The Journal of Clinical Endocrinology and Metabolism
VOLUME 22
AUGUST, 1962
Copyright 1962 by The Endocrine Society
NUMBER 8
Origin of Pregnanetriol in a Patient with Adrenal Carcinoma
DAVID K. FUKUSHIMA, PH.D., H. LEON BRADLOW, PH.D., LEON HELLMAN, M.D. AND T. F. GALLAGHER, PH.D.
Division of Steroid Metabolism and Biochemistry, Sloan-Kettering Institute for Cancer Research, New York, N. Y.
T HE EXCRETION of pregnane-3a, 17,20a-triol may be elevated with some types of adrenocortical hyperfunc- tion, often accompanied by increased amounts of androsterone’ and etiocho-
Received February 23, 1962.
This investigation was supported in part by a grant from the American Cancer Society and a research grant (CY-3207) from the National Cancer Institute of the National Institutes of Health, USPHS.
1 The following trivial names and abbrevia- tions are used in this paper: androsterone, A =3a-hydroxyandrostane-17-one; etiocholano- lone, E=3a-hydroxyetiocholane-17-one; 17- hydroxyprogesterone =17-hydroxy-44-pregnene- 3,20-dione; 17-hydroxypregnanolone = 3a,17-di- hydroxypregnane-20-one; 11-ketoetiocholano- lone, 11 =OE =3a-hydroxyetiocholane-11,17- dione; 11-hydroxyetiocholanolone, OH-E =3a, 118-dihydroxyetiocholane-17-one; 11-hydroxy- androsterone, OH-A =3a,118-dihydroxyandro- stane-17-one; Reichstein’s Substance S =17, 21-dihydroxy-44-pregnene-3,20-dione; hydro- cortisone = 118,17,21-trihydroxy-44-pregnene- 3, 20-dione; THF =3a,118,17,21-tetrahydroxy- pregnane-20-one; ATHF =3a,118,17,21-tetra- hydroxyallopregnane-20-one; THE =3a,17,21- tetrahydroxypregnane-11,20-dione; THS =3a,17, 21-trihydroxypregnane-20-one; THB =3a,11}, 21-trihydroxypregnane-20-one; THA =3a,21-di- hydroxypregnane-11,20-dione; D =36-hydroxy- 45-androstene-17-one; 8-E =36-hydroxyetio- cholane-17-one.
ABSTRACT. The metabolism of 17-hydroxy- progesterone-4-C14 was studied in a patient with metastatic adrenal carcinoma. The daily urine excretion of 119 mg of pregnane- 3a,17,20a-triol by this patient was found to be derived principally from precursors other than 17-hydroxyprogesterone, such as 17- hydroxy-45-pregnenolone or its 20-keto re- duction products. It was estimated that glandular secretion of 17-hydroxy-45-preg- nenolone was between 154 mg and 2 g, with the true value nearer the latter figure. Evidence also was obtained for the hydroxylation of 17-hydroxyprogesterone-4-C14 at C-21 by the isolation of radioactive pregnane-3a,118,20,21- tetrols. The daily production of the urinary steroids is reported.
lanolone (1-9). The peripheral metab- olism of 17-hydroxyprogesterone-4-C14 leads to pregnane-3a,17,20a-triol and 3a,17-dihydroxypregnane-20-one as the major metabolites (10, 11). There is only limited cleavage of the side chain to form androsterone and etiocholanolone, but with elevated production this trans- formation contributed materially to these C19 metabolites (10). The present study was undertaken in a patient with widely disseminated metastatic adrenal carcinoma to examine this pathway and
| Day | Neutral steroid extracts Urine Enzyme hydrolyzed Acid hydrolyzed Total | |||||||
|---|---|---|---|---|---|---|---|---|
| 103 cpm | % of dose | 103 cpm | % of urine | 103 cpm | % of urine | 103 cpm | % of urine | |
| 1 | 520 | 33.9 | 416 | 80 | 58 | 11 | 474 | 91 |
| 2 | 136 | 8.9 | 89 | 65 | 22 | 16 | 111 | 82 |
| 3 | 50 | 3.3 | 16 | 32 | 13 | 26 | 29 | 58 |
| 4 | 36 | 2.3 | 9 | 25 | 9 | 25 | 18 | 50 |
| Total | 742 | 48.4 | 530 | 71 | 102 | 14 | 632 | 85 |
to assess the extent to which other precursors supplied metabolites in com- mon with 17-hydroxyprogesterone. This patient excreted very large amounts of steroids so that it was possible to study a number of metabolites. In particular, further hydroxylation of the exogen- ously administered 17-hydroxypro- gesterone was examined.
