Department of Obstetrics and Gynecology, Medical University of South Carolina, Charleston, S. C., USA, and Reproductive Endocrinology Research Unit, Swedish Medical Research Council, Karolinska sjukhuset, Stockholm
IN VITRO STEROL AND STEROID BIOGENESIS BY A FEMINIZING ADRENOCORTICAL CARCINOMA By R. S. Mathur, H. O. Williamson, L. O. Moody and E. Diczfalusy
ABSTRACT
A post-menopausal woman was found to excrete elevated amounts of urinary oestrogens. An adrenal carcinoma was removed and slices in- cubated in phosphate buffer, pH 7.4 with [14C]sodium acetate in com- bination with either [7a-3H] cholesterol, [7a-3H] dehydroepiandrosterone or [7a-3H] androstenedione. Radiochemically homogeneous tritium labelled oestrone, 178-oestradiol and oestriol were isolated in each of the three experiments. Carbon-14 activity was incorporated into oestrone in each of the three experiments and into oestriol in one case only. No carbon-14 activity was associated with 178-oestradiol isolated from any of the three incubations. Oestrone was found to be the predominant oestrogen synthe- sized in all experiments.
When [14C] sodium acetate and [7a-3H] cholesterol (3H/14C ratio = 0.61) were used as substrates, the following compounds were isolated in a radiochemically homogeneous form (the isotopic ratio in each being shown in parenthesis): cholesterol (13.0), cholesterol sulphate (1.6), pregnenolone (1.0), pregnenolone sulphate (0.2), dehydroepiandrosterone (3.2), dehydro- epiandrosterone sulphate (1.2), 45-androstenediol (3.1), 45-androstene- diol sulphate (isolated as the unconjugated compound following solvolysis of the “disulphate” fraction (0.6), testosterone (1.1), testosterone sulphate (0.6), and oestrone (1.3).
It is concluded that the adrenocortical carcinoma studied was capable of synthesizing oestrone, oestriol and cholesterol sulphate de novo from ace- tate, and that the steroidogenic processes led to the predominant forma-
tion of 45-steroid sulphates. Furthermore, a comparison of the isotopic ratios in the various compounds isolated suggests that a number of un- usual transformations may have occurred during steroidogenesis by the tumour cells in vitro.
Feminizing adrenocortical carcinomata are rare, only 53 cases in males having been reported in as many years (Gabrilove et al. 1965; Rose et al. 1969), since the first report (Bittorf 1919). No case involving an adult female has been reported presumably because of the difficulty to recognize clinically physical findings produced by an extra-ovarian source of oestrogens. This report appears to be the first dealing with an oestrogen-producing adrenocortical carcinoma in an adult female.
EXPERIMENTAL
Case report
This 55-year old G III, P III, A O underwent uneventful menopause in 1965. She had a history of inner ear disease, and experienced vaginal bleeding in April 10-14, 1969 and again 10 days later for 3 days. Uterine curettage revealed a cervical polyp and proliferative endometrium with glandular hyperplasia. Papanicolaou vaginal wall smears showed no basal cells, 65% intermediate cells, and 35% superficial cells. Bleeding recurred September 22, 1969, and approximately every 2 weeks thereafter until December 1969 when a functional ovarian tumour was considered. Laparoscopy revealed an atrophic right ovary and an apparent modestly enlarged left ovary. Re- peated curettage yielded glandular cystic hyperplastic endometrium of more pro- nounced activity than the previous specimen. At laparotomy, an estimated 4 cm left adrenal tumour was palpated. The ovaries were removed in December, 1969 and the gynaecologic surgical specimen revealed atrophic ovaries and a small left par-ovarian cyst. Twenty-four hour urinary steroids on January 8, 1970, were: oestrone 52.8 µg, oestradiol 18.6 µg, oestriol 134.5 µg; 17-ketosteroids 16.3 mg and 17-hydroxycortico- steroids 35.2 mg. Dexamethasone 0.5 mg every 6 hours was given from January 10 through January 12. Urinary steroid values on January 13, 1970 were: oestrone 90.5 ug, oestradiol 26.2 ug, oestriol 20.5 µg, 17-ketosteroids 16.4 mg and 17-hydroxy- corticosteroids 22.6 mg. Urinary gonadotrophins were less than 40 units. She was adrenalectomized on February 28, 1970. The pathological diagnosis was “adrenal cor- tical adenoma with transition to carcinoma and infiltration to the surrounding fat and connective tissue”. She was rehospitalized from July 28 to August 5, 1971. The patient expired October 29, 1971.
