Long-term effects of aminoglutethimide on steroid metabolism in congenital adrenal hyperplasia

135

Clinica Chimicn Acta, 73 (1976) 135-148 0 Elsevier/North-Holland Biomedical Press

CCA 8167

LONG-TERM EFFECTS OF AMINOGLUTETHIMIDE ON STEROID METABOLISM IN CONGENITAL ADRENAL HYPERPLASIA

WILLIAM

HAMILTON

University Department of Child Health, The Royal Hospital for Sick Children, Yorkhill, Glasgow G3 8SJ (U.K.) (Received

June 4th, 1976)

Summary Five cases of congenital adrenal hyperplasia due to CBl-hydroxylase defect were treated with a combination of aminoglutethimide and prednisolone. In the third year of treatment the urinary levels of 17-oxosteroids increased above normal values while the total 17-hydroxy-corticosteroids were normally low. Specifically, urinary pregnanetriol was normal in 3 cases. To determine the reasons for this disparity the adrenal metabolism of cholesterol, as judged by the urinary steroid metabolites, was studied. Fractionation of urinary steroid metabolites was by thin-layer chromatography (TLC) followed by gas-liquid chromatography (GLC). The results indicate that aminoglutethimide inhibits steroidogenesis less than prednisolone; that a pathway from cholesterol via 17o,20a-dihydroxycholesterol to dehydroepiandrosterone is likely to operate after long-term aminoglutethimide therapy; that 11/3-hydroxylase, at least for pregnenes may be inhibited by aminoglutethimide and that the metabolic breakdown of testosterone may be delayed by this drug. Introduction During the evaluation of aminoglutethimide (AG) as an anticonvulsant drug [ 11, it was observed that adrenal steroidogenesis was inhibited [2]. In three epileptic children treated with AG goitrous hypothyroidism developed and this was shown to be due to a blocking of the intrathyroidal organification of iodine [ 31. Such side effects have limited the clinical usefulness of AG as an anticonvulsant. It is now principally used to block steroidogenesis in Cushing’s disease [4], in adrenal carcinoma [5], in endocrine hypertension [6] experimentally for its side-effects [7]. In malignant endocrine disease its use merely protects the patient from the effects of over-production of cortisol or aldosterone, since the drug has no known cytotoxic effects. The precise action of AG is to block C20,22-desmolase in the adrenal gland [8] thus preventing the conversion of cholesterol to pregnenolone, a step normally controlled by cortico-

136

trophin. Aminoglutethimide therapy therefore induces iatrogenic lipid adrenal hyperplasia [ 91, the lipid material being cholesterol [ 81. Because of this action it was suggested [lo] that AG could be used to create complete adrenal steroidogenic blockade. Thus its use seemed indicated in patients with congenital adrenal hyperplasia (CAH) particularly in those children with advanced ossification in whom replacement and suppressive doses of exogenous steroids had inhibited linear growth. With complete adrenal steroidogenic blockade, the patient would be protected from excessive endogenous androgens and only small physiological, rather than suppressive, amounts of replacement steroids would then be required for maintenance. A therapeutic regime consisting of AG by day (30 mg per kg body weight) and prednisolone (1 mg) late at night was devised for five children with CAH whose heights were less than the 3rd centile and whose bone ages were advanced beyond their chronological age. The rationale of this form of treatment and the early results have been published in detail [II]. In summary, during the period of treatment the urinary steroid metabolites were within the normal range, and the patients increased their growth velocity while bone maturation was either static or advanced less than the chronological period of the review. During the third year of treatment the urinary 17-oxosteroid fraction showed a steady increase while the 17-oxogenic steroids and 17-hydroxycorticosteroids (Tables I and II) were maintained within the accepted range (Table III). Pregnanetriol excretion rose in the two older girls. These biochemical changes were reflected clinically by a re-acceleration of bone maturation without a parallel increase in growth velocity. To account for these findings, three possibilities were considered; (a) with increase in age, a greater increase in dosage of both drugs might have been required to maintain adrenal suppression, (b) increased circulating corticotrophin consequent on a fall in plasma corticosteroids might have caused a breakthrough of the adrenal C20,22desmolase blockade and (c) adrenal cholesterol might have been metabolized directly to 17-oxosteroids omitting pregnenolone as an intermediate. The patients were therefore re-investigated and this paper reports the urinary corticosteroid pattern in five cases of CAH after three years of combined AG/ prednisolone treatment. TABLE URINARY

