dexran testosteron

8/7/2019 dexran testosteron

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CL IN . CHEM. 26 /1 . 1 0 1-106 (1 980 )

CLIN ICAL CHEM ISTRY , Vol. 26, No . 1, 1980 101

E valuation and C lin ica l A pplica tion of a D extran-C harcoa l M ethod forM easuring B ound Testosterone in H um an Plasm aRobe rt K . T choia kia n, G e ra ld M . J on es , B arb ara M . S anbo rn , L uis J . R od rig ue z-R ig au , a nd Gera ld G .Re imondoA m ethod for rap id ly determ in ing bound and free testos-terone in human plasma is evaluated and described, inwhich dextran-coated charcoal is used to separate boundand fr ee te sto s te ro ne. Intra-nd inter-assay C Vs w ere 1.9an d 3.8% , respective ly. A t least 50% of the boundtestosteroneas associatedwithspeciesth at b eh av edlike sex steroid horm one-b inding globulin on SephadexG-25 chrom atography and polyacrylam ide ge l e lectro-phoresis.ound and free test oste roneonce ntra tion seredeterminedinnormal men, normal women (duringthem enstrua l cycle and during norm al pregnancy), and inw om en w ith various degrees of h irsutism . W e concludethatdetermin inghe circula ting proportions of bound andfreetestos tero neshelp fulnthediagn osti cvalu atio nfh irs utism in w omen , a nd in th e in ve stig atio n o f n orm al a ndpathologicalond itio ns wher e a nd ro ge n c on ce ntr atio nsm ay be a lt er ed .Ad dI tI on al K ey ph ra se s: h irsutism in w om en . condit ionsa ffe c tin g a n dr og e n c o nc e ntr at io n s n o r ma l values fo r m ena nd w om en c hro ma to gra ph y o n S ep ha de x 0 -2 5 . elec -tro ph ore sis o n p oly ac ry la mid e g el . c ha ng es d urin g p re g-n a n cy , l ac ta ti on , m e n s tr u al p e ri od nature o f b i n d in g p r o te i rs )s te ro id s c lin ic al c o rre la tio n w ith b o u nd a nd free

The bio logical activ ity of testosterone is a function of theconcentration of free testosterone in the blood. This in turnis determ ined by the in teraction of specific b inding proteinsw ith the total c ir cu la tin g te sto ste ro ne . T e st os te ro ne -b in din gglobulin, also called sex steroid horm one-binding globulin(SHBG) , is considered to be the major specific b inding proteinin the circulation; other steroid-binding proteins, such ascorticosteroid-binding globulin and album in, are not specificfor testosterone, thus they contribute less to total testosteronebinding than does SHBG .T echniques used before m easurem ent to separate free frombound hormone include: treatm ent of the sample w ith dex-

tran-coa ted ch arcoal (1, 2), e qu ilib riu m d ia ly sis (3 , 4), gel-filtration (1 , 5 ), polyacrylam ide gel electrophoresis (6), pre-cip itation w ith ammonium sulfate (7, 8) , passage through acolum n of diethylam inoethyl-cellulose (9), and ion-exchangec ol um n c hr om a to gr ap hy (10). Many of these m ethods are t imeconsum ing and thus of lim ited value for routine clin ical use.Furthermore, som e techniques for separating free from boundtestosterone m ay disturb the physio logical equilibrium be-tween them , and the binding proteins involved have not beenidentif ied.

D epartm ent o f R ep rod uctive M edicine and B iolo gy , T he U niversityof Texas M edical School at Houston, P .O . Box 20708, H ouston , TX77025.Received July 16, 1979; accepted O ct. 1, 1979.

A relatively simple and rapid method for determ ining freetestosterone c on ce ntr atio ns in c lin ic al samples is n ee de d. S uc ha method should identify the proteins that collectively bindtestosterone, including S HB G.In our laboratory, as in m any others, dextran-coated char-coal is used in radioimmunoassays to separate free and anti-body-bound steroids. W e decided to utilize sim ilar m aterialsto separate bound from free testosterone in plasm a sam ples.Furthermore, we have attempted to assess what proportionof the bound component is attributable to SHBG . W e reportsuch a method here and have applied it to the determ inationo f b ou nd an d free testosterone in hum an plasm a samples fromv ariou s ty pes o f su bje cts.