In view of the considerable oestrogen excretion in this gonadectomized patient, a study on the de novo steroidogenesis by these tumour cells was undertaken.
Materials and methods
Complete 24-hour urinary oestrogens were measured by the method of Brown (1955) as modified by Diczfalusy & Westman (1956). Seventeen-ketosteroids were measured using the Zimmermann reaction (Zimmermann 1935) and 17-hydroxy-corticosteroids by the Porter-Silber reaction (Porter & Silber 1950).
Abbreviations and trivial names
A: androstenedione (4-androstene-3,17-dione)
45A: 45-androstenediol (5-androstene-38,17ß-diol)
45 ADS: 45-androstenediol disulphate (5-androstene-33,178-yl-disulphate) C: cholesterol (5-cholesten-38-ol)
CAc: cholesterol acetate (5-cholesten-38-yl acetate)
CDAc: cholesterol dibromide acetate (5a,6B-dibromocholestan-38-yl acetate)
CS: cholesterol sulphate (5-cholesten-38-yl sulphate)
DHA: dehydroepiandrosterone (30-hydroxy-5-androsten-17-one)
DHAS: dehydroepiandrosterone sulphate (17-oxo-5-androsten-38-yl sulphate) DPM: disintegrations per minute
E1: oestrone (3-hydroxyoestra-1,3,5(10)-trien-17-one)
E2: 178-oestradiol (oestra-1,3,5(10)-triene-3,178-diol)
E3: oestriol (oestra-1,3,5(10)-triene-3,16a,17ß-triol)
PPC: paper partition chromatography
45P: pregnenolone (38-hydroxy-5-pregnen-20-one)
17aHO-45P: 17a-hydroxy-pregnenolone (38,17a-dihydroxy-5-pregnen-20-one)
45PS: pregnenolone sulphate (20-oxo-5-pregnen-38-yl sulphate)
P: progesterone (4-pregnene-3,20-dione)
17aHO-P: 17a-hydroxy-progesterone (17a-hydroxy-4-pregnene-3,20-dione)
S. A .: specific activity
T: testosterone (178-hydroxy-4-androsten-3-one)
TAc: testosterone acetate (3-oxo-4-androsten-178-yl acetate)
TS: testosterone sulphate (3-oxo-4-androstene-178-yl sulphate)
TLC: thin layer chromatography
Radioactive materials and chemicals
[1,2-14C]sodium acetate1), CFA 229, batch No. 16 with a S. A. of 56.0 mCi/mmol was used without further purification.
[7a-3H] cholesterol2), NEN 66-129 with a S. A. of 5.0 mCi/mmol was purified by chromatography in TLC System No. 1 with two developments. Its purity was checked by recrystallization. The S. A. of this material remained unchanged following acetyla- tion, bromination, and recrystallization.
[7a-3H]androstenedione1), TRA 16, batch No. 2 (S. A. 0.670 Ci/mmol) was purified by TLC in System 2 followed by PPC in System No. 1. Crystallization to constant S. A. indicated a purity of almost 100 %.
[7a-3H] dehydroepiandrosterone1), TRK 163, batch No. 3 (S. A. 4.6 Ci/mmol) was purified by TLC in System No. 2 followed by PPC in Systems No. 3 and 6. Crystal- lization to constant S. A. indicated a purity of 99 %.
Paper partition chromatography
All chromatographies were carried out at room temperature using the following systems:
No. 1. Ligroin (b. p. 60°-90℃), methanol, water (5:4:1)
No. 2. System 1 for 7 h
1) Purchased from the Radiochemical Center, Amersham, Buckinghamshire, England.
2) Purchased from New England Nuclear Corp., Boston, Mass., USA.
No. 3. System 1 for 10 h
No. 4. N-hexane, benzene, methanol, water (33:17:40:10)
No. 5. System 4 for 7 h
No. 6. Benzene, ligroin, methanol, water (6:3:8:2)
No. 7. Isopropyl ether, t-butanol, ammonium hydroxide (30% as NH3), water (6:4:1:9)
No. 8. System 7 for 8 h
No. 9. System 7 for 24 h
Thin layer chromatography
The following solvent systems were used at room temperature:
1. Benzene: ethyl acetate (5:1)
2. Ligroin (b. p. 80-110°C), ethyl acetate (10:3) with two developments
3. Benzene: ethanol (9:1)
4. Cyclohexane: ethyl acetate (1:1)
5. Chloroform: ethanol (1:1)
6. Benzene: ethanol (8:2)
Formation of derivatives
Acetylation was carried out with a pyridine-acetic anhydride mixture (1:1) overnight at room temperature. The formation of cholesterol dibromide was performed according to the method of Schwenk & Werthessen (1952) as modified by Kabara & McLaughlin (1961).
