Urinary Estrogen Titers in a Patient with Feminizing Adrenocortical Carcinoma
H. H. WOTIZ, S. C. CHATTORAJ, AND J. L. GABRILOVE
Department of Biochemistry, Boston University School of Medicine, Boston, Massachusetts, and the Endo- crine Research Laboratory of the Department of Medicine, The Mount Sinai Hospital, New York, New York 10029
ABSTRACT. The urinary estrogens in a patient with a feminizing adrenocortical tumor were partitioned and measured by gas chromatog- raphy. Marked elevations in the urinary titers, particularly of estriol, were observed. In addi- tion to increased quantities of estriol, estradiol
and estrone, high titers of 2-methoxyestrone, 16-hydroxyestrone, 16-ketoestradiol and 16- epiestriol were encountered. The significance of the urinary titers of estrogen in regard to the prognosis of feminizing adrenocortical tumors is discussed. (J Clin Endocr 28: 192, 1968)
F EMINIZING adrenocortical tumors in the male are rare, only 51 such instances having been reported previously. In the majority of the previously published instances the total urinary estrogens were determined either by relatively crude chemical procedures or by bioassay and only two investigators have reported find- ings of more than the three classic estro- gens (estrone, estradiol and estriol) in the urine of such patients. This investigation is concerned with a gas chromatographic study of the urinary estrogens in a patient with this disorder. In addition, the urinary excretion of estrogen in all of the previously reported cases is reviewed.
Case History
A 53-yr-old man was admitted to the Mount Sinai Hospital in February 1962 with a 3-yr history of loss of libido, impotence and gyneco- mastia and a 3-month history of edema. The pertinent findings on physical examination were plethora, a cervical dorsal fat pad, bi- lateral gynecomastia, pitting edema of the lower extremities and small, soft testes. A right adrenocortical tumor was demonstrable on retrorectal gas insufflation. The urinary excretion of the neutral 17-ketosteroids was
Received August 16, 1967; accepted October 6. This investigation was supported by research grants from the NIH (CA-03135) and a Research Career Development Fellowship (GM-3,15,369) from the USPHS.
45.2 mg/24 hr, of the 17-hydroxycorticoids (Porter-Silber) 23.6 mg/24 hr.
Following removal of the tumor the edema disappeared, but the gynecomastia persisted in spite of the fact that he felt markedly im- proved. Libido and potency returned. However, about 1 yr later he noted recurrent pain in the region of the operative scar, increasing gyneco- mastia and decreased libido. On exploratory laparotomy 1} yr after the original operation, a large infiltrating recurrent tumor was demon- strable (6).
Materials and Methods
For the determination of the urinary estro- gens a slight modification of the procedure de- scribed by Wotiz and Chattoraj (1) was used. An aliquot of the urine was treated with 500 U of ß-glucuronidase and 250 U of sulfatase (glusulase) per ml of urine for a period of 48 hr at 37 C. Following this, the steroids were ex- tracted, washed and separated into 4 fractions by thin-layer chromatography (TLC) as de- scribed previously. Gas chromatography on SE-30 elastomer (3.8%) allowed separation and measurement of estrone, 2-methoxyestrone (2- methoxy-3-hydroxy-41,3,5(10)-estratriene-17-one), estradiol, estriol and 16-epiestriol (41,3,5(10)- estratriene-3,166,173-triol) as the acetates as well as the trimethylsilyl ethers (TMSi). The 2 ring D a-ketols were separated and quantified by application of the proper TLC fraction, after acetylation or TMSi formation, to a 3 foot column packed with a mixed phase of 1% ethylene glycol adipate (stabilized)1 and 2%
1 Analabs, Inc., Hamden, Conn.
XE-60 nitrile silicone or on a 6 foot QF-1 (3%) column.
Quantitation was carried out by peak height measurement.
Results
Evidence for the specificity of the pro- cedure as originally applied to pregnancy urines has been presented previously. This was based on the number of separation steps involved, as well as application of physical methods of characterization (countercurrent distribution, infrared and fluorescence spectroscopy) to fractions of the extract trapped following gas chroma- tography.
In view of the different origin of the extracted material analyzed here, addi- tional evidence for specificity for the indi- vidual fractions was obtained by peak augmentation methods and comparison of retention times on three different columns. In no instance did peak augmentation indicate an alteration of peak symmetry, peak broadness, or show the appearance of a shoulder. Retention times of each of the estrogens analyzed, when compared to authentic standards, showed variation of 2% or less.