Ma te ria ls and Me thodsMaterialsA ll solvents used in these procedures w ere of nanograde

quality (M allinckrodt, Inc., S t. Louis, M O 63147). Non-ra-dioactive testosterone was obtained from S teraloids, Inc.,W ilton, NH 03086, and recrystallized before use. [1 ,2,6,7-3H)Testosterone (100 kC i/m ol; cat. no. 1978-79 TRK -402;Am ersham /Searle C o., Arlington H eights, IL 60005) w as pu-rified by thin-layer chrom atography before use. D iluent w asprep ared b y d issolving 1 g of b ov ine g am ma-g lo bulin (F ractionII; S igma Chem ical Co., S t. Louis, M O 63178) in a liter of sa-line (154 m mol/L). Dextran-coated charcoal was prepared bym ixing 0.5 g of dextran (T -70; P harm acia F ine C hem icals, Inc.,P iscataway, N J 08854) w ith 0.5 g of charcoal (D arco G-60;M athe so n C olem an and Bell, C incinnati, O H 45212), in a litero f s al in e .Assay Procedure for Determ in ing Free and BoundTestosterone in P lasm aPlasma samples, 25 ftL , from men and women were deliv-

ered into 10 X 75 mm assay tubes, and to each was added anequal volume of diluent containing 18 000 dpm of the [3H1-testosterone. The samples were incubated at room tem pera-ture for 10 coin, transferred to an ice bath , and 1 mL of dex-tran-coated charcoal suspension was added. The contents ofthe tubes were then vortex-m ixed gently and left in ice for 15m m. The sam ples w ere centrifuged (2500 rpm , 10 mm), thesupernate containing the bound fraction was decanted into3 m L of p-dioxane in scintillation vials, 10 m L of scintilla tionflu id w as a dd ed , an d the radioactivity in th e vials w as co un tedin a Tricarb Liquid Scintilla tion Spectrometer, M odel 2450(Packard Instrument Co., Downers G rove, IL 60515).Bound and free testosterone were determ ined in 20 repli-cates of the same plasma sample in the sam e assay, to deter-m ine in tra-assay variation. Furtherm ore, samples from apooled specim en of plasma from men were incorporated in to15 consecutive assays, to determ ine in ter-assay variation .


2/610 2 C LIN IC AL C HEMIS TR Y, V ol. 2 6, N o. 1 , 1 98 0

The percent bound and free, and the concentration of freetestosterone, were calculated as follows:% bound = (c ou nts in s up ern ate - n on sp ec if ic c ou nts )!(to tal counts added - m achine background counts) X 100% free = 100% - % boundFree testosterone, ng/L = (% free X to tal testo stero ne

co ncn , in ng /L )/1 00E va lu at io n o f t he M et ho d

Sample volume: To establish what sam ple volume wasappropriate to use for the determ ination of free and boundtestosterone, w e selected four plasma sam ples: a pooledspecimen, a sample from one man, and sam ples from twowom en. W e m easured bound and free testosterone in thesesamples by the above m ethod, using 5 to 50 tL of eachsample.

S am ple dilu tio n: To determ ine the effect of various dilu-ents on the separation of free from bound testosterone, w eexam ined four different fresh ly prepared solutions: (a) d ilu-ent, consisting of 1 g of bovine gamma-globulin (F raction II,S igm a) per liter of saline; (b ) d ilu ent p lu s tris(h yd ro xy -methyl)methylam ine (Tris) buffer, 1.21 g/L , pH 7.4 at 4 #{176}C;(c ) isotonic saline plus T ris buffer, 10 mmol/L final concen-tration (pH 7.4 at 4 #{176}C);nd (d ) salin e o nly .

