- Email: [email protected]
Urinary excretion of dehydroepiandrosterone in normal children and in patients with adrenocortical disorders
CLINICA
CHIMICA
ACTA
371
cc_4 4581
URINARY
EXCRETION
CHILDREN S. B. P.4L
AND
OF DEHYDROEPIANDROSTERONE
IN PATIENTS
IN NORMAL
ADRENOCORTICAL
DISORDERS
AND W. M. TELLER
Universitiit Ulm. Abteilung Endokrinologie devheilkunde, D 7 9 Ulm (Donau) , (West (Received
WITH
May
und Stiffwechsel, Germany)
Zentrum
fiir Innere
Medizin
und Kin-
7. 1971)
SUMMARY
A systematic analytical procedure for the determination of urinary dehydroepiandrosterone (DHA) was developed. An aliquot of IOO ml from a 24-h urine collection was subjected to hot hydrolysis at neutral pH for 6 h. Liberated steroids were extracted with diethyl ether and the ketones were separated from the non-ketonic compounds by Girard’s T reaction. Dehydroepiandrosterone was separated by paper chromatography in Bush A system and, after eluting, the concentration of DHA was measured spectrophotometrically by a modified Zimmermann reaction. The overall average recovery of the procedure was 83.3% where DHA-7 a-T(n) sulphate-potassium salt was added to the urine and taken through the analysis. The identity of DHA was confirmed after making various chemical derivatives and checking their chromatographic mobilities against derivatives prepared from authentic steroids. When this procedure was applied to the urines of r8 normal children (IO boys and 8 girls), aged between 5 years I month-15 years 8 months, the average level of DHA was 0.6 mg/z4 h (range 0.22-1.9 mg/z4 h). The excretion of DHA was 26% (range 12.1-47~/~) of total neutral r7-ketosteroids (estimated after enzymatic hydrolysis of urine followed by solvolysis). From IO children, aged 9-15 years, with various adrenocortical disorders, the levels of DHA were found to be above normal. Of these IO patients, 2 had adenoma, 4 carcinoma and 4 nodular hyperplasia; the average value of DHA was 3.6 mg/24 h (range 2.555.1 mg/24 h). The excretion of DHA was 68.4% of total neutral r7-ketosteroids (range 55.5-83.6O/,). All these patients had to undergo surgery and, in each case, DHA was estimated after 3 months, when the average value was found to be 0.7 mg/24 h (range 0.38-1.1 mg/24 11). The average excretion of DHA was 30% (range 15.8-43%) of total neutral r7-ketosteroids. From these results it can be concluded that urinary DHA determination could be used for the diagnosis of adrenocortical disorders.
Part of this work was presented at the 9th Meeting crinology, Lyon I6-20th July, ‘970.
of the European
Society
Clin. Chim.
for Paediatric
Acta,
Endo-
38 (1972) 371-378
PAL, TELLER
372 INTRODUCTION
It is well established that the adrenal is able to secrete steroids which are strongly androgenic in certain disease states, such as in adrenal cancer, adrenal hyperplasia and virilization. Dehydroepiandrosterone (3/I-hydroxy-5-androsten-r7-one) which has weak androgenic potency, is secreted by the adrenal and can also serve as an efficient precursor of urinary androsterone and etiocholanolone. It has been isolated from human adrenal tissue and, furthermore, biosynthesis of DHA from [r-Xlacetate has been reported. It has also been isolated from adrenal venous blood. Dehydroepiandrosterone is found in the urine of normal women and in male castrates. Its urinary excretion increases after the administration of ACTH and decreases after the administration of cortisol. The level of DHA in urine is sometimes very high in patients with various diseases of the adrenal’ and there is little doubt that it derives partly from adrenal precursors. Therefore, it was hoped that an accurate determination of urinary excretoin of DHA in various adrenocortical disorders could be clinically useful. First, a suitable procedure for the determination of urinary DHA alone, had to be set up. Urinary DHA is mainly excreted as a sulphate. As, in our experience, no reliable sulphatase preparation was commercially available that would give efficient hydrolysis of DHA sulphate, various hydrolytic procedures were studied in order to find an effective way of hydrolysing DHA sulphate and of determining this steroid accurately. This communication will be confined to cases of children only, although, during the course of this work, urine samples from adult subjects and patients were used for recovery experiments. MATERIALS
AND METHODS
All chemicals and solvents used were of analytical grade. .%c &Helix pomatia. I ml contains IOOOOOunits of p-glucuronidase (Fishman), 50000 units sulphatase (Whitehead) (Industrie Biologique Franqaise, S.A., Quai du Moulin de Cage, Genevilliers, Seine, France). These quantities of enzymes, contained in a r-ml ampoule, were dissolved into 9 ml of M sodium acetate-acetic acid buffer of pH 5.2 before use. Glass-distilled mater was used throughout the work. Glass-stoppered test tubes 15 x 2.5 cm, approximate capacity 54 ml and 12.5 x 1.5 cm, approximate capacity ~5 ml were used (Quickfit & Quartz Ltd, U.K.). DehydroePiandrosterone was purchased from BDH Chemicals Ltd., Poole, U.K. Dehydroepiandrosterone sulphate-sodium salt and other reference steroids used were obtained as gifts from Steroid Reference Collection (Medical Research Council Chemistry Department, Westfield College, Hampstead, London, U.K.). Dehydroepiandrosterone-TX-T(n) sulphate, dihydrate (potassium salt), (n indicates the nominal position of the isotope where is any uncertainty as to whether the labelling is confined to the normal position) specific activity 930 mC/mmole; n-hexadecane-r,z-T were purchased from The Radiochemical Centre, Amersham, Buckinghamshire, U.K. z,5-Difihenyloxazole Clzx. Chim.
