CLINICAL CHEMISTRY, Vol. 16, No. 2, 1970 111

Quantitationof PlasmaTestosteroneby ImprovedCompetitiveProtein-BindingTechnique

J. A. Demetriou and F. G. Austin

A method is described for quantitation of testosterone in plasma fromfemales and males. The principal operations of extraction, chromatographicfractionation, and competitive protein-binding assay can be completed foreight duplicate samples in a single day. Experimental data used in the devel-opment of the test and the rationale for the specific operations and condi-tions are presented. The specificity of the method was established by com-paring plasma testosterone concentrations determined by the double isotopederivative technique. Concentrations are reported for normal female andmale subjects, and for patients with hirsutism, adrenal hyperplasia, Kline-felter’s syndrome, and other endocrine conditions.

SINCE THE PUBLICATION of the review on theapplication of protein-binding technique for the

quantitation of hormones by Murphy (1), muchresearch has been devoted to development of suchassays for various steroid hormones. Specifically,the development of a method for plasma testos-terone has been under intensive study as evidencedby the appearance of twelve publications in twoyears (2-13). All of these methods include a verysimilar competitive protein-binding step, but differin extraction and purification procedures.

The competitive protein-binding method fortestosterone described in this paper was designedand evaluated from the standpoints of appropriate-ness for multisample processing, speed, and routineusefulness in a clinical laboratory. A detaileddescription of the method is presented along withdata on the accuracy and precision of the method.Concentrations of testosterone found in the plasmaof normal subjects and in patients with knownendocrinopathies are presented.

A section on the variables and rationale used inthe development of the procedure is included.Our procedure, used in this laboratory for the pastyear, has been satisfactory for the routine assayof testosterone in plasma or serum.

From the Research Dept., Bio-Science Laboratories, 7600Tyrone Ave., Van Nuys, Calif. 91405.

Received Oct. 27, 1969; accepted Nov. 25, 1969.

Apparatus and Equipment

A Packard Tri-Carb liquid scintillation spec-trometer (Model 314 or 3375) was used for countingthe tritium-labeled steroid.

Chromatographic equipment consisted of rec-tangular glass tanks (Applied Science Labs),chromatogram support frames and aluminum dry-ing racks (A. H. Thomas) - Stainless steel clips wereused to hold the chromatograms on 0.25 in. alumi-num rods. A plastic template (Fig. 1) was used tomark the chromatograms. An electric kiln (11 X11 X 13 in.) (J. J. Cress Co., El Monte, Calif.)was used to incinerate the chromatograms.

Syringe-type dispensers (Repipet, Labindus-tries) were employed to dispense 1 or 5 ml. Anautomatic dilutor (1-mi aspirator and 10-mi dis-penser, Labindustries) was used to transfer theradioactive steroid-protein solution and liquidscintillation solution to glass counting vials. TheJet-Pipet (York Industries) was also used fordispensing calibrated volumes.

Steroid fractions were eluted from the chromato-grams by the needle-syringe technique. Glasssyringes (1 ml), hooked needles (26 gauge), poly-ethylene bottles (240 ml), and glass funnels (45mm) were used to fabricate the apparatus formultiple elutions. The design of a single unit isshown in Fig. 2.

A Turbomixer (Bel-Art) was used to mix Florisil(Floridin Co.) and protein solution. A Vortex mixer

-SOLVENT FRONT LINE

CHROM-AR 500 SHEET

SAMPLE CUT-OUTSr -

1,2L

SAMPLE CUT-OUTSr - - - --‘- - -

3 4,

SAMPLE APPLICATION SPACES

- - $ -i- $ - -‘1 1

BLANK : STANDARD SPACE ‘ BLANKL _____ L -- --

______ POLYETHYLENEBOTTLE

CHROMARSEGMENT

112 CLINICAL CHEMISTRY, Vol. 16, No. 2, 1970

Fig. 1. Diagrams of plastic template (left) and chromatogram markings (right)

2cm.

15 cm.

3cm.

(Scientific Industries) may be substituted, butrequires longer mixing because of a difference in thedegree of agitation.

