CHARACTERIZATION OF URINARY STEROIDS IN ADRENAL HYPERPLASIA: ISOLATION OF METABOLITES OF CORTISOL, COMPOUND S, AND DESOXYCORTICOSTERONE FROM A NORMOTENSIVE PATIENT WITH ADRENOGENITAL SYNDROME

Hortense M. Gandy, … , E. Henry Keutmann, Anthony J. Izzo

J Clin Invest. 1960;39(2):364-377. https://doi.org/10.1172/JCI104047.

Research Article

Find the latest version: https://jci.me/104047/pdf CHARACTERIZATION OF URINARY STEROIDS IN ADRENAL HYPERPLASIA: ISOLATION OF METABOLITES OF CORTI- SOL, COMPOUND S, AND DESOXYCORTICOSTERONE FROM A NORMOTENSIVE PATIENT WITH ADRENOGENITAL SYNDROME * By HORTENSE M. GANDY,t E. HENRY KEUTMANN AND ANTHONY J. IZZO (From the Departmtent of Medicine, the University of Rochester, School of Medicine anld Dentistry, Rochester, N. Y.) (Submitted for publication August 25, 1958; accepted October 8, 1959) Congenital virilizing adrenal hyperplasia miiay corroborated by Bartter and associates (13). present a varied clinical picture. Patients have Dorfman (14) postulated that there is a deficiency been described who have only excessive virilization in the C-21 hydroxylation mechanism. Eberlein (1); others have an associated Addison-like elec- and Bongiovanni (15) then found that some pa- trolyte disturbance (2, 3), with hypoglycemia (4), tients with congenital virilizing adrenal hyper- or with hypertension (5-9). Recent studies (10- plasia excreted C-21 hydroxylated compounds in 12) indicate that the manifestation of virilism may approximately the same quantities as did normal result from defects in the biosynthesis of cortisol.' individuals. Tetrahydrocortisone in small amounts The first suggestion regarding the nature of the was found. The present authors (16) have stud- error was made by Lewis and Wilkins (12), and ied six patients with virilizing adrenal hyperplasia in addition to the one reported here. Each of these * This study was supported by a grant (C-1003) from was found to be excreting C-21 hydroxylated com- the National Cancer Institute, Bethesda, Md. pounds in amounts equal to those found in nor- t Postdoctoral Research Fellow, United States Public Health Service. Present address: New York Hospital, mals of com)parable age. Tetrahydrocortisol was New York, N. Y. one of the a-ketols present. More recently, both 1 The following trivial names are used in the text: Hy- tetrahydrocortisone and tetrahydrocortisol have drocortisone (Compound F) is 1l11, 17a, 21-trihydroxy- been found in the urine of patients with this dis- 4-pregnene-3,20-dione. Tetrahydrocortisol (tetrahydro F) is 3a, llp, 17a, 21-tetrahydroxy-pregnane-20-one. Corti- order (17-20). sone (Compound E) is 17a, 21-dihydroxy-4-pregnene-3,11,- Abnormally high excretion of precursors of cor- 20-trione. Tetrahydrocortisone (tetrahydro E) is 3a, 17a, tisol, metabolites of precursors, and androgens are 21-trihydroxy-pregnane-11,20-dione. 11-Desoxyhydrocor- well recognized consequences of the derangement tisone (Compound S) is 17a, 21-dihydroxy-4-pregnene-3,- in the pathway for synthesis of normial corti- 20-dione. Tetrahydro S is 3a, 17a, 21-trihydroxy-preg- Further of the amounts nane-20-one. Dihydro S is 17a, 21-dihydroxy-pregnane- costeroids. augmentation 3,20-dione. Corticosterone (Compound B) is 11,, 21-dihy- of such products is to be expected fromii the over- droxy-4-pregnene-3,20-dione. Tetrahydrocorticosterone stimulation by excess corticotropin. The identi- (tetrahydro B) is 3a, lip, 21-trihydroxy-pregnane-20-one. fication of some of the abnormal metabolites has Desoxycorticosterone (DOC) is 21-hydroxy-4-pregnene- led to the clarification of the probable location of 3,20-dione. Tetrahydrodesoxycorticosterone is 3a, 21-di- errors in the process Lab- hydroxy-pregnane-20-one. Dihydrodesoxycorticosterone is the synthetic (7-22). 21-hydroxy-pregnane-3,20-dione. Allo-dihydrodesoxycor- oratory and clinical data reported by many work- ticosterone acetate is 21 acetoxy-allo-pregnane-3,20-dione. ers favor the concept that the defect in some cases Twenty-one desoxycortisone is 17a-hydroxy-4-pregnene-3,- may be at two or more steps of the steroid svn- 11,20-trione. Pregnanetriol is pregnane-3a, 17a, 20a-triol. thesis. Pregnanediol is pregnane-3a, 20a-diol. Androsterone is Eberlein and (7-9) studied an un- 3a-hydroxyandrostane-17-one. Etiocholanolone is 3a-hy- Bongiovanni droxy-etiocholane-17-one. 11-Keto androsterone is 3a-hy- treated patient who had congenital adrenal hyper- droxy-androstane-11,17-dione. 11 Ketoetiocholanolone is plasia associated with hypertension. The plasma 3a-hydroxy-etiocholane-11,17-dione. 11 Hydroxyandro- and urinary 17-hydroxycorticosteroids were imiark- sterone is 3a, 11-dihydroxy-androstane-17-one. 11 Hy- The contained S droxyetiocholanolone is 3a. 1 lp-dihydroxy-etiocholane-17- edly elevated. plasma Comlpound one. and tetrahydro S. The predomiiinant compound in 364 \IETABOLITES OF COMPOUND S AND DOC IN AN ADRENOGENITAL PATIENT 365 the urine was tetrahydro S. Tetrahydrodesoxy- amination were limited to a penis measuring 9 cm in corticosterone was also identified. length and small testes and prostate. The bone age, ac- two cording to Todd's "Atlas of Skeletal Maturation," corre- Three nonketonic C,1 steroids and C19-17- sponded to that of 11 years and 9 months. The neutral ketosteroids were isolated and identified by infra- 17-ketosteroids ranged from 15.7 to 21.7 mg per day red spectroscopy. None of the compounds iso- for 4 successive days before this investigation began. lated had oxygen functions at the 11-carbon. The Urinary a-ketol excretion, measured as blue tetrazolium authors proposed that these findings were evidence reducing substances (23), ranged from 18.6 to 27.7 mg for in C-11 hydroxylation. They also per 24 hours. The normal range for children 5 to 7 years deficiencv of age was found to be 2.5 to 3.5 mg per 24 hours in our suggested that the hypertension may be attributed laboratory. Excretion of pregnanetriol and pregnanediol, to accumulation of desoxycorticosterone, and this determined according to the method of Bongiovanni (24) type of defect has been considered characteristic was 4.5 and 1.5 mg per day, respectively. Serum electro- of congenital adrenal hyperplasia when hyperten- lytes, hemogram, blood sugar anid urinalysis were normal. sion is a complication (21). One week after the administration of 25 mg of cortisone acetate orally every 12 hours, the urinary neutral 17- Later Dyrenfurth and associates (19) reported ketosteroid excretion and total corticoid excretion had de- finding large amounts of tetrahydro S in the urine creased to 2.5 and 12.4 mg per 24 hours, respectively. The of two lpatients with congenital adrenal hyper- excretion of both types of steroids rose to pretreatment l)lasia: one had hypertension and the other did levels 48 hours after the cortisone was discontinued. not. Both of them excreted metabolites of cortisol. The patient was then placed on 37.5 mg of cortisone orally per day in divided doses for the next 9 months. -More recently Birke and colleagues (20) studied During that time the 17-ketosteroid excretion ranged be- the steroid patterns in the urine of two siblings tween 1.2 and 2.5 mg per 24 hours. Urinary pregnanetriol with congenital adrenal hyperplasia. Both pa- and pregnanediol were found to be 1.7 and 0.8 mg, re- tients excreted tetrahydro S, tetrahydrocortisone spectively, per day. No change in the range of systolic and 11-oxygenated 17-ketosteroids. Neither pa- or diastolic blood pressure was noted during the trial of cortisone, when the drug was stopped, or during mainte- tient had hypertension. nance therapy. The study reported here concerns a normoten- sive patient with the adrenogenital syndrome with METHODS a corticoid pattern characterized by the presence Corticosteroids. A 3 day pool of urine was collected of the metabolites of Compound S and of desoxy- before treatment and again a week after cortisone therapy corticosterone as well as of cortisol. had been started. Twenty-four hour aliquots from each pool were buffered at pH 4.8, hydrolyzed for 48 hours at 470 C with p-glucuronidase (250 units per ml of urine), CASE REPORT and then continuously extracted xith ether for 48 hours D.(., a white male, was first seen at 5.5 years of age after the pH had been readjusted to 6.8. The extracted \hen he was admitted to the Pediatric Service of this urine was put aside and the ether extracts were dried and hospital for evaluation of sexual precocity. The parents washed in chloroform by the procedure described by related that he weighed 3.5 kg at birth. Acceleration of Burton, Zaffaroni and Keutmann (25). The number of growth was noted during the first year of life. At 3 washings was reduced to 3 alkali, 1 acid, and 1 water months the patient weighed 4.6 kg, at 6 months 9.6 kg, wash, together with their appropriate back washings. and at 10.5 months 12.0 kg. Acne was noted on his face Preliminary fractionation and identification were done and chest at one year of age. The history was not re- on one of the 24 hour aliquots, the other two were used markable otherwise. for more definite and confirmatory information both as OIi admission the patient had the physical develop- to identity and quantitation. ment of an adolescent; his voice was deeply pitched. The total a-ketol content of the neutral extracts was A papular rash was present on the face and over the measured with blue tetrazolium according to the method shoulders. There was a moderate amount of hair on the described by Izzo, Keutmann and Burton (23) prior to upper lip and in the axillae; pubic hair was abundant. and after chromatographic fractionation on paper. The He weighed 35 kg; his height was 142 cm. total C-21, 17-hydroxyglycol content was determined by The blood pressure was recorded by 3 different ex- the method of Wilson and Fairbanks (26), and total 17- aminers to be 100/65, 105/70 and 120/80, respectively. ketosteroid content by the method of Talbot, Berman and Numerous other observers always found it to be within MacLachlan (27), using alcoholic KOH prepared ac- these limits. After 3 years' treatment with cortisone, cording to Wilson and Carter (28). The amounts of the drug was discontinued for 2 weeks. At the end of the individual fractions or compounds were determined this time the blood pressure was again found to be nor- on known aliquots of eluates from the paper after the mal. The other remarkable features of the physical ex- compound had been located with the appropriate spotting 366 H. M. GANDY, E. H. KEUTMANN AND A. J. IZZO