Bound and free testosterone w ere determ ined according tothe above-described procedure, except for successive use ofeach of the above solutions, each of which w as tested in du-plicate. Two pooled sam ples of plasma from normal men andtw o sam ples from normal women were used. Each solution wastested w ith various volum es of plasma (10 to 100 giL).Incubation T im e and Tem peratureTo determ ine the optim um incubation tim e and tem pera-

ture required for p lasma testosterone to equilibrate w ithadded [3H ]testosterone before separating the free from thebound component, we incubated replicate p lasma samplesw ith 18 000 dpm of [3H]testosterone in 25 zL of diluent forvarious intervals at room tem perature or at 4 #{176}C,hen deter-m ined bound and free testosterone.Variab les in the Incubation w ith C harcoal

Durat ion: To determ ine the optim um duration of incuba-tion for the bound/free separation , we incubated several 25-Lplasma sam ples w ith 25 L of diluent contain ing 18000 dpmof [3H ]testosterone for 10 mm at room temperature, added 1m L of dex tran-co ated charco al susp ension to each sam ple, andincubated for 4, 8, 12, 16, or 20 mm at 4 #{176}C.

Charcoal concentrat ion: To determ ine the effect of vary ingthe concentrations of dextran-coated charcoal used, we as-sayed samples containing 25 to 500 ig of charcoal for bounda nd fre e te sto ste ro ne .S am ple S ta bility

It is not alw ays possib le to determ ine concentrations of freeand bound testosterone im mediately after b lood w ithdrawal,so it was essential to investigate the effect of storage upon theirrelative concentrations in plasma sam ples. W e assayed plasmaspecimens from five normal m en and five normal women, bothwhen fresh and and on six occasions during storage at -20 #{176}Cfor 244 days. For each determ ination the specim ens werethaw ed, sam ples w ithdrawn, and the remaining specimenre-frozen.Nature of the Bound Component

Plasm a sam ples (50 L ) from males were incubated w ith50 iL of distilled water contain ing the 13H]testosterone(36 000 dpm , equivalent to 46 .3 pg), w ith and w ithout 500-fo ld

molar excess of unlabeled steroid , for 10 mm at room tem -perature. The samples were transferred to an ice bath , 2 mLof dextran-coated charcoal was added, and each sample wasgently vortex-m ixed , placed on ice for 15 mm , and then cen-trifuged (10 mm, 1500 rpm , 4 #{176}C ).ndiv idual 0.2-m L aliquotsof the supernates were applied to Sephadex G -25 colum ns,polyacrylam ide gels, or their rad ioactivity w as counted tomeasure bound steroids. The Sephadex G-25 columns (0 .5 x7 cm ) were eluted w ith a pH 7.4 eluent contain ing , per liter,50 mmol of Tris.HCI, 1 mmol of d isod ium EDTA , and 10 0 mLof glycero l, and 1-mm fractions of the eluates were counted.Electrophoresis was performed in 0.5 X 10 cm tubes on the(7 .5% crosslinked) gels at 2-3 mA per tube (1 1, 1 2). This sys-tem separated SHBG from CBG, as judged by m igration ofb ou nd [ 3H ]te st os te ro ne an d [3 H]cortisol as w ell as the re lativeeffectiveness of the respective unlabeled steroids to decreasebinding.C lin ica l App lica tion of the MethodW e used the m ethod described to determ ine free an d bound

plasma testosterone in sam ples ob tained from the fo llow ingtypes of subjects: 11 norm al men, six norm al women, 18wom en w ith variable degrees of hirsutism [m ild , moderate ,or severe by a m odification of the criteria of Thomas andFerriman (13)], a norm al wom an at various times during themenstrual cycle, and a norm al wom an at various times duringpregnancy and lactation. W e also m easured to tal testosteronein all the sam ples and calculated free testosterone concen-trations as described above.Total P lasm a Testosterone Determ ined in Samplesfrom Men and Women

Sa mp le p repa ratio n: One-half m illiliter of plasma fromwom en or 0 .1 mL of plasm a from men was pipetted in to 35-mLglass-stoppered conical centrifuge tubes, followed by 0.5 mLof distilled w ater and 1800 dpm of [1 ,2 ,6 ,7-3H ]testosterone(equivalent to 2 .5 pg), for use in recovery estimations.