STEROID DEFICIENCY BY NIGHT

17-oxosteroids: Case

Age

Clinical

(years)

status

IN THE TREATED

17-O&.%

9

2 3 4 5

9 6 5 4

Salt-loss Nonsalt-loss Salt-loss Salt-loss Salt-loss

OF FIVE BY

WITH 21AND PREDNI-

steroids; 17-Ohcs, l?-hydroxycortisteroids.

Urinary steriod (~mol/vol.) 17-Ohcs

17”ogs

17-0s

1

AND NIGHT A~INOGLUTETHIMIDE

Day

Night 24 h

Day

Night

24 h

IJay

Night

24 h

8.32 6.14 7.6 1.73 5.24

2.64 8.00 1.7 0.24 1.77

11.84 10.5 5.08 1.06 3.21

u.d. 7.4 2.2 0.56 0.41

11.84 17.9 7.28 1.56 3.62

16.0 15.7 8.85 1.0 3.33

1.74 3.8 2.35 0.63 0.3

17.74 18.5 11.20 1.64 3.63

10.96 14.14 9.3 1.97 7.01

137 TABLE II URINARY PREGNANETRIOL, PREGNANETRIOLENE AND PREGNANEDIOL URINES OF FIVE GILRS WITH 21-HYDROXYLASE DEFICIENCY TREATED THIMIDE BY DAY AND PREDNISOLONE

IN DAY AND NIGHT WITH AMINOGLUTE-

BY MIGHT

u.d.. undetected. Case

Age (years)

Clinical

~mol/vol.

. status

1 2 3 4 5

9 9 6 5 4

Salt-loss Nonsalt-loss salt-loss Salt-loss salt-loss

Pregnanediol

Pregnanetriolone

Pregnanetriol Day

Night 24 h

Day

Night

24h

Day

Night

24 h

1.27 1.38 0.43 0.34 0.29

0.39 1.23 0.02 0.26 0.24

0.07 0.18 u.d. u.d. 0.1

0 0.16 u.d. u.d. 0.13

0.07 0.34 0.23

u.d. 0.13 0.07 u.d. 0.11

u.d. u.d. u.d. u.d. 0.05

0.13 0.07 0.16

2.66 2.61 0.45 0.60 0.53

Materials and methods Urine was obtained from five girls, whose ages ranged from 4 to 9 years, and who had CAH due to C21-hydroxylase deficiency. Treatment had been with AG and prednisolone [ll]. Urine collections (16 h and 8 h) were made to correspond with the respective periods of AG (0800 to 2400 h) and prednisolone (2400 h to 0800 h) administration. The steroid metabolites in the urine (50 ml) TABLE IIIA URINARY EXCRETION (MIXED SEX)

OF VARIOUS

STEROID

METABOLITES

17-0s. 17-oxosteroids; 17-Ogs, l’l-oxogenic steroids: l’l-Ohcs, triol: P’triolone, pregnanetriolone: P’diol, pregnanediol.

IN 188

NORMAL

17-hydroxycortosteroids:

CHILDREN

P’triol, pregnane-

pmo1/24 h

Age (years)

l-3 4-10

17-0s

17-ogs

17-Ohcs

P’triol

P’triolone

P’diol

0.3-1.3 3.4-8.5

5.2-18.3 11.0-31.2

6.9-20.8 13.8-38.1

O-O.6 0.5-0.89

o-O.3 0.2-0.4

O-O.4 O-O.6

TABLE IIIB URINARY

EXCRETION

T, testosterone; cholanolone. Age (years)

