Department of Biochemistry, Guy’s Hospital Medical School, London, S. E. 1
STEROID EXCRETION AND BIOSYNTHESIS WITH SPECIAL REFERENCE TO C19-416-STEROIDS IN AN INFANT WITH A VIRILIZING ADRENOCORTICAL CARCINOMA
By D. B. Gower, J. R. Daly*, G. J. A. I. Snodgrass* and Margaret I. Stern **
ABSTRACT
A case of a 15-month old girl with a virilizing adrenocortical carcinoma is described. Three C19-416-steroids, 3a-hydroxy-5a-androst-16-ene, 3a- hydroxy-58-androst-16-ene and 38-hydroxy-androsta-5,16-diene, have been obtained from the glucuronide fraction of the urine and identified by column and gas-liquid chromatographic techniques. Estimation of these compounds, not normally present in infants’ urine, showed that adult female amounts were being excreted, although post-operatively very little was detected. Androsterone, aetiocholanolone and especially de- hydroepiandrosterone were present in excessive amounts preoperatively; approximately one-third of the dehydroepiandrosterone was excreted as glucuronide, the rest as sulphate. The adrenal tumour metabolized [4-14C] pregnenolone and [4-14C] progesterone to 4.7 % and 0.15 % respectively of 30-hydroxy-androsta-5,16-diene. The evidence for biosynthesis of this compound from progesterone supports that obtained by other workers for the production of 45- from 44-steroids. Labelled androstenedione was also obtained from both substrates and 113-hydroxyandrostenedione from progesterone. Only small quantities of radioactivity were recovered in the glucuronide and sulphate fractions from the tissue incubations.
Functioning tumours of the adrenal cortex are rare in childhood. Over the last hundred years only 250 cases have been reported since the original patient of
* Departments of Chemical Pathology and Paediatrics, Charing Cross Group of Hospitals.
** Endocrine Unit, Chelsea Hospital for Women, London, S. W. 3, and Institute of Obstetrics and Gynaecology, University of London.
Pitman (1865) and Ogle (1865). The experience of even large centres has been limited to only a few cases. Four examples were found out of 82 220 in- patients in one hospital over 26 years by Garrett (1951). Twelve further cases occurred in the Mayo Clinic over a 30 year period (Hayles et al. 1966). This group also reviewed 222 other cases from the literature and found that the tumours were usually malignant and that virilization was the commonest clinical presentation. Recently, the case of a girl of 15 months, showing signs of masculinization due to a hormone producing tumour of the right adrenal gland has been described (Sekouri & Dardougia 1968). The tumour was re- moved in toto and histological examination revealed an adrenal carcinoma. The child’s subsequent course was uneventful. No biosynthetic studies were carried out on the tumour, but urinary 17-oxosteroid and 17-hydroxycortico- steroid values were similar to the results presented here, both pre- and post- operatively.
Except for the more recently reported cases, biochemical data has been scanty. In cases of carcinoma where virilization is pronounced and the urinary excretion of 17-oxosteroids high, the main steroid metabolite recovered in the urine is usually DHA *. A considerable number of steroids has been detected in the urine of such cases, and the present report concerns an infant with an adrenocortical carcinoma who excreted C19-416-steroids. Although 3a- androstenol is a normal constituent of the urine of both adult males and females (Brooksbank & Haslewood 1961), it has not previously been detected in the urine of infants (Gower, unpublished) nor of pre-pubertal children (Cleveland & Savard 1964). 3a-androstenol excretion may be increased in cases of adrenal tumour (Burstein & Dorfman 1962; Gower & Stern 1969), adrenal hyperplasia (Mason & Schneider 1950), and luteoma of the ovary (Engel et al. 1953). 3a-androstenol was detected in the urine of our patient together with small quantities of two other C19-416-steroids, aetiocholenol and androstadienol, also not previously detected in the urine of a child. Burstein & Dorfman (1962) have shown that cholesterol and pregnenolone are precursors of 3a-androstenol in vivo in a woman with virilizing adrenal adenoma. Gower & Stern (1969) found that pregnenolone was metabolized to androstadienol, and progesterone to androsta-4,16-dien-3-one by adrenal carcinoma minces. It was of interest, therefore, to measure C19-416-steroid excretion in the present case and to study the formation of this group of steroids by the diseased adrenal tissue.