Blanks and c ontrols : Water and stripped plasma (fromwom en) blanks w ere incorporated into each assay. S trippedplasm a from wom en was prepared by adding activated char-coal (25 gIL ) to the plasm a and vortex-m ixing for 30 mm, thenfiltering and centrifuging at 4 #{176}C.he supernatant flu id wasrecen trifuged three times at 4 #{176}C,hen filtered through a45-zm filter (M illipore Corp ., Bedford , MA 01730). N o tes-tosterone w as detectab le in such stripped plasm a. W e incor-porated tw o groups of controls in to each assay: six samplesfrom a pool of p lasm a from wom en, to determ ine intra-assayvariability; and nine samples of stripped plasma samples, eachthree receiv ing a different am ount of testosterone, rangingfrom low to high concentrations, to determ ine assay preci-sion.

Ext raction: Blanks, controls, and all the plasma samplesw ere adjusted to pH 9-10 w ith 100 mmol/L N aOH and ex-tracted tw ice w ith 5-mL portions of ether/ch loroform (80/20by vol). The respective extracts were com bined and washedtw ice w ith 2-mL portions of d istilled water, evaporated undernitrogen , then reconstitu ted in 0 .5 mL of m ethanol. W heretriplicate assay samples were obtained , three 0 .1-m L aliquotswere transferred in to indiv idual 10 X 75 mm glass assay tubes,evaporated in a vacuum oven set at 40 #{176}C,nd assayed, whilean equal aliquot of the sam ple was pipetted in to a scin tilla tionvial for recovery estimation . W here single assay determ ina-tions were done on samples, 0 .2-mL aliquots were assayed and0.2 mL was used for recovery estimation .

Radioimmunoassay of testosterone: We h ave p rev iou slyreported th e specificity of the testosterone antiserum and theprocedure for radioim munoassay of testosterone (1 4, 15 ). Inthe assay of plasma extracts from both men and women thebound/free separation was done w ith dextran-coated charcoal


3/6

600z0

40

ppppHFPMP*

20

8C

200.

9d

ib - io - 3O - 40 -

80

CLIN ICAL CHEM ISTRY, Vol. 26, No. 1 , 1980 103

SAMPLE VOLUME (p1)Figure 1.Boundtestosterone(%) determined on va ri ous volumes( dilu tio ns) o f f ou r human p la sma sampl esFP . normal woman; PP . plasma p oo l; HFP , h i rs ut e woman ; MP, norm al m an

and the supernate contain ing the bound fraction w as decantedinto 3 mL of p -dioxane in scintillation vials, 10 mL of scin-tilla tion fluid w as added, and the radioactiv ity in the vials wascounted in the liquid scin tilla tion spectrom eter. Intra- andinter-assay coefficien ts of variation were 6 and 9% , respec-tively.ResultsAssay Procedure for Determ in ing Free and BoundTestosterone in P lasm a

Although the assay m easures bound testosterone, it is m oreappropriate to express clin ical data on the basis of the calcu-lated free fraction , because th is is the bio logically activefraction . W hen we assayed 20 replicates of the same plasm asam ple concurrently w e obtained a m ean of 43.3 0.18 (SEM )percent free testosterone, w ith an in tra-assay CV of 1.9% .W hen plasma controls from a pool of plasm a from men wereincorporated in to 15 consecutive assays, w e obtained a m eanof 44.2 0.4 (SEM ) percent free testosterone, w ith an in ter-assay CV of 3 .8% .Evaluationof the Method

Sam ple volume: Figure 1 show s the percent bound testos-terone determ ined in various dilu tions of four samples ofhum an plasm a. The mean value from such dilu tions indicatesthat 25 jzL is the sm allest volum e of plasm a sample thatshould be used in the assay, b ut use of larger sam ple volum esdoes not significantly change the results for percent bound.