Acta,
(PPO), I,+bis-z,4-methyl-5-$henyloxa~olyl
38 (1972) 371-378
bewene
(dimethyl-
URINARY
POPOP),
EXCRETION
naphthalene,
OF DHA
dioxalze
373 and tol,Nene, all scintillator
grade, were used (E. Merck
A.G.). Dioxane scintillator was prepared after dissolving 60 g naphthalene, 8 g PPO and 0.2 g POPOP into a mixture of solvents containing 50 ml ethanol, IOO ml toluene and 900 ml dioxane and stored in amber glass bottles at 4’. Tolurne scintillator was prepared by adding 3.0 g PPO and 0.3 g POPOP in I litre of toluene and was stored in an amber glass bottle at 4O. All the extracts for paper chromatography or liquid scintillation counting were evaporated to dryness throughout this work in a water bath at 40’ under a stream of nitrogen. The extracts were spotted on paper chromatograms under a stream of nitrogen, later on replaced by air. The radioactive extracts were evaporated directly in high quality glass vials (Packard Low-Potassium-I) using 15 ml of scintillation solution. The Packard Liquid Scintillation spectrometer was operated at an average efficiency of 35% for 3H with a background of 14 counts/min. Sample measurements were corrected for quenching by the internal standardization method. n-Hexadecane-r,z-T was used as an internal standard. The background and radioactive samples were counted long enough to collect
roooo counts. A rotary-film evaporator (Rotavapor) R was used (Btichi, Flawil, Switzerland). The paper chromatograms were prepared on a sheet of Whatman No. z filter
paper without
any previous washing and r-cm wide lanes were cut out between
the
strips. A sample of DHA extracted from pooled urines, purified by paper chromatography and repeatedly crystallised, was used as reference steroid at the same time as authentic DHA. Chromatograms were equilibrated for at least 3 h, if not overnight, and developed at room temperature, between zo-22", keeping the tanks in a wooden draft-proof box, and, later on, in a thermostatically controlled hot box at 30’. Bush A system (1000 ml petroleum ether, b.p. 1oo~--12o“: 800 ml methanol: 200 ml water) was used throughout the work. Urine was collected over and stored at 4’ until analysed. were made up to 2 litres if they when the volume was less than
a period of 24 h under 5 ml toluene as preservative In case of adults, volumes of all the urine collections were less than this volume. In the case of children, 500 ml, it was made up to this volume; urine collec-
tions of over 500 ml but of less than I litre were made up to I litre. Distilled
water
was used in each case. The total neutral IT-ketosteroids were estimated after hydrolysing IOO ml urine from 24-h collections with Sue d’Helix pomatia, followed by solvolysis2. The ketonic steroids were separated from the non-ketones by Girard’s T reaction and the concentration of IT-ketosteroids was measured on a 115th portion of the steroid residue by a modified Zimmermann procedure. The total neutral IT-ketosteroids and dehydroepiandrosterone
were determined
in the patients before surgery and three months after surgery. Different procedures were carried out to hydrolyse the urine and to find an efficient condition under which to hydrolyse DHA sulphate and achieve an accurate estimation of this steroid. Urine samples from a normal man, a normal woman and from various patients with endocrine disorders, were subjected to: I. Hot acid hydrolysis (IO ml cont. HC1/1oo ml urine) : (a) extracted with diethyl ether C&z. Chim. Acta, 38 (1971) 371-378
P.4L,TELLER
374 (b) overlay with benzene (c) overlay with toluene 2. Hot hydrolysis under (a) overlay with benzene
during hydrolysis3 during hydrolysis. neutral pH (ref. 4) during hydrolysis
(b) overlay with toluene during hydrolysis (c) extracted with diethyl ether after hydrolysis. 3. Enzyme hydrolysis with Sue d’Helix$omatia, extracted
with ether, followed
by solvolysis of the aqueous phase, and washings. In each case, IOO ml urine was taken from 24-h collections, after hydrolysis, the extracted steroids were separated for ketones from non-ketones by Girard’s T reaction and further purified by paper chromatography. The concentration of DHA was finally measured spectrophotometrically by a modified Zimmermann procedure. As mentioned before, comparison of different hydrolytic procedures was performed and DHA was recovered in each case by adding IOO ,ug of DHA sulphatesodium salt and 15000 counts/min of [3H]DHA sulphate-potassium salt to IOO ml of water and to IOO ml of urine. Ten experiments were performed in each case. Chemical derivatives such as acetate, epoxide and chromic acid oxidation product
of DHA isolated from urine, were prepared
in parallel with pure DHA. The
respective acetate, chromic acid oxidation product and epoxide of the test and pure steroid were chromatographed at the same time. When these paper chromatograms were dipped into Zimmermann reagent to locate the position of steroids, in each case a single spot was found on the chromatogram with the same mobility as the derivative prepared from the reference steroid. The melting point of isolated DHA was checked and agreed with the published figures. A sulphuric acid absorption curve was also prepared from DHA isolated from urine, with agreed with the curve prepared from pure steroid. RESULTS
It was found, by comparing different hydrolytic procedures for urinary DHA, that the maximum amount of DHA was estimated after hot hydrolysis of the urine under neutral conditions overlaid with benzene, toluene or extracting with ether. The pattern was the same for urines from normal human individuals (2 males and z females) and patients with different endocrine disorders (2 adrenal hyperplasia, 2 simple hirsutism, I hirsutism with polycystic ovaries and I hirsutism with ovarian tumour). When the recovery experiments (n = IO) for DHA were performed, applying different hydrolytic procedures by the addition of DHA-sulphate-sodium salt and [aH]DHA-sulphate-potassium salt to water and to urine, in each case, the maximum chemical and radioactive recovery of DHA was found after hot hydrolysis under neutral conditions, overlaid with benzene, toluene or extracting with ether. Overlay with benzene: DHA Water 74.5% (SD & 1.23) range (72%76.2%) Urine 74.1% (SD & 0.91) range (72.7-75.376)
Clin. Chiw.
Acta,
38 (1972)
371-378
[3H]DHA 81.7ojo (SD + 0.67) range (80.8-82.6~~) 81.1% (SD -L 0.9) range (79.4-82.7%)
URINARY EXCRETION OF
DHA
375
Overlay with toluene. DHA Water 74.2% (SD rsI:1.1) range (72.3-75.60/o) Urine 742% (SD f 0.82) range (73.2-75.6%)
[3H]DHA 83.8% (SD & 0.57) range (83.2-84.6%) 80.6% (SD -& 1.28) range (78.6-82.5%)
Ext~~ct~o~~with ether:
DHA Water 73.8% (SD rt 1.26) range (72.3-75.7%) Urine 74.1% (SD -& 0.68) range (73.2-75.1%)
r3H]DHA 83.3% (SD & 0.94) range (82.2-54.7%) 81.4% (SD rir:0.73) range (80.2-82.674)
Table I shows details of the method which was finally adopted for the determination of urinary DHA used throughout the work. The results presented are not corrected for recoveries. TABLE
T
PLOW-SHEET
OF THE
METHOD
FOR DETERMININATION
A roo-ml aliquot of urine from a 24-h collection+
OF URINARY
fH]DHA
DHA
sulphate was taken.