reagent. Quantities of individual compounds other than tion of two different group-specific reacting areas or to the above types were quantitated by methods which will eliminate urinary pigments. Complete separation was at be indicated in each instance. In order to insure against times not achieved until acetates were made and chro- loss of a-ketols, no preliminary separation into various matographed (35). This was especially true for mixtures types of the steroids in this extract was attempted. Dur- of a-ketols and other C-21 compounds which had the same ing treatment with Girard's reagent T, for instance, losses mobility. These mixtures were separated most efficiently have been encountered by ourselves and other investiga- when undiluted formamide was used as the stationary tors (29, 30). The direct chromatography presented phase. some problems, of course, when a-ketols and glycols or Characterization was attempted in the case of each other steroids with similar or identical running rates isolated steroid. The criteria for identification were: were found in greatly differing amounts on the same a) the chromatographic mobility in the free state and as chromatograms. Separation was then accomplished by acetates compared with authentic compounds and their rechromatographing the area with different systems, dif- acetates; b) reactions of the unknown compounds on pa- ferent amounts, or by acetylation and subsequent chro- per with the aforementioned group-specific reagents; c) matography. the absorption spectra in methanol and sulfuric acid, re- Fractionation of each urine extract by paper chromatog- spectively (40) ; and in most instances, d) infrared ab- raphy was similar to that described by Burton and col- sorption spectra. Well defined spectra were obtained leagues (25) with certain modifications as indicated in when the acetates either were partitioned between purified Figure 1. The neutral extract was applied to washed ethyl ether and ice water, or were passed through a Whatman no. 1 filter paper impregnated with freshly pre- micro-column of silica gel, or both. pared 50 per cent solution, by volume, of formamide in Non-a-ketolic C-21 steroids. These steroids were lo- absolute methanol. The chromatograms were developed cated on the toluene/propylene glycol chromatograms by in the benzene/formamide system for 48 hours so as to means of those spot tests, enumerated earlier, which did yield 3 fractions consisting essentially of C2,O3 (Fraction not test for a-ketols but have been found to react with A), C2104 (Fraction B), and C,O5 (Fraction C) com- groupings on the molecule which apply specifically here pounds, respectively. (32-39). In some instances there was overlapping on the The steroids remaining on the paper as Fraction C were chromatograms with a-ketols of similar polarity, and com- eluted off with methanol. This eluate was rechromato- plete separation, sufficiently satisfactory for complete graphed in the toluene/propylene glycol system for 44 identification, was not accomplished until after acetylation. hours. The chromatogram was dried and examined un- Seventeen-ketosteroids. The urines which had been der a fluoroscopic scanner (31). Reference strips 0.25 extracted at pH 6.8 after enzyme hydrolysis were next cm wide were cut from the center of the chromatogram adjusted to pH 1 with 50 per cent sulfuric acid and again and the steroids were located with the following: aqueous continuously extracted with ether for 48 hours. These blue tetrazolium; 2,4 dinitrophenylhydrazine (32); metha- extracts were dried and washed in the same manner as nolic sodium hydroxide (33); the Zimmermann reaction those containing largely the a-ketolic steroids. The total using aqueous KOH; periodic acid oxidation followed by amounts of Zimmermann-reacting substances were then the Zimmermann reaction (34); aqueous iodine (35); and measured. Separation of the individual 17-ketosteroids sulfuric acid fluorescence. In certain instances additional was accomplished by paper chromatography utilizing the spot tests were used to help in the location and identifica- benzene/formamide, cyclohexane/formamide, and cyclo- tion, namely, treatment with phosphomolybdic acid (36); hexane: hexane/formamide systems (25). The Zim- treatment with 70 per cent phosphoric acid (37, 38) ; and mermann reaction, when used to spot the compounds on treatment with trichloracetic acid (39). the paper, was performed with aqueous KOH. For quan- The reference strips treated with blue tetrazolium indi- titation, alcoholic KOH (28) was used. The overflow cated that the a-ketols were grouped in 3 zones at this from Fraction A of the a-ketol procedure was chromato- time as illustrated in Figure 1. These were eluted sepa- graphed with cyclohexane: hexane/formamide, the spots rately and designated as subfractions C-1, C-2 and C-3 located, identified and quantitated, and the results pooled (Figure 1 and Table I). Fractions A and B were re- with those of the pH 1.0 extract (Table II). Six com- chromatographed in the cyclohexane/formamide and the pounds were present in sufficient quantity for ready benzene/formamide systems, respectively, as indicated in separation, quantitation, and identification by comparing Figure 1. Steroids were located on reference strips from their mobilities and infrared absorption spectra with each of these chromatograms with the group-specific authentic compounds. In addition there were 5 com- spotting reagents enumerated above. Just as had been pounds which were present in insufficient quantity to done with Fraction C, these chromatograms were divided identify definitely. into subfractions corresponding to blue tetrazolium reac- tive zones. RESULTS The eluates of each of these were rechromatographed in the appropriate systems (Figure 1). In many instances Corticosteroids. The steroids isolated and iden- it was necessary to rechromatograph a subfraction or an tified prior to any treatment of the patient and isolated steroid several times in order to achieve separa- during cortisone therapy are listed in Table I and METABOLITES OF COMPOUND S AND DOC IN AN ADRENOGENITAL PATIENT 367