Of additional interest was the hypertension in our patient which was re- lieved following removal of the tumour. 1-Oxygenated steroids were detected
* Abbreviations used are as follows: 36-androstenol, 38-hydroxy-5a-androst-16-ene; 3a-androstenol, 3a-hydroxy-5a-androst-16-ene; aetiocholenol, 3a-hydroxy-58-an- drost-16-ene; androstadienol, 30-hydroxy-androsta-5,16-diene; DHA, dehydroepi- androsterone.
in the patient’s urine pre-operatively, but were not present in the post-opera- tive samples. These compounds have been detected previously in human urine on two occasions; both patients were infants with hypertension (Edwards et al. 1968). The details of the 1-oxygenated steroid excretion in the urine of this patient will be published elsewhere.
CASE HISTORY
The patient, a girl, was delivered normally at home at 36 weeks gestation. The birth weight was 2500 g. She was the second child of young parents. The elder sibling, also a girl, is completely normal. The pregnancy had been complicated by a threatened abortion at 8 weeks gestation but no hormonal preparations were given at any time.
The parents thought the clitoris was unduly prominent at birth but the attending personnel did not comment on this. No other abnormalities were noted at this time.
Although the child thrived, her mother noticed that the clitoridal enlarge- ment had increased relative to her general growth by the age of 6 months. She did not seek medical aid then nor even when pubic hair began to appear at the age of 8 months. Under pressure from her relatives, the mother sought paediatric advice when the patient was aged one year.
On admission she was found to be a muscular child of a strikingly out- going temperament. Mental development was normal for her age. A discrete acneiform rash was present over the forehead and hirsuties were noted over the paravertebral region as well as the limbs. Surprisingly, considering the evidence of virilism, her supine length was only 73.6 cm (50th percentile) but she weighed 11.2 kg (90th percentile). The head circumference was normal at 47.9 cm.
Detailed examination of the genitalia revealed scanty, coarse and curly pubic hair of a feminine distribution. The clitoris was 2 cm long. The labia majora and minora were enlarged, the vaginal and urethral orifices normally situated. Rectal examination confirmed the presence of a uterus.
The only other abnormality noted in any system was a persistently elevated blood pressure. At the initial examination this was 160/85 mmHg but had in- creased to 180/110 mmHg prior to operation.
A buccal smear was chromatin positive and 3% of the neutrophils showed drumstick formation.
Operation
The abdomen was explored via a right upper para-median incision. The left adrenal was first exposed and found to be normal in size and appearance.
No recognisable adrenal tissue was seen on the right side. Instead there was a well encapsulated tumour the size of a hen’s egg. This was removed and found to weigh 50 g. No invasion of the para-renal tissues was seen, nor ex- tension into the local vasculature. Peripheral venous and right adrenal vein blood was obtained before removal of the tumour. Microscopical examination of the tumour (Professor J. C. Sloper) showed it to have a thin fibrous capsule and to be composed of sheets and trabeculae of large cells mostly eosinophilic and granular. There was no suggestion of zonal arrangement. Considerable nuclear pleomorphism was seen and also scattered areas of calcification sug- gesting the tumour had been present for some time. The diagnosis was that of adrenocortical carcinoma.
Post-operative management
The patient was maintained on intravenous fluids and gastric suction for 5 days following operation. Cortisol 60 mg was given by this route over the first 24 h and 30 mg and 15 mg over the following 2 days. Simultaneously, ACTH 60 units was given daily by continuous infusion for a total of 5 days.
Recovery was rapid thereafter and she was discharged home three weeks later, well and receiving no medication. She has remained well since that time (2 years).
EXPERIMENTAL Materials and Methods
Purification of solvents, preparation of alumina and Kieselgel G used for chromato- graphy have been described earlier (Gower & Ahmad 1967).
