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Routine Radioimmunoassay of Plasma Testosterone, and Results for by lpx20272

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									 CUN.CHEM.         21/2.   206-210 (1975)




 Routine Radioimmunoassay of Plasma Testosterone,

and Results for Various Endocrine Disorders

Pui-Yuen Wong, Donald E. Wood, and Thomas Johnson


We describe a modification of published methods for ra-                           Materials and Methods
dloimmunoassay of plasma testosterone. This simpler
                                                                                  Materials
method involves no chromatographiC        steps, and the
necessary reagents, including tritiated testosterone and                             Testosterone       antiserum:       This was obtained       from
testosterone   anti-serum, are commercially   available. A                        Endocrine     Sciences,     Tarzana,     Calif. 91356.
digital computer is used for the calculation. Without diffi-                         Testosterone      standard:       This was obtained     from E.
culty, a technician can complete 100 assays in three                              Merck, EM Laboratories            Inc., Elmsford,      N.Y. 10523,
working days. Mean testosterone concentrations (±2                                and [1,2,6,7(n)-3H]-testosterone             (which we call here
SD) in the plasmas of 21 normal men and 26 women                                  “31I-labeled     testosterone”)        from Amersham/Searle,
with  a normal menstrual   cycle were 684 ± 300 and 45
                                                                                  Arlington     Hei’ghts, Ill. 60005 (cat. No. TRK-402,             90
zk 2O ng/dl, respectively.    Within- and between-assay
precision (coefficient of variation) were 5.2% (n = 29)                           Ci/mmol).
and 6.7% (n = 26), respectively.       We have assayed                               Organic     solvents:       All solvents, including      water,
more than 1000 samples during the past year. We give                              were redistilled      in glass.
data on the concentrations of testosterone in plasma of                              Borate    buffer:     1 g of gelatin was dissolved       in 100
patients with various endocrine             disorders.                            ml of hot water. Boric acid, 4.0 g, and 1.0 g of sodium
                                                                                  azide were dissolved          in 500 ml of water and the pH
Addftlonal Keyphrases: diagnostic                aid      endocrine
                                                       #{149}           disord-   was adjusted      to 8.0 with sodium hydroxide.        The gela-
ers       normal
       #{149}       values       steroids
                              #{149}
                                                                                  tin solution     was added        and the mixture     diluted    to
     Assay of plasma testosterone                 is an index of andro-           1000 ml.
                                                                                     Saturated      aqueous      ammonium         sulfate.
genic function         that is useful in evaluating                feminine
hirsutism, virilizing          tumors,        polycystic      ovaries,     and
                                                                                     Dextran-coated            charcoal:      50 mg of dextran            and
the adrenogenital          syndromes,         and in the male in eval-            500 mg of Norit A charcoal               (Schwartz/Mann,           Orange-
uating impotence,           primary       and secondary          hypogona-        burg, N. Y. 10962) were added to 100 ml of the borate
dism, and delayed            and precocious           puberty.     It is also     buffer.
                                                                                     Testosterone          antiserum       solution:     The stock anti-
useful in monitoring            therapy       in several of these con-
ditions.                                                                          serum was diluted            with borate buffer containing              3H-
    Techniques       proposed         for assay of testosterone              in   labeled    testosterone         (about 5000 cpm/ml)              to obtain
plasma     include gas chromatography                    (1), the double-         50-60% bound at 0 pg testosterone                   concentration.
isotope     derivative       method         (2, 3), and competitive                  Liquid      scintillation        fluid:     “Unogel”        (Schwartz!
protein-binding         analysis       (4-6).     Some of these meth-             Mann).
                                                                                     Pipettor:      Model 25000, Micromedic Systems, Inc.,
ods are accurate,          sensitive,       and specific,       but all are
                                                                                  Philadelphia,       Pa. 19105.
tedious.    The recent successful               production      of an anti-
                                                                                     Liquid scintillation           counter:       Unilux II with Tele-
serum for testosterone              provides       a more reliable        and
                                                                                  type print-out         (Searle Analytic         Inc., Des Plaines,        Ill.
facile technique        that retains accuracy              and sensitivity
(8,9).                                                                            60018).
                                                                                     Digital computer:            PDP 11/40 (Digital           Equipment
    In this communication               we wish to report our expe-
rience in routine radioimmunoassay                     of plasma testos-          Corp., Maynard,           Mass. 01754).
terone with use of [1,2,6,7(n)-3H]-testosterone                        for re-    Methods
covery determination              as well as for radioimmunoas-
                                                                                     Extraction     of plasma    testosterone:     The procedure
says.
                                                                                  is summarized       in Figure 1. Generally,       1.0 ml of plas-
                                                                                  ma (or in the case of men, 0.1 ml of plasma)             was pi-
  Toronto    General   Hospital,    101 College St.,      Toronto,    Ontario,
Canada    M5G 1L7.                                                                petted    into test tubes containing         3000 cpm of 3H-la-
  Received   July 8, 1974; accepted    Nov. 6, 1974.                              beled testosterone       (about 10 pg). The contents        were