Although the likelihood that substances not identical with the estrogens indicated should have partition coefficients identical with these steroids on three different columns is remote, further work was carried out to ascertain the specificity of the assays reported here. Verification was obtained by a comparison of the quanti- tative response of each of the fractions following GLC on the nonpolar (SE-30), polar (QF-1) and selective (XE-60) col- umns when two derivatives of differing polarity were applied.
Of the values determined, acceptability was restricted to those where at least three out of six (or 2 out of 4 for the a-ketols) determinations did not differ more than ±7.5% (the limit of methodologic error for multiple sample handling) and no value obtained was less than the lowest of the three.
The individual values obtained for each fraction are recorded in Table 1. It is clear that, even where only three out of six values fall within the ±7.5% limit, the remaining values obtained are not very much greater than the lowest three or more, indicating that relatively few major impurities are carried through the pro- cedure. Notable exceptions to this are samples 2B, 6B, 7B, 11C and 9E. Only two pieces of data were rejected because of non- adherence to the above criteria. For example 5A, only two values out of six showed a variation of 15%. Nevertheless, four out of six values had a variation of ±20% at a level of about 10 µg/24 hr of estrone and no value lower than 9 ug was obtained. Similarly, sample 2D gave two values out of four with a variance of ±25%, with no lower values. In a signifi- cant number of analyses (23 out of 77), particularly when excretion levels were relatively high, all the data obtained were within the accepted variation.
The retention times of the substances analyzed were highly reproducible (±2%) and were within these limits when com- pared to authentic standards.
In view of the fact that such tumors might produce 11-oxygenated estrogens or equilenin (2), the retention times for au- thentic equilenin, 11-ketoestradiol (3,170- dihydroxy-41,3,5(10)-estratrien-11-one) and 116-hydroxyestrone (3,116-dihydroxy- 41,3,5(10)_estratrien-17-one) were deter- mined. None of the urinary fractions produced any gas chromatographic peaks which might reasonably be construed to be identical with these standards. Con- centrations of approximately 1 ug/24 hr would have been detected.
The mean values of 24-hour urinary excretion levels as determined by multiple GLC of estrone, 2-methoxyestrone, estra- diol, 16a-hydroxyestrone (3,16a-dihydroxy- 41,3,5(10)-estratrien-17-one), 16-ketoestradiol (3,170-dihydroxy-41,3,5(10)-estratrien-16-one), estriol and 16-epiestriol in the patient with a feminizing adrenal carcinoma reported
| No. | De- riv.t | A Estrone | 2-Methoxyestrone B | Estradiol C | ||||||
|---|---|---|---|---|---|---|---|---|---|---|
| S | Q | X | S | Q | X | S | Q | X | ||
| 1 | A | 183 | 164 | 172 | 80 | 69 | 103 | 27 | 15 | 13 |
| T | 188 | 169 | 165 | 74 | 69 | 92 | - | 11 | 12 | |
| 2 | A | 20 | 12 | 18 | 20 | 24 | 67 | 36 | 38 | 30 |
| T | 21 | 11 | 10 | 18 | 21 | 34 | 39 | 26 | - | |
| 3 | A | - | 1 | - | 110 | 86 | 143 | 101 | 84 | 83 |
| T | 2 | - | - | 90 | 79 | 103 | 91 | 79 | 86 | |
| 4 | A | 12 | 9 | 9 | 49 | 54 | 82 | 71 | 54 | 61 |
| T | 11 | 10 | 8 | 52 | 46 | 59 | 74 | 60 | 54 | |
| 5 | A | 15 | 9 | 12 | 42 | 36 | 74 | 87 | 84 | 79 |
| T | 21 | 13 | 10 | 39 | 37 | 94 | - | 89 | 80 | |
| 6 | A | 69 | 62 | 67 | 110 | 96 | 164 | 110 | 103 | 96 |
| T | 81 | 59 | 56 | 101 | 89 | - | 92 | 100 | - | |
| 7 | A | 98 | 87 | 86 | 246 | 162 | 204 | 96 | 89 | 86 |
| T | - | 84 | 87 | 186 | 172 | - | 99 | 90 | 94 | |
| 8 | A | 60 | 58 | 63 | 136 | 144 | - | 27 | 36 | 24 |
| T | 69 | 58 | 61 | 152 | - | 162 | 29 | 32 | - | |
| 9 | A | 46 | 40 | - | 128 | 162 | 149 | 51 | 51 | 44 |
| T | 49 | 38 | - | 119 | 121 | 131 | - | 47 | 50 | |
| 10 | A | 43 | 45 | 38 | 128 | 117 | 169 | |||
| T | 40 | 49 | - | 132 | - | 146 | lost | |||
| 11 | A | 101 | 79 | 85 | 124 | 129 | 141 | 66 | 47 | 53 |
| T | 91 | 74 | 79 | 136 | 144 | 162 | 75 | 51 | 46 | |
- = Trace amounts.
