STUDIES ON PYROGENICSTEROIDS. I. SEPARATION, IDENTI-FICATION, AND MEASUREMENTOF UNCONJUGATED

DEHYDROEPIANDROSTERONE,ETIOCHOLANO-LONE, ANDANDROSTERONEIN HUMAN

PLASMA*

By GEORGEL. COHN,t PHILIP K. BONDYAND CARMELACASTIGLIONE

(From the Department of Internal Medicine, Yale University School of Medicine,New Haven, Conn.)

(Submitted for publication June 13, 1960; accepted October 20, 1960)

Previous studies have shown that endotoxin(1-4), viruses (5-7), and pyrogenic substancesfrom polymorphonuclear leukocytes (8-11) arecapable of producing fever. A different group ofpyrogenic substances was described by Kappas,Soybel, Fukushima and Gallagher (12), who re-ported that certain C19 and C21 steroids with a3a-hydroxy, 5/3 configuration produce fever. Theprototype compound is etiocholanolone 1 (13-16).Four to 8 hours after intramuscular or intrave-nous administration of this compound to volun-teers, the temperature began to rise, reached apeak (1030 to 1050 F) at 12 hours, and gener-ally subsided within 24 hours. Chills, anorexia,headache, malaise, myalgia, arthralgia, and leuko-cytosis accompanied the reaction.

Recently, Bondy, Cohn, Herrmann and Crispell(16) demonstrated unconjugated 2 etiocholano-lone in the plasma of two patients during febrileattacks of etiocholanolone fever.3 When the pa-tients were afebrile and asymptomatic, unconju-gated plasma etiocholanolone was not detected.

* Supported by Grants 254 (C-7) and A-4222 from theNational Institutes of Health, Bethesda, Md.

t Advanced Research Fellow of the American HeartAssociation.

1 The following trivial names and abbreviations areused: etiocholanolone, ETIO (3a-hydroxyetiocholane-17-one) ; androsterone, ANDRO(3a-hydroxyandrostane-17-one); dehydroepiandrosterone, DHEA (3,8-hydroxy-5-androstene-17-one); etiocholanolone fever, EF; familialMediterranean fever, FMF.

2 The term uiconjugated refers to those steroids ex-tracted with an organic solvent (viz., chloroform) with-out previous hydrolytic procedures.

3 It was suggested that this type of fever be calledetiocholanolone fever in order to distinguish this syn-drome from familial Mediterranean fever on the onehand and the undifferentiated periodic fevers on the other(17).

The 24-hour urinary 17-ketosteroid patterns werenormal when the patients were without symptoms.However, etiocholanolone was the only major 11-deoxy-17-ketosteroid isolated during the sympto-matic interval.

Since the plasma clearance of 11-deoxy-17-ketosteroids, such as androsterone and etiocho-lanolone (18) is rapid (20 minute half-life), previ-ous attempts to recover these steroids and dehy-droepiandrosterone in an unconjugated form fromplasma of normal subjects has been discouraging,since at most, only trace amounts were present(19, 20).

However, Savard (21) was able to isolate andidentify (but not measure) unconjugated andros-terone from 100 L of extracorporeal dialysis fluidobtained from a 22 year old patient with acutetubular necrosis and anuria. Moreover, Tamm,Beckmann and Voigt (22) analyzed 200 to 300nil of normal human sera and found 17 to 61 ,ugper 100 ml of total unconjugated 17-ketosteroids,measured by micro-Zimmermann reaction, afterchloroform extraction, NaOHwash, and Girardseparation. However, no separation of 11-oxy-and 11-deoxy-17-ketosteroid was attempted. Onthe other hand, Migeon (23) extracted 500 ml ofnormal human plasma and reported that "therewas probably not more than 1 wg per 100 ml eachof unconjugated androsterone and dehydroepi-androsterone."

