Determination of serum testosterone and androstanediol by competitive protein binding

27

DETERMINATION OF SERUM 'PES'IOSTRRORE AND ANDROSTANEDIOL BY COMPJXTIT!IVE PROTEINBIRDING A.L. Strickland,H. Apland,and J. Bmton Departzentof Pediatrics WalterReed General Hospital Washington,D.C.

Received

10/10/72

ABSTRACT A methodfor the sizultaneousneasureaeztof 8eruz testosterozo (T) and androstanediol (Ad) utilizingaluzinuzoxide thin layer chroz~ atographyand competitiveproteinbindinganalysisis presented.The method separatesnot only T from Ad, but also the azdrostanediol isomers.The primaryAd found was 5d4mdrostane-34, 176 diol. Levels of both steroidswere determinedin norzal adultsand children, and in a varietyof endocrinedisorders.The averageT/Ad ratio was higherin fenmle childrenthan other controlsexceptadult zales. However,resultswere too variableand nuzber of patientsinsufficient to draw definiteconclusionsas to the value of determinationof this ratio in patientswith androgen disorders.

The prepubescentrat testis (and ovary)has the capacityfor synthesizingtestosteroneat a rapid rate during_-iz vitro incubation studies(l-3). Thistestosteroneis not secreteddue to its rapid conversionto androstauediol and other reducedzetabolites. At pzbesoencethe reductaselevel diminishesand testosteroneis secreted.

STEROIDS

28

21:l

It thereforeseemed importantto determinewhetherandrostanediol was presentin disproportionately large quantitiesduring the prepubescentperiod as well as in variousdisordersof pubertyin humans. It is also of interestto study androstanediol becausein many respects the androgenicpropertiesresembletestosterone and dihydrotestceterone(&8).This study reportsthe simultaneous measurement of testosterone(T) and androstanediol (Ad) ('jti-androstane-3ti,17Pdiol) in the sera of normal childrenand adultsand disordersof androgenmetabolism. MATERIALS 4-"C-testosterone(New EnglangNuclearCorp.),specificact(NEN)sp. act. ivity 30nC/mmole(5 ug/uCi>and 1,2- H-testosterone was furtherpurifiedby chromatography on 30 C/mmole (O,OO5ug/uCi) paper developedin ligroinermethanolrwater 100:80:20followedby silicagel thin layer chromatrography (TLC) in bensenerethyl acetate (3tl),and then Al 03 TLC in bensenerether(311),and thembenzenet was made by incubatebhyl acetate (3~1f . Tritfatedandrostanediol ing 1,2=3H_testosterone in minced immaturerat testeswith cofactors as describedin Stricklandet al (3). Incubationtime was 3 hours. The labelledAd was purifiedin systemssimilarto testosterone and finally in a sephadex-LH-20 columnas describedby Murphy (9). A portionof the final product (54~androstane-3d,17@diol was crystalieed to cons t specificactivityae proof of its structure. In t"3H-Ad was found to have a constantsp. act. after deaddition,the rivatisationwith trimethylsilyl ether (THSE)and chromatography on silica gel TX in Bensenerethyl acetate (3:1),elutionof the area correspondingto an AdQMSE pilot , and finallyGLC on a 2% XE-60 column.In all instancesthe labelledmaterialappearedto be the same as the authenticunlabelledcompound(Steraloids, Pawling,N.Y.). All non-radioactive steroidswere obtainedfrom Steraloids,Inc., and were used after recrystallization twice in hexanermethanol. Glasswarewas soaked overnightin Alaonoxwater, thoroughly rinsed with distilledwater, then boiled in distilledwater for 15 minutes,and after dryingwas rinsed just beforeuse with freshly distilledmethylenedichloride. Solventswere spectrograde(FisherScient.Co., Fair Lawn,N,J.)

