Adrenocortical Cancer: Steroid Biosynthesis and Metabolism Evaluated by Urinary Metabolites
MORTIMER B. LIPSETT, M.D. AND HILDEGARD WILSON, PH.D. Endocrinology Branch, National Cancer Institute, National Institutes of Health, Bethesda, Maryland
T THE PROFUSION and diversity of the steroids excreted by the patient with adrenal cancer have facilitated the characterization and estimation of many metabolites. These analyses, in turn, have permitted the formation of hy- potheses about the pathways of steroid biosynthesis in the normal adrenal gland, since it was assumed that neoplastic adrenal cortical tissue possessed only those enzymes present in the normal tissue. This assumption was justified, since there is now ample evidence that neoplasia generally is attended by de- differentiation and by a variable deletion of specific metabolic pathways, but not by the development of new enzyme systems.
In order to study the alterations of the biosynthetic mechanisms in adrenal can- cer, we have undertaken the analysis of certain urinary steroids in 10 patients with histologically proven functional metastatic adrenal cortical carcinoma. Using the currently accepted concepts of adrenal cortical steroid biosynthesis, it is then feasible to infer which alterations in normal synthetic mechanisms have occurred with the development of neo- plasia.
Methods
Urines were incubated for 4 days with 8- glucuronidase (Ketodase) acidified to pH 1, then continuously extracted for 48 hr with ether and for 48 hr at 1.ON sulfuric acid con-
ABSTRACT. In order to survey character- istic metabolic abnormalities in adrenal can- cer, we have measured important urinary metabolites of intermediates and end-prod- ucts of steroid biosynthesis. The adrenal can- cer in 9 of 10 cases could not efficiently 118- hydroxylate Substance S, as shown by the elevated excretion of tetrahydro Substance S both absolute and relative to cortisol metabo- lites. In 2 patients, the tumor seemed to have a complete block of 118-hydroxylation of Sub- stance S. Hydroxylation of 17a-hydroxypro- gesterone was relatively inefficient in 6 of 10 cases, as judged by the excretion of preg- nanetriol relative to corticoid excretion. Pregnanediol excretion was high in the 4 sub- jects studied. The neoplasm of one patient was probably lacking in the 36-hydroxysteroid dehydrogenase.
The 11-oxy-17-ketosteroids were excreted in amounts greater than could be predicted from the excretion of cortisol metabolites. The most quantitatively important in this group was 118-hydroxyandrosterone. Urinary dehydroepiandrosterone was increased in each patient, as was the excretion of etiocholano- lone and androsterone.
Analysis of these findings suggests that the urinary metabolites in patients with adrenal cancer reflect overproduction of pregnenolone, inefficient utilization of intermediates at sev- eral stages of steroid synthesis, and abnormal metabolism of the steroids either within the tumor tissue or peripherally.
centration. The neutral fractions were com- bined and separated into groups of metabo- lites by a modification of the silica-alumina partition column (1, 2). The elution sequence of the major metabolites is shown in Fig. 1.
Further fractionation was accomplished by paper chromatography in systems previously described (2-4), by derivative formation using chromate, bismuthate and periodate oxidation, and by acetylation when pertinent. After elution, quantitation of the 17-keto- steroids (17-KS), 45-36-OH compounds, di-
Received March 9, 1962.