For the investigation and analysis of 416-steroids, androsterone, aetiocholanolone and DHA in urine, samples were processed as described earlier (Gower & Stern 1969) and hydrolysed extracts chromatographed on columns of alumina (Brooksbank & Gower 1970). Two fractions were obtained using 41-90 ml and 91-150 ml of a mixture of light petroleum (80-100ºC):benzene (1:1, v/v). The first of these normally contains 3a-androstenol and the second aetiocholenol and androstadienol (Brooksbank & Gower 1970). Gas-liquid chromatography (GLC) of the chloromethyldimethylsilyl (CMDS) ethers of 416-steroid fractions was performed as described by Brooksbank & Gower (1970). For further identification the bromomethyldimethylsilyl (BMDS) ethers were also prepared using a modification of the method of Thomas & Walton (1968). Fol- lowing these non-polar fractions, androsterone, aetiocholanolone and DHA were eluted together from alumina using 75 ml of benzene containing 0.5% of ethanol and were estimated by GLC of their trimethylsilyl ethers (Kirschner & Lipsett 1963).
Other methods used for the estimation of urinary steroids are given in Table I.
Plasma testosterone was measured by the method of Collins et al. (1968).
In vitro studies
1 g portions of minced adrenal tumour tissues were incubated for three hours in
5 ml Krebs bicarbonate buffer, pH 7.3-7.4 with 1.5 uCi each of [4-14C] pregnenolone (specific activity 24 mCi/mM) and [4-14C] progesterone (specific activity 58.5 mCi/mM) and co-factors as described earlier (Gower & Stern 1969). After incubation and ex- traction with 3 X 5 ml ethyl acetate, 50 ug of each of the following carrier steroids was added: 3a- and 30-androstenols, aetiocholenol, androstadienol, 3-hydroxy-oestra- 1,3,5(10),16-tetraene, androsta-4,16-dien-3-one, androstenedione, DHA, progesterone, pregnenolone and testosterone. The pooled ethyl acetate extract was evaporated to dryness under reduced pressure, on a warm water bath. The pH of the remaining aqueous mixture was adjusted to approximately 2.0 with concentrated HCI, ammonium sulphate (1:2, w/v) was added, and extraction carried out three times with ether: ethanol (3:1, v/v) (Edwards et al. 1953). The organic solvent was evaporated to dry- ness, 15 ml of 0.1 M acetate buffer (pH 4.5) and 2 ml Ketodase (William R. Warner & Co. Ltd., Eastleigh, Hants., England) added to each flask and the mixtures were incubated at 37ºC for 48 h. After adjusting the pH to approximately 1.0, the aqueous mixture was extracted three times with 10 ml ether, and the ether evaporated. This extract was designated the G (Glucuronide) fraction. The remaining aqueous portion was solvolysed according to Magendantz & Ryan (1964). The resulting ethyl acetate was evaporated to dryness and designated the S (solvolysed) fraction. The initial ethyl acetate extracts and the G and S fractions were chromatographed on alumina, essen- tially as described earlier (Gower & Ahmad 1967) unless the radioactivity was too small for further studies to be performed.
Separation and tentative identification of radioactive metabolites was achieved by submitting each alumina column fraction to thin layer chromatography followed by radioautography and preparation of derivatives, where possible, as described in detail earlier (Gower 1966; Gower & Stern 1969). It was possible to characterise one 416- steroid metabolite by column chromatography (see Results section).
RESULTS
Urine
Results obtained from analysis of urine are given in Table 1. Total 17-oxo- steroids were markedly raised and gradient elution chromatography showed the presence of a considerable quantity of DHA. GLC revealed that one-third of this quantity was excreted as glucuronide. These values had returned to normal one year after operation. Oestrogen excretion was small in amount and pre- and post-operative values did not differ markedly. Pregnanetriol was found in each urine specimen examined, still being present a year after opera- tion, at which time pregnanediol was only just detectable. Pre-operatively, urinary testosterone gave a value in the normal adult female range but de- creased post-operatively. Pregnenetriol before operation was only just de- tectable.