S am ple d ilu tion : We saw no significant d ifference for thepercent bound testosterone on using the four different d ilu-ents shown in Figure 2. T herefore, for convenience, w e decidedto use the radio immunoassay diluent preparation sim ilar tothat already in use in our radio im munoassays.I nc ub at io n T im e a nd Temperature

There was no statistically significant change in the pro-portions of bound or free testosterone from the first m inuteto 2 h of incubation at room tem perature. O ne-w ay analysisof variance and D uncans m ultip le-range test of the data whenvarious incubation in tervals at room tem perature and at 4 #{176}Cwere compared indicated that the am ount of bound stero idm easured after a 10-mm incubation w ith [8H]testosterone atroom temperature did not significantly differ from thatm easured after incubation for 20 to 50 coin at 4 #{176}C.onve -n ience prom pted our use of a 10-coin incubation at roomtemperature.

/4 42b - 4O 60 80 i#{243}oSAMPLE VOLUME Ipli

Fig . 2 . E ffect of use of various d iluents on results for boundte sto ste ro ne in p la sm a fro m (to p d ata ) a wom an and (bottomdata) a man#{149},IA d ilu en t; U , T ris b uffe r, p H 7 .4 ; 0 . sa lIn e; 0 , R I Ad il uen t + T ris bu ff er ,pH 7 .4

Variablesinthe Incubationwith CharcoalDurat ion: B inding was greatest betw een 1 and 3 coin anddecreased gradually up to 60 mm (Figure 3). W hen these data

were plo tted as log percent bound vs. tim e, the m ajor disso-ciating com ponent exhib ited a t112 of about 100 coin. Thevariab ility in the estimated percent bound during the second10-coin in terval w as consisten tly less than during the first 10coin . In addition, the values for nonspecific samples incorpo-rated in to each assay w ere higher during the first 10-coin in-terval. For these reasons, w e chose an incubation of 15 coinw ith dextran-coated charcoal.

Ch a r co a l c onc entrat ion: Varying the concentration ofdextran-coated charcoal d id not significantly affect the boundor free testosterone concentration , indicating that the charcoald id not adsorb additional am ounts of b inding proteins.S am ple S ta bilityOne-way analysis of variance of the data indicated no sig-

nificant d ifference in the proportions of free and bound tes-tosterone in any of the indiv idual sam ples during the 244-daystorage, nor was any significant d ifference found when wecompared the percent d ifference betw een consecutive deter-m inations. P lasma bound testosterone evidently remainsstable, even w ith repeated freezing (-20 #{176}C)nd thaw ing (+20#{176}C) ,or at least eight months.BindingProteinsinPlasma

The bound-hormone supernate of dextran-charcoal treatedplasma was further characterized by Sephadex G -25 exclusionchrom atography and polyacrylam ide gel electrophoresis.Roughly 50% of the radioactivity in the dextran-coatedcharcoal supernate (bound fraction) of the norm al p lasm afrom men was bound to macromolecules excluded fromSephadex G25 (M r >20000) and which m igrate like SHBGon electrophoresis. Testosterone in 500-fold m olar excesscompeted successfu lly for the bound [3H ]testosterone in bothsystem s; cortisol had only a m inor effect. The data indicatethat a substan tial proportion of the testosterone in thebound fraction is associated w ith SHBG . Further d isso-ciation may have occurred during the subsequent fraction-ation procedures, so 50% is a conservative estimate.C lin ica l App lica tion of the Method

Table 1 summarizes our total p lasma testosterone con-


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0zD0

9080706050

40

30

20

MeanRangeSD

Total

382 31.5n9/L

12080 39.2

MeanRangeSD

24582

419-69290895

3. 0

44.036

38 8

Table 1. Total Testosterone and Percentage andConcentration of Free Testosterone in Plasm a ofM en an d W om en