NEUTRAL EXTRACT I CHROMATOGRAPHED IN B/F 48 HRS. CPDL NO.

- MEOH ELUATE -CHROMAT. IN OF STEROIDS T/PG 44HRS. REMAINING _ C-I -4 IN CHCL3/F F7HRS.7HRS.<4< ACETATES IN. B:CY/F 5HS. -112 ON PAPER * B: CY/F*4HRS. -[ 3 FRACTION C - . (C2105 CPDS.) C-2-4 T/PG 56 HRS. ' *F' 3HRS.. 6 C-3 -4 T/PG 40HRS. - * CY/F 6HRS,- 8

5-48 HRS - RCHROMAT. IN OVERFLOW B/F 5HRS.

B-l q IN T/PG 12 HRS. ACETATES IN 3HRS. -4 B:CY/F* CIO FRACTION B -4 CPDS.) '1 (C2,04 1 2 B-2 -4 " T/PG 7 HRS. CY/F* 5 HRS.-13 14 ,15

0-5 HRS. -C CHROMAT. IN OVERFLOW CY/F 24HRS.

I CY/F 24HRS. IN (C2F03 CPDS.) A-I -4 IN ACETATES H/F' 3HRS.-16

FIG. 1. SCHEME OF THE FRACTIONATION BY PAPER CHROMATOGRAPHY. In each instance the overflow from the chromatograms was added to the subsequent, less polar material before this was chromatographed. This assured against loss of any steroids throughout. Abbreviations: B/F = benzene/formamide. T/PG = toluene/propylene glycol. CY/F = cy- clohexane/formamide. CHCI,/F = chloroform/formamide. H/F = hexane/formamide. B: CY/F = equal volumes of benzene and cyclohexane/formamide. In the case of formamide, when an asterisk appears on the diagram, it denotes that undiluted solvent was used to im- pregnate the paper for the stationary phase; the absence of an asterisk indicates that the form- amide was diluted with an equal part of methanol before impregnating the paper. their properties summarized. The identity of a steroids excreted per 24 hours. The free com- number of them could not be established. The pound as well as its acetate had the same chromato- patient excreted 18.6 to 27.7 mg of a-ketolic ster- graphic mobilities as had authentic tetrahydro S oids per day prior to cortisone therapy. The and its acetate. The free compound and its acetate a-ketolic content of the urine pool used for chro- reacted with blue tetrazolium to give an intense matography was 21.0 mg per day which was six blue color. When the compound was dissolved in times the amount excreted by normal children of methanol, no peak was found in the ultraviolet ab- comparable age (41 ). sorption spectrum. The sulfuric acid chromogen of both the free compound and its acetate gave Identified ketolic steroids an absorption peak at 314 mu and a plateau ex- Tetrahydro S. The predominant a-ketol iso- tending from 400 to 420 m,u. This pattern was lated from the most polar fraction (Fraction C), identical to what we observed with known tetra- designated as Compound 6 in Table I and Figure hydro S (T.H.S.). An infrared absorption spec- 1, accounted for 68 per cent of the total a-ketolic trum of the acetylated compound in carbon di- 368 H. M. GANDY, E. H. KEUTMANN AND A. J. IZZO

TABLE I Quantities and some properties of the steroids isolated from the urine of D. G.*