Underscored values represent the lowest amount for each group of analysis within the acceptable limit of error ( ±7.5%).
Columns: S =SE-30; Q=QF-1; XE =XE-60; X/NGS = XE-60/neopentylglycol sebacate. t Derivatives: A =acetate; T = trimethylsilyl ether.
here are recorded in Table 2. A series of urines covering a period of almost two years were analyzed. The first specimen
was obtained just prior to the first surgical intervention. The second specimen, col- lected soon after adrenal resection, reflects
GLC columns as two derivatives (ug/24 hr)
| D 16-Hydroxy- estrone | E 16-Hydroxy- estradiol | F Estriol | G 16-Epiestriol | ||||||
|---|---|---|---|---|---|---|---|---|---|
| X/NGS | Q | X/NGS | Q | S | Q | X | S | Q | X |
| 845 | 821 | 775 | 742 | 2891 | 2901 | 2842 | - | - | - |
| 859 | 836 | 761 | 757 | 2903 | 2865 | 2872 | - | - | - |
| 13 | 6 | - | - | 132 | 115 | 142 | - | - | - |
| 18 | 9 | - | - | 129 | 117 | - | - | - | - |
| 130 | 113 | - | - | 764 | 749 | 771 | 88 | 69 | 98 |
| 142 | 126 | - | - | 758 | 746 | 760 | 74 | 79 | 103 |
| 111 | 128 | 15 | 16 | 365 | 359 | 341 | - | 2 | - |
| 130 | 119 | 14 | 20 | - | 351 | 349 | 12 | - | 2 |
| 78 | 84 | 2 | - | 796 | 784 | 791 | - | - - | - |
| 93 | 79 | - | - | 773 | 781 | - | 4 | - | 2 |
| 369 | 402 | 35 | 29 · | 307 | 315 | 331 | - | - | - |
| 383 | 370 | 27 | 29 | 298 | 305 | - | - | - | - |
| 312 | 290 | - | - | 1383 | 1361 | 1342 | 394 | 346 | 367 |
| 319 | - | - | - | 1391 | 1400 | 1422 | 329 | 350 | - |
| 201 | 212 | 125 | 103 | 1464 | 1403 | 1492 | 488 | 470 | 498 |
| 223 | 206 | 117 | 108 | 1469 | 1472 | 1503 | 459 | 439 | - |
| 212 | 212 | 540 | 342 | 1293 | 1224 | 1243 | |||
| 243 | 229 | 327 | 338 | - | 1215 | 1286 | -lost- | ||
| 218 | 214 | 101 | 112 | 1678 | 1603 | 1645 | |||
| 209 | 240 | 98 | 99 | 1586 | 1627 | - | -lost- | ||
| 198 | 203 | . 124 | 129 | 2704 | 2812 | 2746 | 244 | 201 | 214 |
| - | 211 | 120 | 126 | 2792 | 2786 | 2779 | 226 | 207 | 292 |
this procedure by the marked drop in total estrogens. Estradiol-178 was the only fraction which was not decreased post-
operatively. It should be noted, however, that, while the preoperative specimen con- tained over 4700 ug of estrogens and the
postoperative one only 188 ug, the latter still represents over ten times the normal value for males.
In general, it appears that quantitatively the most significant single estrogen is estriol. Although all the metabolites are elevated, only estrone, the a-ketols and estriol are present in the urine in extreme quantities. After surgery all of these sub- stances show a gradual though erratic in- crease to high levels but after a few months most tended to level off. 16-Epiestriol, nearly undetected in the early stages, showed very high levels in some specimens. Again, the most significant increase was in estriol, which kept increasing in quantity postoperatively.
Only urine specimens from the cases reported by Slaunwhite et al. (4) and Jayle et al. (5) were analyzed for 16-epiestriol and a-ketols. Both groups reported fairly high values for estrone (between 20 and 80% of total), whereas in our patient only about 4% of the total urinary estrogen was estrone. In contrast, in the case reported here estriol represents over 60%, while in these two earlier cases this fraction repre- sents about 15% of the total estrogen con- tent of the urine.
Discussion
A review (6) of these cases shows that in only 31 of the 51 previously reported cases with feminizing adrenocortical tumors were estrogen assays carried out. In 11, the procedure was bioassay; in 14, chemical; and in six, both types of determinations were employed.