PARTITION COLUMN FOR SEPARATING 42 METABOLITES 15 gm. Silica-Alumina, Stationary Phase =50% Ethanol
| STEROID STRUCTURE | A-3B-OH | C21 | C19 and C21 | |
|---|---|---|---|---|
| ELUANT: mt.20 A: 40 Hexane 80 120 | Androsterone Etiocholanolone Epi androsterone | |||
| DHA Pregnenediol | Pregnanediol | |||
| 100% | 140 | |||
| B: 10 | Androstenediol | 17-OH- | ||
| CHCI3 3% | 50 90 | Progesterone | ||
| BI_130- B2 | 17-OH Pregnenolone | Pregnanetriol | ||
| Hexane | 170 | Il-Ketoetiocholanolone S | ||
| 97% | 210 | |||
| C: | 40 | |||
| CHCI3 | 80 | Pregnenetriol | 118-OH-Etio. | |
| 8 % | CI 120 | |||
| Hexane | C2 160 200 | Cpd. S | ||
| 92 % | 240 | Tet. S | ||
| 280 | ||||
| 320 | ||||
| D: 40 | 200 STRIOL | |||
| CHCI3 15% | II-Ketopregnanetriol | |||
| Hexane 85% 120 | 20B-Tet S TRIOL | |||
| 160 | ||||
| 200 | ||||
| E: 40 | Cpd. F | I13-OH-Pregnanetriol | ||
| CHCI3 | 60 | Tet. E | ||
| 100 | ||||
| 30% | 140 | Tet. F | ||
| Cortolone | ||||
| Hexane | 180 | |||
| 70% | 220 | |||
| 260 F: 40 | Very Polar | |||
| CHCI3 50% Hexane 50% 80 120 | Corticosteroids | |||
hydroxy acetone groups and 17,20-dihy- droxy-21-methyl groups was performed by methods previously noted (2). Ketosteroids have been expressed as mg of dehydroepi- androsterone (DHEA), using the corrections of Wilson (5). In some patients, column
eluates were treated with digitonin and the a- and B-fractions were assayed directly with several group-specific reagents or were subse- quently fractionated by paper chromatog- raphy.
In order to estimate the C-20-hydroxy
| Patient No. | Sex | Age | Cushing's syndrome | Virilization | Average 17-KS, mg/24 hr | Average S-PC, mg/24 hr |
|---|---|---|---|---|---|---|
| la | ? | 30 | + | + | 52 | 28 |
| 1b | ? | 32 | + | + | 129 | 34 |
| 2 | ? | 28 | + | + | 94 | 87 |
| 3 | 9 | 41 | + | + | 91 | 74 |
| 4 | 8ª | 36 | + | - | 162 | 51 |
| 5 | 8ª | 29 | - | 86 | 37 | |
| 6 | 0ª | 59 | - | - | 144 | 11 |
| 7 | 8 | 55 | 1 | + | 46 | 14 |
| 8 | 0 | 50 | 土 | 111 | 24 | |
| 9 | 8 | 8 | - | + | 107 | 4 |
| 10 | 3ª | 1} | - | + | 122 | 0.3 |
metabolites of cortisol,1 the column eluates containing cortisol metabolites were chro- matographed in the Bush C system for 24 hr. The overflow was rerun for 5 hr in the same system. Spot tests for 17,20,21-triols and 17,20-diols (6) on the first paper usually showed strong bands corresponding to cortol and cortolone, as well as a band more polar than cortol. (The 20a- and 200-hydroxy isomers of cortol and cortolone do not sepa- rate in this system.) The reactive areas were eluted and measured by the periodate-KS method.
Reichstein’s Substance U (Compound E- triol) could not be clearly separated from pregnane-3a,113,17a,20a-tetrol, which would also occur in the eluate. Consequently, the contribution of E-triol was assessed by elu-
1 The following abbreviations and trivial names are used in the text: Cortisol (Compound F) = 11 8, 17 a, 21-trihydroxy - 4- pregnene-3, 20- dione; 63-OH-cortisol =68,118,17a,21-tetrahy- droxy-4-pregnene-3,20-dione; cortisone (Com- pound E) =17a,21-dihydroxy-4-pregnene-3,11, 20-trione; 66-OH-cortisone =66,17a,21-trihy- droxy-4-pregnene-3,11,20-trione; cortol and ß- cortol =pregnane-3a,118, 17a,20a (or B), 21- pentol; cortolone and 8-cortolone =3a,17a,20a (or ), 21-tetrahydroxypregnane-11-one; Reich- stein’s Substance U (Compound E-triol) =17a,208,21-trihydroxy-4-pregnene-3,11-dione; Reichstein’s Substance E (Compound F-triol) =118,17 a, 208,21 - tetrahydroxy -4 -pregnene-3- one; tetrahydro F (THF) =3a,118,17a,21-tetra- hydroxypregnane-20-one; tetrahydro E (THE) = 3 a, 17 a, 21 -trihydroxypregnane- 11, 20-dione; 21-deoxycortisol = 118,17 a-dihydroxy -4-preg - nene-3,20-dione; Substance S =17a,21-dihy- oxy-4-pregnene-3,20-dione; tetrahydro S (THS) = 3a, 17a,21-trihydroxypregnane-20-one; 17 a-OH-progesterone = 17 a-hydroxy-4-preg- nene-3,20-dione; pregnanetriol = pregnane- 3a,17a,20a-triol; 17a-OH-pregnenolone =38,17a- dihydroxy-5-pregnene-20-one; pregnenetriol =5-pregnene-38,17a,20a-triol; progesterone =4- pregnene-3,20-dione; pregnanediol = pregnane- 3a,20a-diol; 118-OH-androsterone (11-OHA) =3a,118-dihydroxyandrostane-17-one; 11-keto- androsterone = 3 a - hydroxyandrostane - 11, 17 - dione; 118-OH-etiocholanolone (11-OHE) =3a,118-dihydroxy-etiocholan-17-one; 11-keto- etiocholanolone (11-OE) =3a,hydroxyetiocho- lane-11,17-dione; androstenedione =4-andro- stene-3,17-dione; androsterone =3a-hydroxy- androstane - 17 - one; etiocholanolone = 3 a - hydroxyetiocholane-17-one; dehydroepiandro- sterone (DHEA) =36-hydroxy-5-androstene-17- one.
