PARTIAL CHARACTERIZATION OF URINARYADRENOCORTICAL STEROIDS IN ADRENALHYPERPLASIA

Walter R. Eberlein, Alfred M. Bongiovanni

J Clin Invest. 1955;34(8):1337-1343. https://doi.org/10.1172/JCI103181.

Research Article

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PARTIAL CHARACTERIZATIONOF URINARY ADRENOCORTICALSTEROIDS IN ADRENALHYPERPLASIA'

BY WALTERR. EBERLEIN AND ALFRED M. BONGIOVANNI

(From the Department of Pediatrics, University of Pennsylvania, and the Endocrine Divtision,Children's Hospital of Philadelphia, Philadelphia, Pa.)

(Submitted for publication December 13, 1954; accepted April 6, 1955)

Following the demonstration by Wilkins,Lewis, Klein, and Rosenberg (1) that cortisonecontrols the clinical manifestations and abnormallaboratory findings in virilizing adrenal hyper-plasia, Bartter, Albright, Forbes, Leaf, Dempsey,and Carroll (2) postulated a deficient synthesisof "sugar hormone" (17-hydroxycorticosterone,Compound F) in this disease, reasoning from theunusual response of such patients to the adminis-tration of adrenocorticotropin (ACTH). Thishypothesis has been supported by the demonstra-tion of low blood levels of 17-hydroxylated-corti-costeroids ("Porter-Silber chromogens") in un-treated patients with adrenal hyperplasia and theusual failure of these levels to rise, following ad-ministration of ACTH (3,4). The discoverythat, routinely, large quantities of pregnane-3a,17a,20a-triol are excreted in the urine of patientswith virilizing adrenal hyperplasia (5) has sug-gested a possible site of the metabolic block in thesynthesis of Compound F.

The present studies were undertaken to char-acterize the corticosteroids excreted in the urinein adrenal hyperplasia, in an attempt to definemore precisely the specific defect in steroid bio-synthesis.

METHODS

Twenty-four-hour collections of urine from normalcontrol subjects and from patients with adrenal hyper-plasia were investigated in the following manner:

Urine for 17-ketosteroid determinations was hydrolyzedfirst with beta-glucuronidase2 and manually extractedwith ether; the remaining urine was continuously ex-tracted at room temperature with ether for 36 hoursafter the addition of 10 volumes per cent (v/v) of 40per cent sulfuric acid. The combined extracts werefractionated on alumina columns (6) and suitable frac-

1 This work was made possible by a research grant(A-619 R) from the Division of Research Grants andFellowships, of the National Institutes of Health, UnitedStates Public Health Service.

2 "Ketodase," Warner-Chilcott Co.

tions were submitted to infra-red spectrophotometricanalysis.

The level of urinary neutral 17-ketosteroids producedby bismuthate oxidation ("17-ketogenic steroids") wasdetermined according to the method of Brooks andNorymberski (7).

The quantities of pregnane-3a,17a,20ca-triol were de-termined in urine hydrolyzed with beta glucuronidase2and chromatographed on alumina according to themethod of one of the authors (5).

Crude extracts of urine hydrolyzed with beta glu-curonidase were chromatographed on florisil (8). Thefraction eluted with 25 per cent methanol in chloroformwas examined by the following techniques: a) Thephenylhydrazine-sulfuric acid method of Porter and Sil-ber (9); b) periodate oxidation for the simultaneousdetermination of formaldehydogenic and acetaldehydo-genic steroids (10); c) phosphomolybdate method ofHeard and Sobel (11); d) blue tetrazolium method ofChen, Wheeler, and Tewell (12); e) 2,4-dinitrophenyl-hydrazine method of Gornall and Macdonald (13).

Urines collected from patients during therapy withcortisone were obtained two weeks to six months fol-lowing the introduction of the intramuscular adminis-tration of Compound E acetate in doses ranging from25 to 75 mg. given every third day. In each case, thespecimen studied was obtained at a time when the totalurinary 17-ketosteroids were significantly depressed (1).