Estrogen bioassays are, therefore, avail- able for 17 patients. In all except three the urinary titers were markedly elevated. The urinary excretion of estrogens was normal in one patient with carcinoma and in one with a benign tumor. In a third patient, with a benign tumor, reported by Wilkins (7), the urinary excretion of estrogen was only minimally increased.
Preoperative chemical estimations of
urinary estrogen are available in 18 sub- jects apart from our patient. In addition, in two other patients values for urinary estrogens were obtained only postoper- atively. Most of the assays were carried out by the Brown method (8) or by the “phenolsteroid” assay (5). However, several other chemical methods have been employed in individual instances (6). In all the subjects studied increased titers were encountered. In addition, in 13 patients partition studies of the estriol, estradiol and estrone fractions were per- formed by various methods. Although all fractions participated in the increased urinary excretion of estrogen, a greater proportional increase was noted in the estrone and estriol fractions rather than in estradiol. Estriol was the fraction most strikingly increased in approximately 2/3 of the subjects, whereas in the remainder estrone was the major estrogen. According to Brown (9), in normal men the mean daily excretion of estrone is 5.4, estriol 3.5 and estradiol 1.5 µg/24 hr.
Following removal of the tumor the urinary excretion of estrogen usually falls, as it did in our patient. A failure of the titer to fall or the recurrence of elevated titers is indicative of the presence of functioning tumor tissue and, therefore, an indication that the tumor is a carci- noma and that either part of the tumor remains or functioning metastasis is present. A postoperative fall in titer to normal levels does not in itself indicate the disease is due to a benign tumor or that recurrence will not occur. In general, the more modest the preoperative urinary titer of estrogen in patients with feminiz- ing adrenocortical tumors the more likely is the tumor to be benign. Very high titers suggest the tumor is malignant. Postoperative recurrence of elevated titers or failure of the urinary excretion of estrogen to return to normal postoper- atively should suggest that the tumor is really malignant even though the histo- logic diagnosis is adenoma (6).
| No. | Date | A Estrone | Methoxy- B 2- estrone | C Es- tradiol | D 16- Hydroxy- estrone | Hydroxy- E 16- estradiol | F Estriol | G 16-Epi- estriol | Total estrogens |
|---|---|---|---|---|---|---|---|---|---|
| 1 | 2-26-62 | 172 | 71 | 12 | 840 | 759 | 2879 | - | 4733 |
| 2-28-62 | surgical removal of tumor | ||||||||
| 2 | 3-17-62 | 11 | 20 | 28 | * | - | 123 | - | 188 |
| 3 | 7- 3-62 | - | 85 | 85 | 123 | - | 758 | 74 | 1125 |
| 4 | 1-26-63 | 9 | 49 | 57 | 119 | 15 | 353 | - | 602 |
| 5 | 4-26-63 | * | 39 | 84 | 80 | 785 | - | 998 | |
| 6 | 8-13-63 | 59 | 95 | 98 | 381 | 29 | 311 | - | 968 |
| 7 | 8-14-63 | 86 | 173 | 92 | 307 | - | 1383 | 348 | 2389 |
| 8 | 8-15-63 | 60 | 144 | 27 | 211 | 109 | 1467 | 470 | 2488 |
| 9 | 8-28-63 | 39 | 125 | 48 | 224 | 336 | 1252 | -lost- | 2024 |
| 10 | 8-30-63 | 40 | 126 | -lost- | 220 | 103 | 1628 | -lost- | 2117 |
| 11 | 10-19-63 | 79 | 133 | 49 | 204 | 125 | 2770 | 212 | 3573 |
| 10-30-63 | Laparotomy-recurrent tumor demonstrated | ||||||||
- = Trace amounts.
* Insufficient checking of values.
References
1. Wotiz, H. H., and S. C. Chattoraj, Anal Chem 36: 1466, 1964.
2. Wotiz, H. H., In Paulsen, C. A. (ed.), Estrogen Assays in Clinical Medicine, Univ. of Washing- ton Press, 1965.
3. Salhanick, H. A., and D. L. Berliner, J Biol Chem 227: 583, 1957.
4. Slaunwhite, W. R., Jr., and K. W. Buchwald, J Clin Endocr 20: 786, 1960.
5. Jayle, M. F., J. Decourt, J. P. Michard, E. E. Baulieu, and R. Scholler, Ann Endocr (Paris) 22: 798, 1961.
6. Gabrilove, J. L., D. C. Sharma, H. H. Wotiz, and R. I. Dorfman, Medicine (Balt) 44: 37, 1965.
7. Wilkins, L., J Clin Endocr 8: 111, 1948.
8. Brown, J. B., Biochem J 3: 1, 1955.
9. Mem Soc Endocr 3: 1, 1955.