Clinical Research Centre, Watford Road, Harrow, Middlesex HAI 3UJ, England and Institute of Child Health!), 30 Guildford Street, London WCl

MULTICOMPONENT GAS CHROMATOGRAPHIC ANALYSIS OF URINARY STEROIDS EXCRETED BY AN INFANT WITH A DEFECT IN ALDOSTERONE BIOSYNTHESIS

By C. H. L. Shackleton, J. W. Honour, M. Dillon1) and P. Milla1)

ABSTRACT The urinary steroids excreted by an infant with a salt-wasting syndrome due to a suspected defect in the 18-oxidation of corticosterone have been analysed by gas chromatography-mass spectrometry. The excretion of tetrahydroaldosterone was low (3.5 \g=m\g/24 h) whilst the excretion of 3\g=a\,11\g=b\,21- trihydroxy 5\g=a\ pregnan -20- one (allo-tetrahydrocorticosterone) and other corticosterone metabolites was high (total about 2 mg/24 h). The excretion of cortisol metabolites was apparently normal for age (total about 2 mg/24 h) but 3\g=a\,11\g=b\,17\g=a\,21-tetrahydroxy-5\g=a\-pregnan-20-one (allo-tetrahydrocortisol) rather than tetrahydrocortisone, was the major component of the group. The excretion of an 18-hydroxycorticosterone metabolite 3\g=a\,18,21-trihy-

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droxy-5\g=b\-pregnane-11,20-dione (18-hydroxytetrahydroCompound A) was higher than normal for infants of this age (between 50 and 200 \g=m\g/24h), suggesting that the defect was in 18-hydroxysteroid dehydrogenase rather than 18-hydroxylase. In addition, 18-hydroxytetrahydrocorticosterone,

another metabolite of 18-hydroxycorticosterone was tentatively identified and it was found that the rate of excretion of this compound was of similar magnitude to 18-hydroxytetrahydroCompound A. The salt balance of the infant has been successfully controlled by salt administration (77 mEq./24 h) and treatment with Fludrocortisone

(0.5 mg/day).

The commonest adrenal

of salt-loss in childhood is congenital adrenal of patients with this disorder due to a hyperplasia. Thirty per cent of deficiency 21-hydroxylase are overt salt-losers certainly because of low aldosterone secretion. Apart from Addison's disease other causes of decreased aldosterone secretion are deficiencies of the enzymes required for 18-oxidation of corticosterone (Visser & Cost 1964; Ulick et al. 1964; Degenhart et al. 1966; Royer 1967; Rappaport et al. 1968; David et al. 1968; Jean et al. 1969). This clinical situation may be confused with so-called pseudohypoaldosteronism in which there may be end-organ unresponsiveness to aldosterone (Check & Perry 1958; Donnell et al. 1959; Barakat et al. 1972) and recently a report was published from this laboratory which described a gas chromatographic-mass spectrometric (GC-MS) study of the steroids excreted by an infant with this disorder (Shackleton & Snodgrass 1974). The most noticeable feature was the high excretion of tetrahydroaldosterone, the major aldosterone metabolite. This communication describes the identification by gas chromatographymass spectrometry and quantitation by gas chromatography of the major steroids excreted by an infant with a salt-losing syndrome caused by deficiency or inactivity of one of the enzymes required for oxidation of the C-18 methyl group of corticosterone. cause

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MATERIALS AND METHODS

Reference steroids Almost all reference steroids

were

obtained from the M. R. C. Steroid Reference

Collection, Westfield College, Hampstead, London. Dr. S. Ulick kindly donated

tetrahydroaldosterone

and the etiolactone of

18-hydroxytetrahydroCompound

A.

Steroid abbreviations TetrahydroDOC: 3a,21 dihydroxy 5ß pregnan-20-one;

18 hydroxytetrahydroDOC: 3a,18,21-trihydroxy-5/?-pregnan-20-one; tetrahydroCompound A: 3a,21-dihydroxy-5/?pregnane -11,20 dione; hexahydrocorticosterone: 5f pregnane 3a, 1 l/?,20a,21 tetrol : tetrahydrocortisone: 3a, 17a,21 -trihydroxy-5/9-pregnane-l 1,20-dione; tetrahydrocortisol : 3a,ll/?,17a,21-tetrahydroxy-5/?-pregnan-20-one; «?/o-tetrahydrocortisol: 3a,ll/?,17a,21tetrahydroxy-5a-pregnan-20-one; tetrahydrocorticosterone: 3a,ll/?,21-trihydroxy-5/?pregnan- 20- one; ff//o-tetrahydrocorticosterone: 3a, 11/9,21- trihydroxy-5a-pregnan-20one; tetrahydroaldosterone: 3a,l 1/?,21-trihydroxy-5/?-pregnan-20-on-18-al; 3ß-allotetrahydroaldosterone: 3/?,ll/?,21-trihydroxy-5a-pregnan-20-on-18-al and fludrocortisone: 9a-fluro-ll/?,17a,21-trihydroxy-4-pregnene-3,20-dione. -

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Methodology (a) Urine extraction and-hydrolysis. The methods used for this analysis have been described in a previous communication (Shackleton et al. 1973) but will be summarised here. The urine samples were made up to 300 ml with water and 100 ml was taken -

for steroid conjugate extraction on Amberlite XAD-2 columns (25 g). Following extraction the conjugates were hydrolysed in acetate buffer with Helix pomatia dige¬ stive juice and /?-glucuronidase (Ketodase). After 48 h the freed steroids were ex¬ tracted on 13 g columns of Amberlite XAD-2.

(b) Sephadex LH-20 chromatography. Sephadex LH-20 columns were used to purify the steroids prior to gas Chromatographie analysis. Six g columns were used with the solvent system cyclohexane:ethanol, 4:1; two fractions were collected (A) 12-55 ml (principally Cjg steroids) and (B) 55-160 ml (corticosterone and cortisol metabolites). -

Derivalisation. For multicomponent analysis aliquots of Fractions A and B combined and internal standards were added (5a-androstane-3a,17a-dioI and cholesteryl butyrate). The dried sample was dissolved in 100 fi\ 1 % methoxyamine hydrochloride in pyridine and heated for 1 h at 60°C. Following methyloxime forma¬ tion, 100 fil N-trimethylsilylimidazole was added and the mixture heated for 2 h at 100°C. The reagents were removed by passing the derivatives through a 250 mg co¬ lumn of Lipidex 5000 using the solvent system cyclohexane, 98: pyridine, 1: hexamethyldisilazane, 1: an adaptation of the method described by Axelson 8c Sjövall (1975). For the determination of tetrahydroaldosterone by selective ion monitoring 3ß-allotetrahydroaldosterone was added as internal standard to an aliquot of Fraction B. Following oxime formation silylation was carried out with 0.2 ml hexamethyldisilazane and 25 ;