Each of the methods described is designed to estimatethe quantities of steroids with functional groups as fol-lows. The determination of 17-ketosteroids formed bybismuthate oxidation measures only those compoundswith 17,20-dihydroxy, 21 methyl groupings. The phenyl-hydrazine-sulfuric acid method which determines so-called "Porter-Silber chromogens," measures 17,21-di-hydroxy-20-carbonyl side-chains only. Periodate oxida-tion of urine residues releases formaldehyde from thosecompounds with a 21-hydroxy group having either a20-hydroxy or 20-carbonyl substituent, regardless of thepresence or absence of a 17-hydroxyl function. Periodateoxidation will release acetaldehyde only from steroidswith 17,20-dihydroxy, 21-methyl side-chains and hencehas the same significance as the 17-ketogenic compoundsindicated by bismuthate oxidation. Both the phospho-molybdate and blue tetrazolium techniques measure re-ducing substances or steroids with an alpha-ketolic side-chain, although the former will also react with moleculespossessing an alpha-beta unsaturated system in ring A.The reagent, 2,4-dinitrophenylhydrazine, reacts with vari-ous carbonyl functions on the molecule, resulting in the

1337

WALTERR. EBERLEIN AND ALFRED M. BONGIOVANNI

formation of complexes showing various maxima between400 to 500 ma, depending on the number and location ofsuch groupings, as described by Gornall and Mac-donald (13).

PAPER CHROMATOGRAPHY

Twenty-four-hour collections of urine from a normalcontrol subject, a patient with Cushing's syndrome, anda patient with untreated adrenal hyperplasia were ex-tracted at neutral pH, and again after glucuronidasehydrolysis, with chloroform. The separate washed ex-tracts were fractionated on florisil columns, as above, andapplied to paper. The solvent systems modified afterBush (14) were: Benzene: 50 per cent methanol(B5); iso-octane-toluene: 70 per cent methanol (E1);and iso-octane: 90 per cent methanol (E2). Chromato-grams were equilibrated at 320 and developed at roomtemperature for 3 to 12 hours. The positions of Cpd.F, Cpd. E, pregnane-3a,llp,17a,21-tetrol-20-one (tetra-hydro-F), and pregnane-3a,17a,21-triol-11, 20-dione (tet-rahydro-E) were recognized by scanning chromatogramsunder ultraviolet light and staining central strips witha methanolic-sodium hydroxide solution of triphenyl-tetrazolium hydrochloride (TPZ), permitting simulta-neous detection of reducing steroids, steroids with thedihydroxyacetone side chain (15), and others with theA4-3-keto configuration of Ring A (16). The elutedareas were dried, acetylated, and rechromatographed inthe slower systems E1 and E2 against standards acety-lated in the same manner. Final identification restedon these criteria: Mobility of the free compound and itsacetate compared to standards on the same chromato-gram; the reactions with TPZ and NaOH; the fluor-

escence on paper of Cpd. F treated with concentratedsulfuric acid (17); the blue color given by Cpd. Etreated with iodine-potassium iodide (17); the fluorescentreactions of tetrahydro-F and tetrahydro-E treated with15 per cent phosphoric acid (18); the spectra of the fouracetates in sulfuric acid compared to those of pure com-pounds reported in the literature (17, 19).

RESULTS

Figure 1 illustrates a typical chromatographicstudy of the neutral 17-ketosteroids excreted bypatients with adrenal hyperplasia, before and dur-ing treatment with cortisone. It is clear that notonly is the total amount of 17-ketosteroids elevatedprior to treatment but that the quantities of 11-oxygenated-17 ketosteroids, identified as 11-keto-eti6cholanolone and 1 1-hydroxyandrosterone byinfra-red analysis, are disproportionately in-creased. In this laboratory the 11-oxygenatedcompounds were found to constitute about 10per cent of the total neutral 17-ketosteroids ex-creted by adult male and female controls and 18to 25 per cent of the total in individuals withadrenal hyperplasia.

The results of bismuthate oxidation are indi-cated in Figure 2 for six patients with untreatedadrenal hyperplasia, compared to nine normalcontrols. The 24-hour excretion of pregnane-3a,

CHROMATOGRAPHIC (COLUMN) PATTERN Of ALPHA NEUTRAL 17-KSOf PATIENT WITH ADRENOGENITALSYNDROME

FIG. 1.The ordinate indicates milligrams of 17-ketosteroids per fraction and the abscissa,

the individual fractions in order of their elution from the column. The solid lineillustrates the results before treatment and the dotted line during treatment withcortisone.

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STEROIDS IN VIRILIZING ADRENALHYPERPLASIA

MG.//AY

40

30-

20

10

UNDERI YEAR

2 -34 YEARS

FIG. 2. TWENTY-FOUR-HOUR EXCRETION IN THEURINE OF 17-KETOSTEROIDS LIBERATED BY BISMUTHATEOXIDATION.

The first 4 "normal" columns and the first 2 "adrenalhyperplasia" columns represent children under the ageof nine years. The black columns represent the excre-

tion of these substances during the administration ofcortisone.