Spot test reactions Periodate oxidation Corn- Blue 2,4-Dinitro- + Alpha pound tetra- phenyl- Zimmer- U.V. absorption spectra max. Treatment Fraction ketols no. zolium hydrazine mann in conc. sulfuric acid Steroid mg/24 hr mAt Nonie 'rotal Neutral 21.2 C-1 (I + 0 0 260 (shoulder), 310, 410, iJnideintified 510 (shoulder) 0.8 42 + 0 0 260 (shoulder), 325, 410, 1Tetrahydro F 510 (shoulder) 0.5 3 + 0 0 315,410 Uniiidentified 1.5 4 + 0 0 325-330, 410 Tetrahydro E C-2 5 0 0 + 315 Pregnane-3a, 17a, 20Q, 21-tetrol 14.7 6 + 0 0 315, 400-20 (plateaLl) Tetrahydro S 7 0 0 + 310, 410 (shotilder) Three a, 17a, 20a- trihydroxy preg- iiane-1 1-one C-3 0.2 8 + oranige 0 268, 315, 440, 500 Dihvdro S (shoulder) B-1 0.9 9 + 0 0 315, 440, 500-520 Utniidentitied (shoulder) 10 0 0 + 310, 440, 510 (shouldler) Pregnane-3a, 17a, 20a-triol 11 0 vellowv 0 320 Three a, 17a, dihy- droxy-pregnane- 20-one B-2 12 0 oranige 0 280, 330, 420 Uniidentified 0.1 13 + 0 0 270, 320 Uniidenitified 14 0 vellow 0 265, 295, 310 tUniidenitified 0.2 15 + 0 0 305-10, 375, 400 U'nidenitified A-pig. 0.2 0 0 0 Pigmenits A-1 0.6 16 + vellow 0 285-290, 360-70, 435 Dihvdro DOC 1.0 17 + 0 0 310, 380-410 (plateaui) Tetrahydro DOC A-over- 0.5 trace 0 0 17-Ketosteroids and flow pigmiients Cortisonie Total Neutral 12.4 C-1 0.3 1 + 0 0 260 (shoulder), 320, 410, tUnlidentified 500 (shoulder) 2.0 2 + 0 0 260 (shoulder), 318, 410, Ietrahydro F 510 (shoulder) 0.5 3 + 0 0 320, 410 UTniidenitified 5.0 4 + 0 0 325, 410 'retrahydro E C-2 0.7 5 + yellow + 270, 310, 375, 410, 500 LTUnidenitified (shoulder) B-1 0.2 6 + 0 0 305-10, 420 Tetrahydro B 0.6 7 + 0 0 305 Unidentified 0.4 8 + yellow 0 255, 305, 375, 420-30 Unidentified A-1 0.4 9 + vellow 280, 355-65, 420, 510 Unidenitified 0 (shoulder) A-over- 0.6 0 0 17-Ketosteroids and pigments

* None of the compounds when dissolved in miiethaniol gave ani absorptioni peak in ultraviolet light. All compounds failed to produce fluorescence when treated with sodium hydroxide. None of the compounds gave a blue color when treated with iodine solution (22). sulfide was likewise identical to that of authentic amiiotunts of several of the other a-ketols in Table T.H.S.. A smaller amount (3.5 mg) of T.H.S. I were spotted in this extract. was identified in the urine extract obtained after Tetrahydrocortisol. Compounds 1 and 2 were hot acid hydrolysis. It was quantitated and the not separated in the free state with the toluene/ amount is included in Table I. Insignificant prol)ylene glycol and chloroform/formamide sys- METABOLITES OF COMPOUND S AND DOC IN AN ADRENOGENITAL PATIENT 369 tems. As acetates they were separated from one TABLE II another when chromatographed in the benzene: 17-Ketosteroids isolated from the urine of patient D. G. cyclohexane/formamide system. The chromato- Ex- Ex- graphic mobility of the acetate of Compound 2 tracted tracted at at was identical to that of authentic tetrahydrocorti- pH 1.0 pH 6.8 Total sol diacetate in a mixed chromatogram. An ab- mg/24 hr mg/24 hr mg/24 hr sorption spectrum given by the sulfuric acid chro- Anidrosterone 2.8 2.3 5.1 mogen and the infrared absorption spectrum of the Etiocholanolone 1.3 2.6 3.9 1 1-Keto androsterone 1.8 1.8 acetylated compound in a potassium bromide disc 11 Keto etiocholanolone 0.2 0.2 was identical with that of authentic tetrahydro- 11 Beta hydroxyandrosterone 0.3 0.3 11 Beta hydroxyetiocholanolone 0.2 0.2 cortisol. Unidentified 0.8 0.8 Tetrahydrocortisone. Compound 4 exhibited chromatographic mobility identical to that of au- 12.3 thentic tetrahydrocortisone in the free state and as acetate. Its reaction with blue tetrazolium, hexane: benzene/formamide systems. The sul- its nonreactivity with the other spotting reagents, furic acid chromogen and the infrared absorption and its failure to absorb ultraviolet light charac- spectra were identical to those given by authentic terized it as a saturated a-ketol. The sulfuric acid tetrahydrodesoxycorticosterone. chromogen and the infrared absorption spectra Alpha-ketols not completely characterized. Five were identical to those of known tetrahydrocorti- additional a-ketolic compounds were separated in sone diacetate. varying amounts (Compounds 1, 3, 9, 13, 15). Dihydro S. Compound 8 behaved like dihydro All seemed to be saturated in Ring A. Many of S. In the free form and as acetate it exhibited the characteristics of Compounds 1 and 3 sug- polarity similar to Compound E and its acetate. gested that they were probably "allo" isomers of However, it gave a color, when treated on paper tetrahydro F and tetrahydro E, respectively (43, with 2,4 dinitrophenylhydrazine, which suggested 44). The polarity of the others ranged from the presence of a nonconjugated ketone group in slightly less than that of cortisone to slightly less Ring A. The sulfuric acid chromogen pattern than that of 11-dehydrocorticosterone. Neither was identical to that of authentic dihydro S. their chromatographic behavior as free steroids, or Dihydrodesoxycorticosterone and tetrakydro- as acetates, their sulfuric acid chromogen spec- desoxycorticosterone. The two a-ketols found in tra, nor the various spot tests or derivatives aided Fraction A (Compounds 16 and 17) were of more in their identification. Compound 9 may possibly than ordinary interest. Alpha-ketols have not been have been tetrahydro A contaminated with another found in the urine of normal persons in this frac- steroid. The sulfuric acid chromogen curve did tion (25, 41). In the free form they moved as a not resemble the two identified steroids found next single wide band which was slightly more polar to it. than desoxycorticosterone (cyclohexane/formamide Non-a-ketolic C-21 steroids. Two glycols were and a number of other systems). The more polar found in Fraction C-2 of the toluene/propylene of the two acetates (no. 16) was compared with glycol