Solvolysis was performed by the method of Burstein & Lieberman (1958) as modified by Mathur et al. (1970).
Measurement of radioactivity
This was done by liquid scintillation spectrometry (Nuclear Chicago, Model Mark II). The scintillation fluid consisted of 4 g PPO and 100 mg POPOP dissolved in 1.0 litre of toluene. Corrections for quenching in each vial were made by use of an external standard and results expressed as DPM.
All steroids were obtained from Steraloid Inc., Pawling, N. Y. and were checked for purity by determination of melting points.
Glass plates precoated with Silica Gel (F-254) were purchased from Brinkmann In- struments, Westbury, New York.
Krebs-Ringer phosphate buffer, pH 7.4 was prepared as described by DeLuca & Cohen (1964). The gas phase was 95 % oxygen and 5% carbon dioxide.
Incubation procedure
From the stock solutions of tritiated C, DHA, and A in methanol, aliquots of each were transferred to 50 ml Erlenmeyer flasks, and the solvent evaporated under nitro- gen. Two or three drops of propylene glycol were added to redissolve the residue in each flask. Appropriate amounts of [1,2-14C]sodium acetate dissolved in 20 ml of phosphate buffer, pH 7.4 were added to each flask containing either [7a-3H]C (ex- periment No. 1), [7a-3H]DHA (experiment No. 2), or [7a-3H] A (experiment No. 3). Slices from the adrenal tumour were obtained using a sharp spatula and were washed free of blood in phosphate buffer. Approximately 300-500 mg slices were added to each flask without cofactors, and incubated at 37ºC for 33 hours in a Dubnoff meta-
(90% methanol) “total steroids” fraction
ether
water
PH 9
unconjugated steroid- like material
n-butanol extraction
steroid sulfate-like material
toluene
1 N NaOH
HC1
PH 2
PPC No. 1
ether
extraction
PPC No. 7
P
SP
A
DHA
Polar
benzene : n-hexane
water
CS
SPS
DHAS
Polar
522
acetylation
PPC No. 2
acetylation
PPC No. .
PPC No.
4
(E3)
TLC No. 5
PPC No. 8
PPC No. 9
PPC No. 9
TLC No. 3
x
x
x
I
5A
TLC No.
4
ether
extraction
rs
PADS
X
acetylation
acetylation PPC No. 5
E1
E2
TLC No. 6
x
X
X
X
solvolysis
X
X
X
X
X
X
X
solvolysis
solvolysis
solvolysis
solvolysis
PPC No. 5
acetylation
acetylation
acetylation
X
X
X
X
X
X
5
A
X
X
X
x
bolic shaker. Reactions were then stopped by adding an excess of hot ethanol and the contents frozen at -15°C until processed.
Extraction procedure
The contents of the flasks were filtered, the solid material homogenized using a mortar and pestle and extracted as described previously (Mathur et al. 1970). The filtrate and the extracts were pooled, evaporated to dryness, the residue dissolved in 100 ml of methanol and aliquots assayed for radioactivity.
Isolation of sterols and steroids
Following extraction and precipitation in 70% (v/v) methanol, the material con- tained in the supernatant was subjected to solvent partition (n-hexane: 90 % methanol v/v) in a countercurrent fashion using 6 separatory funnels. The n-hexane fraction was processed as described previously (Mathur et al. 1970) and cholesterol isolated. The 90 % methanol fraction was processed as shown in Fig. 1.