4 Hydrolysed for 6 h (IDO’) at pH 7, cooled. Liberated steroids were extracted washed with 0.1 &’ NaOH and water: evaporated to dryness.
with diethyl ether,
& Ketones were separated from non-ketones by Girard’s T reaction. Steroid residue was chromatographed for DH.4 in Bush A system, eluted and evaporated. j. The concentration
of DWA was determined
spectrophotometrically --.. ~_
by Zimmermann
reaction.
Normal values
From 18 children (IO boys and 8 girls) aged between 5 years I month and 15 years 8 months, all of whom hospitalised, but without endocrine disorders, the excretion of total neutral 17-KS was between 1.0-6.0 mg/z4 h (mean 2.3) and that of DHA was between o.zz-I.9 mg/24 h (mean 0.6); the percentage (“/;i)DHA of total 17-KS being between 12.1% to 47% (mean 26%). Pathological values
In IO children with adrenocortical disorders, DHA was estimated. Of these IO patients, 2 had adenoma, 4 carcinoma and 4 nodular hyperplasia; the average value of DHA was 3.6 mg/24 h (range 2.5-5.1 mg/24 h); the total neutral 17-KS was 5.2 mg/z4 h (range 4.5-6.3 mg/z4 h). The excretion of DHA was 68.4% of total neutral 17-KS (range 55.5-83.60/0). In each case DHA was estimated 3 months after surgery and the average value of DHA was found to be 0.7 mg/24 h (range 0.38-1.1 mg/z4 h). The average excretion of DHA was 30% (range 15.8--43%) of total neutral x7-KS. UISCUSSION
The comparison of sulphate shows that hot established that hot acid dehydration, halogenation
different hydrolytic procedures for dehydroepiandrosterone acid hydrolysis was found to be disappointing. It is well hydrolysis forms artifacts of the steroid molecule due to and other molecular rearrangementP; also, it is not certain C&z. Chinz.Acta, 38 (1972)371-378
PAL,TEl.LER
376
whether hydrochloric acid really completes the hydrolysis of DHA sulphate as poor chemical and radioactive recoveries were found after the addition of DHA sulphatesodium salt and [3H]DHA sulphate-potassium salt. Hot acid hydrolysis after overlay with an organic solvent, such as benzene or toluene, was found to be more satisfactory as it is a simultaneous hydrolysis and extraction process; although recoveries of added steroid conjugates is a highly toxic solvent
were better but the procedure was not adopted as benzene and toluene was found to be inconvenient to handle due to
its high boiling point (109-112"). Dehydroepiandrosterone is mainly excreted as sulphate conjugates in human urine. Although its excretion as glucuronides in normal children of both sexes (aged between 6 months and 16 years) had been reported6, no such information is available from the authors’ laboratory in this respect. This finding appeared, in general, to be very uncertain. It has been reported7 that the glucuronide fraction may be considered as an artifact from DHA sulphate shown to be transformed to 6/Shydroxy-3,5-cyclo5a-androstan-r7-one, even under conditions as mild as glucuronide hydrolysis. This rearrangement occurred particularly to a large extent when urine was directly subjected to enzyme hydrolysis for 3 days. Under these conditions, the glucuronide fraction contained up to 46.5% of iso-androstenolone 8$s. It was much less evident when the conjugates were first extracted from the urine and were then hydrolysed. Enzyme hydrolysis followed by solvolysis2 was also rejected. In the first place, p-glucuronidase does not hydrolyse sulphate conjugates; it has been reported that, unless interfering substances have been eliminated from the urine, total hydrolysis of steroid glucuronosides cannot be achieved with /3-glucuronidase. Secondly, due to the very slow activity of sulphatase, its efficiency in hydrolysing a sulphate conjugate is uncertain, but solvolysis of the aqueous layer (after the extraction of liberated steroids by enzyme hydrolysis) at pH I with sulphuric acid, sodium chloride and ethyl acetate, was found to be an ideal condition for hydrolysing all steroid sulphate conjugates completely. This procedure was not strictly necessary for this study, especially when DHA alone was to be estimated; besides this, epiandrosterone migrates very closely to DHA on a paper chromatogram. If epiandrosterone is not separated by an additional chromatographic step, DHA could be overestimated. As this study was mainly concerned with the estimation of DHA only, the hot hydrolysis procedure4 was adopted, which consists of boiling the urine at neutral pH for 6 h. In this procedure, 3F-sulphates of 3/Lhydroxy-d5 steroids were selectively hydrolysed, androsterone sulphate and epiandrosterone sulphate were not hydrolysed during the boiling of urines. The chemical and radioactive recoveries of DHX by the addition of DHA sulphate conjugates were good. Hot hydrolysis after overlay with benzene or toluene did not improve the recovery significantly. Thus the hot hydrolysis procedure was adopted; after hydrolysis of the urines at neutral pH for 6 11, they were cooled, DHA was extracted with ether and the residue obtained from the ether extract was subjected to Girard’s T reaction to separate the ketones from non-ketones and, after further purification by chromatography, DHA was estimated by the Zimmermann reaction. The specificity of the Zimmermann reaction in human IT-ketosteroids is IOO~/~.This reaction is easier and more rapid in a laborator!, where a large number of samples are analysed. We have not, so far, come across any values published in the literature for urinary DHA in children obtained by hot hydrolysis at neutral pH. The range of Cl&. Chim.
Ada,
38 (197')371-178
DHA
377
values of DHA from normal children given in this communication agreed in general with other published values (Table II) in spite of some differences in methodology. In a few cases, they were lower than the values given here. It has also been reported that DHA was not detected in urines from children, particularly in the pre-pubertal stage. No DHA was found in the urine of children under the age of 9 years, except in sexually premature childrenl”. Similarly, no DHA was detected in 5 boys aged 5 to 7 years and in 7 girls aged 5 to 7 years”. TABLE URIKARY
11 EXCRETION
OF
NORMAL.