chromatogram. The total amount per 24 allo-dihydrodesoxycorticosterone acetate since di- hours was 1.6 mg. hydrodesoxycorticosterone acetate was not avail- Pregnane-3a, 17a, 20$, 21-tetrol. This com- able to us. The movements of the unknown and pound (no. 5 in Figure 1 and Table I) was found reference compounds were identical in hexane/ between tetrahydrocortisone and tetrahydro S; it formamide as well as in cyclohexane/formamide. overlapped with the latter before acetylation. The sulfuric acid chromogen was identical to that When compared with known samples of Com- of dihydrodesoxycorticosterone (42). pounds F and E its chromatographic mobility was The less polar of the two acetates (no. 17) ex- found to be similar to Compound F. It did not hibited chromatographic behavior like the acetate react with blue tetrazolium and gave a positive of tetrahydrodesoxycorticosterone in hexane/ Zimmermann spot after periodate oxidation. formamide, cyclohexane/formamide, and cyclo- With phosphomolybdic acid it gave a blue color. 370 H. M. GANDY, E. H. KEUTMANN AND A. J. IZZO It did not become fluorescent when treated with A, both in the free form and as acetates. The ace- sodium hydroxide and no color was produced with tate was also less polar than desoxycorticosterone dinitrophenylhydrazine. Its sulfuric acid chro- acetate. The free compound, when located on the mogen had a peak at 314. An infrared absorp- paper chromatograms with phosphoric acid, gave a tion spectrum had peaks which were identical to violet color and pink fluorescence (37, 38). When those described by Eberlein and Bongiovanni (9) treated with trichloracetic acid (39) it gave a for this compound. blue color in daylight and blue fluorescence. It When the compound was oxidized a 17-ke- did not react with blue tertazolium and all reactions tosteroid was produced which, when chromato- for 4, 3-keto groups were negative. After pe- graphed in the cyclohexane/formamide system, riodate oxidation, according to the method of Ro- had mobility identical to etiocholanolone. manoff and Hunt (34), it gave a weak Zimmer- Three a, 17a, 20a-trihydroxy-pregnane-11-one. mann reaction. With phosphomolybdic acid it This compound (no. 7 in Figure 1 and Table I) gave a blue color (36). In sulfuric acid it formed was less polar than tetrahydro S when found on a pale orange color (9) and its sulfuric acid chro- the toluene/propylene glycol chromatogram. It mogen absorption spectrum had a major peak was also less polar than Compound E when com- at 440, a minor peak at 310 and a shoulder at 510. pared with a known sample of this compound. It The amount, in addition to having been done on was spotted by treatment with 70 per cent phos- an aliquot of urine as mentioned in the case re- phoric acid, which produced a fluorescence (37, port, was again quantitated by eluting the com- 38). It did not react with blue tetrazolium but pound off the paper and applying the formula of gave a positive Zimmermann reaction after oxi- Allen to the absorption spectrum obtained with a dation with periodate. It gave a strong blue sulfuric acid chromogen. In this manner 4.8 mg color with phosphomolybdic acid (36), did not per 24 hours was calculated to be present. fluoresce when treated with sodium hydroxide, Three a, 17a-dihydroxy-pregnane-20-one. This and 2,4-dinitrophenylhydrazine produced no color. compound (no. 11 in Table I) was detected on the A sulfuric acid chromogen spectrum had a peak paper by its reaction with 2,4-dinitrophenylhydra- at 310 and a shoulder at 410. When the acetates zine, thus characterizing a ketone group in the of this compound and of pregnane-3a, 17a, 20,21- molecule. Its polarity was somewhat less than di- tetrol were chromatographed their polarity was in hydro S and pregnanetriol. It did not reduce blue the reverse order of the free compounds. This tetrazolium and did not have an ultraviolet light suggests that the unknown compound (no. 7) had absorption peak when dissolved in methanol. After fewer hydroxyl groups available for acetylation chromatography of the acetate its sulfuric acid than the identified glycol-3a, 17a, 20$, 21-tetrol. chromogen absorption spectrum consisted of a peak After oxidation with periodate or chromic acid at 320 m,u. This was identical to the spectrum the unknown compound yielded a 17-ketosteroid yielded by authentic 3a, 17a-dihydroxy-pregnane- which, when chromatographed with cyclohexane/ 20-one (42). An infrared absorption spectrum formamide, moved identically to 11-keto-etiocho- also coincided with that given by this compound. lanolone. The quantity was insufficient for fur- While most of the compound was sharply localized ther tests. All the above information, especially in this region of the chromatogram, traces were the comparison with the findings on 3ca, 17ca, 20,21- also distributed to some extent throughout Frac- tetrol, suggests that this glycol did not have an tions B and A in the preliminary fractionation and oxygen function at the 21-carbon but had one on necessitated rechromatography of certain of the carbon-11. The most likely compound to fit all other steroids in order to clean them up satisfac- the above findings is 3ac, 17a, 20a-trihydroxy-preg- torily for identification. Similar difficulties were nane-1 1-one. encountered by Fukushima and Gallagher (17) Pregnane-3ar, 17a, 20a-triol. This compound when a urine extract contained large amounts of (no. 10 in Table I) was found in the upper part pregnane-3ca, 17a, 20a-triol. From the aliquot used of Fraction B, slightly less polar than the blue 8.4 mg of the compound in crystalline form was tetrazolium zone designated B-1 in Figure 1. It isolated, and this accounted for an excretion of was less polar than tetrahydro B and tetrahydro slightly more than 25 mg daily. METABOLITES OF COMPOUND S AND DOC IN AN ADRENOGENITAL PATIENT 371