2-6 7-8 9-10 11-12

OF VARIOUS

AD, androstenedione;

STEROID METABOLITIES DHA. dehydrocpiandrosterone;

IN 88 HEALTHY

BOYS

Andro, androsterone: Aetio, aetio-

nmo1/24 h T

AD

7-34

20-l

20

DHA

Andre

Aetio

1 l@HydroxyAndre

11-Oxo-Andre

40 k 20 50? 30 160 f 80 180 + 110

150i 60 250 f 100 560 k 230 1080 -t 690

150* 40 160 C 40 330 + 50 740 * 350

330 660 980 1470

250 260 520 750

* 70 * 290 ? 330 * 400

+ 130 f 90 f 190 f 260

138

were hydrolyzed first with fi-glucuronidase [12] then with sulphatase 1131 and finally by solvolysis at pH 1 [ 141. Dehydro[ 7a-3H] epiandrosterone (7a-TDHA) and 7a-T-DHA sulphate were added to the urine samples before hydrolysis to check recovery rates. After each hydrolytic step, the urine was extracted with ether, ether and ethyl acetate, respectively. The washed, neutralized extracts from each urine sample were combined and evaporated to dryness before thin-layer chromatography (TLC) on 0.25-mm silica gel plates. The plates were developed for two separate 60-min periods in the system benzene~ethyl acetate (40 : 60, v/v). Cholesterol, testosterone, ll@-hydroxyandrosterone and pregnanetriol were run as standards on side-lanes which latter were sprayed, after development, with 0.1 per cent Rhodamine 6-G in ethanol. A typical chromatographic separation of standard compounds into 3 zones is shown in Fig. 1. The compounds from the 3 zones indicated were eluted with acetone (2 X 5 ml) and dried under a nitrogen stream. 0-Methyl-oximes [15] were formed of the compounds in Zones 1 and 2 before trimethylsilylation 1161. The trimethylsilylethers of compounds in Zone 3 were immediately formed. Subsequent GLC separation was achieved on a ‘I-foot glass column in a Pye 104 SOLVENT ----_____~_-___~____~~~~~~~~~~~----

FRONT __--_.

10 ems ________~______~_________________I______

Ch i%

IWO

Eh

OH

cn

A”dW

_-

1 -_______II-

ZONE

ZONE !l ---_“.._------

20.22 OH Ch

A0 DHA

tuo ---

-_-----,

~--‘S.--,,~.--‘--__-,IK* A1 m

T

6CrnS -.

Zcms ________-___________-------~ drr,pr Pirwbne

ZONE

3

--_-.

ORIGIN

Fig. 1. Chromatographic (TLC) separation of urinary steroids on 0.25~mm silica gel plates developed in benzene/ethyl acetate (40 : 60, v/v) for two separate 60-min periods. Ch, cholesterol: 20 OH Ch. 2Oorhydroxycholesterol; 17.20 OH Ch, 17a,20a-dihydroxycholesterol; 20,22 OH Ch, 20&22-dihydroxyDHA, dehydroepiandrosterone; T, testosterone; cholesterol; Andre, androsterone; Etio, ~etiochofanolone; AD, androstenedione: IlKA. ll-oxoandrosi~rone: llKE, 11-oxoaetiocholanolone; 11 OH Andre, ll@hydroxyandrosterone; 11 OH E, lip-hydroxyaetiocholanolone; P’diol. pregnanediol: As-P’triol, pregnrnetrial; P’triol. pregnanetriol; P’triolone, pregnantriolone.

139

Separation of trimethylsilylethers and methoximetrimethylsilylethers of steroids in TLC Lone 1 7’ - 2 % Np

XE

Flow

F I D

60

rate

on GCQ 25

Attenuotlon

180 ‘C/36

mr

ml

100/120

per

mesh

mfn

100 -

72

1°C/min

increase

60

48

36

24

12

0

Mins

Separation of trimethylsilylethers and methoximetrimethvlsilvlethers of steroids in TLC zone 2

2 % XE 60 on GCQ 100/120 mesh

7’ N,

Flow

FI D 195-C

rate

25 mi per min

Attenuaton Mass

100

0.25

.uS

“a

30

24

12

16

6

0

Mlns 2. Separation by GLC of the 11-deoxy-Cl9 compounds in TLC Zone l/(a) pounds, pregnanediol and pregnenetriol in TLC Zone 2 (b).

