Radioimmunoassay of androsterone and androsterone-3-sulfate in plasma

727

RADIOIMMUNOASSAY

OF ANDROSTERONE IN PLASMA

AND

ANDROSTERONE-3-SULFATE

Jacob Kream, Leon Hellman and R. S. Rose&Id Institute for Steroid Research and Department of Oncology, Montefiore and Medical Center, New York, New York 18467

Received:

Hospital

Y/10/76 ABSTRACT

Details of a sensitive and specific radioimmunoassay for androsterone (1) and androsterone sulfate in plasma have been presented. Benzene extracts of plasma were chromatographed on alumina to isolate the androsterone fraction either (a) directly after extraction (A) or (b) after solvolysis (AS). Following treatment with rabbit anti-A-17-BSA, antibody bound steroid was precipitated by ammonium sulfate. Androsterone concentrations in normal male plasma averaged 57 4 24 (SD.) ng/dl, range 35-135 ng/dl and for normal women, 44 2 21 (S.D.) ng/dl, range 18-98 ng/dl. Androsterone sulfate concentrations were: males 55 2 28 p$dl (range 10-l 14 pg/dI); premenopausal females 52 I! 31 pg/dl (range 16-318 pg/dl). Androsterone,

a major metabolite

of the androgenic hormones dehydroiso-

androsterone and testosterone, is a 17-ketosteroid (2).

with physiologic properties

In man it is rapidly conjugated with sulfuric acid and glucuronic acid; the

glucuronates are excreted

rapidly (3) so that AS, except for DHA sulfate, com-

prises the largest amount of circulating

steroid.

Recently, abnormal concentrations

of androsterone conjugates in blood and urine have been reported in a variety disease states (4-8) which makes their measurements of increasing interest. of their low affinity non-immunologic

for plasma proteins, the 17-ketosteroids

competitive

and generally

cannot be measured by

have been assayed by more tedious

and less sensitive methods such as double isotope derivative and spectrophotometry.

Vo’owne 27, Number 6

Because

protein binding techniques (unless the 17-keto group

is first reduced to a 17-hydroxyl)

chromatography,

of

S

analysis, gas liquid

In view of the methodological

WDICOXDI

difficulties

June,

2976

S

728

'EEIIEtOXDS

encountered above, radioimmunoassay offers the most convenient and sensitive technique for assaying this steroid and its major circulating

metabolite.

Although several RIA procedures have been reported for the analyses of DHA and DHAS in the circulation have appeared (15).

(9-14)

few methods for plasma androsterone

It is the purpose of this ~mmuni~tion

to describe a tech-

nique for the analysis of A and AS in the plasma using an antiserum to an androsterone-BSA

conjugate prepared in this laboratory

(16); a preliminary

report

describing this assay has been presented (17). MATERIALS Benzene (spectroanalyzed), methanol (certified A.C.S.), ethyl acetate (spectroanalyzed), acetone (certified A.C.S.), acetic acid, glacial (reagent grade A,C,S,), boric acid (certified A.C.S.), sodium azide (purified) and sodium chloride (certified AC‘S) were all purchased from Fisher Scientific Company and were used without further purification; toluene and xylene (certified A.C.SJ from the same supplier were redistilled before use. Aluminum oxide, Woelm neutral (M. Woelm Eschwege, distributed by ICN Pharmaceutics, Inc., Cleveland, Ohio 44128) was washed three times with distilled water, three times with methanol, three times with 6% (aq.) acetone, and finally dried at 72OC overnight. Ethyl alcohol, absolute-reagent quality, U.S.P, (U.S. Industrial Chemicals Co., New York, N.Y.) was used without further purification. Sodium sulfate, anhydrous, granular, reagent A.C.S. was obtained from Matheson, Coleman and Bell, Norwood Ohio 45212) and both ammonium sulfate, special enzyme grade (Cat. no. 1946) and albumin, bovine powder fr. V, were supplied by Schwarz/Mann, Orangeburg, N. Y. 10962. Freund’s Bacto Adjuvant, complete, was supplied by Difco Laboratories, Detroit, Michigan; Triton X-l 14 was obtained from McKesson Chemical CO., New York, N.Y. 10017. Human gamma globulin, (Immu-G, Stock 4-619-l) was supplied by Park, Davis and Co., Detroit, Michigan 48232. 2,%Diphenyloxazole (PPO) and butyl-phenyl biphenyl-oxadiazole (Butyl PBD) were purchased from NuclearAssociates, Inc., Westbwy, New York 11590. Androsterone was obtained from Sigma Chemical Company, St. Louis, Missouri, and was recrystallized from acetone-isooctane before use. Androsterone-3-sulfate (3a-hydroxy-So-anbostan-17-one-3sodium sulfate) was obtained from Schwarz/Mann as the hemihydrate. After solvolyzing a sample in the presence of added 1,2-3H-androsterone, the liberated free androsterone was determined by means of isotopic dilution. From the data obtained, the purity ot the androsterone-3-sulfate was determined to be IO0 percent. 1,2-3H-Androsterone Nuclear,