Jan.1973

STEROIDS

29

and were redistilledjust prior to use* Solventswere kept in a refrigeratorbelow 10°C. Al O3 and silicagel plateswere E. Merck from Brinwnn Instrument8o., Westbury,N.Y. Each plate was develaped (wash&) overnightin dist, benmene:meths.nol lti, then dist. bensenex4 (m more) and was not removedfrom the wash tank until just prior to use, Aftertwobenssne washesagrooveuas etched2 cm from the top across the width of the plate and 4x20 cm seotions were cut (glasscutter)from the 20x20 cm plate.Each sectionwas then centrallygroovedwith a needle creatingtwo 2 cm lanes in each sectionfor the sample tracts. One of these small p tes servedfor ahromatography of the labelledl%-testosteroneand FH-androstanediol pilots. These small platescut from the 20x20 cm platesprovided less chance of any sample cross contamination and chromatographyuas more uniform0 A pool of heparinizedplssmawas obtainedfrom women in the third trimesterof pregnancyand storedat -10°C. A dilute sex hormonebindingprotein(SHBP)solution(0.5%)was preparedbiweekly by dilutingwith d st. water. To this solutionwas added 10,000dpm per ml of 1,2-fH-testosterone. Liquid scintilating fluid was made with 5.OgmBBOT (Packard Inst. Co,, DownersGrove,Ill.)per liter and to this was added 0.25 liter of triton-X-100detergentsolubilimer(PalmettoSupplyCo., Greenville,S.C.).Countingefficiencyfor tritiumwas 32%. Samplesmeasuredin this study wmre obtainedfrom normaladult men and women workingin proximityto our unit and their children. Samplesfrom patientswere those being followedin the pediatric endocrineclinic of WalterReed GeneralHospitala Patientswith prematurepubarchewere girls under 8 years of age who had pubis and some axillaryhair, but showed no excessivelineargrowth pattern or increasedbone age and had no increasein urinarybioassayable gonadotropins.Patientswith precociouspubertywere girls under 8 years of age who had ewidenceof breastdevelopmentas well as pubic and/or axillaryhair, increasedbone age and linear growthrate, and hsd positiveurinarygonadotropins by bioassay. Patientswith prematurethelarchehad breastdevelopmentwithoutother evidenceof precocitysuch as increasedbone age or lineargrowthrate and no evidenceof androgeniaation.

METHOD of women and children's Extraction, Two ml of adult male and 8-123111 sera were used in duplicateand extractedwith 20 and 60 ml of methylene dichloride#eC12) respectively0A volume af 1N NaOM equal to l/l0 volume of serum was added to each sampleprior to extr ticm. To 10 ml distilledwater or 2 0 ml serum was added 28,000cpm % -testtosteroneand 225,000cpm 5H- androstanediol and extractionwas carried out along with the SGURP~~S.Identtaalquantitieswere also addsd to

30

STEROIDS

21:l

countingvials to determinerecovery. Another10 ml distilledwater was also extractedandcarriedthroughthe procedureto serve as a blank. The sampleswere placed on an Rberbachshakerfor 1 hour and the aqueouslayer was discarded. Each samplewas washed two times with 5.0 ml water. The @Cl2 was transferredto a 13 ml conicaltube and dried under N2. h t The dried extractswere spottedwith 0.1 ml ethanol mX2 on ~620cm Al203 stripscut from a 20x20 cm plate. Each strip was grooveddown the center so each duplicatehad a 2xi8 cm tract for development. The pilot and recoverylane was &side the water blank lane. After spotting,the plateswere run first in bensene to a line etchedacross the plate 2 cm from the top. Plates were dried and developedin ether,redriedand developedX4 in bensenerether 3~1. After each &VelOpments the plates were dried and replacedin the tank. Each developmenttime was about 5 hours. The initialdevelopmentin ben5ene SerVed to furthereliminateinterfering materialand did not move the steroidsfrom the origin. The pilot strip was scannedin a Packardradiochromatogram scanner (model7200) and the area of each peak corresponding to the testosteroneand androstanediol were alignedand scrapedfrom each strip into a 13 ml conical.tubeto which 8 ml distilledchloroform was added, These tubes were agitatedon a vortex Shakerfor 20 seconds each, allowed to sit at room temperaturefor 1 hour, agitatedagain, placed in a water bath at 45°C for 5 minutes,reagitated,and spun in an IEC Centrifugefor 5 minutesand 7.5 ml of the chloroformwas removedby HeCl2 rinsed pasteurpipets (drawnout to fine tip) in aliquotsof 2.5 ml into 12x90 mm tubes for assay* Centrifugingwas carriedout after removalof each aliquotso as to avoid any traces of Al203 gel in the assay tube. The areas from the pilot plate containingthe labelledSteroid8were scrapedeinllarlyto the sample and were transferredto countingvials,dried under N2 and 10 ml BBOT countingfluid was added. Recoveryrangedfrom 82-92s. Since 2.0 ml of adult male sera Were extractedto measureandrostanediol, an aliquotof 2% was taken after chromatography for the testosterone determination. Channelratio countingrevealedno contamination of the two isotopesindicatingthat the chromatography affordedgood separation. In a few preliminaryassays,quantikiesof eithertritiated testosteroneor andostanediol ismall enough to serve as recovery, but not large enough to interferewith the counts in the binding assay were added to each samplethus makingit possibleto check individualrecoveries. These recoveriesvariedfrom 80 to 92% CompetitiveProteinBindingAssay: One ml of 0.5% SRRP solution was added to each sampleand water blank tube as well as to duplicate standardtubes into nhich 0,0.5,1.0, 1.5, 2.0, 3.0 ng testosteronehad been dried. These were agitatedfor 20 secondseach

