ISOLATION AND IDENTIFICATION OF 16-OH PREGNENOLONE (PREGN-5-EN-38, 16a-DIOL-20-ONE) IN URINE FROM A PATIENT WITH ADRENOCORTICAL CARCINOMA.
Jose Cara
Department of Pediatrics, State University of New York Medical School, Upstate Medical Center Syracuse, N. Y. (1)
Received January 7, 1969
Abstract
The isolation and identification of 16-OH-pregnenolone (pregn-5- en-38, 16a-diol-20-one) in urine from a patient with adrenocorti- cal carcinoma is reported for the first time. The patient is a 12 year 9 month old girl with slight hirsutism, arterial hyper- tension and episodes of hypoglycemia. A 700 gram adrenocortical carcinoma of the right adrenal gland was surgically removed. Urine collected before surgery was submitted to enzymatic hydroly- sis and to solvolysis. The “glucosiduronate” and the “sulfate” fractions were chromatographed separately in alumina column using a gradient elution technique. 16-OH-pregnenolone was present in the urine in the amount of 8.7 mg/24 h., 7.0 mg being excreted as “glucosiduronate” and 1.7 mg. as “sulfate”. Pregn-5-en-38, 16a, 20a-triol was excreted in the amount of 11.0 mg/24 h., almost entirely as “glucosiduronate”. Identification of these steroids was confirmed by infrared spectrography. The 24 hour-urinary ex- cretion of other steroids was as follows: 17-hydroxycorticoids 43.0 mg., tetrahydro “S” 7.2 mg., pregnanetriol 2.4 mg., total 17-ketosteroids 31.0 mg., and the “beta” fraction as measured by Allen’s procedure 18.0 mg. The large amount of urinary 16-OH- pregnenolone and its metabolite pregn-5-en-38, 16a, 20a-triol pre- sumably is related to some of the clinical signs present in our patient, particularly to the episodes of hypoglycemia.
We are reporting here the isolation and identification of 16-OH-pregnenolone (pregn-5-en-38, 16a-diol-20-one) from the urine of a patient with adrenocortical carcinoma, whose salient clinical manifestations were arterial hypertension and episodes of hypogly- cemia. This steroid was first identified in large amount in the urine of patients with the 36-hydroxysteroid dehydrogenase defi-
ciency type of congenital adrenal hyperplasia (2). It was later found to be present in urine from infants with the more common type of congenital adrenal hyperplasia due to a deficiency of 21-hydro- xylase (3,4), and in urine from endocrinologically normal infants, particularly during the first few weeks of life (3,5). It has also been identified in a pool of urine from normal adult males (6). Although several metabolites of 16-OH-pregnenolone have been iso- lated in urine from patients with adrenocortical carcinoma (7,8,9), to our knowledge this is the first report of the isolation of 16-OH- pregnenolone in urine of such patients.
MATERIAL AND METHODS: The patient is a 12 year 9 month old girl, with very slight hirsutism, arterial hypertension of 160/100 and episodes of hypoglycemia. A mass was palpated deeply in the right flank below the liver. Steroid determinations in a 24 hour urin- ary specimen resulted in the following values: 17-hydroxycorticoids 43.0 mg (10), Tetrahydro “S” 7.2 mg (11), pregnanetriol 2.4 mg (12), total 17-ketosteroids 31.0 mg. (13) and the “beta fraction” as measured by Allen’s technique 18.0 mg. (14).
A 700 gm. tumor of the right adrenal was removed surgically and the pathological examination confirmed the clinical diagnosis of adrenocortical carcinoma.
Urine collected before surgery was kept frozen at - 10ºc. A 600 ml. aliquot was processed as previously described (15). The fraction extracted after beta-glucuronidase hydrolysis (“glucosiduronate”) and the fraction extracted after solvolysis (“sulfates”) were chromatographed separately in alumina column with a gradient elution technique and 10 ml. fractions were col- lected (15).