(N),

Boston, Mass. 02118.

40-50

Ci/mM,

Upon receipt

was purchased from New England it was diluted with a benzene to a

final concentration of lOpCi/mi (stock solution) and stored in the dark at room temperature. Its purity was determined periodically by means of a reverse isotope dilution technique. A stock solution of 1,2-3H-anckosterone-3-sulfate in ethyl alcohol was also prepared. The sulfate was obtained from the free labeled androsterone by treatment with the triethylamine-sulfur trioxide complex in pyridine (18). Reverse isotope dilution analysis and evaluation of contamination by free androsterone showed it to be > 99% pure. Counting was performed in a Packard Tricarb Scintillation Spectrometer, Model 3375, using low potassium glass vials (cet. 6(6001009, Packard Instrument Co.,Inc., Downers Grove, III. 60515). For counting of labeled androsterone, a scintillant composed of 2% methanol and 0.5% PPO in toluene was used (19); 10 ml ~cintillant A) provided a two-phase system when mixed with the 025 ml aliquots counted in connection with the RIA for free endrosterone. The counting efficiency for tritiy was 39.3 + -08 (SD.)%. For counting of 1,2-31i-androsterone-3-sulfate and 1,2- H-endrcsts&e which was recovered from aluninun oxide columns after solvolysis in the radloimmunoassay of androsterone-sulfate, the following scintillation fluid was used: 0.6% butyl PBD, 25% (v/v) Triton X-l 14, 5% (v/v) water in xylene. The counting efficiency for tritium was 21.5 f 0.2 (S.D.) % when 18 ml (scintillant B) was mixed with 0.5 ml of aqueous sample. Samples were extracted in 12 ml Pyrex conical centrifuge tubes with ground glass stoppers, Disposable 10 mm x 75 mm (O.D.) glass test tubes were used for the radioimm~~~y and disposable 5 ml serological glass pipettes served as micro columns employed in the p~ifi~ti~ of extracts prior to RIA. All glassware was washed in chromic acid, rinsed with tap, then distilled water, and finally dried in a hot air oven. All non-volumetric glassware (including disposable items) were heated for 4-5 hours in a General Electric Self Cleaning Oven (Model JK 14) set at the self-cleaning mode. Buffer-BSA-Hgg solution (Standard Buffer). All antibody dilutions were made up in a standard buffer having the following composition: 0.05 M borate buffer, pH 8.0, containing 0.5% (w/v) BSA and 0.05% (w/v) Hgg. It is similar in composition to the buffer system described by Fwuyama et al (20) except that it was found necessary to adjust the BSA concentration from 0.06% to 0.5% in order to minimize non-specific binding of i,2-3H-ardrosterone during the RIA procedure. METHODS Preparation of antisera against androsterone-17-BSA

conjugate

The 17”carboxymethoximino derivative of androsterone was prepared and conjugated to BSA by the mixed anhydride technique of Erlanger et al (21). Details of the preparation of the conjugate as well as its properties have been described in a previous report (16).