Age, yeavs
A~thw __-..____K&d&r et al.jz Kid&r et (al.= Vestergaardls Vestergaard13 Paulsen et al I4 Teller’s Teller’s Steen0 et ad.“” Blunckl’ Blunck” Gupta’a Berger et aE.l@ Berger ct al9 Present authors Present authors
DHA IN
5
5
3 3 10
-13 -13 -77 -16 -16
ro6/12-r2p/l* 1P/~~-16~/,,
II -15 -IO 7 -15 14 3 -14 5 -18 5 -16 5’/,,-r56&2 76~,~-14l/~~ ___~____.
CHILDREN,
FROM
S&Z! 18 20 18 18 34
boys girls boys girls (mixed) II (mixed) 8 (mixed) 60 boys 12 (mixed) 11 (mixed) 42 (mixed) ‘74 boys 127 girls IO boys 8 girls ---
THE
LITERATURE
V&ES (range) mgjq h 0.1 -0.5 0.1 -1.1 o -2.54 0 -0.60 0.03 -0.48 0.004-0.07 0.01+0.8
0.04x-0.34 0.008-0.1
I4
0.075-0.48 0 -0.42 0
-0.8
-I.55 0.22 -1.9 0.30 -1.2 _____0
The values of DHA from IO children with adrenocortical disorders were found to be higher than those obtained from 18 normal children. When DHA was again estimated in these children with adrenocortical disorders 3 months after surgery, the level of DHA was within the normal range. It is essential to use great care in interpreting the results obtained from the urinary excretion of steroids in children, especially of androgens, before the onset of puberty. All methods developed for steroid investigation in adults must be reassessed before being applied to children, particularly in early infancy’@. An adult human individual in good physical condition has a fairly stable maximal level of activity, especially in regard to androgen metabolism, whilst the child is undergoing constant ph~Tsiolo~ica1 changes and has sometimes not attained normal adult levels of androgen production even at the age of 15 and 16 years. D~~llydroepiandrostero~e has not invariably been detected as a constituent of urine in infants and in childrenlo$‘lB1a, as it is in normal adults. However, from the present investigation, it is possible to conclude that the determination of urinary DHA in children could be used as an index of abnormal adrenocortical function and was found to be clinically useful. ACKNOWLEDGEMENTS
We wish to thank Professor Dr. E. I?. Pfeiffer and other physicians and surgeons for their interest shown during the course of this work ; Professor W. Kiyne, W’estfield College, London, for generously providing reference compounds from the Xedical C&n. Chint.
Acta,
38
(1972)
371-378
PAL, TELLER
375
Research Council Steroid Reference Section and FrWein B&-be1 SchZfer for performing Zimmermann estimations for dehydroepiandrosterone in normal children. Our thanks also go to Frau Martha Rupp and to Mrs. M. R. Pal for their help in typing and preparing the manuscript, respectively. REFERENCES I H. L. MASOS ASD W. W. ENGSTROM, Physiol.Rev., 30(19p) 213. S. BURSTEIN AND S. LIEBERMAR.,J.B~O~. Chew, 233(rgj8)331. P. \T~~TER~.%.~R~AND B. CLAUSSEN, ~~~~ ~~~do~~~n~~., 39 Suppl. 64 (1962). Ii. FOTHE~BY, Biochem. J., 69 (1958) 596. I?, I. DORFMAX AND F. UEU‘GAR,in Metabolism ofSteroid Normones, Academic
2 3 4 5
IgGj, 6
7 k 9
IO II IL 13 14
15 16
17
18 rg
Press, New York
?. 28.
B. LORAS, w. Roux, B. ChuTENET, C. OLLAGXEN, M. FOREST, E. DE PERETTI ANU J. %R*~~Asu, in h. VER?VIEULF.~~ hND R. ESLEY (Ed%), And~0gen.sin Novmalund Pathological Con&ions, PFOG.Second Symp. Steroid Howmnes, Ghent, Excerpta NIedica Foundation, Rmslerdam. S. ~RSTEIN AND R. I. DORFMAN, Acta EndocrinoE.,40(1962) I&% 1'.VESTERGMRD, Acta Endocrinol,, 39, Suppl. 64 (1962)‘50. J. HAMMERSTEIN, in C. CASSANO, A. KLOPPER AED C. CONTI (Eds.), Research on Steroids, Vol. 111, North-Holland Publishing Company, Amsterdam, I$%, p. 323, B. P. P~u~ser\;AND E. 15. SOBEL, Amer. J. Diseases Children, 1oo (1960) 546. F. I~XUS,IX. P. ZURBR~GCJ, J.CARA AND L. LGARDNER, J.Clin.Endocrinol., 22(1962) rogo. A. K~DAR, T. FEH&R AX‘D 0. KOREF. Arch. DiseasesCh~Zdhood, 39 (1964) 257. P. VESTERGAARD, Acta E&ocrinot., 49 (1965) 436. E. P. PAULSEX, E. H. SO~EL AND M.S. SHAFRAX,J. Clin.Endocrinol., 26(x966) 329. W. 3%.TEI.LER,Z.&S. Exp. Med. ~42 (1967) 222. C. STEENO, W. HEY%, H. VAX BAELEN, A. VAX MERLE AND P. DYE MOIOOR,Ew'. J. .%v'oids, 2 (1967) 273. W. BLIJK~K, ilcta EndacvinoE., 59 Suppl. 134 (1968) 9. D. GUPTA, Clin. Chirn. Acta, 26 (1~69)256. H. BERGER, M. FINK, H. J. FRITZ, H. GLEISPACH, P. HEIDEMAN?: AXD J. WOLF, 2. li'lin
Chew .KEi,z. B&hem., 8 (1970) 354. 20 V.I.,. MITCHELL AND H.L.SWACKELTON,~~I Cf.~oDA~sKYA~DC.~.STEWART(~CIS.),~~W~~~~~ in Clinical Chemistry, Academic Press, New York, London, 1969, p. 141. C&?. Chiiir. Actn, 38 (1972) 371-378
CHIMICA
ACTA
371
cc_4 4581
URINARY
EXCRETION
CHILDREN S. B. P.4L
AND
OF DEHYDROEPIANDROSTERONE
IN PATIENTS
IN NORMAL
ADRENOCORTICAL
DISORDERS
AND W. M. TELLER
Universitiit Ulm. Abteilung Endokrinologie devheilkunde, D 7 9 Ulm (Donau) , (West (Received
WITH
May
und Stiffwechsel, Germany)
Zentrum
fiir Innere
Medizin
und Kin-
7. 1971)
SUMMARY