tion, periodic acid oxidation and estimation of adrenosterone after rechromatography. A similar procedure was used to detect Reich- stein’s Substance E (Compound F-triol). Somewhat less polar triols could be metabo- lites either of Substance S or of 21-deoxy- cortisol (Fig. 1). These mixtures were re- solved by elution of the reactive areas of the paper chromatograms and oxidation with periodic acid to the easily separable deriva- tives, C1902-17-KS from metabolites of Sub- stance S, and 11-oxy-C1903-17-KS from 21- deoxycortisol.
Pregnanediol was isolated from the ap- propriate partition column eluate by acety- lation followed by adsorption chromatog- raphy. It was measured as the sulfuric acid chromogen in the Carey spectrophotometer, as previously described (7). Pregnanetriol was oxidized with either periodate or bis- muthate after previous partition column chromatography and was measured as etiocholanolone. Three of the values for pregnanetriol were reported previously by Finkelstein (8).
These methods generally will suffice for the adequate identification, separation and quantitation of known steroids in the urine of normal adults. In the cases examined here, it may be assumed that the steroids isolated could still have been contaminated with minor amounts of unknown steroids. How- ever, the combination of group-specific color reactions after chromatography and deriva- tive formation makes it unlikely that the presence of other steroids can appreciably alter the analytic values or invalidate the conclusions.
Subjects
Pertinent data regarding the subjects of this study are presented in Table 1. The values for 17-KS and Silber-Porter chromo- gens (S-PC) are averages of many values taken before treatment. The urines taken for analysis had 17-KS and S-PC values close to the average. Patient 1 was studied before the removal of her adrenal carcinoma, and 2 years later, when metastatic disease had developed.
Results and Discussion
17,20,21-Triol Metabolites of Cortisol
In addition to the cortols, the corto- lones and the triol material more polar
than cortol, small amounts of Compound E-triol were found in the three subjects so studied. No definite test correspond- ing to Compound F-triol was obtained, nor was this metabolite detected by elution of the appropriate areas, oxida- tion with periodic acid and rechroma- tography for androst-4-ene-17-one. Occa- sionally, minor amounts of 17@,20,21- trihydroxypregnane-3,11-dione and of 17a,116,20,21-tetrahydroxypregnane-3- one were suggested when less polar frac- tions were similarly treated, and etio- cholanetrione and 113-hydroxyetiocho- lanedione were seen on rechromatog- raphy.
In one subject (Patient 5) who ex- creted large amounts of tetrahydro Substance S (THS), the techniques de- scribed above indicated substantial ex- cretion of pregnane-3a,17a,20a,21-tetrol, of which about 40% was in the eluates containing the metabolites of cortisol.