Fig.

and the 11-oxy-Cl9

com-

140

Separation of trinlethylsiiyletilers of steroids in TLC zone 3

7’

!%

SE

Np Flow FI

D

200

30

rate

on

GCQ

25

ml per min

Attenuation -c

Ma55

100

720

mesh

100 05

j/g

--_L..

_-__-.L_.__-__-

30

24

18

12

6

0

MiW.

Fig. 3. Separation of GLC of compounds in TLC Zone 3. principally pregnmetrio] and pmgnaneteolone_

Separation of trimethylsilylethers of hydroxylated cholesterols in TLC zone 1

StolnlessSteelcolumn

5

feet

Support gas ttlr0m Q 120 Coottng 2% ov- :7 Comer

Chart

nitrogen 78 ml per

gas

DetectIon speed

FI

D 1 mm

oven per

temp

rmn

200°C

msnute

Fig. 4. Separation of GLC! of cholesterol and its hydroxylated derivatives in TLC Zone 1. %Oa-Hydroxycholesterol is seen contaminating the cholesterol peak but is better separated as in Fig. 2a.

141

System using 2 per cent XE60 on GasChrom Q 100-120 for compounds in Zones 1 and 2 and 1 per cent SE30 on GasChrom Q 100-120 for compounds in Zone 3. Typical tracings and operating conditions are shown in Fig. 2 and Fig. 3. Better separation of the hydroxylated cholesterols, which were in Zone 1 of the TLC was achieved on a 5-foot stainless steel column using 2 per cent OV17 on GasChrom Q 100-120. The oven temperature was 200°C and detection was by flame ionization. A typical tracing is shown in Fig. 4. Overall recovery rates as judged by the recovery of 7a-T-DHA were between 60 and 75 per cent. Results The results of the analyses are presented in Tables IV and V in which the excretion values for the various compounds are related to AG (day) and prednisolone (night) therapy. From Table II it will be observed that the excretion of pregnanetriol, pregnanetriolone and pregnanediol is, on average, greater by day than by night. Only in the two older girls is the excretion of pregnanetriol unacceptably high (upper normal : 0.89 pmo1/24 h) and in one of them, it is in excess by day and by night. By contrast, pregnanetriolone and pregnanediol excretion is acceptably low throughout both periods for all patients. In Table IV are the day and night excretion rates for testosterone, androstenedione, aetiocholanolone and androsterone. Testosterone is excreted more by day and in the 5 cases is in excess of our laboratory normal values for boys (7-34 nmo1/24 h). Likewise also, androstenedione excretion is greater by day and is in excess of normal in Cases 2, 3 and 4 (20-120 nmo1/24 h; adults 49296 nmo1/24 h [17]). Androsterone excretion, while greater by day than by night, is normal in four cases (Cases 1, 3, 4 and 5) and raised in one case (Case 2). Aetiocholanolone excretion is normal in three cases (Cases 1, 4 and 5) but increased in two cases (Cases 2 and 3) and uniformly greater by day than by night. Normally in this age group, androsterone and aetiocholanolone are excreted approximately in equal quantities but with a slight shift to the 5cu-reduced compound, androsterone. These findings therefore might suggest that the metabolism of testosterone to its metabolites, androsterone and aetiocholanolone is altered by the AG therapy in favour of 5@-reduction to aetiocholanolone. Table V gives the urinary excretion rates for cholesterol and other compounds retaining the As-3fl-hydroxy structure in Rings A and B of the steroid nucleus. Noteworthy points are that night excretion values exceed day excretion values, except for DHA, this being the reverse of the distribution of the other urinary ClS-compounds. Also the excretion of DHA is uniformly greater than normal. Since there are no standard values available for the urinary excretion of cholesterol in children, and since there is a paucity of information on the excretion of intermediate compounds between cholesterol and pregnenolone, a full interpretation of the data is not permissible although they could be interpreted as favouring the existence in these patients treated with AG, of a metabolic pathway from 17,20-dihydroxycholesterol to dehydroepiandrosterone.