Jan.1973

STEROIDS

and set aside for IO minutes,then placed in a water bath at 45OC for 5 minutes. They were again agitatedand placed in an ice bath, put in the refrigeratorfor 25 minutes,and afterwardswere removed end consecutivelyto each was added 2 drops of 5% !&SO4 followed by 2 drops of 0.3N Ba.(OH)2solutionto precipitatethe binding protein. The standardswere run at the beginningand the end, of the samples. The tubes uere centrifugedin the IEC machineat a setting of 20 for 5 minutes. Then 0.3 ml aliquotswere transferredfrom each tube into countingvials, 10 ml BBOT countingfluid was added, and the vials were placed in a Packard'I%-carbscintilationspectrometer(model3320) and after coolingfor 1 hour were countedovernight for 20 minuteseach. Countingefficiencyfor tritumwas 32%, When the SHBP solutionwas made with the additionof 3H-androstanediolinsteadof 3H - testosterone,the standardcurve differed only slightly. For a number of assays both curveswere used and the calculatedvaluesfor both testosterone and androstanediol differed by less than IO%. Sampleresultswere calculatedfrom the standardcurve (figure 1) as follows: ng/lOOml= (x - B) x E x 100 V -‘iiWHEREX= B = V* R=

ng steroiddeterminedfrom standardcurve non-specificblank volume of sampleextracted recoveryof sample

RESULTS Recoverythroughthe entireprocedurefor added labelledtestosteroneand androstan&diol was between82 to 92%. Precisionwas determinedin two ways. The variationbetween35 pairs of duplicateswas 0.094+ 0,077 (SD) ng per sample. When an adult male'sserum was analyzedin quadruplicate, the results were 466.0 + 2.9 (SD) ng/lOO ml for testosterone and 57.8 2 0.91 ng/lOO ml for androstanediol.A pooledsampleanalyzedin quadruplicaterevealed52.8 + 4.8 and 35.0 + 1.5 ng/lOO ml resepectively

31

32

21:l

STEROIDS

for T and Ad.

In each instance the coeff. of variation was less than

10%. Accuracy of the method tiasdetermined by the addition of 0, 1.0, I._#, 2.0, and 3.0 ng of both testosterone and anthostanedio?.to 10 ml of distilled water and carried through the procedure. shown in table I.

Results are

The average interassay variability through all

ranges tested ;sas3.7% for T
com~~letely

exclude this steroid, it was isel:ted by different TLC systems as shown in table II and measured similarly to T and Ad except that the standard curve utilized non-isotopic andrustenediol ( in5d) instead of T or Ad. Table III also shows that the variation betwem T and Ad levels analyzed by different systems is not significantlydifferent. Some androstenediol levels are also presented. Blank of method: 10 ml of distilled water was extracted and run with each assay. Table I shows the blanks as found in 4 such determinations. Blank values in 20 assays varied from 0 to 0.26 ng with a mean of 0.088

+ 0.064

(SD) for the testosterone area and 0.092 + 0.066

(SD)

for the androstanediol area eluted from the Al,03 plates. Normal controls: Table IV shows results for T and Ad in male and female children and adults. Some male children were in the early

Jan. 1973

STEROIDS

33

1.82 2.10 I,98

::g 2.05

2elO zcoo 2011

1.50

3000

2.00 3.00 3.10

0.96

1.9 2.20

1059

0.98 0.98

0.95

1.11

(BOtik)

1.00

ng Androstanediol (Ad) found

0.00 1.00

t

0.00 1.08

0.12

ng Testosterone (T) found 0.10 0.08 0.12

1

0.06 0.08

10 ml H20

added to

ng T & Ad

cf known amountsof testosterone(T) TABLEI. Determination . _ and androstanediol (Ad) extractedfrom water.