In Fig. 2, the excretion of the triol metabolites of cortisol is compared with the excretion of the Silber-Porter chro- mogens (S-PC) derived from cortisol. It is noteworthy that the three patients ex- amined excreted about 75% of their cortisol metabolites as 17,20,21-triols. The total cortisol metabolites were thus three to four times greater than the sum
40
Polar Triols
35
17,20,21 - Triols
Cortols
17,21,-di-OH-20-Ketones
30
( S-P Chromogens)
MG / 24 HRS 25
20
Cortolones
15
10
5
E- Triol
0
PT
3
7
10
Normal
Normal
ď
ㅎ
Cushing’s Syndrome
+ + + + +
30
Tetrahydro F
25
Tetrahydro E
20
MG /
24 HRS 1 5
10
5
0
0
25
44
Tetrahydro S
20
MG /
24 HRS 15
ío
5
0
0
PT. Normal
la
1b
2
3
4
5
6
7
8
9
10
of the S-PC. In two normal subjects, nearly equal amounts of these products were found.
Although the total of the cortisol metabolites was high for all three pa- tients, only Patient 3 had Cushing’s syn- drome. This is consistent with the hy- pothesis that the cancer tissue may have reduced the C-20 ketone of cortisol be- fore it was secreted. This reduction can be carried out by normal human adrenal slices (9). Most of the 17,20,21-triols derived from cortisol were excreted as 3-hydroxy compounds. Since 44-hydro- genases are probably not active in hu- man adrenal tissue, this transformation presumably took place in the liver after reduction at C-20 had occurred.
The proportion of cortols to cortolones (Fig. 2) is similar to that noted by Fukushima et al. (10). However, the appreciable amounts of 17,20,21-triols more polar than cortol in the urine of patients with adrenal cancer has not been noted previously. This material was neither 66-hydroxycortisol nor 60- hydroxycortisone, but could be the C- 20-hydroxy derivative. Similar analysis of the urine of a normal subject receiving
PT 6
4’-90 F - F 10 mg daily
6
MG / 4
24 HRS
2
0
Tetrahydro E &
90 - F -F 30 mg daily
Tetrahydro F
PT. 5
31
34
Tetrahydro S
25
20
MG /
24 HRS
15
10
5
0
1
2
3
4
5
6
DAYS
ACTH showed that the polar triol(s) were low, but a reducing area corre- sponding to 66-hydroxycortisol was noted. These polar triols, therefore, may also result from the metabolism of cor- tisol within the neoplastic tissue.
Tetrahydro E (THE), Tetrahydro F (THF), Tetrahydro Substance S
In Fig. 3, the excretion of the above compounds has been presented. The correlation between the excretion of THE and THF and the clinical manifes- tations of cortisol excess was good, al-
11-OHE
16
11-0 E
II-OHA
14
* Total C190317-KS
12
10
MG /
24 HRS 8
6
4
*
*
2
0
0
0
PT la
1 b
2
3
4
5
6
7
8
9
10
though there were only suggestive clini- cal features of Cushing’s syndrome in Patients 7 and 8. The excretion of THE and THF by Patients 5 and 6 was within the normal range and thus could have been derived from the cortisol secreted by the remaining normal adrenal gland. In order to determine the origin of the THE and THF, the patients were given suppressive doses of corticoids (Fig. 4). In Patient 5, 30 mg of 9a- fluorohydrocortisone daily reduced the excretion of THE and THF to low levels, but the excretion of THS re- mained elevated, indicating that the THE and THF originated from the re- maining normal adrenal gland. In Pa- tient 6, however, neither the excretion of THE and THF nor the excretion of THS was suppressed by 41-9a-fluorohy- drocortisone. In this case, then, it seemed that the adrenal cancer fortuitously pro- duced cortisol in amounts resulting in normal urinary levels of THE and THF. The study in Patient 5 strongly suggests that the adrenal carcinoma was inca- pable of hydroxylating Substance S. It seems probable that this also occurred in Patient 9. However, in order to prove this, similar detailed studies of THE, THF and THS excretion would be neces- sary.
The fact that only five of the ten patients in this series had Cushing’s syn- drome should not imply that this series is not representative of patients with adrenal cancer. Of 39 patients with metastatic adrenal cancer (Lipsett et al., unpublished), only 23 had Cushing’s syn- drome.