IV

9

9

6

5

4

9

9

6

5

4

1

3

4

5

1

2

3

4

5

IN

THE

Salt-loss

salt-loss

Salt-loss

Nonsalt-loss

Salt-loss

23

40

10

150

40

Night

104

85

421

1115

319

Day

BY

AND

59

24

355

244

119

Night

Aetiocholanolane

32

30

120

Salt-loss

1 IO

70

Day

Testosterone

nmol/vol.

Salt-loss

Salt -loss

DAY

PREDNISOLONE

Nonsalt-loss

Salt-loss

status

AND

CliniCal

DAY

Age

BY

CIS-COMPOUNDS

(years)

2

Case

TETHIMIDE

URINARY

TABLE FIVE

170 75 30

30 160 55

1

3

56

13

253

Night

28 876 -

109 163

436

9

1312 9

28

-

762

-

-

24h

438

Night

h

WITH

1359

Day

33

131

183

556

14

24

GIRLS

IlpHydroxy-androsterone

13 303

110 320

Day

24

OF

Androstenedione

URINES

24h

h

NIGHT NIGHT

9 9

15

367

19

Night

38

-

841

229

303

Day

-

14

117

928

58

Night

11-Oxo-androsterone

60

76

87

523

386

Day

DHA

21-HYDROXYLASE

69

h

140

91

38

14

964

1151

361

24

95 104

96

196 1475

Day

890

h

405

24

71

61

29

121

45

Night

TREATED

Androsterone

DEFICIENCY

h

AMINOGLU-

211

165

124

1596

241

24

WITH

V

9 9 6 5 4

(Years)

Age

* See Fig. 6.

1 2 3* 4 5

Case

Salt-loss Nonsalt-lake Salt-loss Salt-loss salt-loss

Clinical status

385 523 87 76 60

Day

DHA

nmol/vol.

19 367 9 15 9

Night 83 27 163 155 120

Day 199 386 783 882 531

Night

Cholesterol

26 u.d. 20 u.d. 8

Day 104 u.d. 123 63 26

Night

HOLY-Hydroxycholesterol

URINARY DEHYDROEPIANDROSTERONE. CHOLESTEROL AND ITS HYDROXYLATED GIRLS WITH Pl-HYDROXYLASE DEFICIENCY TREATED WITH AMINOGLUTETHIMIDE

TABLE

u.d. u.d. 10 8 12

Day u.d. u.d. 29 38 34

Night

20.22-Dihydroxycholesterol

u.d. u.d. 7 9 ud.