Y

N

F;

TABLE

35

STEROIDS

Jan. 1973

II

Thin Layer

Chromatographic Systems Mobilities relative to testosterone TLC systems*

Steroid

I

II

5&androstane-3,

1%dione

1.26

1028

4- androstene-3,

17--dione

2.02

1.58

l’lg-hydroxy-5o(-androstane-3-one

1.57

3&hydroxy-5-androsten-17-one

III

IV

in five

1

V

1.90

1.48

1.34

1. ‘78

1.38

1.40

1.44

1.60

1.15

1.55

1.00

l-43

1.45

3Ot-hydroxy-5+androstan-17-one

1.48

1.31

1.20

5p-androstane-3#3,

1.15

1.28

1.08

1.00

1.00

1.15

-I -I

17 j3-diol

17j3-hydroxy-4-androsten-3-one(

T)

5$-androstane-38,

17 $3-diol

0.74

0.37

5 - androstene-3J3,

17J3-diol

0.72

0.48

l’la-diol

0.72

0.53

0.55

0.41

0.29

0,19

1,3,5-estratriene-3,

1.00

1.00

1.00

0.84 1.28

0.90

0.75 0.82 -

5+androstane-34, 5JGandrostane-34,

17j3-diol 17@diol

(Ad)

1.1.5

0.75

0.75

0.38 -

*

I II III IV V

A1203 (F-254) TLC developed Xl in A1203 (F-254) TLC developed X3 in Sihca gel (F-254) TLC developed X3 Silica gel (F-254) TLC developed in Silica gel (F-254) TLC developed in

ether, then X4 in benzenetether (3:l) benzene:ethyl acetate (3:l) in cyclohexanezethyl acetate (3:l) ye 0.35% ethanol in chloroform at 4°C QKS ) chloroformzmethanol 98.25:1.75 (19)

36

STEROIDS

TABLE

III

- Eiamples

I

Analyzed

Steroid

Levels

bg Different ng/lOO

Testosterone

CR Adult male RR Hyp ogonadal male MA Adult female

ml

I

I

A5d*

Ad

480 460 460 380 408 450 480 310

SA Adult male

TLC Systems

-

tSample

21:l

62 68 74 70 54 70 35 48

140 105 128 17 11

18. 6 1

I

14.3 5, 5 4.7 10.5 16.4 12.4 12.5 11.4 12.5 15.5 10.0 24 20

44 44 65 #3 53 I 5-andros’ kl ae- 38, 17”g -dial isolated from 60 separate systems #2

* **

28 24 17 20 46.0 45.6 47.0 53.0 --

(Table IV)

I

:: TS Prepubertal female Pooled women and children #i

System

24

19.7

prior

28 45 19 18. 5 17.2 to analysis

IV III -=-+1v** I IV III IV I II IV I III -+IV I II v --sp III I I II I v --g III I III -+Iv II v -+-III

13

6. WM 7.DO

**pubertal

6,9 16,O

male

drawn and run at different

8

7

11,KS

2505

2l.*4

mean

9 9

26,O 17,o 17,O 17,o 26.0 L8,8L3,1

_-u_9

Ad

times

2, L

I*9

2*0 1.0 7,6 8,61-X$

3,6

4.4 5.0 3.7 %*6 3*6 1.5 5.7 7.0 3*5 IO,7 z5.5

6,O 4.7 JI 3. !

Q,7

I.06 2.6 i, 2

206 t2.5

5*3 2.9

12.3 2,3

MdW

Adults

13. P 14. OR 15. CS

8, EC 9,JL 10, LM Ll, MS 12. EN

14*3 15.0 S-4 18.0 19,3 23,4 17,o 23.5 27.4 17.5 28.2 30.7 35,o 24*6 23 18 40 15.