Table 1. Distribution (%) of radioactivity in the various fractions following incubation with tritium labelled substrates. Figures in parentheses represent corresponding incorporation from [14C]sodium acetate. The figures are not corrected for methodological losses.
| Fractions | Substrate | ||
|---|---|---|---|
| C (Experiment 1) | DHA (Experiment 2) | A (Experiment 3) | |
| 1 Initial extract | 100 | 100 | 100 |
| (100) | (100) | (100) | |
| 2. 70 % MeOH | 54.6 | 90.5 | 98.5 |
| (55.1) | (50.8) | (71.7) | |
| 3. Solvent partition: a. Sterol | |||
| 47.1 | 1.22 | 1.68 | |
| (4.65) | (7.01) | (11.5) | |
| b. Total steroids | 6.62 | 89.2 | 96.2 |
| (34.10) | (43.10) | (61.2) | |
| 4. Steroid sulphate-like | 1.26 | 63.1 | 17.7 |
| (1.48) | (1.41) | (3.93) | |
| 5. Unconjugated steroids | 0.71 | 10.6 | 65.2 |
| (0.39) | (0.71) | (1.73) | |
| 6. Unconjugated neutral steroids | 0.59 | 6.62 | 57.1 |
| (0.22) | (0.45) | (1.27) | |
| 7. Unconjugated oestrogens | 0.08 | 3.69 | 8.77 |
| (0.13) | (0.19) | (0.46) | |
RESULTS
The distribution of carbon-14 and tritium labelled material in the various fractions is shown in Table 1. Of the carbon-14 activity, 30-50 % was lost in the 70% methanol precipitation step. The corresponding figure for tritium was 45 % (experiment No. 1) and only 1.5% in experiment No. 3. In all experiments, more carbon-14 activity was associated with the steroid sulphate fractions than was found in the unconjugated steroid fractions. Crystallization data from experiment No. 1 are given in Tables 2, 3 and 4. The total amount of radioactivity (uncorrected for procedural losses) associated with each of the
| 3H 14C | 3H | 14C | ||||||
|---|---|---|---|---|---|---|---|---|
| X | ML | X | ML | X ML | X | ML | ||
| C S. M. 10 200 S. M. 1300 | CS S. M. 20 900 S. M. 19 500 | |||||||
| 1 | 9590a) | 14 400 | 1040 | 2580 | 5070e) | 33 400 | 3620 | 102 000 |
| 2 | 9080b) | 9440 | 790 | 1680 | 2550f) | 17 600 | 1040 | 13 200 |
| 3 | 9880c) | 9420 | 840 | 1360 | 336g) | 11 700 | 172 | 5780 |
| 4 | 9470d) | 9570 | 761 | 999 | 188b) | 997 | 117 | 511 |
| 5 | 9680a,r) | 9600 | 764 | 782 | 145a,s) | 376 | 89 | 273 |
| 6 | 9610b,r) | 9580 | 762 | 774 | 140b,s) | 150 | 90 | 110 |
| 7 | 141c,s) | 146 | 87 | 93 | ||||
| 45 P S. M. 122 S. M. 148 | 45PS | |||||||
| S. M. 1080 | S. M. 7120 | |||||||
| 1 | 68a) | 178 | 82 | 222 | 717a) | 35 300 | 3950 | 27 500 |
| 2 | 73b) | Z | 72 | Z | 527e) | 1010 | 4071 | 5240 |
| 3 | 64e) | 99 | 69 | 90 | 629f) | 716 | 3570 | 3790 |
| 4 | 73f) | 68 | 66 | 73 | 597g) | 621 | 3380 | 3900 |
| 5 | 71g,y) | 54 | 68 | 56 | 593b,s) | 587 | 3320 | 3390 |
| 6 | 70c,y) | 70 | 68 | 56 | 595c,s) | 602 | 3330 | 3380 |
X: Crystals, ML: Mother liquor, S. M .: Starting material.
Solvents: a) methanol, b) methanol-water, c) ethanol-water, d) methanol-ethyl acetate, e) ethanol-toluene, f) ethanol-n-hexane, g) ethanol-ligroin, h) ethanol-acetone, r) re- crystallized as acetate dibromide, s) recrystallized as unconjugated compound fol- lowing solvolysis, y) recrystallized as acetate, z) insufficient material for weighing.
Table 3. Experiment No. 1. Recrystallization of dehydroepiandrosterone (DHA), dehydroepi- androsterone sulphate (DHAS), 45-androstenediol (45A) and 45-androstenediol (45 A) isolated following solvolysis of the 45-androstenediol “disulphate” fraction. Figures are in DPM/mg of steroids.