Methods and Materials
The 17-hydroxyprogesterone-4-C14 used was at least 96% pure, as judged by chroma- tography on paper in System D. A 38-yr-old man with metastatic adrenal carcinoma re- ceived approximately 0.05 mg of 17-hydroxy- progesterone-4-C14, specific activity 32 uc/ mg, in 1 ml of propylene glycol injected into the polyethylene tubing leading from a 5% glucose solution into the cubital vein. The syringe and needle containing the radioactive solution were weighed before and after in- jection for measurement of the amount actually delivered. Urine was collected for 4 days following administration of the hor- mone and the radioactivity present was determined (Table 1) by methods previously reported (12). Neutral fractions from ß- glucuronidase2 hydrolysis were prepared by the methods employed in these laboratories (13). The extracted urine and the aqueous washes were combined, acidified to IN with 50% sulfuric acid and continuously ex-
2 B-Glucuronidase, known as Ketodase, was obtained from Warner-Chilcott Laboratories, a division of Warner-Lambert Pharmaceutical Company, New York, N. Y.
tracted with ether for 48 hours. The neutral extract was prepared as before. A portion representing 45% of the combined enzyme and acid-treated extracts (CE) from the 4 days’ urine collection was separated by Girard’s reagent T into a ketonic (K-CE) and a nonketonic fraction (NK-CE) (Fig. 1). The K-CE fraction was separated into “a” and ”(” steroids by precipitation with digitonin (13). Ten per cent of the NK-CE fraction also was separated by digitonin into “a” and """ steroids for measurement of pregnane-3a,17,20a-triol and 45-pregnene- 36,17,20a-triol by the method of Cox (2) after separation by chromatography on paper (18 ×118 cm) in System B for 28 hours. The following quantitative analyses were em- ployed: 17-ketosteroids by the micro Zim- mermann reaction (14); 17-hydroxypreg- nanolone by the enzymatic procedure of Hurlock and Talalay (15) using the “a” steroid dehydrogenease from Pseudomonas testosteroni; reducing steroids by the method of Weichselbaum, Margraf and Mack (16). The values reported were corrected to mg equivalents of the individual compounds analyzed. The separation and estimation of the quantities of endogenous steroid metabo- lites have been described (7).
Solvent systems for paper chromatography:
A. Toluene-propylene glycol.
B. 2,2,4-Trimethylpentane: toluene (3:5) methanol : water (4:1).
C. 2,2,4-Trimethylpentane : toluene (1:3) methanol: water (4:1).
D. 2,2,4-Trimethylpentane: toluene (3:1) methanol : water (3:1).
E. Benzene : methanol : water :ethyl ace- tate-1:1:1:0.1.
August, 1962 ORIGIN OF PREGNANETRIOL IN ADRENAL CARCINOMA 767
Results
Ketonic Steroids
An 80% portion of @K-CE, 78,000 cpm, was chromatographed on 40 g of silica gel containing 16 ml of ethanol at a rate of 10 ml per 30 minutes. Elution with 1% ethanol in methylene chloride- petroleum ether (3:7) afforded 11 mg of androsterone and 31 mg of etiocho- lanolone. Elution with 1% ethanol in methylene chloride yielded fractions con- taining predominantly 11-ketoetiochol- anolone (9,000 cpm), 17-hydroxypreg- nanolone (18,000 cpm), 11-hydroxy- androsterone (6,000 cpm) and 11-hy- droxyetiocholanolone (8,000 cpm), as judged by infrared spectrometry. No other known steroids were eluted with a higher concentration of ethanol.
The androsterone and etiocholanolone fractions were recrystallized to constant specific activity, 76 and 155 cpm per mg, and mp 182-182.5 and 150 C, respec- tively (Table 2).
The fractions containing steroids with three atoms of oxygen proved to be complex mixtures. Each of these frac- tions was chromatographed on What- man No. 1 papers (18×55 cm) in Sys- tem A for 16 hours. The areas containing 17-hydroxypregnanolone were eluted and combined but could not be crystal- lized. The fraction was acetylated, chromatographed on alumina and eluted with benzene. The acetate thus obtained was an oil and was reduced with sodium
borohydride and sodium hydroxide for 18 hours at room temperature. The re- sulting pregnane-3x,17,200-triol was chromatographed on Whatman No. 1 paper in System C for five hours. Elu- tion afforded 1,900 cpm and 261 ug of pregnane-3a,17,200-triol by the method of Cox (2). The specific activity of 17- hydroxypregnanolone thus characterized was 7,300 cpm per mg.