206     CLINICAL CHEMISTRY, Vol. 21, No. 2, 1975
                             Plasma            +                           taming 0, 10, 25, 50, 75, 100, and 150 pg of testoster-
                          3H-Testosterone                                  one and 200 cpm of 3H-labeled                   testosterone      were
                                                                           added. The contents         of these tubes were evaporated.
                                                                           The tubes containing           the dried extracts        were stored
                             1 h      at 37’C                                            until
                                                                           at -20 #{176}C assayed.
                                                                               Radioimmunoassay:            Figure 2 summarizes          the ex-
                             lh       at       4#{176}C
                                                                           perimental      procedure.      Testosterone       antiserum      solu-
                                                                           tion 0.5 ml was added to the test tubes containing                  the
                                  (NH4)2S04                                dry residue, and tubes were incubated                           for
                                                                                                                                at 37 #{176}C an
                                                                           hour followed       by an additional          hour at 4 #{176}C.  Cold
                           Precipitation
                                                                           dextran-coated       charcoal,      0.5 ml, was added to each
                                                                           tube. They were vortex-mixed               and then centrifuged
                                                                           at 3000 rpm for 10 mm at 4 #{176}C. supernatant  The
                                  extraction                               fluid was poured into counting             vials containing      10 ml
                                                                           of Unogel. The activity in all the vials was measured.

                                                                           Calculation
                                                                               Using a digital PD? 11/40 computer           off-line in the
   very
                                                                           batch processing      mode, we constructed        the standard
                                                                           curve by plotting     percent antibody      bound vs. concen-
                                                          determinatio
                                                                           tration   of unlabeled     testosterone.    Percent      antibody
Fig. 1. Flow chart    for the procedure for extracting testoster-          bound testosterone      for standards    is
one from plasma
                                                                                   = [C/(A        + S)] X 100
                                                                           and for samples
                                                                                  = [C/(A         + E)]    x 100

                                                                            where C      =   cpm of supernate         after addition       of dextran
                                                                                             coated charcoal,
                                                                                     A   =   cpm in 0.5 ml of 3H-labeled-testosterone
                                                                                             antiserum  solution,
                                                                                     S   =   cpm added to standard   tubes,
                                                                                     E   =   cpm in the aliquot used for assay, and
                                                                                     T   =   total cpm added to plasma samples before
                                                                                             extraction.