From each tube a 1 ml. aliquot was taken, and both this aliquot and the remnant 9 ml. were dried under nitrogen. The residue in the 1 ml. fraction was reacted with Oertel-Eik Nes reagent, and the absorption spectrum of the reaction recorded in a Bausch and Lomb Spectronic 505, at 10, 30 and 60 minutes. The color of the reaction was also noted. The 9 ml. fractions were inspected for the presence of residue. The quantitative determinations were done using the Oertel-Eik Nes reagent, and applying Allen’s correction (16). Readings were made in a Beckman D.U. spectrophotometer at 382 mu, 407 mu, and 432 mu for 16-OH-pregnenolone and at 380 mu, 405 mu, and 430 mu for pregn-5-en-38, 16a, 20a-triol.
RESULTS: In tubes 59-61 of the “glucosiduronate fraction” a white crystaline residue was obtained, in the amount of about 1.7 mg. The polarity of this compound in the alumina column was between that of 16-OH-dehydroepiandrosterone and that of pregn-5-en-38,17a, 20a-triol. With Oertel-Eik Nes reagent a yellow color was produced and the absorption spectrum showed a sharp peak at 407 mu. Neither the color nor the absorption spec- trum changed appreciably for at least one hour. On paper chroma- tography and on thin layer chromatography this compound re- solved in only one spot. The spot on the latter developed a pink color when sprayed with sulfuric acid. There was no UV absorption on the paper chromatogram, and the Zimmermann reac- tion was positive. Staining with phosphotungstic acid produced an orange color. Porter-Silber and blue tetrazolium reactions carried on with an aliquot of the compound were negative.
The chromatographic behavior of this compound in alumina column, on paper and on thin layer chromatography, and the ab- sorption spectra both with Oertel-Eik Nes reagent and sulfuric acid, were essentially the same as those of 16-OH-pregnenolone. The infrared spectrum performed by Dr. David K. Fukushima and Mrs. Beatrice S. Gallagher, confirmed the presumptive identifi- cation of this steroid as 16-OH-pregnenolone. In the “sulfate fraction” a much smaller amount of 16-OH-pregnenolone was eluted in tubes 58-60. The total daily excretion of 16-OH-pregnenolone was 8.7 mg., 7.0 mg. being excreted as “glucosiduronate” and 1.7 as “sulfate”.
In the “glucosiduronate fraction” another substance was eluted in tubes 70-75. With Oertel-Eik Nes reagent this com- pound gave an immediate yellow color that changed to yellow- green in about 15 minutes and to blue in about 45 minutes, re- maining blue for the next 24 hours. The absorption spectrum with the Oertel-Eik Nes reagent showed a sharp peak at 405 mu and a minor one at 620 mu at 15 minutes. As the color changed the peak at 405 mu diminished in size and the peak at 620 mu became more prominent. On paper and on thin layer chroma- tography the compound resolved in only one spot. There was no UV absorption and the blue tetrazolium, Porter-Silber and Zimmermann reactions were negative. Staining with phospho- tungstic acid gave an orange spot. The melting point of the com- pound was 247ºC. Dr. D. K. Fukushima and Mrs. Beatrice S. Gallagher identified this compound by the infrared spectrum as pregn-5-en 38, 16a, 20a-triol. The total urinary excretion of this steroid in 24 hours was 11.0 mg., nearly all being eliminated as a “glu- cosiduronate”.