In the present study unconjugated plasma 17-ketosteroids were measured in normal subjectsand in patients with fever of known origin, familialMediterranean fever and etiocholanolone fever.Unconjugated plasma androsterone, dehydroepi-androsterone, and etiocholanolone were separated,measured and identified as 2,4-dinitrophenylhy-drazone derivatives. An isotope dilution method

400

UNCONJUGATEDNEUTRAL17-KETOSTEROIDS IN HUMANPLASMA

with tritium-labeled etiocholanolone, an(lrosterone.and dehydroepiandrosterone was used to correctfor losses during the procedures. The method issufficiently sensitive to be useful in measuring un-

conjugated neutral 17-ketosteroids in plasma.

MATERIALS AND METHODS

Subjects

1. Afebrile subjects. Five hundred ml of heparinizedblood was collected between 8 and 10 a.m. from 4 healthymen and 2 healthy women, ranging in age from 23 to 45years. The plasma was separated by centrifugationwithin 15 minutes of collection and frozen until analyzed.Ten determinations were performed on 110- to 120-mlportions of this plasma. In addition, 3,000 ml of freshplasma, obtained from untreated patients with polycy-themia vera, was used for isotopic and chemical recovery

experiments.2. Febrile patients. Unconjugated neutral 17-ketoster-

oids were measured on 12 hospital inpatients with fever(101° F or higher) of known origin. The clinical dataare listed in Table I. Heparinized blood was obtained atthe time of the indicated temperature elevation. Theplasma was frozen until processed. Liver and kidneyfunction tests and an endocrinological evaluation revealedno abnormalities. M.J. (ulcerative colitis, Table I) hadreceived no steroid therapy for at least 3 months prior toentry to the hospital. None of the other patients hadever received steroids.

3. Patients zwith etiocholanolone fever and familialMediterranean fever. The clinical data are listed in TableII. The patients with familial Mediterranean feverpresented the classical family history, signs and symp-toms described by Heller, Sohar and Sherf (24) andLawrence and Mellinkoff (25). The patients with etio-cholanolone fever were not of Mediterranean or MiddleEastern origin. Repeated cultures, skin tests, and pre-

cipitin and agglutination tests for a wide variety of in-fectious agents were unrevealing. Liver function tests,lymph node and liver biopsies were within normal lim-its. Total and differential white blood counts were

normal except during acute febrile attacks, when a poly-morphonuclear leukocytosis with a shift to the left(without eosinophilia) usually occurred.

Plasma samples were collected during the prodrome ofan attack, while the temperature was rising (1000 to102° F). Plasma was also collected during an afebrileand asymptomatic interval when the patients were notreceiving therapy. These plasma samples were alsofrozen until analyzed.

Preparation of radioactive 17-ketosteroids for isotopedilution

Two mg of etiocholanolone, mp 149° to 151° C, and2.5 mg of androsterone,4 mp 1810 to 183° C, were eachexposed to 3 curies of tritium at 27° C and 0.39 atm for14 days.5

The readily labile tritium was removed by a modifica-tion of a method described by Peterson (27) using 250to 500 ml of methanol. After the methanol was removedby vacuum distillation, the dry impure steroid was dis-solved in 20 ml of freshly distilled chloroform. One mlof 0.05 N NaOH was added, mixed, and the alkalinechloroform extract washed three times with 1 to 2 mlof distilled water. The aqueous phase was carefully re-moved by aspiration and the chloroform extract evapo-rated to dryness under nitrogen. The residue was dis-solved in 1 ml of ethyl acetate: methanol (2: 1) andapplied as a streak adjacent to reference compounds ofandrosterone and etiocholanolone on washed (28) What-man no. 1 chromatographic papers in the heptane: propy-

4 Etiocholanolone and androsterone were generouslydonated by Dr. Seymour Lieberman, Dept. of Biochem-istry, Columbia University, New York, N. Y.