Day

u.d. 29 29 25 31

Night

17,20-Dlhydroxy cholesterol

DERIVATIVES IN THE DAY AND NIGHT BY DAY AND PREDNISOLONE BY NIGHT

URINE

OF FIVE

144

Discussion In the text, aliquots of urine have been referred to as “day” and “night” urine. Day urine consisted of a 16-h collection from 0800 h to 2400 h and represented the period of AG administration. The “night” urine from 2400 h to 0800 h was collected during the period of prednisolone therapy. As was expected, the day urine volume exceeded the night volume and to establish the relevance of such a comparison as intended for this investigation, the distribution of urinary creatinine in the two samples was assayed. Assuming a constant rate of creatinine clearance, the day/night creatinine ratios were satisfactorily in a 2 : I. ratio. Further, since the circadian cycle for steroid production is greater during the night hours, low levels of urinary steroid metabolites in the “night” urine could therefore be significant. In most studies, urinary steroid values are expressed per 24 h, thus the fluctuations due to the diurnal rhythm are obliterated. When assessing the significance of the results presented here, it is first necessary to consider whether the combined form of therapy had maintained adequate adrenal suppression to normalise the excretion values for the urinary steroids and secondly, whether AG or prednisolone provided better control in the respective periods. It will be seen from Table II that pregnanetriol and pregnanetriolone were satisfactorily low in the three younger children, but in the two 9 year old girls the daily values were abnormally high. Only in one of these (Case 2) were the day and night values both increased. However, it can be assumed that in these two girls, the combined adrenocortical suppression (prednisofone) and steroidogenie blockade (AG) did not prevent the metabolism of cholesterol through the pregnenolone/progesterone pathway to give rise to increased elaboration of pre~anetriol, Lack of adequate suppression is also reflected in the high values for testosterone and DHA in these two patients. Androstenedione, androsterone and aetiocholanolone were increased only in Case 2 (Table IV) and the DHA presumably derived from 17a-hydroxypregnenolone. Cases 3, 4 and 5 were well controlled in that urinary pregnanetriol was less than 0.6 pmo1/24 h even although the greater portion of this was excreted by day. It is therefore concluded that suppression was more effective with prednisolone than with AG in these patients. Nonetheless, urinary DHA and testosterone were present in excess amounts in all three while the excretion of aetiocholanolone was elevated in one (Case 3). The possibility of the existence of alternative pathways from cholesterol via 20a-hydroxy-cholesterol, 17a,20a-dihydroxycholesterol or ~OCY ,22R -dihydroxycholesterol with subsequent side-chain cleavage to DHA (Fig. 5) was therefore considered to explain the normally low pregnanetriol in the presence of increased DHA and testosterone. Finding in the urine of these three cases 2Oa-hydroxycholesterol, 17a,20a-dihydroxycholesterol and 200~, 2~-dihydroxycholesterol, supported the hypothesis, whereas in the two unsuppressed cases (Cases 1 and 2) these compounds were not isolated in significant quantity. From incubation studies [l&-20] on the steroidogenic activities of an adrenal adenoma the direct formation of DHA from cholesterol without a C21intermediate would seem to be possible, the theoretical intermediate being 1701, 20a-dihydroxycholesterol. Incubation studies using human fetal adrenal slices

145 CHOLESTEROLSEQUENCE

20.22R-dlhdroxycholestero !

Pregnenolone

Fig. 5. Cholesterol and its hydroxylated derivatives showing the accepted pathway via 20,22-dihydroxycholesterol to pregnenolone and an alternative pathway via 20u-hydroxycholesterol and l*l,ZO-dihydroxycholesterol to dehydroepiandrosterone.

R P R P

Nqht

Day url”e

“n”e

Fig. 6. GLC tracings of urine extracts from Case 3 to show increased excretion of 17,2O-dShydroxycholesterol and 20.22-dihydroxycholesterol in night urine (a) over that in the day urine (b).