Pregnane-3a, 20a-diol was found in Fraction A have been due to contamination of the administered by spotting with phosphomolybdic and silico- cortisone by Compounds A or B. The metabolites tungstic acids. It was cleaned up by chromatog- of Compound S and desoxycorticosterone noted raphy using a toluene: methyl cyclohexane/propyl- prior to cortisone therapy were not found. Some ene glycol system and identified by its sulfuric of the unidentified compounds exhibited properties acid and infrared absorption spectra. identical to some of the unidentified compounds Two other nonketolic substances were isolated found during the pretreatment period. This phe- from Fraction B (Compounds 11 and 13). These nomenon may have been due to the cortisone dos- substances, like Compound 9, reacted with 2,4- age being insufficient to suppress completely dinitrophenylhydrazine. From the chromato- ACTH production. graphic mobility of Compound 13 before and after DISCUSSION acetylation we inferred formation of a diacetate. Further identification was not possible. A num- Adrenal gland perfusions and adrenal vein blood ber of lesser spots were found during treatment analyses indicate that cortisol and cortisone are with phosphoric acid and phosphomolybdic acid, the major products of adrenocortical secretion in but their nature was not determined. man. The synthesis of Compound F via pregnen- Seventeen-ketosteroids. The three 24 hour olone (46) is dependent upon hydroxylating en- urine specimens contained an average of 17 mg of zymes which introduce oxygen function at C-17, 17-ketosteroid after hydrolysis with hot hydro- C-21 and C-11 positions, usually in sequential chloric acid. In order to avoid the confusion fashion in the order named, according to Hechter caused by artifacts produced by the hot acid (45) and Pincus (47). the chromatography and identification were per- Patients exhibiting congenital adrenal hyper- formed on extracts prepared as described in plasia with virilization are considered to have a "Methods." This, we believe, assured us that the metabolic defect which does not allow synthesis identified metabolites were excreted as such by the of cortisol at a normal rate. All of the clinical patient. and laboratory findings which have been reported In Table II are recorded the six C19 compounds are in keeping with this hypothesis. In the plasma, which were isolated, quantitated and identified by low levels of 17-hydroxycorticoids and high levels their infrared absorption spectra. Five additional of ACTH have been reported (11). Administra- compounds were found in minute amounts, too tion of adrenocorticotropin usually has not aug- small to identify. These are all included in the mented appreciably the levels of corticoids in the table under "unidentified." The sum of the 17- serum or urine (10-13, 48, 49). Exceptions to ketosteroids thus found was 12.2 mg. According this have been reported (16, 50). These varia- to the work of Brooks (29) this is a reasonable tions of response from case to case probably de- amount when compared with the quantity found pend upon whether or not the adrenals are re- after hot acid hydrolysis. sponding maximally to intrinsic ACTH before ad- Cortisone treatment period. The excretion of ministration of the extrinsic material. This in 17-ketosteroids reverted to normal levels while turn may depend upon differences in the quanti- the patient was receiving cortisone. There was tative extent of the defect. In the cases described also a decrease in the daily excretion of a-ketols prior to 1955 the known metabolites of cortisol and there was a marked change in their pattern. were not found (7, 8, 11). Since then, however, The predominant a-ketols were tetrahydrocortisol both tetrahydrocortisone and tetrahydrocortisol and tetrahydrocortisone which accounted for ap- have been isolated from the urines of untreated pa- proximately 57 per cent of the total a-ketols ex- tients with adrenogenital syndrome (15-18). The creted. A small amount of tetrahydro B was also explanation proposed by Bongiovanni and Eber- found. It is possible that its presence in small lein, and which has been supported by others amounts during the pretreatment period was (18), namely, that the fault is relative and not ab- missed because of the large quantities of abnor- solute, explains these findings in a highly satis- mal steroids located in the same regions of the factory manner. Indeed, the ability to synthesize, chromatogram (Fraction B-1). It could also with sufficient stimulation by ACTH, adequate 372 H. M. GANDY, E. H. KEUTMANN AND A. J. IZZO