This high specific activity was con- firmed by the isolation of 3a,17aß-dihy- droxy-17ax-methyl-D-homoetiocholane- 17-one, a rearrangement product of 17- hydroxypregnanolone when the latter is subjected to prolonged chromatography (17). The effluents from System A were rechromatographed in System D for 24 hours and afforded 2,060 cpm in 270 µg of the D-homosteroid, as measured by the macro Zimmermann method (17). This represented a specific activity of 7,350 cpm per mg for 17-hydroxypreg- nanolone, from which it was derived.
Further confirmation of the amount of radioactivity present in 17-hydroxy- pregnanolone was obtained by reverse isotopic dilution analysis. For this pur- pose 9% of the combined unfractionated neutral steroid extracts of days 1-4 was added to a solution of 50.1 mg of carrier 17-hydroxypregnanolone. After chroma- tography and recrystallization of the free steroid, the compound was acetyl- ated and the acetate was further re- crystallized. At constant specific ac- tivity the product had 410 cpm per mg (as the free steroid). Therefore the neu-
| Constant spe- cific activity cpm/mg | Total activity* 103 cpm | % of neutral extract | % of dose | |
|---|---|---|---|---|
| Androsterone | 76 | 3.1 | 0.5 | 0.2 |
| Etiocholanolone | 155 | 24.7 | 3.9 | 1.6 |
| 17-Hydroxypregnanolone | 7,300 | 254 | 40.2 | 16.5 |
| Pregnane-3a,17,20a-triol | 342 | 163 | 25.8 | 10.6 |
* Calculated from specific activity and endogenous production.
| C21 reducing steroids | 17-ketosteroids | ||
|---|---|---|---|
| Compound mg/24 hr | Compound | mg/24 hr | |
| THE | 23.6 | A | 10.4 |
| THF | 12.9 | E | 42.1 |
| ATHF | 2.1 | OH-A | 2.7 |
| THS | 39.0 | OH-E | 3.7 |
| THB | 1.8 | 11=OE | 4.0 |
| THA | 6.1 | D | 48.4 |
| 6-E | 1.4 | ||
| Compound | mg/24 hr |
|---|---|
| Pregnane-3a,20a-diol | 12* |
| Pregnane-3a,17,20a-triol | 119 |
| Pregnane-3a,17,208-triol | 1* |
| 45-Pregnene-36,17,20a-triol | 40 |
| 45-Pregnene-38,17,208-triol | 3 |
| 45-Pregnene-38,16a,20a-triol | 1* |
| 45-Androstene-36,16a,178-triol | 4* |
| Etiocholane-3a, 16a,178-triol | 6* |
| 3a,17-Dihydroxypregnane-20-one | 8.7 |
| Pregnane-3a,118,17,20a-tetrol | 1.3 |
| Pregnane-3x,118,17,208-tetrol | 0.6 |
| 11-Ketopregnane-3a,17,20a-triol | <0.3 |
| Pregnane-3a,17,20a,21-tetrol | 12.1 |
| Pregnane-3a,17,20,21-tetrol | 5.8 |
| Pregnane-3x,113,17,20x +3,21- pentols | 3.4 |
| 11-Ketopregnane-3a,17,20a +8,21- tetrols | 10.1 |
* Semiquantitative estimates based on amount isolated in pure form.
tral steroid fraction of the total collec- tion contained 242,000 cpm as 17-hy- droxypregn -molone. This was 38.3% of the total radioactivity and a calculated specific activity of 7,000 cpm per mg, in agreement with the other estimates.
The 11-oxygenated 17-ketosteroids, 3a,113-dihydroxyetiocholane-17-one and 3a-hydroxyetiocholane-11,17-dione, proved very difficult to purify and failed to crystallize despite repeated efforts. Al- though the oily materials appeared to contain small amounts of radioactivity, there was insufficient material to estab-
lish whether this was in the com- pounds under examination or in asso- ciated impurities.