Investigation of C19-416-steroids
Figs. 1 and 2 show the GLC tracings obtained by chlorosilanisation of the >/16-steroid« fractions obtained from alumina. The »androstenol« fraction contained a compound with a retention time (RRT) similar to that of the
| Steroid | Pre-operative | Post-operative, within 2/52 | Post-operative one year | Normal values for infants | Method |
|---|---|---|---|---|---|
| Total 17-OS | 13.5-28.0 | 0.3-0.6 | 1.6-2.4 | 1.4 ± 0.7 | Drekter et al. (1952) with modified Zimmermann |
| DHA G | 7.5 | - | |||
| S | 14.1 | 6* | 0.1 | ||
| Androsterone G | 1.3 | ||||
| S | 1.3 | 25* | 0.1 | Kirschner & Lipsett (1963) modified | |
| Aetiocholanolone G | 1.4 | - | |||
| S | 0.2 | 50* | 0.1 | ||
| Sa-Androstenol | 0.51 | - | 14* | ||
| Aetiocholenol | 6* | - | N. D. | N. D. | Brooksbank & Gower (1970) |
| Androstadienol | 25* | - | N. D. | ||
| Testosterone | 10.0* | 6.4* | <5.0* | <5.0 ** | |
| Epitestosterone | <5.0* | <5.0* | <5.0* | <5.0* | Brooks (1964) modified |
| Total 17-OHCS | 1.5-3.5 | 0.3-0.6 | 1.6-2.4 | <2 | Few (1961) modified James & Caie (1964) |
| Pregnanetriol | 0.3 | 1.1 | 0.5 | <0.1 | Stern (1957) |
| Pregn-5-en-38, | <0.1 | - | - | <0.1 | Stern & Barwell (1963) |
| 17a,20a-triol | |||||
| Pregnanediol | 1.6 | 0.3 | <0.1 | <0.1 | Klopper et al. (1955) |
| Oestradiol | <1.0* | <1.0* | - | <0.1* | |
| Oestrone | 2.2* | <1.0*, 2.6* | - | <0.1* | Brown (1955) modified by Brown |
| Oestriol | 1.1* | 1.2*, 1.1* | - | <0.1* 1 | et al. (1957) |
N. D. = not detected
G = Glucuronide
- = not measured
S = Sulphate
(a)
(b)
1
1
Recorder response
3
4
4
20
10
0
20
10
0
Time(min.)
(a) GLC of a mixture of the CMDS ethers of 3a-androstenol (peak 1) and androsta- dienol (peak 3);
(b) »3a-androstenol« fraction (eluted from alumina and chlorosilanised) obtained after hydrolysis of urinary glucuronides of an infant with a virilising adrenocortical car- cinoma. Cholestane (peak 4) was added as internal standard. Column: CDMS/JXR (0.6 %/0.75 %) and conditions as Table 2.
(a)
(b)
Recorder response
3
4
2
3
4
30
20
10
0
30
20
10
0
Time(min.)
Fig. 2. (a) GLC of the CMDS ethers of aetiocholenol (peak 2) and androstadienol (peak 3); (b) »androstadienol« fraction (eluted from alumina and chlorosilanised) obtained as in Fig. 1. Cholestane (peak 4) was added as internal standard. Column: QF1 (5 %) and conditions as Table 2.
CMDS ether of authentic 3a-androstenol, relative to the internal standard, cholestane (Fig. 1). The second alumina column fraction contained compounds which had RRTs corresponding to the CMDS ethers of aetiocholenol and androstadienol. A trace of the CMDS ether of 3a-androstenol, not separated
on alumina, was also present in this fraction (Fig. 2). Proof of identity was obtained by GLC on two columns of the BMDS ethers prepared from further portions of the alumina column fractions. In each case the RRTs were identical to those of the authentic derivatives (Table 2). The data also show that 38- androstenol was absent in this urine, a finding in keeping with the earlier work of Brooksbank & Gower (1964).