A systematic analytical procedure for the determination of urinary dehydroepiandrosterone (DHA) was developed. An aliquot of IOO ml from a 24-h urine collection was subjected to hot hydrolysis at neutral pH for 6 h. Liberated steroids were extracted with diethyl ether and the ketones were separated from the non-ketonic compounds by Girard’s T reaction. Dehydroepiandrosterone was separated by paper chromatography in Bush A system and, after eluting, the concentration of DHA was measured spectrophotometrically by a modified Zimmermann reaction. The overall average recovery of the procedure was 83.3% where DHA-7 a-T(n) sulphate-potassium salt was added to the urine and taken through the analysis. The identity of DHA was confirmed after making various chemical derivatives and checking their chromatographic mobilities against derivatives prepared from authentic steroids. When this procedure was applied to the urines of r8 normal children (IO boys and 8 girls), aged between 5 years I month-15 years 8 months, the average level of DHA was 0.6 mg/z4 h (range 0.22-1.9 mg/z4 h). The excretion of DHA was 26% (range 12.1-47~/~) of total neutral r7-ketosteroids (estimated after enzymatic hydrolysis of urine followed by solvolysis). From IO children, aged 9-15 years, with various adrenocortical disorders, the levels of DHA were found to be above normal. Of these IO patients, 2 had adenoma, 4 carcinoma and 4 nodular hyperplasia; the average value of DHA was 3.6 mg/24 h (range 2.555.1 mg/24 h). The excretion of DHA was 68.4% of total neutral r7-ketosteroids (range 55.5-83.6O/,). All these patients had to undergo surgery and, in each case, DHA was estimated after 3 months, when the average value was found to be 0.7 mg/24 h (range 0.38-1.1 mg/24 11). The average excretion of DHA was 30% (range 15.8-43%) of total neutral r7-ketosteroids. From these results it can be concluded that urinary DHA determination could be used for the diagnosis of adrenocortical disorders.
Part of this work was presented at the 9th Meeting crinology, Lyon I6-20th July, ‘970.
of the European
Society
Clin. Chim.
for Paediatric
Acta,
Endo-
38 (1972) 371-378
PAL, TELLER
372 INTRODUCTION
It is well established that the adrenal is able to secrete steroids which are strongly androgenic in certain disease states, such as in adrenal cancer, adrenal hyperplasia and virilization. Dehydroepiandrosterone (3/I-hydroxy-5-androsten-r7-one) which has weak androgenic potency, is secreted by the adrenal and can also serve as an efficient precursor of urinary androsterone and etiocholanolone. It has been isolated from human adrenal tissue and, furthermore, biosynthesis of DHA from [r-Xlacetate has been reported. It has also been isolated from adrenal venous blood. Dehydroepiandrosterone is found in the urine of normal women and in male castrates. Its urinary excretion increases after the administration of ACTH and decreases after the administration of cortisol. The level of DHA in urine is sometimes very high in patients with various diseases of the adrenal’ and there is little doubt that it derives partly from adrenal precursors. Therefore, it was hoped that an accurate determination of urinary excretoin of DHA in various adrenocortical disorders could be clinically useful. First, a suitable procedure for the determination of urinary DHA alone, had to be set up. Urinary DHA is mainly excreted as a sulphate. As, in our experience, no reliable sulphatase preparation was commercially available that would give efficient hydrolysis of DHA sulphate, various hydrolytic procedures were studied in order to find an effective way of hydrolysing DHA sulphate and of determining this steroid accurately. This communication will be confined to cases of children only, although, during the course of this work, urine samples from adult subjects and patients were used for recovery experiments. MATERIALS
AND METHODS
All chemicals and solvents used were of analytical grade. .%c &Helix pomatia. I ml contains IOOOOOunits of p-glucuronidase (Fishman), 50000 units sulphatase (Whitehead) (Industrie Biologique Franqaise, S.A., Quai du Moulin de Cage, Genevilliers, Seine, France). These quantities of enzymes, contained in a r-ml ampoule, were dissolved into 9 ml of M sodium acetate-acetic acid buffer of pH 5.2 before use. Glass-distilled mater was used throughout the work. Glass-stoppered test tubes 15 x 2.5 cm, approximate capacity 54 ml and 12.5 x 1.5 cm, approximate capacity ~5 ml were used (Quickfit & Quartz Ltd, U.K.). DehydroePiandrosterone was purchased from BDH Chemicals Ltd., Poole, U.K. Dehydroepiandrosterone sulphate-sodium salt and other reference steroids used were obtained as gifts from Steroid Reference Collection (Medical Research Council Chemistry Department, Westfield College, Hampstead, London, U.K.). Dehydroepiandrosterone-TX-T(n) sulphate, dihydrate (potassium salt), (n indicates the nominal position of the isotope where is any uncertainty as to whether the labelling is confined to the normal position) specific activity 930 mC/mmole; n-hexadecane-r,z-T were purchased from The Radiochemical Centre, Amersham, Buckinghamshire, U.K. z,5-Difihenyloxazole Clzx. Chim.