Nonketonic Steroids
Ninety per cent of NK-CE, 1.31 ×105 cpm, was chromatographed on 160 g silica gel containing 64 ml of ethanol. Dihydroxysteroids were eluted with 4% ethanol in methylene chloride, 12,600 cpm, from which 23 mg of pregnane-3a, 20a-diol was obtained; it was charac- terized as the diacetate, mp 164.5- 165 C, polymorph of the usually melting form, mp 178.5-179.5 C. It was devoid of radioactivity. The trihydroxysteroids were eluted with 5% ethanol in methy- lene chloride, 75,000 cpm. Recrystal- lization of fractions containing preg- nane-3,17,20a-triol from methanol- ethyl acetate gave 45 mg, mp 251-251.5 C, with constant specific activity, 342 cpm per mg. Very polar steroids, 148 mg, 11,200 cpm, eluted with 6 and 7% ethanol in methylene chloride were combined and treated with digitonin.
The mother liquors from the recrystal- lization of pregnanetriol and the re- mainder of the chromatographic frac- tions containing mixtures of trihydroxy- steroids were combined and separated by precipitation with digitonin. The ” fraction (430 mg, 56,700 cpm) was acetylated and rechromatographed on silica gel; pregnane-3a,17,20a-triol 3,20- diacetate was the only known steroid isolated. The “B” fraction, 86 mg, 910 cpm, was acetylated and chromato- graphed on 40 g of silica gel containing 20 ml of tert-butyl alcohol. 45-Pregnene- 36,17,20a-triol diacetate (62 mg) was eluted and recrystallized, mp 201-201.5 C; the compound was devoid of radio- activity. A small amount (2 mg) of 45-pregnene-36,16x,20a-triol triacetate was obtained and identified by its in-
Neutral Steroid Extract (CE) 2.85 ×105 cpm
Girard’s reagent T
Ketonic
Nonketonic
K --- CE 1.21 ×105 cpm
NK-CE 1.46 ×105 cpm
Digitonin
Digitonin on 10% Precipitate
Soluble
Precipitate
Soluble
@K CE 9.80×104 cpm @K-CE 8.50X103 cpm @NK-CE 1.37X104 cpm @NK-CE 310 cpm
frared spectrum in carbon disulfide solution.
The ”(” fraction obtained by the digitonin precipitation of the very polar fractions of the above NK-CE chroma- togram was acetylated and chromato- graphed on alumina. Elution with pe- troleum ether-benzene (1:1) yielded 11 mg of 45-androstene-38,16a,173-triol tri- acetate, mp 182-184 C; the infrared spectrum in the CS2 solution was iden- tical with that of the reference steroid. The “a” fraction, 10,000 cpm, was acetylated and chromatographed on 40 g of silica gel containing 20 ml of tert- butyl alcohol. Elution with 1% tert- butyl alcohol in methylene chloride yielded 13 mg of oil (390 cpm) contain- ing etiocholane-3x,16a,173-triol triace- tate, as judged by infrared spectrometry. Neither the acetate nor the free triol obtained from it could be crystallized. Further elution with 2% tert-butyl alcohol in methylene chloride afforded fractions (65 mg, 6,800 cpm) containing a mixture of the 20 epimers of pregnane- 3a,17,20,21-tetrol 3,20,21-triacetate. The mixture was saponified and chromato- graphed on Whatman No. 1 paper (18 X118 cm) in System E for 18 hours at 23 C. Recrystallization of the steroid iso- lated from the pregnane-3x,17,20a,21- tetrol area gave 1.4 mg, mp 254-256 C,
with a specific activity of 265 cpm per mg; a double mp, 251-253.5 and 257-258 C, has been reported for this compound (7). The specific activity was unchanged after acetylation and crystallization of the triacetate. The area corresponding to pregnane-3,17,206,21-tetrol had 925 cpm, but the steroid could not be crys- tallized. The product was acetylated and chromatographed on alumina to give 3.4 mg of pregnane-3x,17,203,21-tetrol 3,20,21-triacetate (180 cpm per mg), which had an infrared spectrum identical with that of the reference sample in CS2. The triacetate could not be crystallized. The specific activity for pregnane- 3a,17,203,21-tetrol was 246 cpm per mg, corrected to the free steroid. It is uncer- tain that strict radiochemical homo- geneity of this compound was achieved, but it is highly probable that the com- pound contained C14.