17a,20a-triol is indicated in similar fashion inFigure 3.

Table I lists the results of the various assay

methods applied to the steroid fractions obtainedfrom florisil columns. The fractions employedwere free of 17-ketosteroids but contained crystal-line mixtures of several C-21 steroids. It isevident that the amount of acetaldehydogenicsteroids excreted in adrenal hyperplasia is more

than 10 times the amount found in the urine ofnormal controls. These patients also excrete

FIG. 3. TWENTY-FOUR-HOUREXCRETION OF PREG-NANE-3a,17a,20a-TRIOL IN FIvE NORMALADULTS, CON-TRASTED WITH CHILDREN AND ADULTS WITH ADRENALHYPERPLASIA

The suppressed excretion during cortisone treatmentis indicated for two individuals.

greater than normal quantities of material de-tected by the phosphomolybdate reagent.

Figures 4 and 5 illustrate in condensed form theresults of a series of 27 chromatograms run on ex-

tracts of unhydrolyzed and glucuronidase-hydro-lyzed urine from a patient with adrenal hyper-plasia, compared to a normal control subject andan individual with Cushing's syndrome. No Com-pound F or Compound E was detected in the un-

hydrolyzed extract of the patient with adrenalhyperplasia: 30 liters of urine from another pa-

tient so studied also contained no detectable

BLE I

Twenty-four-hour excretion of corticosteroids in urine *

Normal Adrenal hyperplasia

No. Average Range No. Average RangeCorticosteroid assay method subjects mg. mg. subjects mg. mg.

Phenylhydrazinet 7 8.5 3.8-13.1 7 6.1 3.8-8.6Phosphomolybdatet 7 13.0 5.5-24.4 7 29.7 19.8-44.0Blue Tetrazoliumt 4 6.5 2.9-9.0 4 7.7 5.0-10.62,4-Dinitrophenylhydrazinet 5 8.8 2.5-18.4 8 4.5 1.8-11.8

mcg. mcg. mcg. mcg.

Formaldehydogenict 8 374 210-671 8 371 235-618Acetaldehydogenict 7 179 50-456 7 2,014 1,249-3,244

* Comparison is made of the urinary excretion by normal individuals and untreated patients with virilizing adrenalhyperplasia, of corticosteroids measured by the procedures described. These are to be contrasted with the findingsobtained by paper chromatography (Figures 4 and 5) as described in the text.

t Calculated as milligrams of Cpd. E.I Micrograms of formaldehyde or acetaldehyde released by periodate oxidation.

PREGNANETRIOL

NORMALADULTS OADRENAL HYPERPLASIA

CONTROL0CORTISONE U

1339

WALTERR. EBERLEIN AND ALFRED M. BONGIOVANNI

amount of these substances. Although tetrahy-dro-E was present in the hydrolyzed extract ofthis patient, the amount was small compared tothe normal.

The results are in contrast to the findings inTable I that urine residues from patients withvirilizing adrenal hyperplasia contain normalquantities of steroids detected by phenylhydrazine,blue tetrazolium and formaldehydogenic methods.Each of the latter methods will measure com-pounds E or F and their reduced derivatives. Butin view of the results obtained by paper chroma-tography, it is apparent that the substances meas-ured by the methods shown in Table I are notCompounds E or F or their reduced metabolites,as is the case in the urine of normal individuals.Nonetheless, unidentified 21-hydroxylated ster-oids are present in the urine of untreated patientswith adrenal hyperplasia.

FIG. 4. CONDENSATION OF MULTIPLE PAPER CHRO-MATOGRAMSRUNON EXTRACTSOF UNHYDROLYZEDURINEFROMA NORMALSUBJECT, A PATIENT WITH CUSHING'SSYNDROME,AND A PATIENT WITH VIRILIZING ADRENALHYPERPLASIA TO ISOLATE COMPOUNDF, COMPOUNDE,AND THEIR TETRAHYDROMETABOLITES

The first column indicates the relative position on thechromatograms of the 4 steroids. The rectangles incolumns 2 to 4 indicate the relative amount of the in-dividual steroid excreted, estimated by spot size andstaining intensity on the original chromatograms. Notethe excess excretion of the 4 steroids in Cushing's syn-drome and the complete absence of all in adrenal hyper-plasia.