Estimation of 416-steroids in the pre-operative urine revealed that the ex- cretion of 3a-androstenol (0.51 mg/24 h) was equivalent to that of an adult woman. In normal infants, the urinary 3a-androstenol is known to be either very low or undetectable (Gower, unpublished), and in the present case the values of urinary 3a-androstenol post-operatively were very low (Table 1). The levels of aetiocholenol and androstadienol pre-operatively were 6 and 25 µg/24 h which are approximately those found in adult women (Brooksbank & Gower 1970). These compounds have not so far been detected in the urine of normal infants (Gower, unpublished).
| Retention times relative to cholestane (= 1) | ||||
|---|---|---|---|---|
| CDMS/JXR* | QF1 ** | |||
| Authentic steroid | Urine | Authentic steroid | Urine | |
| CMDS ethers | ||||
| 3a-Androstenol | 0.415 | 0.417 | 0.606 | 0.604 |
| Aetiocholenol | 0.488 | 0.484 | 0.610 | 0.613 |
| Androstadienol | 0.580 | 0.582 | 0.730 | 0.731 |
| 3ß-Androstenol | 0.580 | 0.582 | 0.795 | - |
| BMDS ethers | ||||
| 3a-Androstenol | 0.580 | 0.583 | 0.750 | 0.744 |
| Aetiocholenol | 0.677 | 0.670 | 0.786 | 0.780 |
| Androstadienol | 0.840 | 0.830 | 0.910 | 0.916 |
| 38-Androstenol | 0.840 | 0.830 | 1.16 | - |
Columns used: Cyclohexane dimethanol succinate (CDMS)/methyl polysiloxane (JXR) (0.6 %/0.75 %) and fluoralkyl silicone, QF1 (5 %) with carrier gas 50 ml/min. Cholestane times: * 32.5 and 29.8 min at 190℃ and 200°C respectively.
** 35.9 and 33.0 min at 190°C and 200°℃ respectively.
Plasma
Pre-operatively the level of peripheral plasma testosterone was 142 ng/100 ml and the adrenal venous plasma concentration was 263 ng/100 ml.
Biosynthetic studies
The amounts of radioactivity extracted from the adrenal tissue incubations with ethyl acetate and obtained after hydrolysis and solvolysis are shown in Table 3. Virtually all the radioactivity was extracted with ethyl acetate and there was no residual radioactivity in the aqueous layers after hydrolysis and extraction of conjugates. In view of the low content of radioactivity of the conjugate fractions, they were not investigated further. The ethyl acetate- extractable material, however, was chromatographed on alumina columns as described earlier (Gower & Ahmad 1967). Only 66.5 % and 22.9 % respectively of the radioactivity from the pregnenolone and progesterone incubations was eluted using solvents with increasing polarity up to benzene containing ethanol (1 %) (see Gower & Ahmad 1967). Elution with ethanol (50 ml) resulted in the recovery of a further 6-10 % of radioactivity. Only when 50 ml of aqueous methanol (1:1, v/v) was used was the remaining radioactivity recovered (Table 4). Over 20 % was obtained from the pregnenolone and as much as 58 % from the progesterone incubations respectively. This has been noticed repeatedly using adrenal tissue especially with radioactive progesterone as pre- cursor.
Biosynthesis of 416-steroids
Fractions which might contain C19-416-steroids were obtained by alumina column chromatography of the ethyl acetate extracts using 41-150 ml of a benzene-light petroleum mixture (1:1, v/v) (Gower & Ahmad 1967). These
| Precursor | Ethyl acetate- extractable | Glucuronides | Sulphates | Aqueous layer |
|---|---|---|---|---|
| Pregnenolone | 98.3 | 1.4 | 0.21 | nil |
| Progesterone | 98.8 | 1.0 | 0.07 | nil |
Adrenal carcinoma minces were incubated with [4-14C] pregnenolone and [4-14C]- progesterone and extracted with ethyl acetate (3 X 5 ml). Conjugates remaining were extracted, hydrolysed and solvolysed separately and extracted with ethyl acetate (3 × 5 ml).
| Substrate | Fractions I-VI of Gower & Ahmad (1967) | Ethanol (50 ml) | Aqueous methanol (1:1, v/v, 50 ml) | Total |
|---|---|---|---|---|
| [4-14C] pregnenolone | 66.5 | 6.2 | 20.8 | 93.5 |
| [4-14C] progesterone | 22.9 | 10.3 | 58.2 | 91.4 |
Values given are percentages of radioactivity added to the columns.