A measuring spoon for dispensing 40 mg ofFlorisil was fashioned from stainless steel tubing.The precision of the spoon was 39 0.5 mg. Plasticspoons were unsuitable because of the electrostaticcharge exhibited by the dry Florisil.

Migration of the standard testosterone spot onthe chromatograms was visualized in a dark boxwith a short-wavelength ultraviolet lamp (Miner-alite UVS-11).

FUNNEL

COLLECTIONTUBE

SUPPORTRACK

Fig. 2. Chromatogram elution unit

Reagents and Materials

All solvents were glass distilled through aVigreaux column and tested for interfering sub-stances by the competitive protein-binding assay.A value equivalent to 0.2 ng of testosterone was themaximum permissible amount of interfering mate-rial from 4 ml of evaporated solvent; otherwise, thesolvent was redistilled and retested.

1,2-3H-Testosterone with a specific activity of42.3 MCi per mmol was obtained from New EnglandNuclear Corp. The benzene was removed byevaporation under a nitrogen stream and 10 ml ofethanol was added to provide a solution with 25iCi per ml. This solution was stored at -15#{176}C.Late-pregnancy serum, which contains a proteinwith high and specific affinity for testosterone, wasobtained from women in the third trimester of preg-nancy. This serum was pooied and 3-mi aliquotswere distributed into plastic vials and frozen. Thelate-pregnancy serum-steroid solution for thecompetitive protein-binding assay was prepared byadding 0.50 ml of tritium-labeled testosterone to a250-mi volumetric flask and evaporating the eth-anol under a nitrogen stream. After the addition of2.5 ml of late-pregnancy serum, the contents wereincubated and mixed intermittently in a water bathat 45#{176}Cfor 5 mm. The solution was diluted to themark with distilled water, gently mixed, and storedat 4#{176}C.The final concentrations of late-pregnancyserum and tritium-labeled testosterone were 0.01ml serum and 0.05 jiCi per ml.

Chromatography was performed on 8 X 8 in.sheets of ChromAR-500 (silicic acid/glass fiber,Mallinckrodt Chemical Works). The sheets weremarked and interfering material removed before

CLINICAL CHEMISTRY, Vol. 16, No. 2, 1970 113

use as follows: (a) each sheet is placed on a cleanglass plate, spaces for application of the standardand serum extracts are demarcated with a softpencil and a plastic template (Fig. 1), and (b) thesheets are attached to aluminum rods (0.25 x 9.75in.) with stainless steel clips, hung on a chromato-gram support rack, and incinerated for 3 h at350#{176}C.After cooling, the chromatograms arestored in an airtight cabinet until needed.

Florisil, 60/100 mesh, was used to adsorb thefree steroid in the competitive protein-binding step.Fines were removed by washing and decanting withdistilled water (1 liter for each 100 g) repeatedly un-til all the particles settled in 30 s. The Florisil wasdried at 100#{176}Cfor 24 h and stored in an airtightcontainer.

The liquid scintillation solution was composed of4 g BBOT [2,5-bis(2,5-tert-butylbenzoxazolyl)-thiophene, Packard Instrument Co.] dissolved in1 liter of a solution of 1 ml Triton-X-100 (Rohm &Haas Co.) in 100 ml of toluene (BBOT-Triton-tolu-ene). A counting efficiency of 45% was obtainedwith the heterogeneous mixture of 0.5 ml aqueousprotein solution plus 10 ml of BBOT-Triton--toluene.

Disposable culture tubes, 10 X 75 mm (Kimbleno. 73000), were used for the competitive protein-binding reaction. Plasma extracts requiring adjust-ment of the eluant to a measured volume werecollected in 2-mi calibrated test tubes (Kimbie no.28014-P).

Steroids from commercial sources were dissolvedin distilled absolute alcohol. Testosterone solutions(10 ng/ml) used to prepare the standard curvewere stored at 4#{176}Cand used for two weeks. Solu-tions stored for longer periods deteriorated.