5 The Wilzbach procedure (26) was performed by theNew England Nuclear Corp., Boston, Mass.

.E1 I

Clinical data in patients with fever of known origin

Patient Sex Age Diagnosis Rectal temp. Vol. of plasma

yrs OF* mlM.S. F 22 Viral pneumonia 105 28B. B. F 28 Acute follicular-tonsillitis 106 63L.C. M 29 Acute cholecystitis 103 41H.J. F 33 Viral pneumonia 102.5 40M.R. M 36 Acute gasteroenteritis 104 55E.N. F 44 Malignant melanoma 101.2 45M.J. F 45 Ulcerative colitis 101 65E.W. M 51 Bronchopneumonia 103 45S.G. F 61 Lung abscess 102 50A.C. M 64 Acute appendicitis 103 55M.I. M 68 Chronic bronchitis; emphysema 101 47M.Y. F 73 Bronchopneumonia 103 33

* Plasma samples were obtained at the time of the indicated temperature. No sample contained as much as 0.5 Agetiocholanolone per 100 ml of plasma.

401

GEORGEL. COHN, PHILIP K. BONDYAND CARMELACASTIGLIONE

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Constant specific activity was observed following re-verse isotope dilution of the free tritium-labeled steroidsusing paper chromatography, and column chromatog-raphy of the 2,4-dinitrophenylhydrazone derivatives (32)on 3.9 and 5.0 per cent alumina micro-columns as de-scribed below (Table III). The constant specific ac-tivities were interpreted as evidence for the radiochemi-cal purity of the steroids.

Plasnmta 17-ketosteroids

Separatiot. Approximately 5,000 cpm (0.2 ml) oftritium-labeled etiocholanolone (5.2 ,uc per ,ug), andros-terone (2.6 /xc per Lg), and dehydroepiandrosterone (4.4,uc per uog) were pipetted into a 250 ml centrifuge tube andevaporated to dryness. From 45 to 50 ml of plasma wasadded and the tube was shaken for about 2 minutes to in-sure complete mixing of the tritium-labeled steroids andthe plasma. Two ml of 1 N NaOHper 40 ml of plasmawas added, mixed, and the plasma extracted three timeswith 2 vol of freshly distilled chloroform. Emulsionswere broken by centrifugation. The chloroform extractwas dried in vacuio and transferred quantitatively withchloroform to washed Whatman no. 1 filter paper in theheptane: 96 per cent methanol system of Bush and Wil-loughby (30). Twenty-five Ag each of reference com-pounds androsterone, etiocholanolone, and dehydroepi-androsterone were applied together in a single spot, oneach side of the extract area. A sheet of filter paper19 cm wide conveniently accommodated three referencearea spots and two extract spots. The paper was equili-brated for 4 hours and the mobile phase allowed to de-

6 Paper chromatography is carried out in a room withconstant temperature of 200 C and relative humidity ofless than 50 per cent.

Delhydroepiandrosterone-7a-H3-acetate, SA 2.46 mcper mg, lot no. 15-258-9, from New England NuclearCorp.

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UNCONJUGATEDNEUTRAL17-KETOSTEROIDS IN HUMANPLASMA

TABLE III

Specific activities of standard tritium-labeled 17-ketosteroids following reverse isotope dilution

Specific activity

Method Solvent system DHEA ETIO ANDRO

cpm/1.gFree compounds*

Paper chromatographyHeptane: 96% methanol (1:1) 57,781 74,475 78,024

2,4-Dinitrophenyihydrazone derivativesColumn chromatography

3.9% Alumina Benzene: chloroform (4:1) 58,685 77,369 82,4505.0% Alumina Benzene: chloroform (4:1) 59,700 77,694 83,390

* The radioactive steroids were separated by paper chromatography after the addition of 10 jug of each referencestandard. The tritium-labeled free steroids were converted to the 2,4-dinitrophenylhydrazones and measured spectro-photometrically. Radioactivity was determined with corrections for quenching. The (formed)steroid 2,4-dinitrophenyl-hydrazones were rechromatographed on columns and specific activity determined as described.

velop for 12 to 14 hours, after which the paper was air-dried. The three standard strips were cut off and thereference compounds located by staining with Zimmer-mann reagent, according to the technique of Migeon andPlager (20). Appropriate areas corresponding to thereference standards and paper blanks were cut out, placedin glass-stoppered test tubes, and eluted with 4 ml of ab-solute ethanol for 45 minutes at 370 C. Paper blankswere usually obtained below the androsterone (least polarcompound) area. However, if the androsterone mi-grated to the bottom of the paper, a paper blank wastaken either above the dehydroepiandrosterone (mostpolar compound) area, well away from any ultraviolet-absorbing material or below the etiocholanolone area.