                                                                              The testosterone      content    of samples   was read off
                                                                           the standard     curve in picograms        per sample = (pg
                                                                           read off standard    curve       water blank) X (T/E)
                                                                                                              -




                                                                           Results
Fig. 2. Flow chart of the radioimmunoassay procedure                       Analytical        Variables
                                                                              Recovery          of 3H-labeled            testosterone         added     to
                                                                           plasma       sample.        The mean percent            recovery      ±SD of
vortex-mixed       for 30 s and incubated                       for
                                                  at 37 #{176}C 1 h        3H-labeled         testosterone         added to 143 samples             from
and for an additional          hour at 4 #{176}C. volumes of
                                                 Nine                      women and 125 samples from men was 68.7% ± 10.2%
53% saturated        ammonium         sulfate  (4 #{176}C) added
                                                          was              and 55.0% ± 10.1%, respectively.
to each tube and the contents             were mixed. The tubes                Antibody        titration      curve. We made serial dilutions
were centrifuged        at 3000 rpm for 10 mm. The super-                  ranging from 10 000- to 70 000-fold of the antiserum
natant fluid was discarded.                                                in borate buffer containing,                  per milliliter,      5000 cpm
   The precipitate was dissolved in 2 ml of water                          of 3H-labeled           testosterone.       Figure 3 shows the titra-
(room temperature)           and extracted     once with 3 ml of           tion curve.
hexane-ethyl        acetate      (9:1 by vol). The             organic         Water       blank.      Distilled     water was used to investi-
(upper)     phase was separated            by centrifugation         and   gate blank values obtained                  in this system. The mean
0.3 ml aliquots      of it were transferred       to three 10 X 75         ±SD, expressed             as pg equivalents          of testosterone       per
mm glass disposable           tubes and a liquid-scintillation             tube, was 4 ± 2 (n = 20).
counting     vial. The contents        were evaporated       in a vac-         Linearity.         The linearity           of the procedure            was
uum oven at 45 #{176}C.        Ten milliliters      of Unogel was          checked        by assaying          increasing      volumes       of the ex-
added     to the counting         vials and the activity          mea-     tracts of plasma              sample.     The results        are shown in
sured. To another          set of test tubes, standards             con-   Figure 4.

                                                                                                 CLINICAL CHEMISTRY,        Vol. 21, No. 2, 1975      207
                                                                                                     were sent to Inter Sciences       Institute      (Los Angeles,
              BOUND
                                                                                                     Calif. 90025) for determination         of testosterone     con-
                    80                                                                               centrations,  with use of A1203 chromatography              as a
                                                                                                     purification  step. The testosterone        concentrations      of
                    70                                                                               these samples     were also determined         by assay after
                                                                                                     the described    (NH4)2S04   precipitation       in our labora-
                    60
                                                                                                     tory. The results are summarized        in Table 1.

                                                                                                     Clinical   Studies
                    50
                                                                                                         Normal       subjects.      Mean     testosterone         concentra-
                                                                                                     tions (±2 SD) for 26 women (whose menstrual                          cycles
                    40                                                                               were normal)          and 21 normal men, as assayed                 by the
                                                                                                     present     method,        were 44.5 ± 19.8 and 684 ± 300 ng/
                    30                                                                               dl, respectively.
                                                                                                         Pregnancy.         The meafl testosterone            concentration
                                                                                   1/110             (±2 SD) fOr nine pregnant                  women       (gestation       >16
                                        ANTISERUM           DILUTION                                 weeks), as measured            by the present        method,      was 122
Fig. 3. Antibody titration curve                                                                     ± 94 ng/dl.
Endocrine     Sciences’      testosterone       antiserum   T3-1 25 was used
                                                                                                         Hirsutism        and adrenogenital             disorders.        For a
                                                                                                     group of 13 patients            with hirsutism       and adrenogeni-
                                                                                                     tal disorders,       the mean testosterone           concentration         ±
                                                                                                     2 SD was 103 ± 30 ng/dl (range, 78.8-127 ng!dl).
                                                                                                         Hypogonadism.            For a group of 21 patients           with ei-
                                                                                                     ther primary         or secondary       hypogonadism,          the mean
                                                                                                     (2 SD) was 245 ± 190 ng/dl (range, 80.8-390 ng/dl).
                                                                                                         Treatment        of hypogonadism.           After i.m. adminis-
                                                                                                     tration    of 2.0 ml of “Depo-Testosterone”                (100 mg/mi,
                                                                                                     Upjohn)       to each of a group of hypogonadal                 patients,
       0                                                                                             plasma      testosterone       was determined          at 0, 7, 14, 21,
            500
                                                                                                     and 28 days. Generally,             the plasma testosterone            con-
       w                                                                                             centration       was highest       at day 7, and it had dropped
       z
       0    ‘100                                                                                     to the original        value by day 28. A typical              pattern     is’
                                                                                                     shown in Figure 5.
            300