The excretion of increased amounts of tetrahydro S in adrenal cancer has been noted previously (4, 11) and is an indica- tion of the relative or absolute inability of the neoplasm to synthesize cortisol from Substance S. In our experience, this evidence of impairment of 113-hydroxy-
| Patient | P'diol | P'triol | 45-P'triol | P'triol | THS |
|---|---|---|---|---|---|
| THE +THF +THS | THE +THF | ||||
| la | 6 | 0.51 | 0.6 | ||
| 1b | 8.3 | 0.35 | 0.8 | ||
| 2 | 6.5 | 0.15 | 0.4 | ||
| 3 | 21.4 | 12 | 5.1 | 0.12 | 1.6 |
| 4 | 4.4 | 0.13 | 0.8 | ||
| 5 | 4.7 | 8 | 6.3 | 0.17 | 4.6 |
| 6 | 8.5 | 6.7 | 4.8 | 0.64 | 1.2 |
| 7 | 2.5 | 3.6 | 3.7 | 0.33 | 0.6 |
| 8 | 11.3 | 20.0 | 0.48 | 2.0 | |
| 9 | 16.6 | 10.1 | 0.6 | ||
| 10 Normal | 0.9 | 0.9 | 0.1 | 0.15 | 0.02 |
lation has been a constant finding in patients with adrenal cancer and a high excretion of S-PC. In the eight patients presented here, and in five additional patients studied less extensively, the excretion of tetrahydro S was above 4 mg per 24 hours and in several instances exceeded the combined excretion of THE and THF.
Pregnanediol, Pregnanetriol and Preg- nenetriol
The excretion of these urinary metabo- lites was almost invariably increased in those subjects in whom there was active corticoid synthesis by the adrenal tumor (Table 2). The titers presented here for the 3a,17a,20a-pregnanetriol are min-
imal values, since appreciable amounts of the 30- and 200-isomers have been noted. Pregnanetriol excretion was high- est in Patient 9, in whom there was severe impairment of both 116- and 21- hydroxylation. The excretion of one milligram of THS by this patient is evidence of the tumor’s inability to utilize Substance S normally, since the S-PC were low.
The excretion of pregnanediol and pregnenetriol was also well above the normal range in the patients studied. In Patient 3, pregnanediol excretion was 21 mg and pregnanetriol only 12 mg. Since the normal subject excretes small amounts of pregnanetriol at a lower level of total adrenal synthetic activity, we
| Patient | mg/24 hr | ETIO/ANDRO | ||
|---|---|---|---|---|
| DHEA | ETIO | ANDRO | ||
| 1a | 21 | 8.0 | 7.0 | 1.1 |
| 1b | 69 | 26 | 15 | 1.8 |
| 2 | 36 | 22 | 11 | 2.0 |
| 3 | 29 | 43 | 18 | 2.4 |
| 4 | 76 | 33 | 4.1 | 8.2 |
| 5 | 19 | 28 | 4.2 | 6.8 |
| 6 | 44 | 39 | 12 | 3.3 |
| 7 | 19 | 9.1 | 5.1 | 1.5 |
| 8 | 22 | 40 | 12 | 0.33 |
| 9 | 4 | 15 | 28 | 0.5 |
| 10 | 34 | 33 | 28 | 1.2 |
| Normal | 0.7 | 3.8 | 4.2 | 0.9 |
| ? | 0.5 | 1.9 | 1.8 | 1.1 |
have elected to compare its excretion with the excretion of THE plus THF plus THS in order to establish whether or not the utilization of pregnanetriol is as efficient in adrenal cancer as in the normal adrenal gland.
In column 5 of Table 2, the excretion of pregnanetriol is expressed as a fraction of the sum of THE, THF and THS, since this is a measure of corticoid production after 17a-hydroxylation. Normally this ratio is about 0.15, since most of the 17a-hydroxyprogesterone and Sub- stance S is utilized by the normal adrenal gland in the synthesis of cortisol. Four of the ratios are within the normal range, indicating that 21-hydroxylation is rarely impaired to the same degree as 11-hydroxylation. The occurrence of high ratios in some cases of adrenal can- cer demonstrates that, even though there is a greater production of cortisol, the excretion of pregnanetriol is proportion- ately greater than normal. This type of comparison would be more accurate, of course, if more of the metabolites of cortisol had been measured or if the secretion rate of cortisol were known. However, it is a reasonable first approxi- mation.
The origin of the excess pregnanetriol in the urine is not defined by these studies. Although pregnanetriol is a major metabolite of 17a-hydroxyproges- terone, it has also been shown that it can originate from 17a-hydroxypregnenolone (12, 13), although in lesser yield.