Measurement. Two-ml portions of the eluates werepipetted into acid-cleaned 15 X 85 mmPyrex tubes anddried in vacuo for the 2,4-dinitrophenylhydrazine reaction(32). The samples were dissolved in 0.5 ml of absoluteethanol and 0.04 ml of freshly prepared 2,4-dinitrophenyl-hydrazine reagent (2.0 mg per ml of absolute ethanolacidified with 0.05 ml 12 N HCI) was added. The mix-ture was allowed to stand overnight at room temperature.The following day, 0.5 ml distilled water and 0.5 mlBenedict's reagent were added. The tubes were heatedfor 15 minutes in a vigorously boiling water bath, and,after cooling, 0.5 ml of freshly distilled chloroform wasadded. The tubes were stoppered quickly to prevent evapo-ration of the chloroform. Transfer of the steroid-dini-trophenylhydrazone to the chloroform layer was facili-tated by vibrating the test tube containing the Benedictreagent: chloroform mixture for 1 minute with a Dremelhand drill, model 2 (Dremel Mfg. Co., Racine, Wis.),which was fitted with a one-eighth inch rod-like bit, bentat a 20° to 25° angle. The tubes were allowed to standat room temperature for 1 hour. The aqueous layer wascarefully removed by aspiration. The chloroform layer(0.5 ml) was transferred to 0.8 ml microcuvets with 1.0cm light path, and absorbance was measured at 368 mgin a Beckman model DU spectrophotometer. A standardcurve was constructed on each occasion with etiocholano-lone-2,4-dinitrophenylhydrazone. Since androsterone and

dehydroepiandrosterone-2,4-dinitrophenylhydrazones. havethe same absorbance maxima and extinction coefficients at368 miu (32), these compounds may be substituted inconstructing the standard curve. The absorbance fol-lowed Beers' law from 0.3 Mug (A = 0.030) to 3.0 jug (A =0.300). The optical density of the samples was at least2.5 to 4 times that of the paper blanks.8

A 0.5 ml portion of the ethanol eluate was counted toa standard error of ± 2 per cent in a liquid phosphorscintillation counter (Technical Measurements Co., modelLP-2, New Haven, Conn.) which had a counting effi-ciency of 13 to 15 per cent for tritium. Since aliquots ofthe eluates from the paper blanks had only backgroundradioactivity, no radioactive correction for paper blankswas necessary. The final value was calculated, takinginto account the absorbance of the blank, the dilutionfactor, and the isotope recovery (28).

RESULTS

Plasma determinations. Normal values andisotopic and chemical recoveries are listed in TableIV. Twelve patients with fever of known cause(Table I) had less than 0.5 ,tg per 100 ml uncon-jugated etiocholanolone, dehydroepiandrosteroneand androsterone.

8 It has been observed that the steroid 2,4-dinitrophenyl-hydrazones can be further purified by the method of micro-alumina chromatography. In this way, the mean +

standard deviation elution blank readings at 368 myu areA = 0.005 + 0.002. After chemical measurements areperformed, radioactive recovery is determined by directplating at infinite thinness of the steroid-2,4-dinitrophenyl-hydrazones on planchets and counting the tritium in awindowless gas-flow counter (Automatic SampleChanger, model C 100 B, Nuclear-Chicago Corp., DesPlaines, Ill.). The efficiency for tritium is 28 to 30 percent. This modification has increased the sensitivity from0.5 ,Ag to 0.2 /Ag per 100 ml.