DISCUSSION: The urinary excretion of 16-OH-pregnenolone and pregn-5-en-38,16a, 20a-triol is very high in our patient Rather large amounts of the latter compound have been found previously in urine from patients with adrenocortical carcinoma, and it is con- sidered to be a metabolite of 16-OH-pregnenolone (9). On the other hand 16-OH-pregnenolone has not been isolated hitherto from the urine of these patients. However, this compound is found in significant amounts in urine from normal newborns, par- ticularly in the first week of life, diminishing thereafter and
becoming undetectable after 5 months of age (5). In adult life its excretion is very small (6). Both in newborns and in adults 16-OH-pregnenolone is excreted as a “sulfate”. In our patient the large amount of 16-OH-pregnenolone present in urine was excreted predominantly as a “glucosiduronate”. Whether this difference in the mode of conjugation is due to the known relative deficiency of the glucuronidation mechanism in the newborn, or whether this represents a different pattern of secretion of the compound by the adrenal is not clear from our data. Bradlow et al, have speculated about the possible relationship between the mode of conjugation of the urinary product and the nature of the parent substance secreted by the adrenal either as a sulfate or as a free compound (17). We do not know in our case whether the ad- renal secreted the parent compound as a free or as a conjugated product, nor can we state with certainty whether the 16-hydro- xylation was performed by the adrenal or by peripheral tissues. Hydroxylation of steroids at C-16 has been shown to occur in vitro in human liver, particularly from the fetus (18,19) and new- born (20-21); in human fetal adrenal gland (22,23); in hyper- plastic adrenal tissue from patients with Cushing’s syndrome (24) ; as well as in placenta (25), ovary (26), corpus luteum (27) , and testes (28,29). However the 16-OH-pregnenolone originates in our patient, the amount must be very large considering the quantity of 16-OH-pregnenolone and its metabolite pregn-5-en-38, 16a, 20a- triol in the urine. Fukushima et al. have studied the urinary metabolites of 16-OH-pregnenolone administered to human beings (9). These authors were able to isolate and identify 2.5 mg. of
pregn-5-en-3a, 16a, 20a-triol in urine collected during a 4 day period following the continuous intravenous administration of 60 mg. of 16-OH-pregnenolone for two days to a 56 year old man with rheumatoid arthritis. Pregn-5-en-38, 16a, 20a-triol could not be isolated in sufficient amount for characterization. However after oral administration of 500 mg. of 16-OH-pregnenolone to a 49 year old normal man, the same authors isolated and characteri- zed 11.0 mg. of pregn-5-en-3a, 16a, 20a-triol and 2 mg. of pregn-5- en-38, 16a, 20a-triol in urine collected over the 3 following days. The amount of 16-OH-pregnenolone eliminated in urine is not men- tioned. They concluded that pregn-5-en-3a, 16a, 20a-triol was the principal metabolite isolated from urine following administration of 16-OH-pregnenolone and that the 36-isomer was excreted in lesser amounts. We did not attempt to isolate the 3a-isomer and there- fore we do not know whether this steroid represents an important metabolite in our patient as in the subjects studied by Fukushima et al. However, if we assume that the 16-OH-pregnenolone pro- duced by our patient follows the same metabolic pathway as in their two subjects, the amount of 16-OH-pregnenolone produced by our patient would be extremely high, considering the large excre- tion of 16-OH-pregnenolone and pregn-5-en-38, 16a, 20a-triol. It is possible, however, that the enzymatic transformation of 16-OH- pregnenolone, among other factors, may depend upon the length of time it is present in high concentration in the body, and also upon the free or conjugated nature of the adrenal secretion of 16-OH-pregnenolone or upon its production in peripheral tissues.
The biological role of the 16-OH-steroids is not yet clear and we do not know the biological significance of the large amount of 16-OH-pregnenolone produced in the newborn organism. The large amount of urinary_16-OH-pregnenolone, not previously described in association with adrenocortical carcinoma, presum- ably bears a relationship with some of the clinical signs pre- sented by our patient, particularly with the episodes of hypo- glycemia. Further discussion of the possible relationships be- tween the peculiar urinary steroid pattern and the clinical pic- ture presented by our patient will be presented elsewhere.
ACKNOWLEDGEMENTS
This work was supported in part by a grant from the National Institutes of Health (#AM-02504), and in part by a gift from Dr. and Mrs. J. Lamar Davis. Miss Frances O’Connor provided excellent technical assistance.
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