Procedure

Extraction

(a) Pipet 2.5 ml of plasma or serum into a 15-miconical centrifuge tube (Teflon- or glass-stoppered).

(b) Add 5.0 ml cyclohexane-ethyl acetate (1: 1)with a Jet Pipet or Repipet.

(c) Shake tubes 10 mm at high speed of ahorizontal shaker (approximately 5 cycles/s).

(d) Centrifuge tubes at 1500-2000 rpm for 5mm.

(e) Transfer 4.0 ml of the organic phase to aconical test tube and evaporate the contents todryness under a nitrogen stream at 45#{176}C.

(f) Consecutively, with rotation, add 0.15 ml ofdichioromethane and 0.5 ml of ethanol, dropwise,to each tube. Repeat the drying step.

Chromatography

(a) Dissolve the residues in the tubes with 0.02 mlof dichioromethane and 0.09 ml of ethanol. Incu-bate at 45#{176}Cfor 10 mm.

(b) Transfer each sample, in repeated 10-Mlportions, to the 3-cm space on the chromatogram.

(c) Apply 10 il of the testosterone standardsolution (0.5 mg/ml) to a single spot at the centerof the 1-cm space of the chromatogram.

(d) Dry the application lanes for 2 mm with awarm air blower.

(e) Develop the chromatogram in the chroma-tography tank, which contains 100 ml of benzene-ethyl acetate (80:20), for a distance of 15 cm(approximately 25 mm).

(f) Remove the chromatograms, hang them onthe support rack, and dry for 20 mm in a draft ovenat 40#{176}C.

(g) Repeat steps e andf.

Elution of Chromatograms

(a) Locate the standard testosterone zone withthe ultraviolet lamp and mark a line at the top ofthe spot.

(b) Align the template with this line and de-lineate, with a soft pencil, the corresponding 20 X40 mm areas in the sample lanes and blank sections(Fig. 1).

(c) With an X-acto knife, excise each sectionand use forceps to hang it onto the needle of theelution unit.

(d) Fill each syringe with ethyl acetate (approx-imately 1.5 ml) and collect the eluate in the appropri-ate test tubes. Use the 10 X 75mmtubes for samplesfrom women and children and the 2-mi calibratedtubes for the samples from men. Dilute specimensfrom men to 2 ml and transfer 0.4 ml to a 10 X 75mm tube for the assay.

(e) Evaporate the content of the tubes to dry-ness in a nitrogen stream at 45#{176}C.

Competitive Protein Binding Assay

(a) Pipet, in duplicate, 0.0, 0.05, 0.1, 0.2, 0.3,0.4, 0.5, and 1.0 ml of the testosterone standard(10 mg/mi) into tubes and evaporate to dryness.

(b) Add 1.0 ml of the late-pregnancy serum-tritium-labeled testosterone mixture with theRepipet to the testosterone standards, serum frac-tions, and blanks.

(c) Mix the contents of each tube for severalseconds and place in a water bath at 45#{176}Cfor5 mm.

(d) Remix each tube and place in an ice bathfor 10 mm.

(e) Add 40 mg of Florisil with the measuringspoon.

(f) Mix contents of each tube with the Turbo-mixer for exactly 30 s and immediately returnthe tube to the ice bath.

(g) Remove 0.5 ml of the supernatant fluid withthe automntic dilutor and rapidly expel into the

93%

82%

95%

87%86%76%

101%

95%

95%

114 CLINICAL CHEMISTRY, Vol. 16, No. 2, 1970

counting vial along with 10 ml of the BBOT-Triton-toluene.

(h) Mix the vials thoroughly and cool in therefrigerator for a minimum of 1 h. Count for 5 mm,or 20,000 counts.

Graph for Standard Curve and Calculations

(a) On a graph, plot the counts per mm vs. thenanograms of testosterone per tube for the knowns.

(b) Align a large French curve and draw a curvethat best joins the plotted points.

(c) Read the nanograms in the sample from thecurve.

(d) Calculations:

P-B 1

X X 100 = nanograms/100 ml plasma

where P = nanograms in plasma, B = namogramsin blank, 1? = recovery (= 85%), V = volume ofplasma (2 ml for samples from women and childrenand 0.4 ml for samples from men).