403

GEORGEL. COHN, PHILIP K. BONDYAND CARMELACASTIGLIONE

TABLE IV

Normal unconjugated plasma 17-ketosteroid values; isotopicand chemical recoveries

Isotopic Chemicalrecovery [92] recovery [121t

Range [101* Mean ±SD

Pg/loo ml % >DHEA <0.5$ - 1.2 70 i 10.4 81.0 ± 21.8ETIO <0.5 - 1.2 78 i 13.0 66.7 :1 19.6ANDRO <0.5 - 0.8 67.6 + 18.6 57.5 :1 21.8

* Figures in brackets represent the number of determinations per-formed. The normal range represents 10 measurements performed onthe plasma of 6 volunteers.

t Twelve experiments were performed for each of the steroids, withthe following amounts of reference compounds added to 100 to 200 mlof plasma: 1 lug [5]; 2 ,ug [3]; 5 pug [2]; 10 pg [l]; 20 pg [1]. Theaccuracy of the method is reproducible at the 1.0 pug per 100 ml level(mean -SD = 1.0 40.25).

$ The sensitivity of the method permits 0.5 pug to be interpolated fromthe standard curve. However, absorbance of the samples correctedfor paper blank absorbance corresponding to lower than 0.5 pug cannotbe interpolated. Thus the actual lower limit of the normal range hasnot been defined.

In Table II are listed the unconjugated plasma17-ketosteroid measurements in three patientswith familial Mediterranean fever and in five pa-tients with etiocholanolone fever. It was not pos-sible to perform duplicate determinations becauseof the limited amount of plasma available. Ascan be seen, etiocholanolone was not detected inthe plasma of the three febrile patients with fa-milial Mediterranean fever or in the plasma of thepatients with etiocholanolone fever when they wereafebrile.

However, elevated amounts of plasma etiocho-lanolone were detected during the febrile episodesof etiocholanolone fever. Moreover, a significantincrease of plasma dehydroepiandrosterone wasalso demonstrated during the same symptomaticattack. During symptomatic episodes, three ofthe patients (W.H., H.P. and V.E.) had a 2-,3- and 13-fold increase, respectively, of unconju-gated androsterone, compared with the afebrileperiod.

Identification of plasma 17-ketosteroids. The2,4-dinitrophenylhydrazone derivatives of uncon-jugated etiocholanolone, androsterone, and de-hydroepiandrosterone isolated from all of thesamples of plasma analyzed (a total of 10 L) werepooled separately and chromatographed on 1,000mg of neutral aluminum oxide (Woelm) columns(12.5 x 0.3 cm). A stationary phase of benzene:petroleum ether (1: 1) was used for the adsorp-tion chromatography on 3.9 and 5.0 per cent hy-drated alumina columns. The individual com-

pounds were isolated by elution with benzene:chloroform, 4: 1 (32). They were then reap-plied to fresh 3.9 and 5.0 per cent alumina andeluted with 0.05 per cent ethanol in benzene (33).Identification of the steroid-dinitrophenyihydra-zones was verified by characteristic position of thepeaks on elution from the columns and mainte-nance of constant specific activities calculated onthe basis of the absorbance of the eluted materialat 368 mp and the radioactivity, counted as de-scriled above, and corrected for quenching (TableV).

DISCUSSION

It is well established that isotopic labeling canbe used to measure and to correct for losses ofsmall amounts of steroids in analyzing plasma.The use of radioactive tracer(s) for correctionis justified provided that certain conditions aremet. First, the radioactive steroids must be asfree as possible from contamination. This is es-pecially important when the steroids are maderadioactive by exposure to tritium gas. Second,radiochemical purity must be established andmaintained. Third, the manipulations of extrac-tion, paper chromatography, elution, and measure-ment of plasma steroids must affect equally bothendogenotus and added steroids. These conditionswere strictly adhered to in the course of the devel-opment of the method described, and they facili-tated an accurate chemical measurement.

