several infants. Methoxime-trimethylsilyl ether derivatives (MO-TM%) were used in these studies, and although the gas-chromatographic records were satisfactory, the mass spectrometric analyses indicated that many of the gas-chromatographic peaks contained several steroidal components (Fig. 5). Recently a new profile procedure has Clkn.Chim. Acta, 34

(1971) 261-268

STEROIDS AFTER BIRTH

267

j!-SLA

URINARY

DAY

STEROIDS

MO-TMSI

I I ml

I% SE-30

40

TP

I”/MIN

60

ll90”)

80 MIN

Fig. 5. Urinary steroid profile for a newborn infant (baby SLA) on day 2. The GC conditions were the same as those for Fig. 2; MO-TMSi derivatives were prepared. Mass-spectrometric analysis indicated that many of the gas-chromatographic peaks contained several steroidal components.

URINARY

B-SLA DAY

2

STEROICS

BO-TMSi I%

I 3 ml

TP

SE-30

I’/MIN

(190”)

669 T;IB

610

“2

610 610

I

40

-

I

60

80

too

MIN

Fig. 6. Urinary steroid profile for the same infant (baby SLA) for day 2 using BO-TMSi derivatives. The GC conditions were the same as those described for Fig. 2. The ketohydroxysteroids (I, 5-androsten-3p-r6a,diol-r7-one; androsten-triolones, M = 641; and THB) are separated from the polyhydroxysteroids (androstene-triols, M = 522; androsten-tetrols, M = 610; and the IIketohydroxysteroids) and cholesterol (CHOL).

been developed for infants and adults3r4 using benzyloxime-trimethylsilyl (BO-TMSi) derivatives. When this procedure is applied to urines from newborn infants, the androsten-diolones and androsten-triolones are separated from the androsten-triols and androsten-tetrols. In the resulting profiles better resolution is obtained because of the separation of the ketohydroxysteroids from the polyhydroxy and Ir-ketopolyhydroxy steroids (Fig. 6). There is still some peak overlap, depending on the steroid composition of the urine specimen. This procedure is now being applied in an attempt Clin. Chim. Acta, 34 (1971) 261-268

268

HORNING

et at.

to reach a better understanding of factors affecting steroid metabolism in the newborn infant. ACKNOWLEDGEMENTS

This work was supported by grant GM-16216 of the National Institute of General Medical Sciences. REFERENCES I M. G. HORNING, E. C. CHAMBAZ, C. J. W. BROOKS, A. M. Moss, E. A. BOUCHER, E. C. HORNING AND R. M. HILL, Anal. Biochem., 31 (1969) 512. z E. M. CHAMBAZ, C. J. W. BROOKS, M. G. HORNING, E. C. HORNING AND R. M. HILL, Compt. Rend., 268 (1969) 2817. 3 P. G. DEVAUX, M. G. HORNING, R. M. HILL AND E. C. HORNING, Anal. Biochem., in press (1971). 4 P. G. DEVAUX, M. G. HORNING AND E. C. HORNING, Anal. Letters, in press (1971). 5 E. C. HORNING, W. J. A. VANDENHEUVEL AND B. G. CREECH, in D. GLICK (Ed.), Methods of BiochewLical Analysis. Vol. XI, Interscience, New York, 1963. 6 E. C. HORNING, M. G. HORNING, E. M. CHAMBAZ, P. I. JAAKONMAKI AND C. J. W. BROOKS, J. Gas Chromatog., 5 (1967) 283. CL&. Chiwa. Ada,

34 (1971) 261-268