were evaporated to small bulk and portions removed for measurement of radio- activity. The extract from the pregnenolone incubation contained approximately nine times as much radioactivity as that from the progesterone incubation. The remaining portions were chromatographed on thin layers of Kieselgel G using benzene-ether (9:1, v/v) as solvent (Gower 1964). Radioautography revealed radioactive zones in both extracts which ran with the mobility of authentic androstadienol (Fig. 3 a). Other minor radioactive zones have not been iden- tified. The two major zones were investigated further by running them on thin layers of silver nitrate-impregnated silicic acid, using benzene-ethyl ace- tate (1:1, v/v) as solvent since, under these conditions, androstadienol can be resolved easily from 38-androstenol (Lisboa & Palmer 1967). Radioautography revealed that in both cases the radioactivity was associated only with the marker androstadienol (Fig. 3 b). In order to confirm the identity of this metabolite it was eluted from the TLC plate and submitted to chromatography on a column containing 3 g alumina (partially deactivated with 4.5 % water (Gower & Ahmad 1967)) and 3 g of Kieselgel H which had previously been impregnated with silver nitrate (0.66 g). Using a mixture of authentic androsta- dienol and 38-androstenol, it was known (Katkov, unpublished) that the latter could be eluted using approximately 60 ml of a mixture of benzene-ethyl acetate (2:1, v/v). Androstadienol was then eluted with approximately 60 ml of benzene-ethyl acetate - ethanol (40:20:1, by volume). Further details of this separation will be published elsewhere *. After an initial bulk fraction (30 ml), 2 ml fractions were collected until all radioactivity had been eluted. In each fraction the radioactivity was measured by liquid scintillation counting and the weight of added carrier steroid by GLC (Gower & Thomas 1968). Figs. 4 and 5 show that there was no radioactivity associated with 33-androstenol, and
* Katkov T. & Fower D. B .: Biochem. J. (in press).
(a)
(b)
5-P
P
15-P
P
- S.F.
+ — S. F.
0 0
-1
0
-2
+3
-4
D
☒
+5
D
+6
-7
+7
1
0
-6
---- Origin
-Origin
Fig. 3. (a) Radioautograph (tracing) of radioactive 416-steroid fractions (eluted from alumina) obtained from incubation of [4-14C] pregnenolone (45-P) and [4-14C] progesterone (P) with virilising adrenocortical carcinoma tissue. Extracts were run on Kieselgel G three times in benzene-ether (9:1, v/v).
(b) Radioautograph (tracing) of the »androstadienol« zones after TLC as in Fig. 3 (a) run on Kieselgel G impregnated with silver nitrate. Solvent was benzene-ethyl acetate (1:1, v/v). Marker steroids: 1, 5a-androst-16-en-3-one; 2, 3-hydroxy-oestra-1,3,5(10),16- tetraene; 3, androsta-4,16-dien-3-one; 4, 3a-androstenol; 5, aetiocholenol; 6, androsta- dienol; 7, 38-androstenol. S. F. = solvent front.
that radioactivity and mass peaks for androstadienol coincide. The specific activity of androstadienol (counts/min/ug) was constant over the peak in each case (Table 5) indicating that radiochemical purity had been achieved. Fig. 5 also shows that there was a small peak of radioactivity eluted from the column just after the carrier 36-androstenol. It is possible that this may be due to androsta-4,16-dien-3-one formed by oxidation of androstadienol. The ketone is known to be incompletely separated from 30-androstenol on this column.
Analytical losses were calculated by expressing the total weight of carrier androstadienol eluted from the column as a percentage of the amount originally added to the tissue incubation. It was then found that the adrenocortical tissue had metabolized radioactive pregnenolone and progesterone to androstadienol in yields of 4.7 % and 0.15 % respectively.
Biosynthesis of other steroids
(a) From [4-14C]pregnenolone. By running the extracts in three different solvent systems on TLC, formation of derivatives and comparison with the
SPECIFIC ACTIVITY (cpm/ug.)
650
3000
450
2500
5
RADIOACTIVITY (cpm)
WEIGHT OF CARRIER STEROID (ug. )
2000
-
4
1500
3
o
1000
2
I
II
500
1
0
0
0
0
40
50
0
10
20
30
40
50
60
benzene-ethyl acetate
(2:1, v/v)
benzene-ethyl acetate-ethanol (40 : 20 : 1, by vol. )
VOLUME OF ELUTING SOLVENTS (ml.)