146

[21] also have demonstrated the conversion of 20a-hydroxycholesterol to 170(, 20@-dihydroxycholesterol and further that on incubating fetal adrenal slices with this latter substrate, DHA was formed. While these reactions have not been demonstrated in normal adult humans, sulphate esters of 20,2‘&dihydroxycholesterol and of 22-, 23- and 24-hydroxycholesterol among other metabolites of cholesterol have been isolated from meconium and faeces from newborns. Further, deficiency of cholesterol 20a-hydroxylase in the adrenal glands of an infant who died from congenital lipoid adrenal hyperplasia has been demonstrated [23]. It is of interest that male infants with this syndrome have incompletely masculinized external genitalia and this has been attributed to a failure of adequate adrenal androgen synthesis (DHA) during the period of differentiation. Even although there is a direct pathway from cholesterol to DHA not through pregnenolone, in congenital lipoid adrenal hyperplasia it is therefore likely to be blocked by virtue of defective cholesterol ZOa-hydroxylase, the important intermediate being 17~,2Off-dihydroxycholesterol (Fig. 5). In the case of CAH treated with AG, abnormal adrenal storage of cholesterol is likely and in this pathological state a As-pathway from cholesterol to DHA seems possible. In three of the patients, DHA was excreted in quantities beyond what might have been expected, since the action of AG is to block 20,22-desmolase and not 17,2O~esmolase. The finding of 20~-cholesterol, l?a, 20~-dihydroxycholesterol and 20,22~-dihydroxycholesterol in the urine of these patients supports the thesis that an intermediate C21-compound was not necessarily involved since pregnanetriol was satisfactorily low. To account therefore for the high urinary values of active androgens, DHA and testosterone, in Cases 3, 4 and 5, it is suggested that DHA, so formed directly from 17,20dihydroxycholesterol is the immediate precursor for androstenedione, testosterone and their metabolites. Aminoglutethimide has been shown to have other inhibiting effects on enzyme systems in addition to its action on the desmolase complex. It inhibits adrenal 11/3-hydroxylase. In a case of Cushing’s syndrome a rise in tetrahydro-S and pregnanetriol but not of pregnanetriolone has been observed [24] after administration of AG. The findings reported here of absent or low pregnanetriolone confirm that observation. AG also has peripheral actions [ 251. In acute 3 day studies AG was found to inhibit the excretion of testosterone and its peripheral conversion to aetiocholanolone and androsterone. Long-term effects however are not yet precisely known. In the patients reported here treatment with AG had been continuing for more than 3 years. Even so the urinary excretion of testosterone and DHA were increased but only in Case 2 were aetiocholanolone and androsterone also correspondingly increased. No uniform pathway of peripheral degradation could be defined. In three cases aetiocholanolone was within the normal range while in four cases androsterone excretion was likewise normal. This might suggest impaired metabolism of testosterone to its de~adation products. This altered metabolism of testosterone may be due to changes in smooth endoplasmic reticulum of the liver cells induced by AG [26] but clearly alterations in protein-binding of steroids and alternative metabolic pathways as yet unknown are possible drug-induced mechanisms to account for the observation. From these studies it is concluded that in the two 9 year old patients (Cases

147

and 2) the dosage schedule with aminoglutethimide and prednisolone was inadequate to maintain adrenal suppression and increases in both AG and prednisolone dosage could have been justified. However, in the other patients, adrenal suppression could be judged adequate when pregnanetriol levels alone were considered but nonetheless a rise in urinary testosterone and DHA occurred, the rise in these compounds occurring more by day than by night. Thus it is possible that when adrenal steroidogenesis is blocked by aminoglutethimide a time comes when there is a direct conversion in the adrenal of cholesterol to DHA via 17,20-dihydroxycholesterol and not via pregnenolone. That intermediate compounds 20a-hydroxycholesterol, 20,22-dihydroxycholesterol and 17,20dihydroxycholesterol are synthesized has been demonstrated by detecting these compounds in the urine of three patients. It is noteworthy that these hydroxylated cholesterols appeared more in the night urine which is not inconsistent with the view that as long as aminoglutethimide blockade is effective the pathway is directly to DHA through 17,20dihydroxycholesterol but when its effects are withdrawn during the night, and the other pathway via 20,22dihydroxycholesterol to pregnenolone becomes operative, then there is’ an adrenal discharge of the intermediate hydroxylated cholesterols previously formed. This is the most likely explanation for the findings. It would appear that this is the first time that an attempt to isolate dihydroxycholesterols from human urine has been made. The findings therefore must be regarded qualitatively significant rather than of quantitative value. 1

Acknoweldgements I am indebted to Professor J.H. Hutchison for his encouragement and helpful criticism. Parts of the equipment used were obtained by monies from the Rankin Memorial Fund of Glasgow University, and research assistance from a Research Grant from the Scottish Home and Health Department. I am grateful to Dr. Marcel Gut, The Worcester Foundation for Experimental Biology, Shrewsbury, Massachusetts, for the gift of the cholesterol derivatives used as standards in this work. CIBA Laboratories, Horsham courteously made available aminoglutethimide for this study. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

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148

17

Schubert.

18

Burstein,

S. and

K. and

Dorfman,

19

Goal,

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