Nevertheless, the sensitivity of the microchemi-cal method prevented measurement below 0.5 /Agper 100 ml of plasma since, if less than this amount

of plasma 17-ketosteroid were present, it wouldproduce an optical density reading indistinguish-

TABLE V

Specific activities of pooled unconjugated 17-ketosteroid2,4-dinitrophenylhydrazones

Alumina Specific activityt%' hy-

dration* Solvent system DHEAETIOANDRO

cpin/lg3.9 Benzene:CHC13 (4:1) 4,820 5,244 7,5305.0 Benzene:CHCl3 (4:1) 4,792 5,120 7,4003.9 0.05 %Ethanol in benzene 4,780 5,100 7.3205.0 0.05 %O Ethanol in benzene 4,768 5,086 7,220

* The per cent hydrated alumina and solvent systems were employedin the order listed.

t Before column chromatography the specific activities of the pooled(10 L of analyzed plasma) tritium-labeled 17-ketosteroid 2,4-dinitro-phenylhydrazones were: DHEA, 4,760; ETIO, 5,208; ANDRO, 7,480.The 2,4-dinitrophenylhydrazones were measured at 368 mp in a spectro-photometer as previously described. Corrections for quenching were

performed.

404

UNCONJUGATEDNEUTRAL17-KETOSTEROIDS IN HUMANPLASMA

abic froiu that of the paper blanks. Since the ac-tual lower limit of the noriiial range has not l)eendefined, statistical analysis of the data was notundertaken. However, the method did afford, byits sensitivity and reproducibility, an opportunityto investigate possible aberrations of androgenmetabolism during fever.

Recently it was shown that the administrationof certain steroids produces fever in human sub-jects. Kappas and co-workers (34) reported thatthe febrile response to steroids with a 3a-hydroxyl,5,8 configuration differed from that produced bybacterial pyrogens (35). Steroid pyrogens, unlikebacterial pyrogens, were associated with a muchlonger latent period (4 to 6 hours) and did notproduce tolerance after repeated administration(13). Moreover, these steroids did not producefever in certain experimental animals (e.g., thedog or rabbit) which commonly respond to bac-terial pyrogens (1, 2, 36). According to recentobservations of Kappas and associates (34), thepyrogenic response to steroids was not due to therelease of endogenous pyrogen.

This report describes five patients with acuteattacks of etiocholanolone fever who had elevatedunconjugated plasma etiocholanolone levels dur-ing the prodrome of chills, anorexia, abdominaland/or chest pain, myalgia, arthralgia, headache,and fever. Normal amounts of unconjugated etio-cholanolone were detected in the plasma duringthe afebrile intervals. These observations sup-port the thesis that the presence of the pyrogenicsteroid may in some way be responsible for thedevelopment of this type of fever. However, theactual mechanism is still obscure. Unconjugatedetiocholanolone was not found in the plasma ofpatients with fever of known origin or in threepatients with familial Mediterranean fever. There-fore it seems unlikely that fever itself causes thepresence of the steroid.

The plasma concentrations of dehydroepiandro-sterone, an adrenal androgen (37), and (in threeinstances) androsterone were also increased inthe febrile patients with etiocholanolone fever.

Although liver enlargement was present in thepatients with etiocholanolone fever, liver functiontests and biopsies were normal. Nevertheless,our data suggest that one hepatic defect occurs inthis syndrome, i.e., a change in the pathway ofandrogen metabolism toward excessive etiocho-

laniolone pro(ltction. Toiinkinis (3(S) (lescribecltwo separate an(l specific steroid reductases whichcatalyze the reduction of the double bond on theA ring of steroids, to produce, in one case, 58-steroids and, in the other, 5a-steroids such asandrosterone. The acute attacks of etiocholano-lone fever may be associated in some way with adecrease of the activity of the 5a-steroid reductasesand concomitant increase of 5,8-reductases. Theanalogous situation of increased etiocholanoloneexcretion, as opposed to androsterone excretion,has been described by Bradlow, Hellman, Zumoffand Gallagher (39) and Hellman and associates(40) in hypothyroidism. The administration oftriiodothyronine reversed the clinical status andrestored the androsterone, etiocholanolone (5a/5,8)ratio to normal. Thyroid function measured byprotein-bound iodine or butanol-extractable iodineand radioactive iodine uptake was within the nor-mal range in the five patients with etiocholanolonefever. It would thus seem in the present studythat this defect in C1, metabolism is unrelated tothyroid function. The precipitating causes andpathogenesis of etiocholanolone fever remain to beelucidated.