Totaltestosterone,ng/L

1 1 n orm al m enFree testosterone

Table 3. P lasm a Steroid C oncentrations on D ays10-19 of a Normal Menstrual Cycle

MeanRangeSD

57302470-9040

1880

Freetestosterone% ng/LDay ofcycle

10111213

Totaltestosterone,ng/L

19321 824928 5

S ix n orm al w om enMean 155Range 133-173SD 15

47.239.7-53.1

4. 1

29.627.4-34.7

2.8

ng/L

27201030-4420

94 0

4640-53

5

Estradlol ,n9/L58 14065 14680 20992 161

30.029.832.332.434.128.430.829.330.232.2

14 33215 51916 35017 23418 31819 300

Pro9es-terone,ng/L14 912 920213 717 757 788 9

347127023553

113147108699697

24 644 8227477258

10 0

1 04 CL IN ICA LCHEM IS TRY ,V ol. 2 6, N o . 1 , 1 98 0

T ab le 2 . T ota l T es to ste ro ne a nd P erc en ta ge a ndConcentration of Free Testosterone in P lasm a of

H irs ute W om entes tosterone, Free testosteroneng/L

M ild ly h ir su te ( n = 6 )252-466 26.3-34.4 77-147

39.2 22734.9-42.2 173-262

M o de ra te ly h irs ute ( n = 6 )

S ev er ely h irs ute ( n = 6 )MeanRange 673-121 34.5-53.7 280-446SD 191 6. 8 63

10 20 30 40 50 60TIME (Mn)

Fig. 3 . E ffe ct o f d ex tra n-co ate d c ha rco al o n th e ra te o f d isso -cia tion of bound testosterone in p lasm a sam plesEachpo int represen tsa meano f s ix separa tede te rmina ti onscentrations, percent free, and concentrations of free testos-terone in normal m en and non-hirsu te normal wom en. Table2 show s our resu lts for women w ith various degrees of h irsu-tism . The percent of free testosterone for norm al m en (47.2% )was significantly higher than those for norm al non-hirsute(29 .6%, p


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B e fo re p re g na n cy 242P r e g n a n c y

3 months4 m on th s5 mo nth s6 months7 months

37 254 246 267 886 5

8 months 6969 months 769

Lactat ion10 days 1563 months

A f te r l ac ta ti on10 632 5

CL IN ICA LCHEMIS TRY .V ol. 2 6. N o . 1 , 1 98 0 1 05

T ab le 4 . T ota l T es to ste ro ne a nd P erc en ta ge a ndC oncentration of Free T estosterone at V ariou sIntervals during a Norm al Pregnancy andLactation

Totaltestosterone,ag/I

SHBG-like pro tein . Extrapolation of the dissociation rate dataafter exposure to charcoal back to zero tim e (F igure 3) p lacesthe estimate at about 63% SHBG-bound stero id in the plasmafrom normal men, indicating that the fraction m easured bythis m ethod includes the contribution of SHBG plus addi-tional b inding com ponents.

Free testosterone Although our data on the percentage of free testosteroneag/I in plasm a of normal men and women are sim ilar to those re-31 .3 76 ported by others (2, 1 6), our data on total testosterone con-

centrations in the plasm a of norm al women are low er than18.5 69 those published (2 , 16, 17). This reflects the specificity of the16.5 89 testosterone antibody and of the assay technique used . C ross16 8 111 reactiv ity of the testosterone antiserum w ith 5a-d ihydrotes-177 120 tosterone is 6 .6% , w ith 5a-androstene-3a,17(-d io l is 2.2% , andw ith other stero ids