3H
3H
14C
X
ML
X
14C ML
X
ML
X
ML
DHA
DHAS
S. M. 2780
S. M. 438
S. M. 3210
S. M. 2920
1
2760a)
3560
320
880
3210f)
3270
2570
3220
2
1580b)
17 500
204
1800
3300g)
3380
2590
2840
3
810c)
5960
200
282
3240h)
3320
2640
2640
4
798d)
878
180
282
3210d,s)
3190
2580
2500
5
702e)
1580
179
245
3210e,s)
3200
2570
2550
6
634c,y)
896
174
202
7
580d,y)
864
178
194
8
570e,y)
580
177
182
| 45 S. M. 530 | A S. M. 809 | 45 A (From "disulphate fraction" following solvolysis) | ||||||
|---|---|---|---|---|---|---|---|---|
| S. M. 58 | S. M. 248 | |||||||
| la) | 348 | 1140 | 273 | 1700 | 14 | 115 | 61 | 575 |
| 2b) | 182 | 596 | 84 | 548 | 12 | 20 | 26 | 98 |
| 3c) | 163 | 346 | 62 | 218 | 12 | 10 | 22 | 50 |
| 4d) | 154 | 241 | 60 | 163 | 14* | Z | 20 | Z |
| 5e,y) | 137 | 168 | 47 | 81 | 14 ** | 15* | 23 | 22 |
| 6a,y) | 140 | Z | 48 | Z | 14* | 14* | 22 | 23 |
| 7b,y) | 140 | Z | 42 | Z | ||||
X: Crystals, ML: Mother liquor, S. M .: Starting material.
Solvents: a) methanol-water, b) methanol, c) ligroin, d) n-hexane, e) ethanol-water, f) ethanol-n-hexane, g) ethanol-ligroin, h) ethanol-toluene, s) recrystallized as uncon- jugated steroid following solvolysis, j) recrystallized as acetate and z) insufficient material for weighing.
*: Based on 15 000 DPM net counts.
compounds, the 3H/14C ratio and the proportion of each compound to the total amount isolated is given in Table 5.
Radioactivity initially associated with the following steroids dissociated during the process of purification or crystallization: P, 17aOH-P, A, cortisol, and the sulphates of E1, E2, and E3.
In experiments No. 2 and 3, only C and E1, E2, and E3 were looked for. Crystallization data on E1 and E3 are given in Table 6.
| 3H | 14C | 3H | 14C | |||||
|---|---|---|---|---|---|---|---|---|
| X | ML | X | ML | X | ML | X | ML | |
| T S. M. 309 S. M. 639 | TS S. M. 123 S. M. 349 | |||||||
| 1 | 138a) | 1470 | 251 | 2810 | 118a) | 213 | 276 | 1022 |
| 2 | 118b) | 1360 | 222 | 2390 | 81c) | 142 | 160 | 355 |
| 3 | 119c) | Z | 198 | Z | 65d) | 128 | 138 | 323 |
| 4 | 46d) | 418 | 83 | 864 | 67e) | Z | 132 | Z |
| 5 | 69e) | Z | 83 | Z | 68b,s) | Z | 114 | Z |
| 6 | 47a) | 136 | 72 | 331 | 66c.s) | 68 | 112 | 115 |
| 7 | 23b,y) | Z | 21 | Z | ||||
| 8 | 24c,y) | 23 | 21 | 22 | ||||
| E1 S. M. 236 | S. M. 305 | |||||||
| 1 | 70a) | 429 | 60 | 1170 | ||||
| 2 | 64b) | 2 | 42 | Z | ||||
| 3 | 60f) | Z | 30 | Z | ||||
| 4 | 53g) | 72 | 20 | 50 | ||||
| 5 | 54a,y) | 68 | 21 | 44 | ||||
| 6 | 35b,y) | 58 | 20 | 20 | ||||
| 7 | 33f,y) | 38 | 24 | 25 | ||||
| 8 | 33g.y) | 36 | 25 | 24 | ||||
X: Crystals, ML: Mother liquor, S. M .: Starting material.
Solvents: a) methanol, b ) methanol-water, c) n-hexane-ethanol, d) ligroin-ethanol, e) toluene-ethanol, f) ethanol, g) ethanol-water, s) recrystallized as unconjugated steroid following solvolysis, y) recrystallized as acetate and z) insufficient material for weighing.
DISCUSSION
Feminizing adrenocortical carcinomata are rare. Gabrilove et al. (1965) re- viewed 52 cases in males reported in the literature since 1919, when the first case was reported (Bittorf 1919). Recently, Rose et al. (1969) have reported another case in a male.