In the last column of Table 2, the excretion of THS has been expressed as a fraction of the excretion of THE plus THF to compare these ratios of utiliza- tion with those for pregnanetriol. It is readily apparent that the excretion of THS is proportionately greater than the excretion of pregnanetriol in all in- stances but one (Patient 9).
The finding that urinary pregnene-
triol, a metabolite probably arising uniquely from 17a-hydroxypregneno- lone, is also elevated suggests that the intra-adrenal utilization of this normal intermediate is poor and that some of the pregnanetriol therefore may have origi- nated from the hepatic metabolism of 17a-hydroxypregnenolone. However, the excretion of large amounts of preg- nanetriol and the fact that 17a-hydroxy- pregnenolone is metabolized to 45-preg- nenetriol to a considerably greater extent than to pregnanetriol (12) strongly sup- port the assumption that the excretion of large amounts of pregnanetriol is due to that fraction of the 17a-hydroxypro- gesterone not utilized by the cancer.
It does not seem profitable to try to compare the efficiency of utilization of progesterone and 17a-hydroxypregneno- lone by the above techniques, since the uncertainties of estimating the contribu- tion of each to the corticoids synthesized subsequently are too great. Patients 3 and 8 excreted 21 and 9 mg of pregnane- diol, respectively, strongly suggesting that 17a-hydroxylation was impaired. The elevation of the urinary pregnene- triol is difficult to interpret in view of the evidence that 17a-hydroxypregnenolone can serve as a precursor of cortisol (14) as well as dehydroepiandrosterone (15).
The 11-Oxy-17-ketosteroids
Eight of the ten patients excreted large amounts of 11-oxyketosteroids (Fig. 5). In Patients 6 and 7 the values represent the total C19-ketosteroids after chromate oxidation of the C1903 fraction from the partition column. Although 11-OHA was the largest component of this fraction, the 11-oxy-56 derivatives were likewise increased. This increased excretion is considerably in excess of what might be expected from the metab- olism of cortisol, even in view of the high cortisol production. Similar findings are
apparent in several of the patients studied by Vande Wiele et al. (16).
The excess of the 56-ketosteroids may occur because of several factors. As we have suggested above, the excretion of THE and THF does not represent as great a fraction of the secreted cortisol as in the normal subject, so that estimates of the amount of cortisol metabolized by these patients may be low. Second, if the neoplasm can carry out the oxidation of the 116-hydroxy group, the resulting 11- ketoandrostenedione may be metabo- lized to appreciable amounts of the 58- derivatives (17) in contrast with the metabolism of 113-hydroxyandrostene- dione (18). Finally, the almost invariable occurrence of intra-abdominal metas- tases and proximity to the bowel may increase the enterohepatic circulation of cortisol and its metabolism to the 56- ketosteroids by the enteric bacteria (19).
Patient 5, whose excretion of THE and THF was normal, excreted increased amounts of 110-hydroxy- and 11-keto- etiocholanolone. The most plausible ex- planation is that they were derived from the metabolism of 113-hydroxyandro- stenedione.
The most abundant of the three 11- oxyketosteroids was 118-hydroxyandro- sterone. This compound is the principal metabolite of 118-hydroxyandrostene- dione which is presumably synthesized via the “androgen” pathway in the adrenal from dehydroepiandrosterone. The fact that 116-hydroxyandrosterone was excreted in greater amounts than the 50-derivatives is evidence that most of it was derived from the metabolism of 118- hydroxyandrostenedione, since the me- tabolism of cortisol leads to a high ratio of 56-11-oxyketosteroids to 5x-11-oxy- ketosteroids.
The excretion of 4.0 mg of 116-hydroxy- androsterone by Patient 5 is of inter- est. Evidence was presented in Fig. 3
that the adrenal cancer in this case was incapable of carrying out 116-hydroxyla- tion of Substance S. The finding of an increased excretion of 116-hydroxyan- drosterone demonstrates that either identical conditions are not required for the hydroxylation of Substance S and androstenedione or separate enzymes are involved.
Androsterone, Etiocholanolone and Dehydroepiandrosterone
The levels of the above compounds are tabulated in Table 3. The excretion of DHEA was elevated in all patients, al- though the 4 mg per 24 hours excreted by Patient 9 occasionally has been noted in the normal adult. It is probably for- tuitous that DHEA was elevated in each of these patients, since normal levels of DHEA have been seen in patients with adrenal cancer (16, 20).