--- - .- - - AL.1 . . - FUW1-wwiu UUa - ow L.s M tefrahj*e -00 I1 of 6 fdiJhydre-DOCoe

PREGNENOLONE PROGESTERONE +21-OH DOC + CPD , 1OH - B+?

>_pinie -3m, l?@,*@v-tZril- 1-OSe °Affl

+ 170H

17- F

prepone-30, l7e-dvtiol-- n 50.mg tefolY&-, O.5ON prepane-s3a, /?0,toa-triol 4.5mg tfr*yo-f P.5mg

lIp diy*dr- s - w.omg proonam -s-a. tI?a4 terdio4. I-I tetral 1.0ow FIG. 2. A SCHEME TO EXPLAIN THE ALPHA-KETOLIC METABOLITES FOUND IN THE URINE OF A NORMOTENSIVE BOY WITH ADRENOGENITAL SYNDROME. The steroid metabo- lites which were separated and measured are printed in small slanted type. The prob- able precursors and their interrelations are printed in capitals. Impaired 11- and 21- hydroxylase activities are postulated. amounts of cortisol probably explains why most more 3a, 17a, 20 ?, 21-tetrol. In both cases the of these patients remain in good general health. excretion levels of these compounds were of such The urinary steroid pattern in the case described magnitude that they very definitely implied failure here had certain similarities to that found in the of 11,8-hydroxylation (47). The inference that case with hypertension reported by Eberlein and these amounts of metabolites imply formation by Bongiovanni (9); however, there were marked the adrenals of the same amounts of the parent differences in both kind and amount of metabolites compounds is perhaps plausible, but not certain. isolated. In Figure 2 we have indicated the most In normal individuals the above metabolites are probable precursors of the various C21 metabo- not found to any great extent. Touchstone, Bula- lites found in the urine of our patient. Implica- schenko, Richardson and Dohan (51) thought that tions as to the abnormalities in synthesis are de- a group of normal men and women excreted about rived from the amounts and types of these me- 20 ug of tetrahydro S daily. It was not always tabolites. At the same time the presence of the present, however, even in such small amounts, in 17-ketosteroids listed in Table II should be normal persons, pregnant women or diabetics. taken into consideration. Less than 10 ,ug per day has been found in the The levels of excretion of metabolites of Com- urine of a group of normal children studied in our pound S and of desoxycorticosterone in the two laboratory. Romanoff and Wolf found none in a patients were of roughly the same order. In ours series of normal adults or patients with schizo- we found slightly more tetrahydro S but in the phrenia (52). case of Eberlein and Bongiovanni (9) there was It is interesting that Hechter and colleagues MIETABOLITES OF COMPOUND S AND DOC IN AN ADRENOGENITAL PATIENT-T 373 predicted some years ago (53) that if a clinical was no correlation of blood pressure with age or condition should arise in which there is an in- sex. sufficiency of 11-hydroxylating enzyme, such per- Of equally great interest and perhaps imiportance sons would execrete metabolites of Compound S. was the finding in our case of greater quantities of Several such conditions, in addition to the sub- metabolites of 17-hydroxyprogesterone (25 mg of ject of this paper, can be cited. When Compound 3a, 17a-dihydroxy-pregnaine-20-one, and 4.5 mg of S was administered to patients with adrenal in- pregnane-3a, 17a, 20a-triol) than metabolites of sufficiency. tetrahydro S was found in the urine Compound S, as indicated in Figure 2. The in considerable quantities (54). In some patients amiiounts were verified by repeated analyses on sev- wN-ith adrenal cortical tumlors considerable quan- eral aliquots of the three 24 hour specimens. tities of tetrahydro S have been found in the urine Three a, 1 7a, 20-trihydroxypregnane- 11-one, a (54-56), probably because the production of Com- metabolite of 21-desoxycortisone, was almost cer- pound S was so great that the capacity of the 11- tainly also found. All these metabolites have been hydroxylating mechanism was exceeded, or the found in cases of adrenogenital syndrome with de- subjects hadl defects in the 11-hvdroxylating ficiency of C-21-hydroxylation (16). In the light mechanism. of these findings a relative insufficiency of 21- The most striking (lifference between the case hydroxylating function is strongly suggested in reported here an(l that of Eberlein and Bongio- our case. It is plausible, therefore, that this boy vanni (9) w-as the tinquiestionable evidence for the had partial insufficiency of both 11- and 21-hy- preselnce in ouir patient of 11,8-hydroxylating en- droxylating enzymes. zymiie. This conclusion is based on the isolation It is of slight interest that the 20-keto metabo- of tetrahydro F, tetrahydro E, and the four 11- lites of 17-hydroxyprogesterone were more abtun- oxygenated 17-ketosteroids recorded in Table II. dant than the 20-hydroxy metabolites, as is ustually The overall pattern of the urinary metabolites as the case (17). This could imply some difference well as clinical evidence indicate that the capacity of metabolism from the uisual in either adrenals or on the part of the adrenals to perform this func- liver. The proportions are the same, of course, tion was reduced. as in the case of metabolites of hvdrocortisoine The possibility exists that the (legree or se- uinder normal conditions. verity of the block is of importance and that the Hypertension has never been described in pa- amotunt of DOC synthesized and its effect on blood tients with adrenogenital syndrome who have de- pressure would depend on this. The finding of fects in 21-hydroxylation. The hypertension, similar quantities of miietabolites of DOC may be therefore, appears to occuir only in some of those against this but not definitely so. In addition, platients with defects in 11,8-hydroxylation. Otur one would expect that the amount of 11-oxy- stui(lies, however, as well as several other reports, genated metabolites excreted would give an indi- show that the occurrence of normal blood pressure cation of the degree of the defect. No conclusions in this syndrome does not rule out a defect in in reference to this point can be drawn from pub- 11-hydroxylation or establish that the imajor de- lished cases. To our knowledge there have been fect is at the 21 position. reporte(l five cases of adrenogenital syndrome in Somiie additional remarks concerning observa- whom the mlajor metabolite found in the urine tions which have been made on some of the adrenal w-as tetralhydro S (9, 19, 20, 57). Metabolites of cortical steroids that are made consequent to the DOC were reported in only the case of Eberlein defect of C-11 are in order. In no sttudies w-ith and Bongiovanni (9). One can assume that they Compouind S was there evidence that hypertension were present in some of the other cases if not in cotuld be attribuited to it. Bergenstal, Huggins and all. In all cases except that of Eberlein and Dao (58) gave 25 to 50( mg of Comiipound S in- Bongiovanni evidence of some degree of 11,1/-hy- tramuscularly to adrenalectomized patients. It (Iroxvlation was found in the form of either 11- was followed only by minimal salt retention and oxygenated C21 or C,9 steroids or both. There no carbohydrate effect. The patients were weak was no correlation between the presence or ab- and anorectic and exhibited fall in their blood sence of these metabolites and hypertension. There pressulre. Other clinical studies indicated that 374 H. M. GANDY, E. H. KEUTMANN AND A. J. IZZO quantities as large as 200 mg per day had to be administered 5 mg of desoxycorticosterone acetate given to induce detectable changes in sodium twice daily to 15 individuals for 15 days. The re- chloride balance (59). Still other investigators sponse to injection of epinephrine and norepineph- (60, 61), studying the metabolism of Compound S, rine was measured repeatedly before and after have given this orally and intramuscularly in doses treatment with desoxycorticosterone acetate. up to 1 g per day without noting alteration in While the resting pressures were not significantly blood pressure. altered, there was invariably an increase of pres- While there is no good evidence for etiologic sor response after pretreatment with desoxycorti- relationship between desoxycorticosterone per se costerone acetate. and most clinical hypertensive states, there is evi- Recently Liddle and associates (68) and Jenk- dence which indicates that there may be some cir- ins and associates (69) gave an 11,8-hydroxylase cumstances in which desoxycorticosterone can inhibitor (2-methyl- 1,2-bis [3-pyridyl] -1-propa- cause hypertension. Some authors think that none) to normal nonstressed persons. Compound when this occurs it is a consequence of the sodium S and other 11-desoxycorticosteroids were isolated retention caused by the hormone; others have pre- from the serum and large quantities of their me- sented data which contradict this. tabolites were isolated from the urine of these sub- In almost all studies involving the administra- jects. The blood pressure was not affected; it is tion of desoxycorticosterone to animals or man entirely possible that it would have been with there have been wide variations in the degree of longer administration or larger doses. Experience response by individuals or groups of individuals. with administration of desoxycorticosterone to pa- This was noted in the earliest reports concerning tients with Addison's disease certainly showed that, the