General Considerations

In developing this method for the assay oftestosterone, the practical aspect of productivity ina clinical laboratory was a primary consideration.Not only were recovery, precision, and reproduci-bility evaluated, but also the simplicity and easeof performance of each step were studied.

Extraction. Various solvent mixtures and serum-to-solvent ratios were examined to obtain quanti-tative recovery with a single extraction. Table 1shows the recovery of added radioactive testos-terone to serum after shaking on a horizontal bedshaker for 10 mm. The solvent mixture cyclo-hexane-ethyl acetate (1: 1) and a serum-to-solventratio of 1:2 gave an average recovery of 95% andwas the solvent of choice. Furthermore, the systemhad the added features of low solvent volatilityand a minimum emulsion formation.

The extraction volumes of 2.5 ml of serum with

Table 1. Recoveries of‘H-Testosterone Extracted from Serum

Solvent/serum ratio. mi/mi

Solvent 2:1 4:1

Diethyl etherEthyl acetateDichioromethaneEthyl acetate-

dichioromethane(2:1)

Cyclohexane-ethylacetate (7:3)

Cyclohexane-ethylacetate (1:1)

5 ml of cyclohexane-ethyl acetate were decidedupon to allow the removal of 4 ml of the organicsolvent, equivalent to 2 ml of serum; this circum-vents the tedious quantitative transfer of theentire organic phase or multiple extractions.

A widely used practice is to add various amountsand concentrations of sodium hydroxide to serumbefore extracting, to decrease the amount ofextraneous material extracted. Our studies showedthat the weight of serum extract was indeed less,but recovery of testosterone was also lower in theaikalinized serum samples. By omitting thisalkalimization step, we eliminated the need for acidand aqueous washes of the organic phase.

Chromato graphic media, preparation and tech-nique. Selection of the chromatographic system forseparating testosterone from a serum extract re-quired investigation into hold-up capacity, inter-fering materials in the chromatographic media,separation of steroids, and elution of chromato-grams.

Two criteria for the selection of the chromato-graphic medium were (a) that it would be thickenough to hold an extract equivalent to 2 ml ofserum on a 3-cm application line, and (b) that therewould be no overloading effects after chromato-graphic development. Three chromatographicmedia were studied, cellulose-MN 300 (Brinkmannor Anaitech), silica gel (Eastman K3O1R), orsilicic acid/glass fiber (ChromAR 500, Mallinc-krodt); only the latter met these requirements.

A common problem in competitive protein-binding assays is the presence of nonspecific inter-fering materials in the chromatograms which mustbe removed by extensive washing. Pretreatment ofthe ChromAR 500 by incineration, recommendedby the manufacturer, decreased the amount ofinterference to a value equivalent to 0.3 ng or lessof testosterone for 20 X 40 mm segments of thechromatograms. The chromatograms were incin-erated either by hanging on support racks andheating for 3 h at 350 #{176}C,or incinerated by placing4 to 8 sheets of ChromAR on heavy aluminum foilfor 1 hat 450#{176}C.

The chromatographic mobilities of testosteroneand various other steroids on ChromAR 500 withseveral solvent systems are shown in Table 2. Thesolvent system benzene-ethyl acetate (80:20)afforded good separation of most of the steroids.None of the solvent systems gave an adequateseparation of testosterone and epitestosterone, buta second development with this solvent systemimproved the resolution.

Varying techniques for eluting testosterone fromthe chromatograms were explored but the needle-syringe method was the most practical (Fig. 2).The bell-jar unit with the polyethylene bottle re-duced evaporation of the eluting solvent from thesurface of the chromatogram strip and comse-

Steroid

WATER

75-

50 -

1% LPS

25 -

LU

0U)

LUz0

LUI-U,01-Cl,LUI.;.

K,

I-zUi0

LU0.