23 45

35,O 38.0 20.0 29,o

28 24 25 23

2 3

7.32 6.3: 24.0 36,O 39.0 9-o 14.0

27.0 22, c 3800

39 27 21

3, cw* 4,MM 5, CS 6. EU 7.ED

31 26

I.SS 2. MA*

2

I-H.0 n=3

n=3

65.0

722

31

6, TT lO,S+

7,9 11,l

6200

492

30

5. PT

29,o 23,s 22.4 f3,C

9,9 807

67.0 60,O

520

4, BR

660

8.9

3.1

2Ql l-6 1,8 2,L 1.6 1.6 I.*5 1,4 Z,Of-O-86

2-2

1,6 1.0 100

2*7 3*1 4.3

n.=

Ratio

10,5If:

16.6

56.5 2 506 38.6

640

Ad

500 3 33

T

ml)

30 27 30

@g/100

36

(Ad)

2, LH 3,JA

l.AS*

Patient

(T} and Aladrostanediol Ratio

5*5

Testosterone

5*4 5,6 4,7 II. 0 3.0 2.3 5,3 L3.7(SD

5.5, 3,7

I*0

6,O

4,o

5*7 6.0

6,t

9.0 11.6

12,5

24,o I%. 6 20.0

12. SDS

9. VW 10. BL

3. TS* 4, ” 5. It 6. ” 7. ‘t 8, I!

1. SAS 2. CD

If

9 6

11.RB 12, BA

10, DS

12 9 8

8. RM 9. EB

i-

45.0 17.8 16.0

12

WMc

5.

*samples

3,6 10*4

34.0

26.0

12

4. CA

11

10.0

48,O

13

3.JR

4,3 22,o

52.7 50,o

12

67,O

260,O

13

T

Serum

1. KF**

c

-

2,JW

-F

Controls

Males

Normal

Patient

IV

Children

TABLE

6

9

10 9

12 1 6 4 1 10

1. TE

2. BH 3. BW

1, 2. 3. 4. 1. 1.

8 11 17

3. LH 4. DS 5. GR

F F F M

F F

F F F F F F

6/12 9/12 6/12 6/12 l/12

F F F

F

M F F F F F

O/12 F O/12 F

5/12 F

6/12 2/12 5/12 O/12 11/12 O/12 3/12 6/12 9/12 11/12 O/12 O/12 O/12 6/12

*.x,.,-Y

L

“_LIIUI”Y

37.0 103 70

640.0 10.5 10.0 14.0 10.5 40.0 26.0 13.0

25.0 12.0

24.0

37.0 27.0 6.7 27.0 34.0 30.5 31.0 27.6 13.0 10.6 35.0 9.0 10.5 14.0

Sex Testosterone

U&AU

r.

a/u&III.A.J.

“b”II

5.7 10.0 14

14.8 3.1 2.0 1.5 3.6 40 5 1.0 2.7

4.6 6.5

12.3

11.0 5.5 6.3 4.5 12.0 7,9 16.0 1.5 6. 5 4.3

4.0 8.5 203

Androstanediol

* 1 . 1 ,I...,11. *samples arawn ana run at arrrerent times

8

JM’R YG PS KO SD Lc*

MS KJ BW CM

2. DA

9, 10. 11. 12.

7. KW 8. OE*

VAUAUL”AL

Age

3 ‘7 7 6 7 6 6 4 5 5 7 4 9 7

.

MB JC WW DB HS MJ*

1. 2. 3. 4. 5. 6.

Patient

L**YYa..

Disorder pubarche 1, I, (1 II If II If 11 !I II 11 IV II

Y

NSL - AGS on treatment I, I,

precocious puberty and diabetes mellitus precocious puberty precocious puberty precocious puberty premature thelarche SL - AGS on treatment ,l If

pubic and axillary hair more than 2 years without breast development II I?

prematie

YII-rVIUUI

7

17

21

33

12

1. CL

2. PP

3. SA*

4. RM M

F

F

F

M

M M

M F

M

Sex 27.0 77.0 22.0 30.0 40.0 19.0 40.0 27.6 32.0 27,2 15.2 24.0 17.0 7-7 83 88 62 65 76 40 25 31

Testosterone 1.0 14.0 1.4 2.8 4.0 10.0 11, 6 4.5 4.7 4.2 12.8 2.5 1.0 1.4 14.5 30.0 33.0 23.0 10.5 10.5 1.0 1.0