FIG. 5. CONDENSATIONOF MULTIPLE CHROMATOGRAMSOF GLUCURONIDASE-HYDROLYZEDURINE AFTER INITIALEXTRACTIONAT NEUTRALPH

Patients and symbols as in Figure 4. Note the smallexcretion of tetrahydro-E in adrenal hyperplasia, theexcessive amount of the same steroid in Cushing'ssyndrome.

DISCUSSION

The present investigation of the excretion ofCompound F, Compound E, and their known end-metabolites, tetrahydro-F and tetrahydro-E, stud-ied by means of paper chromatography, firmlysupports the theory that there is a deficient syn-thesis of Compound F in adrenal hyperplasia. Atthe same time, and in agreement with previousstudies on the response of the blood "Porter-Silber chromogen" levels to ACTH administra-tion (4), the findings indicate that at least someof these patients can synthesize a small amountof Compound F, evidenced by the excretion ofa small amount of tetrahydro-E derived fromCompound F.

The most characteristic, and hence diagnostic,laboratory finding in virilizing adrenal hyper-plasia remains the excretion of large amounts ofpregnane-3a,17a,20a-triol in the urine. Thissteroid and its recently isolated relative, pregnane-3a,17a,20a-triol-11-one (20), appear to accountfor the excessive amounts of 17-ketosteroids liber-ated by bismuthate oxidation and for the largeamounts of acetaldehydogenic steroids detected

lOOmi. URINE AFTERGLUCURONIDASE

STANDARDS CUSNING'S ADRENALSTAN*DARDS USYNDROME NORMAL NYPERPLASIA

|TE._<|r"- I

VTRMMRO-E] I I

r CPOF I

I EZIZ

1200ml.URINE. EXTRACTEDpH 7.0

STANDARDS GUSHING'S NORMAL ADRENALSYNDROME L HYPERPLASIA

1" l I I

|CPD.Ft|EFEJEEI~~'EI I

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STEROIDS IN VIRILIZING ADRENALHYPERPLASIA

by periodate oxidation in the urine of patientswith virilizing adrenal hyperplasia. These resultsare interpreted to indicate a block in the synthesisof Compound F, according to the scheme ofHechter, Zaffaroni, Jacobsen, Levy, Jeanloz,Schenker, and Pincus (21), at some point be-tween 17-hydroxyprogesterone and Compound F,since pregnane-3a,17a,20a-triol is probably a de-gradation product of 17-hydroxyprogesterone(22, 23).

The results of the various methods used inthis study to characterize the corticosteroids ex-

creted in adrenal hyperplasia do not specificallyindicate the nature of the defect in Compound Fsynthesis. According to Hechter's scheme, basedupon adrenal perfusion experiments, cholesterol,under the stimulus of ACTH, is converted viapregnenolone into progesterone, which is thensuccessively hydroxylated at carbons 17, 21, and11 to yield Compound F. It has been shown thatadrenal tissue contains enzymes capable of in-troducing hydroxyl groups into suitable sub-strates at these three positions in vitro (24, 25),although none of these enzymes has as yet beenisolated in a pure state. It has already beenshown that endogenous ACTHproduction is highin virilizing adrenal hyperplasia (26), hence one

cannot incriminate the absence of an adequatestimulus to Compound F production. Judgingfrom the present study, there is no deficiency ofthe enzyme which regulates 17-hydroxylation.The excretion of normal quantities of steroidspossessing an alpha-ketolic side chain, measuredby formaldehyde liberation, as well as the bluetetrazolium and Porter-Silber methods, does notreadily suggest a deficiency of a 21-hydroxylatingenzyme. Nor does the presence in the urine oflarge amounts of 1 1-oxygenated-17-ketosteroidsand of pregnanetriol-11-one point to the lack ofan 11-hydroxylating enzyme.

Indeed, in crude extracts of urine from patientswith adrenal hyperplasia one can detect normal or

excessive amounts of steroids, which in the ag-

gregate possess all the necessary molecular con-

figurations of Compound F itself. Unidentifiedsteroids unsaturated in Ring A are demonstrableon paper chromatograms and presumably accountfor the high levels detected with the phospho-molybdate reagent.

The possibility of an abnormal metabolism ofpre-formed Compound F may be ruled out by theease with which small doses of Compound F orE reverse the clinical and chemical abnormalitiesfound in the untreated disease. In addition, Com-pound E administered to patients with the diseasecauses the expected increased excretion in theurine of its known end-metabolite, tetrahydro-E(27).