0 20 40 60 80 100

MILLIGRAMS OF FLORISiL

Fig. 3. Florisil adsorption of ‘H-testosterone from waterand 1% late-pregnancy serum (LPS) at 4#{176}Cwith a 30-smixing interval

I I I I

CLINICAL CHEMISTRY, Vol. 16, No. 2, 1970 115

Table 2. Chromatographic Mobilities (Rf)of Steroids on ChromAR-500

Benzene/ethyi acetate ratio. mi/mi

50:50 60:40 75:25 80:20 80:20’

Cortisol . 0.32 .. 0.11 0.18Cortisone .. 0.33 . . ..

Testosterone 0.56 0.49 0.26 0.23 0.38Epitestosterone 0.63 0.56 0.33 0.30 0.4717a-Hydroxyprogesterone 0.74 0.66 0.41 0.38 0.57Dehydroepiandrosterone .. 0.68 .. . -

Estradiol . 0.80 0.68 ..

Estrone 0.94 0.90 0.84 0.73#{176}Double development.

quently a smaller volume of solvent was needed toelute the testosterone from the strip.

Ethyl acetate, methanol, and ethanol were triedas eluting solvents and gave 94 to 100% recoveryof radioactive testosterone from ChromAR 500strips. The amount of silicic acid particles displacedby ethyl acetate was considerably less; thereforethis solvent was selected for this step.

Conditions for competitive protein-binding. Factorssuch as concentration of late-pregnancy serum,amount of Florisil, and mixing and standing timeswere investigated to establish optimal conditionsfor the assay.

We first established the minimal amount ofFlorisil required to adsorb the maximum expectedamount of free steroid. Conversely, with thisamount of Florisil, the amount of steroid removedfrom the steroid-protein complex should be at aminimum. To establish this point, the adsorptionof radioactive testosterone from water and fromlate-pregnancy serum (1 mI/100 ml water) by in-creasing amounts of Florisil was studied. A 30-smixing time and 4#{176}Cincubation temperature wasused. Forty milligrams of Florisil per ml of solu-tion is the optimal amount (Fig. 3).

The mixing interval and the amount of Florisilare inversely related. For example, 20 mg ofFlorisil and a 60-s mixing interval resulted in anequivalent amount of testosterone adsorption.After mixing, no appreciable change in percentadsorption occurred over 60 mm.

We then investigated the range of assayabletestosterone with different concentrations of late-pregnancy serum in the solution. Of the threeserum concentrations studied, the 1% solutionprovided the most useful range (Fig. 4).

Liquid scintillation solvent system. The countingof the 3H-testosterone-late-pregnancy serum solu-tion was initially explored with liquid scintillationsolvent systems that were dioxane- or toluene-based. Interestingly, the biphasic water-toluenesamples gave a higher counting efficiency than themonophasic dioxane system, indicating either thetestosterone is readily extracted or there is con-

siderably less quenching by the toluene-phosphorsolution.

One problem with the toluene scintillation solu-tion was the time required (3 to 5 h) for the countsto become constant. Addition of Triton X-100reduced the stabilization time for counting to lessthan 1 h.

Results

Recovery. Average recovery of radioactive testos-

- - terone added to plasma and processed through chro-matography was 85%. One or 2 ng of testosteroneper ml was added to a pooled plasma sample witha testosterone concentration of 2.98 0.23 mg/mi.The net recoveries of the added testosterone were1.12 * 0.14 ng/ml and 2.20 0.14 mg/mi.

Precision. The precision obtained with pooledplasma divided into 15 samples and analyzed fourtimes over a 7-day period was 9.02 1.00 (SD)

mg/mi (coefficient of variation, 11%).

LUz0

LUI-

75

II0

505545545

50

2%

1340760

10501130785840460

a Each value is for a different subject.1%

85

8049

10675

12960

115108

0 2 4 6 8NANOGRAMS TESTOSTERONE

Fig.4. Effect of late-pregnancy serum concentration onthe range of assayable testosterone

886 53 186610 48 200

100

116 CLINICAL CHEMISTRY, Vol. 16, No. 2, 1970

Specificity. Concentrations of testosterone inplasma determined by a double isotope derivativemethod (14) and the present assay showed goodagreement (Table 3).