Androsknediol

38.5 11.6 47.0 12.5 41.0 8.8 51.0 12.5 46.0 16.4 53,o 11.4 49.0 17.0 39.0 12.6 48.0 12.1 44.0 9.2 mean 45.624.96 12.4 t 1.4 * __ -I__ orawn Z#__.... and _..J run a~ _L aurerent II_- _.~. L.~~ _I.samples times ** human menopausal gonadotropin

pooled sera (women, children, and bank blood sera)

Age

15 O/12 15 10/U 12 12 12 3/12 7 13

2. Lw*

4. RH 1, BW*

2. Al3 3. DR”

1. RR*

Patient

anorchia

separate determinations to show reproducibility

pre-HMG** pos t-HMG**

of same sample

pre-ACTH post-ACTH

feminization

feminization

polycystic ovary syndrome (PCC) PC0 pre-ACTH po&ACTH idiopathic hirshutism

incomplete testicular (gonads intact)

Noonan’s syndrome incomplete testicular (post gonadectomy)

hypogonadism (? delayed adolescence) 11 (puberty beginning) hypogonadism (craniopharyngioma removed) hypopituitarism (secondary to neurofibromatosis)

Disorder

-

STEROIDS

40

21:l

pubertalstage and had some testosteroneelevation,i.e. KF. In general,when the testosteroneis high the androstanediol is also were repeatedIn elevated. Determinations a mean of 18.8 + 3.8 (SD) n&O0 Ad on 6 determinations.A.S.

one

femalechild T,S. with

ml for T and 5.3 + 3.7 (SD) for

(an

adult male) had an averageT level

of 500 + 33 (SD) ng/lOO ml on 6 determinations,M.A.,anadult female, had a mean of 22.4 f 3.0 for T and 7.3 + 1.9 for Ad

on 10

determinations.In the normalcontrols,the lowestandrostanediols were found in the youngerfemale childrenwho also had the lowest testosteronelevels. Childrenand adults with androgendisordersr Table V shows results in such conditionsas prematurepubarche,derangedpubertalonset, precociouspuberty,adrenogenital syndrome,hypogonadism, incomplete T.F.,PC0 syndrome,idiopathichirsutism,and anorchia. Mean

T/Ad Ratios (+,SD) in Controlsand AndrogenDisorders

1.

adult males

2.

10.5 + 3.1

n m 6

prepubertalmales

4.7 + 3.9

n = 12

31

adult females

2.0 2 0.9

n = 15

4.

prepubertalfemales

8.6 + 7.9

n=

7

5.

AGS (females)

10.2 + 7.4

n=

5

6.

precociouspuberty(females)

6,0+ 1.9

n-

3

7.

hypogonadism

8.

prematurepubarohe(females)

15.8 + 7.8

n= 4

4.0 + 2.3

n - 11

Jan.1973

t

41

STEROIDS

Teats

T/Ad Ratios

t Score

vs. Fepubertal females

0.72

NS

vs. adult females

2.04

NS

3. prepubertalfemales vsa adult males

0.41

NS

4. prepubertalfemales vs. adult females

1.95

NS

5. prepubertalmales

VS~ adult males

6.02 p<.OOl

6. adult males

VS* adult females

6.70 p<.OOl

10

prepubertalmales

2. prepubertalmales

DISCUSSION This study marks the first time androstanediol has been measured in the blood of children. Murphy (9) found levels in adult nonen of 24.0 t_15.0 (SD) comparedto our 17.2 + 9.8 (SD) ng/lOO ml. Since formationof derivativesof androstanediol destroysits reactivitywith the SRBP and derivativeremovalsignificantly increasesthe sampleblank,we attemptedto prove the existence of this steroidby isolatingit using five differentthin layer chromatographicsystemswith Al203 and silicagel. The resultswere not significantly differentwith any method, In our primaryTLC system (I), androstenediol and Ad are not ideallyseparatedand under poor developmentconditionsmight be et al (10) found levels of measuredsimultaneously.Rosenfield-17pdiol of 135 ng/lOO ml in adult males and s-androstene-3j3, 69 ng/lOOml in adult females.Murphy (11) found levelsof 120 ng/lOO ml in men and 39 ng/lOO ml in women0 Althoughwe have measured