Discussion
This investigation of 17-hydroxypro- gesterone in vivo in adrenocortical car- cinoma shows that at least two, and perhaps more, precursors contributed to pregnane-3a,17,20a-triol, the major uri- nary metabolite. This is evident from comparison of the specific activity of 17- hydroxypregnanolone and pregnane-3x, 17,20a-triol, with the former more than
20 times greater than the latter. The radioactive tracer, in contrast, was transformed to these same two metabo- lites in approximately the proportion of 17-hydroxypregnanolone : pregnane- 3a,17,20a-triol of 1.5:1. At least one cther, and probably major, precursor of pregnane-3a,17,20a-triol was 36,17-di- hydroxy-45-pregnene-20-one (6, 18), since 40 mg per day of its principal metabolite, 45-pregnene-33,17,20a-triol, were found in the urine extract.
It is difficult to suggest effective pre- ursors of pregnane-3a,17,20a-triol other than these two known adrenal secretory products, except perhaps the glycols from bio-reduction of the C-20 carbonyl function. It is therefore of interest to consider the relative contribution of 17- hydroxyprogesterone and 36,17-dihy- droxy-45-pregnene-20-one to both 17- hydroxypregnanolone and pregnane-3a, 17,20a-triol. If we assume (contrary to fact, but in order to obtain a maximal estimate) that 17-hydroxypregnanolone was derived solely from 17-hydroxypro- gesterone, the secretion rate of the pre- cursor can be calculated from the specific activity of the metabolite, the counts in- jected and the term of study (19). The value obtained is 52 mg per day. From the tracer study of 17-hydroxypro- gesterone, 52 mg of that compound in this patient would have been converted to an average daily production of 5.5 mg of pregnane-3a,17,20x-triol (i.e., 10.6% of the amount administered). This amount was the maximum that could have been derived from 17-hydroxyprogesterone; as a consequence, at least 114 mg of the daily urinary pregnane-3a,17,20a-triol must have come from some other pre- cursor, in all probability from 36,17- dihydroxy-45-pregnene-20-one or the de- rived 20a-hydroxysteroid. The minimal amount secreted by the metastatic car- cinoma was 154 mg per day (114 mg calculated to be converted to pregnane-
33a,17,20a-triol plus 40 mg accounted for as 45-pregnene-33,17,20x-triol). The true secretory rate was probably much greater.
In two other studies (6; and unpub- lished results from this laboratory) in which labeled 36,17-dihydroxy-45-preg- nene-20-one was administered by vein and pregnane-3x,17,20a-triol was meas- ured, the metabolite represented 5.1% of the amount given in one study and 6.4% in the other. Fotherby and Love (18) studied five patients given non- isotopic36,17-dihydroxy-45-pregnene-20- one in amounts varying from 25 to 47.5 mg in multiple doses intravenously. They measured from 2 to 12% of the dose as pregnane-3x,17,20a-triol in urine, with an average recovery of about 7%. If we assume conversion occurred in the carcinoma patient to the extent reported in these studies, 114 mg of urinary preg- nane-3a,17,20a-triol then represented 1.8 to 2.0 grams of glandular precursor. The true secretory value is probably in the range between the lowest estimate of 154 mg and the higher value of 2 g; a more precise estimate of the true secretory rate is precluded from the nature of the study. It is suggested that the biochemical reactions leading from adrenal carcinoma production of 36,17- dihydroxy-45-pregnene-20-one to preg- nane-3a,17,20a-triol were more efficient in this patient than might have been anticipated from the other studies re- ported.
The results of this study in many respects are complementary to those of Roberts et al. (6) from their inves- tigation of the fate of 30,17-dihydroxy- 45-pregnene-20-one in a patient with adrenal adenoma. These authors con- cluded that 17-hydroxyprogesterone was a negligible precursor of urinary preg- nane-3a,17,20a-triol in their subject; the major precursor of the saturated triol was the compound administered. The
August, 1962 ORIGIN OF PREGNANETRIOL IN ADRENAL CARCINOMA 771
pattern of endogenous metabolites of their patient differed significantly from that of the carcinoma patient, since the daily production of 45-pregnene-36,17, 20a-triol was more than three times that of pregnane-3a,17,20a-triol, in contrast with the reverse ratio with the present subject. Yet it is evident that both pa- tients secreted a considerable quantity of 36,17-dihydroxy-45-pregnene-20-one and relatively little 17-hydroxypro- gesterone.