It has also been suggested (17) that an inter-mittent defect in conjugation might occur con-comitantly with increased etiocholanolone produc-tion at the time of an attack of etiocholanolonefever. Kappas and colleagues (12) reported thatsubstitution of an acetate radical on the third car-bon atom of etiocholanolone inhibits pyrogenicactivity. Kappas' observations suggest that etio-cholanolone in plasma must be unconjugated ifit is to account for the pyrogenic activity duringattacks of etiocholanolone fever. Since the dataindicate that unconjugated plasma etiocholanoloneis associated with attacks, a possible aberration inconjugation has been hypothesized. In fact, pre-liminary data suggest that an attack is associatedwith decreased glucuronide conjugation of plasmatetrahydrocortisone and tetrahydrocortisol with-out increased P-glucuronidase activity.

Although the present studies have failed to findan increase in the unconjugated plasma etiocho-lanolone in patients with fever of known cause,only 12 such patients were studied. It is stilltoo early to be certain that abnormalities of steroidmetabolism do not occur in association with typesof fever other than those studied in this brief series.

405

GEORGEL. COHN, PHILIP K. BONDYAND CARMELACASTIGLIONNE

SUM MARY

A method is presecntedl for the sel)aration of Lin-conjugated plasma 17-ketosteroids and micro-measurement of their 2,4-dinitrophenyihydrazonederivatives. An isotopic radiochem-nical dilutiontechnique with pure tritiumi--labeled etiocholano-lone, dehydroepiandrosterone, and androsteroneis applied to correct for losses during the proce-

dure. The normal plasma ranges are: etiocho-lanolone < 0.5 to 1.2 pg per 100 ml; androsterone,

< 0.5 to 0.8 Kug per 100 ml; dehydroepiandro-sterone, < 0.5 to 1.2 tug per 100 ml. The totalmean SD isotopic and chemical recoveries are

72 14 and 67 + 21 per cent, respectively.Each of the measured 2,4-dinitrophenylhydra-

zone derivatives of unconjugated etiocholanolone,dehydroepiandrosterone, and androsterone was

identified by characteristic elution mobility andmaintenance of constant specific activity after fourseparate column chromatographic procedures.

Elevation of unconjugated plasma etiocholano-lone (a known pyrogenic steroid) and dehydro-epiandrosterone was demonstrated in five patientsduring attacks of etiocholanolone fever. In addi-tion, three of the five patients had elevated uncon-

jugated plasma androsterone levels. Dehydroepi-androsterone and etiocholanolone were within thenormal range during the afebrile intervals. Un-conjugated etiocholanolone was not detectable(< 0.5 ltg per 100 ml) in the plasma of 12 pa-

tients with fever of known cause and 3 febrile pa-

tients with familial Mediterranean fever.It has been suggested that in acute attacks of

etiocholanolone fever, an intermittent decrease ofhepatic 5a-reductases may be associated with a si-multaneous increase in 5[3-reductases, resulting inthe production of excessive amounts of etiocho-lanolone at the expense of androsterone. Conclu-sive evidence of a defect in conjugation of 17-ketosteroids in etiocholanolone fever remains to

be determined.

REFERENCES

1. Atkins, E., and Wood, W. B., Jr. Studies on thepathogenesis of fever. I. The presence of trans-ferable pyrogen in the blood stream following theinjection of typhoid vaccine. J. exp. Med. 1955,101, 519.

2. Atkins, E., and Wood, W. B., Jr. Studies on thepathogenesis of fever. II. Identification of an

endogenous pyrogen in the blood stream following

the injection of typhoid vaccine. J. Cxp. Mud.1955, 102, 499.

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