            200



            100
                                                                                                        Table 1. Comparison of Plasma Testosterone
                                                                                                           Values for 13 Subjects, as Determined
                                                                                                          by Radioimmunoassay, with Use of A1203
                                        100                       200                       300         Chromatography and (NH4)1S04Precipitation
                                            i    EXTRACT         ASSAYED
                                                                                                                    as Purification Steps
                                                                                                                  AIzO, chromatography              (NH42S04 precipitation
Fig. 4. Relationship between the aliquots of extract used in
the radioimmunoassay and their testosterone content                                                                                         ng/dl
                                                                                                                                        Males
                                                                                                                          606                              589
                                                                                                                          442                              420
                                                                                                                           20.0                             14.6
     Precision.Within-assay       precision     was examined                                                                                                35.8
                                                                                                                           33.0
by measuring     the testosterone        concentrations        of a                                                       123                              120
pool of plasma from women. The coefficient               of varia-                                                         52.0                             52.6
tioh for 29 samples   was 5.2% and the thean value (±2                                                                    932                              979
SD) was 47.3 ± 2.5 ng/dl. Between-assay                 precision                                                         727                              714
was determined     by measuring      the testosterone       in the                                                        182                              218
same pool plasma      on 26 separate        days. The coeITi-                                                             465                              555
cient of variation was 6.7% and the mean value (±2                                                                        402                              473
SD) was 45.8 ± 3.1 ng/dl.                                                                                                             F’emales
     Comparison                of plasma            testosterone            concentration                                  34.0                             40.7
in     subjects           as     determined                 by     radioimmunoassay                                        23.0                             30.4
with       and     without        chromatography.                       Thirteen           samples

208        CLINICALCHEMISTRY, Vol. 21, No. 2, 1975
          ng%                                                                            Table 2. Plasma Testosterone Values for a
     TES1OSTERJE                                                                         29-Year-Old Man with Hypogonadism upon
                                                                                             Choriogonadotropin                Stimulation         Test
                1501                                                                                                                             ng /d I
                                                                                                 Diagnosis: Hypogonadism
                                                                                                            Baseline:                             60.0
                                                                                                3 days after HCG:
                                                                                                             0800h                               125
                                                                                                             2000h                               123
                1001

                                                                                     Table 3. The Laboratory Results for a 26-Year-Old
                                                                                               Man with Panhypopituitarism
                                                                                                  HGH:    j          TSH:     1.
                                                                                                  13                 14      ,
                500                                                                               Plasma cortisol:    0800h      1000 h
                                                                                                                                      --#{176}      -