Relatively few studies have been done on the capability of feminizing adrenal tumours to synthesize steroids. Formation of oestrogens from C-21 and C-19 steroid precursors has been shown (Gabrilove et al. 1965; Rose et al. 1969; Baggett et al. 1959). However, conversion of radioactive acetate to
oestrogens could not be demonstrated (O’Donnel et al. 1962; Stewart et al. 1964). The important finding of the present investigation is the demonstration that this tumour synthesized CS, E1 and E3 de novo from acetate. This is the first report on the de novo synthesis of CS and oestrogens by a feminizing adrenocortical carcinoma.
A comparison of the isotopic ratios in the various compounds isolated in experiment No. 1 seems to suggest that in the steroidogenic processes, a number of unusual transformations could have occurred. The isotopic ratio in CS (1.6) may indicate that it was partly synthesized from C (13.0) and partly from acetate by a pathway bypassing C, or alternatively, that only the C present in a certain compartment was available for CS formation. Pathways of CS syn- thesis in adrenocortical carcinoma have not yet been established, though lano- sterol sulphate as a possible precursor has been suggested (Baulieu et al. 1967). We have found no report in the literature on the isolation of CS from normal
| Compound | Total radioactivity isolated (DPM) ** | 3H/14C | % of the total | ||
|---|---|---|---|---|---|
| 3H | 14C | 3H | 14C | ||
| C | 578 000 | 45 700 | 13.0 | 82.0 | 21.6 |
| CS | 3220 | 2020 | 1.6 | 0.5 | 1.0 |
| 45PS | 15 100 | 84 800 | 0.2 | 2.1 | 40.0 |
| DHAS | 83 000 | 66 600 | 1.2 | 11.8 | 31.4 |
| TS | 1800 | 3040 | 0.6 | 0.3 | 1.4 |
| 45 ADS* | 402 | 645 | 0.6 | 0.1 | 0.3 |
| 45P | 1640 | 1580 | 1.0 | 0.2 | 0.8 |
| DHA | 15 400 | 4740 | 3.2 | 2.2 | 2.2 |
| 45 A | 3590 | 1150 | 3.1 | 0.5 | 0.5 |
| T | 618 | 552 | 1.1 | 0.1 | 0.3 |
| E1 | 1360 | 1030 | 1.3 | 0.2 | 0.5 |
| Total | 704 000 | 212 000 | 100.0 | 100.0 | |
* 45 ADS: Crystallized as the unconjugated steroid (45A) following solvolysis of the 45-androstenediol “disulphate” fraction (see Fig. 1, methodology).
** Figures were calculated from the mean of the last two sets of crystals and are not corrected for methodological losses.
Table 6. Recrystallization of oestrone (E1) and oestriol (E3) in experiments No. 2 and 3. The figures are expressed as DPM/mg. In experiment No. 2, [14C]sodium acetate (84.8 x 106 DPM) in combination with [7a-3H] dehydroepiandrosterone (DHA) (31.8 x 106 DPM) (3H/14C) = 0.4) was used as substrate, while in experiment No. 3, 32.3 x 106 DPM of [14C] sodium acetate and 24.3 x 106 DPM [7a-3H] androstenedione (A) (3H/14C = 0.8) were used.
| 3H 14C | 3H | 14℃ | ||||||
|---|---|---|---|---|---|---|---|---|
| X | ML | X | ML | X | ML | X | ML | |
| E1 (Experiment No. 2) | E1 (Experiment No. 3) | |||||||
| S. M. 19 100 | S. M. 304 | S. M. 36 000 | S. M. 84 | |||||
| 1a) | 18 700 | 30 200 | 214 | 810 | ||||
| 2b) | 17 900 | 28 300 | 160 | 600 | 30 200 | 61 000 | 34 | 164 |
| 3c) | 17 000 | 22 100 | 154 | 210 | 30 900 | 39 900 | 45 | 70 |
| 4d) | 16 500 | 21 200 | 150 | 203 | 40 600 | 39 600 | 76 | 69 |
| 5e) | 16 500 | 21 000 | 146 | 198 | 35 000 | 35 500 | 49 | 51 |
| 6a,y) | 16 500 | 15 000 | 142 | 136 | 35 600 | 35 800 | 58 | 60 |
| 7b,y) | 16 500 | 16 100 | 145 | 142 | 35 400 | Z | 61 | 2 |
| E3 (Experiment No. 2) | ||||
|---|---|---|---|---|
| S. M. 2720 | S. M. 1080 | |||
| 1a) | 1270 | 7240 | 406 | 1820 |
| 2b) | 608 | 1540 | 182 | 1140 |
| 3c) | 406 | 1100 | 160 | 222 |
| 4d) | 265 | 2020 | 89 | 1480 |
| 5e) | 358 | 486 | 99 | 96 |
| 6a,y) | 297 | Z | 63 | Z |
| 7b,y) | 244 | 236 | 88 | 90 |
| 8c,y) | 228 | 224 | 80 | 86 |
X: Crystals, ML: Mother liquor, S. M .: Starting material.