6/6106 CL IN ICALCHEM ISTRY,Vo l. 2 6 , N o. 1, 1980

8. H eyns, W ., and D eM oor, P ., T he binding of 17 /3-hydroxy-5a-an-drostan-3-one to the stero id-b inding a-g lobulin in hum an plasm a,as stu died b y m ean s o f am m on ium sulfate p recipitatio n. Steroids 18 ,7 09 -7 30 (1 97 1).9 . M ic ke ls on , K . E ., and Petra , P . H ., A filter assay for the sex stero idbinding protein (SBP) of hum an serum . FEBS Lett. 44 , 34-40(1974).10. H arm an, S . M ., and D anner, R . L ., Rapid measurem ent of anin dex o f testostero ne bin din g to serum bind ing globulin using ione xc ha ng e c olu mn s. J. Clin. Endocrine!. M etab. 4 5, 9 53 -9 59 (1 97 7).11. D avis, B . J., D isc electrophoresis-lI. M ethod and applicationto hum an serum proteins. Ann. N.Y. Aced. Sci. 1 21 , 4 04 -4 27(1964).1 2. E lk in gton , J. S . H ., S anb orn , B . M ., an d S tein berg er, E ., T he effectof testosterone propionate on the concentration of testicu lar andep id idy mal and rog en b in ding activity in the h yp oph ysecto mized rat.Mo!. C ell. E nd o crin ol. 2 , 1 51 -1 70 (1 97 5).13 . Thom as, P. K ., and Fernim an, D . G . P ., Variation in facial andpubic hair grow th in white women. A m. J. P hys. A nthropol. 15 ,1 71 -1 75 (1 95 7).1 4. T cho lak ian, R . K ., C ho wd hury , M ., an d C how dh ury , A ., R eco veryo f testicular and pitu itary fu nction s in adu lt m ale rats after cessationo f sh ort and lo ng term estrad io l treatm en t. B io l. R e pr o d. 19,431-438(1978).15. Rae, P . N ., M oore, P. H ., Peterson , D . M ., a nd T ch ola kia n, R . K .,Synthesis of new steroid haptens for radio imm unoassay. Part V .

1 9-O -C arb oxy meth yl ether deriv ativ e o f testostero ne. A h igh ly sp e-cific antiserum for im munoassay of testosterone from both m ale andfem ale plasm a w ith out ch ro matog rap hy. J. Steroid Biochem. 9,5 39 -5 45 (1 97 8) .16 . W iest, W . G ., Paulson, J. D ., K eller, D . W ., and W arren , J. C ., Freetestosterone concentration in seru m: A m etho d fo r d eterm in atio n.A m. J. O bstet. Gynecol. 130, 321-328 (1978).17. C lark , A . F ., M arcellus, S., DeLory , B ., and Bird , C . E., P lasm ate sto ste ro ne free in de x: a b ette r in dic ato r o f p la sm a a nd ro ge n ac tiv ity .F er til. S te ril. 2 6, 1 00 1-1 00 5 (1 97 5).18 . Paulson , J. D ., K eller, D . W ., W iest, W . G ., and W arren, J. G ., Freetestosterone concentration in serum : elevation is the hallmark ofhirsutism. A m. J. O bstet. G yn eco l. 1 28 ,8 51 -8 57 (19 77 ).19. A braham , G . E ., O varian and adrenal contribution to peripheralandrogens during the m enstrual cycle. J. C lin . E ndo crin ol. M etab .3 9, 3 40 -3 46 (1 97 6).20. Dawood, M . Y ., and Saxena, B . B ., P lasma testosterone and di-hydrotestosterone in ovulato ry cycles. A m. J. O bstet. G yn eco l. 126,4 30 -4 35 ( 19 76 ).21. K im , M . H ., Rosenfield , R . L., and Dupon, C ., The effects ofdexam ethasone on plasm a free androgens during norm al m enstrualcycle. A m. J. O bstet. G ynecol. 1 26 ,98 2-98 6 (19 76).2 2. D eM erto gh , R ., T hom as, K ., an d V an derhey den , I., Q uantitativ edeterm ination of sex-horm one-binding globulin capacity in theplasm a of norm al and diabetic pregnancies. J. C lin . E nd ocr in e!.Me t a b . 42 , 7 73 -7 77 ( 19 76 ).

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dexran testosteron