use of this hormone in the treatment of Ad- when larger doses of this drug were given, it took dison's disease (62-64). The patients varied several months in some patients for the blood greatly in the amount of hormone esters required pressure to reach hypertensive levels. to alleviate the manifestation of adrenal insuffi- ciency. With the same doses wide variations SUM MARY were noted in sodium chloride and water retention, in body weight changes, edema formation, and The urinary steroid pattern of an untreated effects on blood pressure. normotensive 5.5 year old boy with congential When desoxycorticosterone was given to nor- virilizing adrenal hyperplasia has been studied. motensive, spontaneously hypertensive, and Gold- Abnormally large amounts of metabolites of blatt-hypertensive dogs, 9 out of 12 dogs developed Compound S and of desoxycorticosterone were hypertension; 3 did not (65). Perera and Blood found. This indicates that hydroxylation at C-11 (66) administered desoxycorticosterone to 10 of the steroid nucleus was impaired. normotensive and 15 hypertensive individuals while The patient also excreted normal or near nor- they were under strict metabolic control. The mal amounts of tetrahydro F, tetrahydro E, and normotensive individuals developed no change of other metabolites possessing an oxygen grouping basal blood pressure, while in the hypertensives at C-11. This is interpreted to indicate that the both systolic and diastolic pressures increased. patient was capable of hydroxylation at this posi- The authors were of the opinion that changes in tion when the adrenals were subjected to excessive body weight and salt balance were the same in stimulation by endogenous ACTH. both groups; therefore, they did not accept elec- Large amounts of metabolites of 17-hydroxy- trolyte retention as being the crucial factor re- progesterone were also found. This was inter- sponsible for the increase in blood pressure. preted to indicate a relative insufficiency of C-21- Some of the work by Davies and Clark (67) hydroxylation when this system was working un- suggests that increase of blood pressure after ad- der a load. ministration of desoxycorticosterone may not re- When hypertension accompanies the adreno- sult from a direct effect of the steroid on blood genital syndrome it probably indicates a defect in vessels, but the hormone may condition them for hydroxylation at C-11 of the steroid nucleus and an increased response to pressor substances. They resulting excessive manufacture of DOC. A nor- METABOLITES OF COMPOUND S AND DOC IN AN ADRENOGENITAL PATIENT 375 mal blood pressure does not rule out the existence 12. Lewis, R. A., and Wilkins, L. The effect of adreno- of this defect. The factors which determine the corticotropic hormone in congenital adrenal hy- difference in blood pressure response are not perplasia with virilism and in Cushing's syndrome treated with methyl testosterone. J. clin. Invest. known. 1949, 28, 394. 13. Bartter, F. C., Albright, F., Forbes, A. P., Leaf, A., ACKNOWLEDGMENTS Dempsey, E., and Carroll, E. The effects of The authors are grateful to Dr. Gilbert Forbes of the adrenocorticotropic hormone and cortisone in the Department of Pediatrics of Strong Memorial Hospital for adrenogenital syndrome associated with congeni- allowing them to study the patient. They are indebted to tal adrenal hyperplasia: An attempt to explain Drs. T. F. Gallagher and G. Roberts of the Sloan-Ketter- and correct its disordered hormonal pattern. J. ing Institute and Drs. A. Sandberg and R. Slaunwhite of dlin. Invest. 1951, 30, 237. the Roswell Park Memorial Institute for infrared spectra. 14. Dorfman, R. I. The adrenogenital syndrome in Tetrahydrodesoxycorticosterone was kindly supplied by Adrenal Function in Infants and Children, L. Dr. A. M. Bongiovanni. Gardner, Ed. New York, Grune and Stratton, 1956, p. 90. REFERENCES 15. Eberlein, W. R., and Bongiovanni, A. M. Partial characterization of urinary adrenocortical steroids 1. Wilkins, L. The diagnosis of the adrenogenital syn- in adrenal hyperplasia. J. dlin. Invest. 1955, 34, drome and its treatment with cortisone. J. Pediat. 1337. 1952, 41, 860. 16. Gandy, H. M. Partial characterization of Co steroids 2. Darrow, D. C. Congenital adrenal cortical insuffi- in the urines of children with the adrenogenital ciency with virilism; case report. Yale J. Biol. syndrome (abstract). J. dlin. Endocr. 1956, 16, Med. 1944, 16, 579. 948. 3. Barnett, H. L., and McNamara, H. Electrolyte bal- 17. Fukushima, D. K., and Gallagher, T. F. Steroid iso- ance in a male infant with adrenocortical insuffi- lation studies in congenital adrenal hyperplasia. ciency and virilism. The effect of desoxycortico- J. biol. Chem. 1958, 229, 85. sterone acetate and salt therapy with special refer- 18. Bongiovanni, A. M., and Eberlein, W. R. Defective ence to potassium. J. dlin. Invest. 1949, 28, 1498. steroidal biogenesis in congenital adrenal hyper- 4. White, F. P., and Sutton, L. E. Adrenogenital syn- plasia. Pediatrics 1958, 21, 661. drome with associated episodes of hypoglycemia. 19. Dyrenfurth, I., Sybulski, S., Notchev, V., Beck, J. C., J. dlin. Endocr. 1951, 11, 1395. and Venning, E. H. Urinary corticosteroid ex- 5. Shepard, T. H., II, and Clausen, S. W. Case of cretion patterns in patients with adrenocortical adrenogenital syndrome with hypertension treated dysfunction. J. dlin. Endocr. 1958, 18, 391. with cortisone. Pediatrics 1951, 8, 805. 20. Birke, G., Diczfalusy, E., 6. Williams, L., Plantin, L. O., Robbe, H., Crigler, J. F., Jr., Silverman, S. H., and Westman, A. Familial congenital hyperplasia Gardner, L. I., and Migeon, C. J. Further studies of the adrenal cortex. Acta Endocr. (Kbh.) 1958, on the treatment of congenital adrenal hyperplasia 29, 55. with cortisone. III. The control of hypertension with cortisone, with a discussion of variations in 21. Wilkins, L. The Diagnosis and Treatment of Endo- the type of congenital adrenal hyperplasia and re- crine Disorders in Childhood and Adolescence, 2nd port of a case with probable defect of carbohydrate- ed. Springfield, Charles C Thomas, 1957, p. 332. regulating hormones. J. dlin. Endocr. 1952, 12, 22. Jailer, J. W., Gold, J. J., Vande Wiele, R., and 1015. Lieberman, S. 17a-Hydroxyprogesterone and 21- 7. Eberlein, W. R., and Bongiovanni, A. M. Congeni- desoxyhydrocortisone; their metabolism and pos- tal adrenal hyperplasia with hypertension; unusual sible role in congential adrenal virilism. J. clin. steroid pattern in blood and urine. J. dlin. Endocr. Invest. 1955, 34, 1639. 1955, 15, 1531. 23. Izzo, A. J., Keutmann, E. H., and Burton, R. B. 8. Bongiovanni, A. M., and Eberlein, W. R. Clinical Special considerations in the use of blue tetrazolium and metabolic variations in the adrenogenital syn- for the measurement of alpha-ketols in mixtures drome. Pediatrics 1955, 16, 628. with other steroids. J. clin. Endocr. 1957, 17, 889. 9. Eberlein, W. R., and Bongiovanni, A. M. Plasma 24. Bongiovanni, A. M. The detection of pregnandiol and and urinary corticosteroids in the hypertensive pregnantriol in the urine of patients with adrenal form of congenital adrenal hyperplasia. J. biol. hyperplasia, suppression with cortisone. A pre- Chem. 1956, 223, 85. liminary report. Johns Hopk. Hosp. Bull. 1953, 10. Jailer, J. W. Virilism. Bull. N. Y. Acad. Med. 1953, 92, 244. 29, 377. 25. Burton, R. B., Zaffaroni, A., and Keutmann, E. H. 11. Kelley, V. C., Ely, R. S., and Raile, R. B. Hormone Corticosteroids in urine of normal persons de- patterns in patients with congenital adrenal hyper- termined by paper chromatography. J. biol. Chem. plasia. Pediatrics 1953, 12, 541. 1951, 193, 769. 376 H. 'M. G.\XDY. E. H. KFL'TAI..N'N\ AND A. J. IZZO) 26. Wilson, H., and Fairbanks, R. A micro-method for hydrocortisol (3a, 11,8, 17a-21 -tetrallydroxy-allo- the detection and assay of steroid C2,-17-hydroxy- pregnan-20-one) in lhuman urine. J. clin. Endocr. alpha-glycols. Arch. Biochem. 1955, 54, 457. 1957, 17, 434. 27. Talbot, N. B., Berman, R. W., and MacLachlan, E. A. 44. Bush, I. E., and Willoughby, M. The excretion of Elimination of errors in colorimetric assay of neu- allotetrahydrocortisol in humani uritne. Biochetmi. tral uriniary 17-ketosteroids by means of a color J. 1957, 67, 689. correction equation. J. biol. Chem. 1942, 143, 211. 45. Lieberman, S., Mond, B., and Smyles, E. Hydrolysis 28. \VilsoII, H., and Carter, P. Stabilization of the al- of urinary ketosteroid conjugates. Recent Progr. colholic potassium hydroxide in colorimetric 17- Hormone Res. 1954, 9, 113. ketosteroid determinations. Endocrinology 1947, 46. Saba, N., Hechter, O., and Stone, D. The conver- 41, 417. sion of cholesterol to pregnenolone in bovine adrenal 29. Brooks, R. V. A method for the quantitative frac- homogenates. J. Amer. chem. Soc. 1954, 76, 3862. tionation of urinary 17-oxosteroids with some ob- 47. Hechter, O., and Pincus, G. Genesis of adrenocorti- servations on steroid excretion during administra- cal secretioni. Physiol. Rev. 1954, 34, 459. tion of ACTH and in the adrenogenital syndrome. 48. Mason, A. S. Adrenal virilism: Abnormal response Biochem. J. 1958, 68, 50. to ACTH. Proc. roy. Soc. Med. 1951, 44, 162. Taylor, W. Isolation of oxo-steroids under neutral 30. 49. Christy, N. P., \Vallace, E. Z., and Jailer, J. W. The conditions. 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