Purification of radioactive androstadienol formed by incubating [4-14C] pregnenolone with adrenocortical carcinoma tissue. Radioactive androstadienol was isolated by TLC (Figs. 3 a and b), carrier 38-androstenol (I) and androstadienol (II) added, and the mixture eluted from a column of Kieselgel H impregnated with silver nitrate (see text). The weight (o) of carrier steroids was measured by GLC and radioactivity (.) by liquid scintillation counting.
SPECIFIC ACTIVITY (cpm/µg.)
130
700
100
0
9
6
600
RADIOACTIVITY (cpm)
WEIGHT OF CARRIER STEROID (pg.)
5
500
a
400
d
4
P
300
9
3
1
II
200
2
100
1
0
0
0
40
50
0
10
20
30
40
50
benzene-ethyl acetate
benzene-ethyl acetate-ethanol
(2:1, v/v) -+ (40: 20: 1, by vol.) VOLUME OF ELUTING SOLVENTS (ml.)
| Data from | Number of estimations | Mean (counts/ min/ug) | Range | SD | Coefficient of variation (%) |
|---|---|---|---|---|---|
| Fig. 4 | 15 | 560.3 | 525-650 | 34.5 | 6.1 |
| Fig. 5 | 12 | 117.7 | 106-128 | 5.8 | 4.9 |
relevant authentic steroids, tentative evidence for the formation of androstene- dione was obtained, and for the presence of unchanged pregnenolone. There were several other compounds in small quantities. No evidence was obtained for the presence of testosterone, progesterone, DHA, or 11-hydroxyandro- stenedione in detectable quantities.
(b) From [4-14C]progesterone. Tentative evidence was obtained for the presence of androstenedione and unchanged progesterone as well as 118- hydroxyandrostenedione, by formation of derivatives and running in three different TLC systems with the relevant authentic steroid. No testosterone could be detected in the extract after incubation.
DISCUSSION
To our knowledge, this is the first case of an infant with an adrenal carcinoma in which 416-steroid excretion and biosynthesis have been investigated. Pre- vious studies have shown that in two virilized women who had an adrenal adenoma (Burstein & Dorfman 1962) or carcinoma (Gower & Stern 1969), abnormally high amounts of 3a-androstenol were excreted in the urine. The urine of our patient contained adult female amounts of 3-androstenol and two other 416-steroids (aetiocholenol and androstadienol), not usually detect- able in infant urine (Gower, unpublished).
Moreover, evidence was obtained to show that the diseased adrenal could produce in vitro relatively large yields of androstadienol from pregnenolone. These results are in agreement with those obtained from a former patient although the yield of androstadienol was only 0.2 % (Gower & Stern 1969) and from recent in vivo experiments (Brooksbank & Wilson 1969).
The formation of a small amount of radioactive androstadienol from pro- gesterone is unexpected, and if this is correct it is difficult to interpret. This observation would be explicable if progesterone were converted to pregneno- lone, by a reversal of the 45-30-hydroxysteroid dehydrogenase-isomerase
system. In recent years, evidence has accumulated to suggest that this can occur (Ward & Engel 1964, 1966a,b; Rosner et al. 1965).
It is noticeable that, despite the urinary excretion of DHA, this compound could not be detected in biosynthetic studies after incubation with [4-14C]- pregnenolone.
ACKNOWLEDGMENTS
The authors are grateful to Dr. Hugh Jolly and Mr. Peter Philip who had clinical charge of the patient. Dr. D. B. Gower thanks Mr. D. C. Bicknell for valuable assistance and the Medical Research Council (Grant no. G. 967/305/B) and Guy’s Hospital Endowments Fund for financial support. Dr. M. I. Stern thanks members of the Endocrine Unit who carried out some of the analyses.
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Brooksbank B. W. L. & Wilson D. A .: Acta endocr. (Kbh.) Suppl. 138 (1969) 71.
Brown J. B .: Biochem. J. 60 (1955) 185.
Brown J. B., Bulbrook R. D. & Greenwood F. C .: J. Endocr. 16 (1957) 49.
Burstein S. & Dorfman R. I .: Acta endocr. (Kbh.) 40 (1962) 188.
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Received on September 22nd, 1969.