Plasma testosterone levels. The concentrations oftestosterone in the plasma of normal subjects inthe laboratory are shown in Table 4. The ages ineach group ranged from 20 to 45 years.

To assess the clinical usefulness of the method,plasma testosterone was determined in patientswith known endocrinopathies (Table 5). Concen-trations of plasma testosterone were consistentwith both clinical diagnosis and the concentrationsreported for testosterone by other investigators(14-16).

Table 3. Comparison of Plasma TestosteroneConcentrations in Seven Subjects as DeterminedbyCompetitive Protein-Binding (CPB) and Double

Isotope Derivative (DID) Methodsng/100 ml plasma

CPB 42 210 33 160DID#{176}43 200 12 160

a Plasma samples assayed by DID method made available byDr. J. R. Kent, Long Beach Veterans Hospital.

Table 4. Plasma TestosteroneConcentrations in Normal Subjects’

Discussion

ng/100 mi of piasmaMales Females

760 3251

8274

In developing the competitive protein-bindingmethod for the quantitation of plasma testosterone,several other factors besides recovery, precision,and specificity had to be evaluated. The otheraspects of the method examined were the smallestvolume of plasma adequate for the assay, simpli-fication of the various operational aspects of themethod, and the maximum productivity pertechnician.

Most published methods, including the presentone, utilize extraction, purification, and competi-tive protein-binding steps (3-12). They differ inthe solvents and solutions used for extraction, thechromatographic media (paper, silica gel, silicicacid) used in purification of the testosterone frac-tion, and the way unbound and protein-boundfractions are separated (Florisil, Sephadex, Dex-tram-Charcoal, precipitation).

If this test is done in a clinical laboratory, thereare certain problems that are not considered inresearch laboratories. First, there is the time re-quired to report a value. With the describedmethod, eight serum samples in duplicate can beprocessed by a technician in one day. Radioactivityis measured at night and the calculations arecompleted during the next day’s sample run. Manyother methods require 2 to 3 days for completionand, in some cases, fewer samples can be assayed.Second, automatic and precalibrated measuringdevices replace most of the hand pipetting opera-tions. This reduces errors in pipetting and transfersteps. Third, solvents and materials of a consistentquality and purity, which require only a minimumamount of purification or special treatment, mustbe available to a clinical laboratory. ChromAR 500requires only a single incineration of 3 h to removenonspecific interfering materials. All the solventsor chemicals for the method are commercially avail-

Diagnosis

Hirsute 393023143386

40 117641739

3419

22349

6027

26

375

46

22

16

35

53563920

CLINICAL CHEMISTRY, Vol. 16, No. 2, 1970 117

Table 5. Plasma TestosteroneConcentrations in Patients#{176}

Testosterone.ng/100 miAge

FEMALES

2235311821

Obese, hirsute 28

Cushing’s syndrome

Polycystic ovary 15

PanhypopituitarismPanhypopituitarism

(brain tumor)Primary testicular

atrophyKlinefelter’s

syndrome

MALES

229575319536

#{176}Plasma samples from patients at the Los Angeles County-University of Southern California Medical Center. Each sampleis from a different subject.

able. Fourth, the manipulations of the proceduremust be amenable to production-type operation.The use of automatic dispensing devices, a plastictemplate for marking the chromatograms, and thesimultaneous handling of multiple samples (elutionunits and chromatogram racks) are the principalfeatures of this method.

The specificity of the various competitive pro-tein-binding methods for testosterone has beencompared with the results for the 85Sthiosemicarbazone derivative (7), double isotope derivative(8, 9), and multiple chromatographic derivativetechniques (5, 12). Although testosterone is notunequivocally identified in our assay, the chroma-tographic purification of the testosterone extractand the relative specificity of the testosterone-binding protein interaction yields measurementsthat are equivalent to the longer, more tediousisotope derivative techniques.