Since it appears that the adrenal in this diseasecan synthesize 17-hydroxyprogesterone, as meas-ured by excessive pregnane-3a,17a,20a-triol ex-cretion, but cannot produce an adequate amountof Compound F, a deficiency in the enzymes con-cerned with hydroxylation either at carbon 11 orat carbon 21 might be postulated. The presentstudy offers several objections to the theory of"111,-hydroxylase" deficiency in adrenal hyper-plasia. Since 1 1-hydroxylation follows 21-hy-droxylation (21), one would not expect a blockat this site to lead to the marked excretion ofpregnane-3a,17a,20a-triol but rather of 21-hy-droxylated steroids, such as 17-hydroxy-11-des-oxycorticosterone and its reduced derivative, whichshould be measurable as alpha-ketolic steroidsboth in blood and urine. The latter compoundshave not so far been detected in the urine orblood of patients with virilizing adrenal hyper-plasia.

Another objection to "l l/-hydroxylase" lackis the excess excretion of 11-oxygenated steroidswhich occurs. In respect to this, however, inadrenal perfusion experiments, but not in studiesusing adrenal homogenates, some li-hydroxyla-tion of 21-desoxy steroids, such as progesterone,can occur (21). This suggests that in the intactadrenal there may be normally two enzyme sys-tems concerned with 11,8-hydroxylation, one de-pendent upon, and the other independent of, priorhydroxylation at carbon 21. If this is true, the11-oxygenated steroids excreted in adrenal hyper-plasia might derive primarily from 21-desoxy pre-cursors through the action of the latter enzyme.

Dorfman (28) has suggested a deficiency ofa 21-hydroxylating enzyme in adrenal hyper-plasia. The theory is compatible with the highexcretion of pregnane-3a,17a,20a-triol in the urineand the low levels of Porter-Silber chromogens inthe blood. The present study reveals that pa-

1341

WALTERR. EBERLEIN AND ALFRED M. BONGIOVANNI

tients with this disease excrete approximately thesame amount of alpha-ketolic (i.e., 21-hydroxyl-ated) steroids as do normal individuals, and thatmost of these steroids detected with the Porter-Silber reagent are hydroxylated at carbon 17, al-though some may have 16-hydroxyl groups (29).Since endogenous ACTHproduction is high, theamount of alpha-ketolic steroids excreted may berelatively low. But it is difficult to explain why,in the presence of more than adequate amountsof enzymes capable of inducing hydroxylation atcarbon 11, the adrenal cortex is unable to synthe-size more respectable quantities of Compound Ffrom precursors already oxygenated at C-17 andC-21. In other words, the amount of CompoundF and its metabolites constitutes a significantlysmaller proportion of the total alpha-ketolic ster-oids excreted in the urine of subjects with thisadrenal disorder than in normal controls.

The present study, therefore, does not supportthe theory of either a single "11,8-hydroxylase"or "21-hydroxylase" deficiency in adrenal hyper-plasia, reasoning from the current concepts ofsteroid biosynthesis in the adrenal cortex. Thepresumed inborn error of metabolism is apparentlymore complicated and may well involve a combina-tion of enzymes and their cofactors. Work inprogress in this laboratory, including final iden-tification of the alpha-ketolic steroids excreted inthe urine, may help to clarify the underlying dis-turbance of Compound F synthesis.

SUMMARY

A study, utilizing both paper and columnchromatographic techniques, revealed an abnor-mally low excretion of Compound F and itsmetabolites in the urine of patients with adrenalhyperplasia. This strongly supports the conceptof a deficient synthesis of Compound F by the dis-eased adrenal cortex. The characteristic excre-tion of pregnane-3a,17a,20a-triol in large quan-tities suggests a deficient conversion of 17-hy-droxyprogesterone into Compound F. Largeamounts of steroids oxygenated at C-i1 and atC-17 and "normal" quantities of 21-hydroxylatedsteroids were detected in the urine of several af-fected individuals. The nature of the underly-ing enzymatic defect in the synthesis of Com-pound F in this disease awaits precise definition.

Addendum

A patient with congenital adrenal hyperplasia compli-cated by hypertension, since studied, excreted in theurine predominantly tetrahydro-S (pregnane-3a,17a,20-triol-20-one, confirmed by infra-red analysis), a findinghitherto unreported in this disease. Complete absencein the urine of the normal 11-oxygenated C-21 and 17-ketosteroids suggests "l1,B-hydroxylase" deficiency inthis form of the disease. (Eberlein, W. R., and Bongio-vanni, A. M., J. Clin. Endocrinol. & Metab., Submittedfor publication.)