Acta,
(PPO), I,+bis-z,4-methyl-5-$henyloxa~olyl
38 (1972) 371-378
bewene
(dimethyl-
URINARY
POPOP),
EXCRETION
naphthalene,
OF DHA
dioxalze
373 and tol,Nene, all scintillator
grade, were used (E. Merck
A.G.). Dioxane scintillator was prepared after dissolving 60 g naphthalene, 8 g PPO and 0.2 g POPOP into a mixture of solvents containing 50 ml ethanol, IOO ml toluene and 900 ml dioxane and stored in amber glass bottles at 4’. Tolurne scintillator was prepared by adding 3.0 g PPO and 0.3 g POPOP in I litre of toluene and was stored in an amber glass bottle at 4O. All the extracts for paper chromatography or liquid scintillation counting were evaporated to dryness throughout this work in a water bath at 40’ under a stream of nitrogen. The extracts were spotted on paper chromatograms under a stream of nitrogen, later on replaced by air. The radioactive extracts were evaporated directly in high quality glass vials (Packard Low-Potassium-I) using 15 ml of scintillation solution. The Packard Liquid Scintillation spectrometer was operated at an average efficiency of 35% for 3H with a background of 14 counts/min. Sample measurements were corrected for quenching by the internal standardization method. n-Hexadecane-r,z-T was used as an internal standard. The background and radioactive samples were counted long enough to collect
roooo counts. A rotary-film evaporator (Rotavapor) R was used (Btichi, Flawil, Switzerland). The paper chromatograms were prepared on a sheet of Whatman No. z filter
paper without
any previous washing and r-cm wide lanes were cut out between
the
strips. A sample of DHA extracted from pooled urines, purified by paper chromatography and repeatedly crystallised, was used as reference steroid at the same time as authentic DHA. Chromatograms were equilibrated for at least 3 h, if not overnight, and developed at room temperature, between zo-22", keeping the tanks in a wooden draft-proof box, and, later on, in a thermostatically controlled hot box at 30’. Bush A system (1000 ml petroleum ether, b.p. 1oo~--12o“: 800 ml methanol: 200 ml water) was used throughout the work. Urine was collected over and stored at 4’ until analysed. were made up to 2 litres if they when the volume was less than
a period of 24 h under 5 ml toluene as preservative In case of adults, volumes of all the urine collections were less than this volume. In the case of children, 500 ml, it was made up to this volume; urine collec-
tions of over 500 ml but of less than I litre were made up to I litre. Distilled
water
was used in each case. The total neutral IT-ketosteroids were estimated after hydrolysing IOO ml urine from 24-h collections with Sue d’Helix pomatia, followed by solvolysis2. The ketonic steroids were separated from the non-ketones by Girard’s T reaction and the concentration of IT-ketosteroids was measured on a 115th portion of the steroid residue by a modified Zimmermann procedure. The total neutral IT-ketosteroids and dehydroepiandrosterone
were determined
in the patients before surgery and three months after surgery. Different procedures were carried out to hydrolyse the urine and to find an efficient condition under which to hydrolyse DHA sulphate and achieve an accurate estimation of this steroid. Urine samples from a normal man, a normal woman and from various patients with endocrine disorders, were subjected to: I. Hot acid hydrolysis (IO ml cont. HC1/1oo ml urine) : (a) extracted with diethyl ether C&z. Chim. Acta, 38 (1971) 371-378
P.4L,TELLER
374 (b) overlay with benzene (c) overlay with toluene 2. Hot hydrolysis under (a) overlay with benzene
during hydrolysis3 during hydrolysis. neutral pH (ref. 4) during hydrolysis
(b) overlay with toluene during hydrolysis (c) extracted with diethyl ether after hydrolysis. 3. Enzyme hydrolysis with Sue d’Helix$omatia, extracted
with ether, followed
by solvolysis of the aqueous phase, and washings. In each case, IOO ml urine was taken from 24-h collections, after hydrolysis, the extracted steroids were separated for ketones from non-ketones by Girard’s T reaction and further purified by paper chromatography. The concentration of DHA was finally measured spectrophotometrically by a modified Zimmermann procedure. As mentioned before, comparison of different hydrolytic procedures was performed and DHA was recovered in each case by adding IOO ,ug of DHA sulphatesodium salt and 15000 counts/min of [3H]DHA sulphate-potassium salt to IOO ml of water and to IOO ml of urine. Ten experiments were performed in each case. Chemical derivatives such as acetate, epoxide and chromic acid oxidation product
of DHA isolated from urine, were prepared
in parallel with pure DHA. The
respective acetate, chromic acid oxidation product and epoxide of the test and pure steroid were chromatographed at the same time. When these paper chromatograms were dipped into Zimmermann reagent to locate the position of steroids, in each case a single spot was found on the chromatogram with the same mobility as the derivative prepared from the reference steroid. The melting point of isolated DHA was checked and agreed with the published figures. A sulphuric acid absorption curve was also prepared from DHA isolated from urine, with agreed with the curve prepared from pure steroid. RESULTS
It was found, by comparing different hydrolytic procedures for urinary DHA, that the maximum amount of DHA was estimated after hot hydrolysis of the urine under neutral conditions overlaid with benzene, toluene or extracting with ether. The pattern was the same for urines from normal human individuals (2 males and z females) and patients with different endocrine disorders (2 adrenal hyperplasia, 2 simple hirsutism, I hirsutism with polycystic ovaries and I hirsutism with ovarian tumour). When the recovery experiments (n = IO) for DHA were performed, applying different hydrolytic procedures by the addition of DHA-sulphate-sodium salt and [aH]DHA-sulphate-potassium salt to water and to urine, in each case, the maximum chemical and radioactive recovery of DHA was found after hot hydrolysis under neutral conditions, overlaid with benzene, toluene or extracting with ether. Overlay with benzene: DHA Water 74.5% (SD & 1.23) range (72%76.2%) Urine 74.1% (SD & 0.91) range (72.7-75.376)
Clin. Chiw.
Acta,
38 (1972)
371-378
[3H]DHA 81.7ojo (SD + 0.67) range (80.8-82.6~~) 81.1% (SD -L 0.9) range (79.4-82.7%)
URINARY EXCRETION OF
DHA
375
Overlay with toluene. DHA Water 74.2% (SD rsI:1.1) range (72.3-75.60/o) Urine 742% (SD f 0.82) range (73.2-75.6%)
[3H]DHA 83.8% (SD & 0.57) range (83.2-84.6%) 80.6% (SD -& 1.28) range (78.6-82.5%)
Ext~~ct~o~~with ether:
DHA Water 73.8% (SD rt 1.26) range (72.3-75.7%) Urine 74.1% (SD -& 0.68) range (73.2-75.1%)
r3H]DHA 83.3% (SD & 0.94) range (82.2-54.7%) 81.4% (SD rir:0.73) range (80.2-82.674)
Table I shows details of the method which was finally adopted for the determination of urinary DHA used throughout the work. The results presented are not corrected for recoveries. TABLE
T
PLOW-SHEET
OF THE
METHOD
FOR DETERMININATION
A roo-ml aliquot of urine from a 24-h collection+
OF URINARY
fH]DHA
DHA
sulphate was taken.