Plasma testosterone levels in normal subjectsand patients with various clinical conditions re-ported here (Tables 4 and 5) are similar to thosereported by other investigators (8, 15). Clinicalcorrelation of testosterone levels in the subjectswith panhypopituitarism, testicular atrophy, andKiimefeiter’s syndrome are noteworthy. Althoughidiopathic hirsutism is thought to be caused by

high concentrations of testosterone, neither thedata of Maeda et al. (8) and Gandy and Peterson(15) nor the present values support this viewentirely.

The present method affords a relatively simple,specific, and sensitive method for the quantitationof plasma testosterone in males and females. Theneed for small volumes of plasma, the ease of per-forming the quantitation of nanogram amounts ofthis hormone, and the ability to process multiplesamples are the specific advantages of this method.

The authors wish to thank Dr. B. E. P. Murphy for providinga preliminary method for the determination of testosterone,Dr. J. R. Kent for supplying plasma samples previously assayedfor testosterone by the double isotope derivative method, andDrs. John Bethune and M. Stuart-Bentley for clinical samplesfrom the Los Angeles County-U.S.C. Medical Center. Thetechnical a.ssistance of Mr. J. Fries is gratefully acknowledged.

References1. Murphy, B. E. P., Application of the property of protein-binding to the assay of minute quantities of hormones and othersubstances. Nature 201, 679 (1964).

2. Murphy, B. E. P., Studies of the protein-binding of testos-terone and estrogens in human plasma. Program of the Forty-ninth Meeting of the Endocrine Society. Abstr. no. 110 (1967).

3. Fritz, G. H., and Knobil, E., The measurement of testosteronein plasma by competitive protein-binding analysis. Fed. Proc.25, 757 (1967).

4. Horton, H., Kate, T., and Sherins, H., A rapid method forthe estimation of testosterone in male plasma. Steroids 10, 245(1967).

5. Mayes, D., and Nugent, C. A., Determination of plasmatestosterone by the use of competitive protein-binding. J. Clin.Endocrinol. Metab. 28, 1169 (1968).

6. Hallberg, I’d. C., Zorn, E. M., and Wieland, H. G., A sensitivetestosterone assay by protein-binding. Steroids 12, 241 (1968).

7. Kato, T., and Horton, H., A rapid method for the estimationof testosterone in female plasma. Steroids 12, 631 (1968).

8. Maeda, H., Okamoto, M., Wegienka, L. C., and Forsham,P. H., A clinically useful method for plasma testosterone deter-rnination. Steroids 13, 83 (1969).

9. Frick, J., and Kinel, F. A., The measurement of testosteronein plasma by competitive protein-binding. Steroids 13,495 (1969).

10. Heyns, W., Verhoeven, G., van Baelen, H., and De Moor, P.,Measurement of testosterone in plasma by competitive protein-binding. Ann. Endocrinol. 30, 153 (1969).

11. Crepy, 0., Schwob, A., Ducret, M. A., Gueriguian, J. L.,Mowszowicz, I., and Dray, F., Critical study of some methodsfor measurement of plasma testosterone. Ann. Endocrino!. 30, 165(1969).

12. Rosenfield, II. L., Eberlein, W. R., and Bongiovanni, A. M.,Measurement of plasma testosterone by means of competitiveprotein-binding analysis. J. Clin. End.ocrinoi. Metab. 29, 854(1969.)

13. Pizarro, It. A., and Arredondo, R., Measurement of plasmatestosterone by competitive protein-binding. Ann. Endocrinol.30, 159 (1969).

14. Burger, H. G., Kent, J. R., and Kellie, A. E., Determinationof testosterone in human peripheral and adrenal venous plasma.J. Clin. Endocrinot. Metab. 24, 432 (1964).

15. Gandy, H. M., and Peterson, H. E., Measurement of tes-tosterone and 17-ketosteroids in plasma by the double isotopedilution derivative technique. 1. Clin. Endocrinot. Metab. 28, 949(1968).

16. Casey, J. H., and Nabarro, J. D. N., Plasma testosteronein idiopathic hirsutism, and the changes produced by adrenaland ovarian stimulation and suppression. .1. Clin. Endrocrinol.Metab. 27, 1431 (1967).