ACKNOWLEXGMENTS

The authors acknowledge with pleasure the helpfulsuggestions of Doctors Lawson Wilkins and NathanTalbot. The infra-red analyses were performed throughthe kindness of Doctor Seymour Lieberman. Samples ofpure steroids were generously supplied by Doctors JohnSchneider, Hirsch Sulkowitch, Harold Mason, The G. D.Searle Company, The Squibb Institute for MedicalResearch and Merck and Company.

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2. Bartter, F. C., Albright, F., Forbes, A. P., Leaf, A.,Dempsey, E., and Carroll, E., The effects ofadrenocorticotropic hormone and cortisone in theadrenogenital syndrome associated with congenitaladrenal hyperplasia: An attempt to explain andcorrect its disordered hormonal pattern. J. Clin.Invest., 1951, 30, 237.

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5. Bongiovanni, A. M., and Clayton, G. W., Jr., Asimplified method for the routine determinationof pregnanediol and pregnanetriol in urine. Bull.Johns Hopkins Hosp., 1954, 94, 180.

6. Dingemanse, E., Huis in't Veld, L. G., and Hartogh-Katz, S. L., Clinical method for the chromato-graphic-colorimetric determination of urinary 17-ketosteroids. II. Normal adults. J. Clin. Endo-crinol. & Metab., 1952, 12, 66.

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STEROIDS IN VIRILIZING ADRENALHYPERPLASIA

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13. Gornall, A. G., and Macdonald, M. P., Quantitativedetermination of the steroid hormones with 2,4-dinitrophenylhydrazine. J. Biol. Chem., 1953, 201,279.

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15. Axelrod, L. R., A specific test differentiating betweena-ketol and dihydroxy-acetone groups of Cft-steroids on paper chromatograms. J. Am. Chem.Soc., 1953, 75, 4074.

16. Bush, I. E., and Sandberg, A. A., Adrenocorticalhormones in human plasma. J. Biol. Chem., 1953,205, 783.

17. Zaffaroni, A., and Burton, R. B., Identification ofcorticosteroids of beef adrenal extract by paperchromatography. J. Biol. Chem., 1951, 193, 749.

18. Neher, R., and Wettstein, A., tTber Steroide. Far-breaktionen mit Steroiden, insbesondere Corticos-teroiden, im Papierchromatogramm. Helvet. chim.acta, 1951, 34, 2278.

19. Bernstein, S., and Lenhard, R. H., The absorptionspectra of steroids in concentrated sulfuric acid.I. Method and data. J. Org. Chem., 1953, 18, 1146.

20. Finkelstein, M., Euw, J. V., and Reichstein, T.,Isolierung von 3a,17,20a-trioxy-pregnanon- (11)aus pathologischem menschlichem Ham. Helvet.chim. acta, 1953, 36, 1266.

21. Hechter, O., Zaffaroni, A., Jacobsen, R. P., Levy, H.,Jeanloz, R. W., Schenker, V., and Pincus, G., Thenature and the biogenesis of the adrenal secretoryproduct. Rec. Prog. Hormone Res., 1951, 6, 215.

22. Mason, H. L., and Kepler, E. J., Isolation of steroidsfrom the urine of patients with adrenal corticaltumors and adrenal cortical hyperplasia: A new17-ketosteroid, androstane-3a,11-diol-17-one. J.Biol. Chem., 1945, 161, 235.

23. Bongiovanni, A. M., Unpublished data.24. Hayano, M., Dorfman, R. I., and Yamada, E. Y.,

The conversion of desoxycorticosterone to glyco-genic material by adrenal homogenates. J. Biol.Chem., 1951, 193, 175.

25. Plager, J. E., and Samuels, L. T., Enzyme systeminvolved in the oxidation of carbon-21 in steroids.Federation Proc., 1952, 11, 383.

26. Sydnor, K. L., Kelley, V. C., Raile, R. B., Ely, R. S.,and Sayers, G., Blood adrenocorticotrophin inchildren with congenital adrenal hyperplasia.Proc. Soc. Exper. Biol. & Med., 1953, 82, 695.

27. Eberlein, W. R., Unpublished data.28. Dorfman, R. I., The metabolism of adrenal steroids

in Adrenal Cortex. Transactions of the FifthConference, 1953, New York, Josiah Macy, Jr.Foundation, 1954, p. 27.

29. Mattox, V. R., Mason, H. L., and Albert, A., Isola-tion of a sodium-retaining substance from beefadrenal extracts. Proc. Staff Meet., Mayo Clin.,1953, 28, 569.

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