From this study and other similar in- vestigations, 17-hydroxyprogesterone was found to be a minor precursor of the C19 metabolites, androsterone and etio- cholanolone. In this carcinoma patient not more than 0.1 mg of androsterone nor more than 0.85 mg of etiocholanolone was derived from 17-hydroxyproges- terone, as compared with a daily total production of 50 mg of both products. Comparison with the adenoma patient of Roberts et al. (6) is pertinent; they concluded that 36,17-dihydroxy-45-preg- nene-20-one contributed 1.8 mg per day to the 27 mg of androsterone and etio- cholanolone. If a similar conversion (3.0%) of 38,17-dihydroxy-45-pregnene- 20-one occurred in the carcinoma pa- tient, an interesting possibility emerges. Using the minimal estimate for the daily secretion rate of 36,17-dihydroxy- 45-pregnene-20-one of 154 mg for the carcinoma patient, 4.5 mg of andro- sterone plus etiocholanolone were de- rived from the C21 steroid; using the higher estimate of 2.0 g per day of precursor and the same percentage of conversion, 60 mg per day of the two C19 metabolites would have resulted. The latter figure is somewhat higher than the 50 mg measured. It is possible, then, that between 10 and 100% of the androsterone and etiocholanolone of this carcinoma patient was derived from a C21 steroid precursor. The possibilities outlined indicate that further work is
needed on this problem, since the situa- tion is analogous to that of the individual with congenital adrenal hyperplasia in whom high production of a minor pre- cursor of androgen metabolites resulted in a very substantial contribution to these urinary steroids.
One of the principal aims of the pres- ent investigation was to ascertain whether or not the patient’s large mass of functional glandular tissue, or, less likely, other tissues not directly in- volved with hormonal biogenesis, were able to achieve a characteristic step in hydrocortisone synthesis. In the normal sequence this would be hydroxylation of 17-hydroxyprogesterone at C-21 to yield Reichstein’s Substance S. This trans- formation was recognized from the presence of C14 in the reduced metabo- lites, pregnane-3x, 17,20a,21-tetrol and its epimer pregnane-3x,17,200,21-tetrol, both of which contained small but significant radioactivity. Moreover, both products had almost identical specific activities, so that the initial product formed must have been introduced into a pool that led to the formation of the epimers at C-20 without causing any considerable change in the relative specific activity of either of the pair. This is reasonable if the initial product was Reichstein’s Substance S, but un- likely if hydroxylation at C-21 had oc- curred after reduction of the C-20 carbonyl. While the mechanism may be uncertain, the fact of C-21 hydroxyla- tion seems to be on a firm basis. The large mass of metastatic tissue prob- ably was responsible for the quite small amount of steroid metabolized over this route. This situation is similar to that found by Solomon, Carter and Lieber- man (20), who were able to demonstrate the formation of dehydroisoandrosterone from 36,17-dihydroxy-45-pregnene-20- one in vivo in a patient with extensive metastatic adrenocortical carcinoma but
were unable to demonstrate any signif- icant transformation in a normal sub- ject. However, the possibility of hy- droxylation of 17-hydroxyprogesterone by peripheral tissues should not be overlooked, since Vermeulen et al. have reported the isolation of labeled 3a,17, 21-trihydroxypregnane-11,20-dione fol- lowing administration of 17-hydroxy- progesterone-4-C14 to a normal subject (11).
The endogenous steroid production of this patient, shown in Table 3, was sufficiently unusual to merit comment. The very large daily production of THS is noteworthy. The amount of the major hydrocortisone metabolites THE, THF and ATHF indicated daily secretion of approximately 150 mg of that hormone (12). Despite this very high production, the patient exhibited only slight signs of typical Cushing’s syndrome. The very great production of other steroids may have had some influence in this connec- tion, but this must be considered more as a possibility for investigation than as an explanation. It is pertinent to note also the very unusual finding of con- siderably more THE than THF in the presence of the very great amount of hydrocortisone metabolites. All other carcinoma patients whose endogenous steroids have been examined in these laboratories have shown higher levels of THF than THE when hydrocortisone secretion was elevated. The reverse in this patient also may have been re- sponsible in part for the slight clinical manifestations of hydrocortisone excess, since THE appears to be a measure of the biologically much less active cor- tisone (21-23). The situation is rem- iniscent of the hyperthyroid patient stimulated with ACTH, as described earlier (23), but there was no evidence for excessive thyroid hormone in this patient, nor was any administered prior to or during these studies.
Acknowledgment
The authors gratefully acknowledge the in- valuable technical assistance of Mrs. Shirley Dobriner and Miss Ruth Jandorek and the determination and interpretation of the infrared spectra by Mrs. Beatrice S. Gallagher.
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