4 Hydrolysed for 6 h (IDO’) at pH 7, cooled. Liberated steroids were extracted washed with 0.1 &’ NaOH and water: evaporated to dryness.
with diethyl ether,
& Ketones were separated from non-ketones by Girard’s T reaction. Steroid residue was chromatographed for DH.4 in Bush A system, eluted and evaporated. j. The concentration
of DWA was determined
spectrophotometrically --.. ~_
by Zimmermann
reaction.
Normal values
From 18 children (IO boys and 8 girls) aged between 5 years I month and 15 years 8 months, all of whom hospitalised, but without endocrine disorders, the excretion of total neutral 17-KS was between 1.0-6.0 mg/z4 h (mean 2.3) and that of DHA was between o.zz-I.9 mg/24 h (mean 0.6); the percentage (“/;i)DHA of total 17-KS being between 12.1% to 47% (mean 26%). Pathological values
In IO children with adrenocortical disorders, DHA was estimated. Of these IO patients, 2 had adenoma, 4 carcinoma and 4 nodular hyperplasia; the average value of DHA was 3.6 mg/24 h (range 2.5-5.1 mg/24 h); the total neutral 17-KS was 5.2 mg/z4 h (range 4.5-6.3 mg/z4 h). The excretion of DHA was 68.4% of total neutral 17-KS (range 55.5-83.60/0). In each case DHA was estimated 3 months after surgery and the average value of DHA was found to be 0.7 mg/24 h (range 0.38-1.1 mg/z4 h). The average excretion of DHA was 30% (range 15.8--43%) of total neutral x7-KS. UISCUSSION
The comparison of sulphate shows that hot established that hot acid dehydration, halogenation
different hydrolytic procedures for dehydroepiandrosterone acid hydrolysis was found to be disappointing. It is well hydrolysis forms artifacts of the steroid molecule due to and other molecular rearrangementP; also, it is not certain C&z. Chinz.Acta, 38 (1972)371-378
PAL,TEl.LER
376
whether hydrochloric acid really completes the hydrolysis of DHA sulphate as poor chemical and radioactive recoveries were found after the addition of DHA sulphatesodium salt and [3H]DHA sulphate-potassium salt. Hot acid hydrolysis after overlay with an organic solvent, such as benzene or toluene, was found to be more satisfactory as it is a simultaneous hydrolysis and extraction process; although recoveries of added steroid conjugates is a highly toxic solvent
were better but the procedure was not adopted as benzene and toluene was found to be inconvenient to handle due to
its high boiling point (109-112"). Dehydroepiandrosterone is mainly excreted as sulphate conjugates in human urine. Although its excretion as glucuronides in normal children of both sexes (aged between 6 months and 16 years) had been reported6, no such information is available from the authors’ laboratory in this respect. This finding appeared, in general, to be very uncertain. It has been reported7 that the glucuronide fraction may be considered as an artifact from DHA sulphate shown to be transformed to 6/Shydroxy-3,5-cyclo5a-androstan-r7-one, even under conditions as mild as glucuronide hydrolysis. This rearrangement occurred particularly to a large extent when urine was directly subjected to enzyme hydrolysis for 3 days. Under these conditions, the glucuronide fraction contained up to 46.5% of iso-androstenolone 8$s. It was much less evident when the conjugates were first extracted from the urine and were then hydrolysed. Enzyme hydrolysis followed by solvolysis2 was also rejected. In the first place, p-glucuronidase does not hydrolyse sulphate conjugates; it has been reported that, unless interfering substances have been eliminated from the urine, total hydrolysis of steroid glucuronosides cannot be achieved with /3-glucuronidase. Secondly, due to the very slow activity of sulphatase, its efficiency in hydrolysing a sulphate conjugate is uncertain, but solvolysis of the aqueous layer (after the extraction of liberated steroids by enzyme hydrolysis) at pH I with sulphuric acid, sodium chloride and ethyl acetate, was found to be an ideal condition for hydrolysing all steroid sulphate conjugates completely. This procedure was not strictly necessary for this study, especially when DHA alone was to be estimated; besides this, epiandrosterone migrates very closely to DHA on a paper chromatogram. If epiandrosterone is not separated by an additional chromatographic step, DHA could be overestimated. As this study was mainly concerned with the estimation of DHA only, the hot hydrolysis procedure4 was adopted, which consists of boiling the urine at neutral pH for 6 h. In this procedure, 3F-sulphates of 3/Lhydroxy-d5 steroids were selectively hydrolysed, androsterone sulphate and epiandrosterone sulphate were not hydrolysed during the boiling of urines. The chemical and radioactive recoveries of DHX by the addition of DHA sulphate conjugates were good. Hot hydrolysis after overlay with benzene or toluene did not improve the recovery significantly. Thus the hot hydrolysis procedure was adopted; after hydrolysis of the urines at neutral pH for 6 11, they were cooled, DHA was extracted with ether and the residue obtained from the ether extract was subjected to Girard’s T reaction to separate the ketones from non-ketones and, after further purification by chromatography, DHA was estimated by the Zimmermann reaction. The specificity of the Zimmermann reaction in human IT-ketosteroids is IOO~/~.This reaction is easier and more rapid in a laborator!, where a large number of samples are analysed. We have not, so far, come across any values published in the literature for urinary DHA in children obtained by hot hydrolysis at neutral pH. The range of Cl&. Chim.
Ada,
38 (197')371-178
DHA
377
values of DHA from normal children given in this communication agreed in general with other published values (Table II) in spite of some differences in methodology. In a few cases, they were lower than the values given here. It has also been reported that DHA was not detected in urines from children, particularly in the pre-pubertal stage. No DHA was found in the urine of children under the age of 9 years, except in sexually premature childrenl”. Similarly, no DHA was detected in 5 boys aged 5 to 7 years and in 7 girls aged 5 to 7 years”. TABLE URIKARY
11 EXCRETION
OF
NORMAL.