Solvents: a) methanol, b) methanol-water, c) ethanol, d) ethanol-water, e) methanol- benzene, y) recrystallized as acetate and z) insufficient material for weighing.
adult human adrenals. Gurpide et al. (1966) have shown that blood borne CS is not an important intermediate in the synthesis of DH AS in normal adrenals; however, conversion of CS to DHAS and 45 AS without prior hydrolysis of the substrate has been demonstrated in an adrenocortical carcinoma (Roberts et al. 1964). Direct conversion of 45PS to DHAS had been demonstrated earlier in the same tissue (Calvin et al. 1963). The isotopic ratio in DHAS (1.2) isolated in the present study suggests that it was not formed exclusively from 45PS (0.2). It is
possible that some DHAS may have been derived directly from CS (1.6). This assumption is further supported by the observation that the isotopic ratio in DHA (3.2) was considerably higher than those of 45P (1.0) or DHAS (1.2), suggesting that some DHA could have been formed directly from C bypassing 45P (1.0). Direct conversion of C to DHA has been postulated in animal tissues (Jungman 1968a,b). The possibility of such a pathway operating in the human foetus at midgestation has also been discussed (Archer et al. 1971).
The very low isotopic ratio of 45PS (0.2) seems to indicate that a major part of this compound could not have been derived from C (13.0), CS (1.6) or 45P (1.0).
In vitro conversion of [17a-3H] 45A to [17a-3H]T by a virilizing adrenal tumour has been reported (Baulieu et al. 1963). In the present study, the iso- topic ratio in 45 A (3.1) isolated is almost identical with that found in DHA (3.2), but considerably higher than that in T (1.1). It seems that even though 45 A was almost exclusively derived from DHA, some T must have been pro- duced from precursor(s) other than DHA/45 A. It is conceivable that part of T may have been formed from TS (0.6). Finally, it seems that E1 (1.3) was almost exclusively derived from T (1.1).
Lack of P, 17aHO-P, A and large amounts of 45PS and DHAS formed show that the 45-steroid sulphate pathway seems to be the preferred route of steroid biogenesis in this feminizing adrenocortical carcinoma.
The experimental approach used in this study imposes certain major limita- tions on the interpretation of the results obtained. One disadvantage of using tissue slices is that the permeability differences of the precursors used can complicate the results of the incubation. Furthermore, the endogenous pool size of acetate, C and the various compounds isolated is not known; therefore, uni- form labelling of the entire pool cannot be assured. It has also been observed (Telegdy et al. 1970) that exogenously administered cholesterol is metabolized differently than that synthesized intracellularly de novo.
Last, but not least, it is realized that under the experimental conditions used the isotopic ratios of the various intermediates may change continuously during the incubation period and that the ratios observed after a fixed incubation time may lead to erroneous conclusions, unless dynamic studies are carried out using various incubation times. Thus, utmost caution should be exercised in extra- polation of the results obtained in an in vitro experiment to in vivo situations.
,
ACKNOWLEDGMENTS
Preoperative evaluation and diagnosis was by Dr. Sture Cullhed of Regionsjukhuset, Linköping, Sweden whose incentive made this study possible. Adrenal surgery was by Docent H. Tera. Dr. Arne Victor provided facilities to carry out the incubations in Linköping. Miss A. B. Leaming provided technical assistance.
Expenses of this investigation were partly defrayed by research Grants from the Ford Foundation, Swedish Medical Research Council, Swedish International Develop- ment Authority, Grant HD 00173 of the National Institute of Health, United States Public Health Service, Bethesda, Md., USA and Institutional Grant NIH GRS RR 420.
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Received on August 11th, 1972.