21:l

STEROIDS

42

levels in only a small number of adults, our androstenediollevels in women have not been quite this high, but have been consistent by a number of TLC systems. 5'o(-androstane-36,176 -dial is adequately separated from so(-androstane-3v(,17g -dial (Ad) in system I (table II); however, if present in sufficient quantity, it could erroneously elevate the androstenediol levels as reported in table II since both have somewhat similar reactivity with SHBP and migrate together in system I. The systems used isolate all the known androgenic steroids which react with the SJBP, thus it is reasonable to assume that the steroid we have measured is s~J,-androstane-3lo(, 17@-diol. In animal (rat) experiments, it was found that 30 minute incubations of immature rat testes with labelled progesterone (1) or cholesterol (3) produced conversion to relatively large quantities of testosterone. If however, the incubation was carried out for 3 hours, the testosteronewas converted to androstanediol.The mature rat test&S, on the other hand, produces chiefly testosterone during both incubation periods, suggesting that the testis contains a

re-

ductase enzyme which becomes inactive as the testis matures or

per-

haps a reductase inhibitor develops. The inhibitor conceivably might be pituitary gonadotropin.We therefore felt it important to see if the blood of prepubertal children contained a proportionatelygreater quantity of androstanediolto

testosterone than adults.

It would appear from the ratios of testosterone to androstanediol found in this study that reductase activity is relatively low in adult males and prepubertal females whereas the reverse is true

Jan. 1973

43

STEROIDS

for adult femalesand prepubertalmales. The reduotaseactivitywithin the testisdecreaseswith maturitysimilarto that found in male rats (1,3),

whereasthe activityin femalesincreaseswith maturation.

The reductaseactivitythereforeseems

sex dependent,however

furtherspeculationwould be prematurewithoutmore data. Measurementof static levels of androstanediol in blood does not adequatelyassess the dynamicsof testosterone metabolism.Secretion rates for androstanedioIl from testes,ovaries,and adrenalswould providemore definitiveinformationregardingreductaseactivity. Mahoudeauet&12)

found productionrates for 5oi-androstanediol .ti

73 ng/day in men and 5 ng/day in women. They felt that some of the androstanediol may come from tissumprecursors*Thereforegonadal reductaseactivitymay not directlydeterminethe quantityof Ad in the blood. Unfortunately, in the many varieddisordersstudiedin thhs paper as well as in some normalwomen and children,the androstanediol levelsare rather low and the sensitivityof the assay at these concentrationsmay not be criticalenough to discernsubtledifferences.It appearedhowever,that the androstanediol levels in childrenwith early evidenceof androgenization were slightlyhigherrelativeto their testosteronelevel.A patientwith incompletetesticularferninization(L.W.),whose gonads were intact,had a high T/Ad ratio possibly consistentwith the postulatethat these patientsmay have low reductase activity(1347). In general,the androstanediol level in normaladult males and females,as well as in childrentends to parallelthe testosterone.

STEROIDS

44

21:l

Statisticalevaluationrevealsthat there is a highlysignificantdifferencein T/Ad ratiosbetweenprepubertalmales and adult males (p ( 0.001)and betweenadult males and females (p (0,001). Differencesbetweenother controlswere not signficant. The average (T/Ad)ratio is higher in controlfemalechildren than in other controlsexcept in adult males. The T and Ad levels are too variableand the number of patientsinsufficiently large to draw any definiteconclusionsat this time as to the value of androstansdiollevels or the T/Ad ratio in differentiating betweenpatients with androgendisorders.

The followingsteroidsare referredto in this study; I II III IV V VI VII VIII IX X XI XII

testosterone (T) androstenedione dehydrospiandrosterone androsterone androstanedione androstansdiol(Ad) 5+-androstanediol 5$-androstanediol 3@-androstanediol androstenediol dihydrotestosterone estradiol-17.3

17p-hydroxy-4-androsten-3-one 4+&ndrostene-3, 17 dione 3fl-hydroxy-fi-androsten-17-one 3z$-hydroxy-5-androstan-17-one 54-androstane-3,lll_dione 5oi-androstsne-3~4,17@-diol 544~androstane-3p,17~-diol 5$-androstane-3%,17$-diol 5$-androstane-39,17;3 4.01 5-androstene-3$,17iS-diol 17@-hydroxy-54-androstane-3-one 1,3,5-estratriene-3, 17~-diol

Acknowledgement This work was supportedby grantsfrom the U.S. Army Research and DevelopmentCommand. We wish to thank Dr, BeverlyE.P. Murphy,M.D.and Dr. Judson J. Van Wyk,M.D.for their helpfulassistanceand review of the manuscript.

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Jan.1773

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