If secreted DHEA is normally metabo- lized to the extent of 90% or better (21), it is curious that, in adrenal cancer, a significant fraction of the DHEA is excreted unchanged. This is not due to hepatic metastases, since several of these patients either werefree of hepatic metas- tases or had only a few nodules. It has been suggested (22) that, as increasing amounts of DHEA are presented to the liver, larger amounts escape metabolism to androsterone, etiocholanolone and other products. The few data presently available do not support this hypothesis, however.
The increased excretion of etiocho- lanolone and androsterone in adrenal cancer has been noted frequently (16, 20) and the increase in the ratio of etio- cholanolone to androsterone has been commented upon (16). It has been shown (23) that some of the etiocholanolone in these patients is derived from sources other than DHEA, in contradistinction to the origin of etiocholanolone in normal
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subjects (21). The occurrence of Cush- ing’s syndrome per se may also favor the 56-reductase (24). These two findings adequately account for the increased ratio of etiocholanolone to androsterone.
Comments
The patient with functional adrenal cancer not only excretes larger amounts of most steroids than the normal subject does but excretes them in widely varying proportions as well. These larger amounts of steroids are probably simply a reflection of the increased mass of tissue engaged in steroid synthesis.
The occurrence of metabolites not normally encountered in the urine may be due to several factors: first, the extra- adrenal metabolism of large quantities of the normal steroids may yield sufficient amounts of what are generally minor metabolites to permit detection; second, the characteristically large tumor nod- ules may favor intratumoral metabolism of the steroids prior to their being pre- sented to the liver; third, the presence of Cushing’s syndrome or virilization can alter the relative activities of enzyme pairs such as the C19-5a- and 56-reduc- tases; fourth, substances that normally comprise only minor fractions of the pre- cursors of common metabolites may be important precursors in adrenal cancer.
Apart from these alterations in the urinary steroid excretion in adrenal car- cinoma, there are more specific changes resulting from decreased capacities of the neoplasm to utilize the normal inter- mediates of steroid synthesis. This may be due to such causes as lack of specific cofactors, alterations in spatial relation- ships within the cancer cell that are necessary for orderly metabolic trans- formations of the steroids, or simply a decreased amount of a specific enzyme. Whatever the basis, the result is the
excretion of disproportionately increased quantities of metabolites such as tetra- hydro Substance S. In fact, the relative inability of adrenal carcinoma to carry out 118-hydroxylation of Substance S has been so uniform that this finding provides an excellent diagnostic feature.
Other transformations within the adrenal cancer are less frequently im- paired but, on occasion, may fully ex- plain the observed steroid pattern. In Patient 9, it is apparent that there was a marked inability to perform 21-hydroxy- lation, resulting in the excretion of 16 mg of pregnanetriol daily. This patient’s tumor tissue also was probably incapable of performing 116-hydroxylation, since the excretion of THS was well above the predicted amount. The tumor tissue of Patient 3 showed a major impairment in the utilization of progesterone. In Pa- tient 10 it is probable that the 36-ol dehydrogenase was absent or inactive, since corticoids were not synthesized via either progesterone or 17a-hydroxypreg- nenolone. In this instance, only precur- sors of the C1902-17-ketosteroids could be synthesized by the tumor.
The cases just cited represent the extreme examples of the several areas in which the cancer has been shown to per- form, less efficiently, the synthetic work of the normal tissue. Further analysis of these areas may not only yield better diagnostic methods but throw further light on the alterations in metabolism that accompany neoplasia.
Finally, it is of interest that, in Patient 1, the steroid excretion was only quantita- tively altered with the development of metastatic disease. This is in contrast with a similar study (20), in which there was a striking increase in DHEA excre- tion with development of metastatic disease, suggesting qualitative changes in the pattern of either steroid synthesis or metabolism.
Acknowledgments
We are greatly indebted to Mrs. Barbara Riter and to Mr. David Ryan for their im- portant technical contributions to this study. We thank Dr. Roy Hertz for his suggestions and criticisms and Dr. Michael Finkelstein for per- mission to quote several of the pregnanetriol values.
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