Age, yeavs
A~thw __-..____K&d&r et al.jz Kid&r et (al.= Vestergaardls Vestergaard13 Paulsen et al I4 Teller’s Teller’s Steen0 et ad.“” Blunckl’ Blunck” Gupta’a Berger et aE.l@ Berger ct al9 Present authors Present authors
DHA IN
5
5
3 3 10
-13 -13 -77 -16 -16
ro6/12-r2p/l* 1P/~~-16~/,,
II -15 -IO 7 -15 14 3 -14 5 -18 5 -16 5’/,,-r56&2 76~,~-14l/~~ ___~____.
CHILDREN,
FROM
S&Z! 18 20 18 18 34
boys girls boys girls (mixed) II (mixed) 8 (mixed) 60 boys 12 (mixed) 11 (mixed) 42 (mixed) ‘74 boys 127 girls IO boys 8 girls ---
THE
LITERATURE
V&ES (range) mgjq h 0.1 -0.5 0.1 -1.1 o -2.54 0 -0.60 0.03 -0.48 0.004-0.07 0.01+0.8
0.04x-0.34 0.008-0.1
I4
0.075-0.48 0 -0.42 0
-0.8
-I.55 0.22 -1.9 0.30 -1.2 _____0
The values of DHA from IO children with adrenocortical disorders were found to be higher than those obtained from 18 normal children. When DHA was again estimated in these children with adrenocortical disorders 3 months after surgery, the level of DHA was within the normal range. It is essential to use great care in interpreting the results obtained from the urinary excretion of steroids in children, especially of androgens, before the onset of puberty. All methods developed for steroid investigation in adults must be reassessed before being applied to children, particularly in early infancy’@. An adult human individual in good physical condition has a fairly stable maximal level of activity, especially in regard to androgen metabolism, whilst the child is undergoing constant ph~Tsiolo~ica1 changes and has sometimes not attained normal adult levels of androgen production even at the age of 15 and 16 years. D~~llydroepiandrostero~e has not invariably been detected as a constituent of urine in infants and in childrenlo$‘lB1a, as it is in normal adults. However, from the present investigation, it is possible to conclude that the determination of urinary DHA in children could be used as an index of abnormal adrenocortical function and was found to be clinically useful. ACKNOWLEDGEMENTS
We wish to thank Professor Dr. E. I?. Pfeiffer and other physicians and surgeons for their interest shown during the course of this work ; Professor W. Kiyne, W’estfield College, London, for generously providing reference compounds from the Xedical C&n. Chint.
Acta,
38
(1972)
371-378
PAL, TELLER
375
Research Council Steroid Reference Section and FrWein B&-be1 SchZfer for performing Zimmermann estimations for dehydroepiandrosterone in normal children. Our thanks also go to Frau Martha Rupp and to Mrs. M. R. Pal for their help in typing and preparing the manuscript, respectively. REFERENCES I H. L. MASOS ASD W. W. ENGSTROM, Physiol.Rev., 30(19p) 213. S. BURSTEIN AND S. LIEBERMAR.,J.B~O~. Chew, 233(rgj8)331. P. \T~~TER~.%.~R~AND B. CLAUSSEN, ~~~~ ~~~do~~~n~~., 39 Suppl. 64 (1962). Ii. FOTHE~BY, Biochem. J., 69 (1958) 596. I?, I. DORFMAX AND F. UEU‘GAR,in Metabolism ofSteroid Normones, Academic
2 3 4 5
IgGj, 6
7 k 9
IO II IL 13 14
15 16
17
18 rg
Press, New York
?. 28.
B. LORAS, w. Roux, B. ChuTENET, C. OLLAGXEN, M. FOREST, E. DE PERETTI ANU J. %R*~~Asu, in h. VER?VIEULF.~~ hND R. ESLEY (Ed%), And~0gen.sin Novmalund Pathological Con&ions, PFOG.Second Symp. Steroid Howmnes, Ghent, Excerpta NIedica Foundation, Rmslerdam. S. ~RSTEIN AND R. I. DORFMAN, Acta EndocrinoE.,40(1962) I&% 1'.VESTERGMRD, Acta Endocrinol,, 39, Suppl. 64 (1962)‘50. J. HAMMERSTEIN, in C. CASSANO, A. KLOPPER AED C. CONTI (Eds.), Research on Steroids, Vol. 111, North-Holland Publishing Company, Amsterdam, I$%, p. 323, B. P. P~u~ser\;AND E. 15. SOBEL, Amer. J. Diseases Children, 1oo (1960) 546. F. I~XUS,IX. P. ZURBR~GCJ, J.CARA AND L. LGARDNER, J.Clin.Endocrinol., 22(1962) rogo. A. K~DAR, T. FEH&R AX‘D 0. KOREF. Arch. DiseasesCh~Zdhood, 39 (1964) 257. P. VESTERGAARD, Acta E&ocrinot., 49 (1965) 436. E. P. PAULSEX, E. H. SO~EL AND M.S. SHAFRAX,J. Clin.Endocrinol., 26(x966) 329. W. 3%.TEI.LER,Z.&S. Exp. Med. ~42 (1967) 222. C. STEENO, W. HEY%, H. VAX BAELEN, A. VAX MERLE AND P. DYE MOIOOR,Ew'. J. .%v'oids, 2 (1967) 273. W. BLIJK~K, ilcta EndacvinoE., 59 Suppl. 134 (1968) 9. D. GUPTA, Clin. Chirn. Acta, 26 (1~69)256. H. BERGER, M. FINK, H. J. FRITZ, H. GLEISPACH, P. HEIDEMAN?: AXD J. WOLF, 2. li'lin
Chew .KEi,z. B&hem., 8 (1970) 354. 20 V.I.,. MITCHELL AND H.L.SWACKELTON,~~I Cf.~oDA~sKYA~DC.~.STEWART(~CIS.),~~W~~~~~ in Clinical Chemistry, Academic Press, New York, London, 1969, p. 141. C&?. Chiiir. Actn, 38 (1972) 371-378