                                                                                                  FSH:      -


                                                                                                       Testosterone:     614 ng/dl




                       0            7          hi         21           20            blank      values      obtained       in this system.           The mean
                                             DAYS                                    (±SD) was 4 ± 2 pg equivalents                   of testosterone          (n =
Fig. 5. A typical plasma testosterone concentrations of a hy-
pogonad patient after i.m. administration of 200 mg of testos-                       20).
terone cypionate                                                                         Cross-reactivity.          Data on the cross reaction              of the
                                                                                     antiserum        with other steroids          was obtained         from the
                                                                                     supplier.       Generally,         dihydrotestosterone,             delta-i-
                                                                                     dehydrotestosterone              and delta-i-testosterone              cross-
                                                                                     react significantly           with the antibody            because       cross
   Pituitary     tumors.    A group of five male patients                            reactions      with these compounds                would be expected
with pituitary      tumors gave values of 12.7, 14.6, 20.5,                          with antibodies          to testosterone        conjugated        at the no.
25.0, and 829 ng/dl.                                                                 3 carbon. A separate             experiment        has shown that 300
    Choriogonadotropin       stimulation    test. For a male                         pg of 17-methyl-testosterone                and 1-dehydrotestoster-
patient     with secondary    hypogonadism,      the testoster-                      one were processed              through      the percent        method       as
one values       before   and after a choriogonadotropin                             though they were equivalent                 to 118 and 199 pg of tes-
stimulation     test are shown in Table 2.                                           tosterone,      respectively.
   Panhypopituitarism.          Results  are summarized         in                       Linearity.       Figure 4 shows the linearity               of the pro-
Table 3 for a male patient       with panhypopituitarism.                            cedure. From this, it may be assumed                      that the mate-
                                                                                     rial assayed        in plasma samples           and that used in the
Discussion                                                                           standard       curve are identical           in their reactivity          with
     A globulin       fraction    rich in bound testosterone                 was     the antibody.
 separated        from serum by ammonium                  sulfate precipi-               Interlaboratory           comparison.         Comparison           of re-
 tation before extraction.             This preliminary             precipita-       sults for 13 samples where testosterone                     assay was de-
tion step enables            the radioimmunoassay                 to be done         termined        by using a chromatographic                    purification
without a chromatographic                 purification        step.                  (Inter Science Laboratories)               and the present method,
     Recovery.       3H-labeled      testosterone        is used to deter-           are summarized            in Table 1. An excellent              agreement
 mine recovery          as well as for radioimmunoassay.                     Be-     was found between these two methods.
cause of the specific activity               of [1,2,6,7(n)-3H]testos-                   Normal       range.      The plasma         testosterone         concen-
terone is greater than that of [1,2(n)-3H]testosterone,                              trations     (±2 SD) of normal subjects                were established
 lessthan 10 pg of labeled steroid                   is required        for re-      by assaying          samples       from 26 women             with normal
covery determination,              an amount         can be ignored            in    menstrual        cycles and 21 normal               men. As shown in
the final calculations.            In addition,        less antiserum           is   Table 4, the normal               ranges are in good agreement
 required      when this labeled compound                  is used.    As Fig-       with several published               methods,      such as double-iso-
 ure 3 shows, maximum                 sensitivity       was obtained           at    tope dilution        (4), competitive         protein-binding          analy-
about a 30 000-fold dilution.                                                        sis (5), radioimmunoassay                 with (8) and without              (9)
     Specificity       of the method was examined                    by analy-       chromatography.
sis of blanks,         determination         of antibody          specificity,           Plasma      testosterone         concentrations         increase      dur-
and by comparison              of results obtained           by the present          ing pregnancy.           Clinically,     these patients         showed no
 method        with those obtained              by using established                 signs of hirsutism          or virilization,      probably       because of
 methods         (3, 5, 8,, 9). Water was used to investigate                        the increased          concentration         of testosterone-binding

                                                                                                         CLINICAL CHEMISTRY,         Vol. 21, No. 2, 1975      209
                            Table 4. Plasma Testosterone                       Values (ng/ dl) for Normal Persons
                                                                                                              Female                                   Male
                  Author                                            Method                           No.            Mean ± 2 SD           No.          Mean ± 2 SD
      Rivarola and Migeon (3)                      Double.isotope dilution                             9             47 ±   30             13           551 ±     302
      Mayes and Nugent (5)                         CPBA                                               18             40 ±   28             16           680 ±     360
      Furuyama et al. (8)                          RIA with chromatography                            12             33 ±   14             13           590 ±     298
      Ismail et al. (9)                            RIA without chromatography                          6             46 ±   42              9           596 ±     404
      Present authors                              RIA without chromatography                         26             45 ±   20             21           684 ±     300




globulin.       Separate          experiments          showed        that     the    References
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specimens       were also increased.                                                 mination    of plasma    testosterone     in human peripheral     blood using
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210     CUNICALCHEMISTRY,Vol. 21, No. 2,              1975

								
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