Immunization

of animals

New

Zealand

antisera

against

antigen

which

of complete

white,

four different

(wt.

animals

were

Adiuvant.

four weeks; Blood

every

specimens

means of a central

puncture

serum (0.25

titers

were

estimated

6000

of saturated for

in the cold.

mlaliquot

determine

A 0.25 the amount

calculated

for all

(0.1

buffer

distributed

containing

solution).

of at least

K value

Preparation

of plasma

plot

tubes.

androsterone

mixer could

cpm/pg)

was then pipetted before

and after

recovery,

in benzene

under a stream into

with

each

water.

standing

of the animals

and diluted in sterile titer

150

in

vaccine

vials

was observed

used for radioimmuno-

constant of 2.2

method

at -2OOC

(K) was determined

x lo9

(23)

M-l.

was 1.7x

Alternatively lo9

M-l.

for radioimmunoassay

Plasma was extracted

at 45OC

to this volume

a value

by the saturation

extracts

dilution

above;

schedule

for radioimmunoassay.

in the antibody

The affinity

was

of the

glass tubes and stored and stored

to

ultimately

described

All

suitable

was thawed

the antiserum

(22) and gave

For estimating

(135

was evaporated

of the immunization

azide

change

solution.

obtained

1. Androsterone: centrifuge

No

3 months;

from this stock

the average

week

by the 5th week.

sodium

of 65%

the conditions

in screw-capped

0.1%

in order

The antiserum

apparently

An

standing

by centrifugation

under

one tube of the antiserum

by means of a Scatchard

ally,

by

radioactivity

bound an average

the 37th

antibodies

and after

was counted

counts added.

antibody

produced

(stock antiserum a period

brought

obtained

long sterile

Antibody-bound

of 1:20,000 after

mixing

was removed fluid

radioactivity.

a detectable

ml) were

assay was made

plasma

were

inch

for 48 hours at 4’C.

while

the precipitate

of the total

from an animal

As needed,

the standard during

of unbound

eventually

used.

was added

of the supernatant

at a dilution

had produced

inoculated

1,2-3H-androsterone

of the assays in this report,

radioactivity

it was obtained

with

15 minutes

as the percentage

employed

at 4’C

and once a month

titers

1 l/2

at

once a week

ity of serially diluted antisera 5 H-androsterone. Diluted anti-

by the abi

(NH4)2S04

at room temperature

until

six weeks,

19 gauge

2 ml

intradermally

schedule:

of antibody

cpm (45 pg) of 1,2-

ml) was incubated

volume

Aliquots

made

1.0 mg of with

needles.

Antibody

which

were

for the next

using

with

and emulsified

to the following

week

used to produce

immunized

NaCl

for the estimation

ear artery

to bind approximately

added

third

Ibs) were

each

Inoculations

sites on the back according

thereafter.

lo-15

in 0.5 ml of 0.9%

Bacto

for the first

equal

rabbits

Three

was dissolved

Freund’s

disposable

mature

A-178SA.

in 12 ml glass stoppered

approximately

was added

1000

to each

of dry nitrogen.

tube;

Samples

were

mixed

at room temperature

the plasma

was extracted

immediately;

however,

be frozen

and extracted

on a subsequent

day.

tube and the solvent

0.5-2-O

if less than 2.0

conical

cpm of 1,2-3H-

ml, briefly

for about

ml of heparinized

the samples

were

on a “vortex” 15 minutes.

at this point

Gener-

the material

Extraction was carried out with two 4 ml portions of benzene by mixing on a “vortex” mixer for 1 minute and centrifuging in order to separate the aqueous phase. Both benzene extracts were combined (about 8 ml) prior to column chromatography. The columns were made using 5 ml disposable pipets containing a small plug of glass wool in the constricted tip. Alumina was added and packed down by tapping so that it occupied a volume of 0.6 ml above the glass wool. The columns were prewashed with 5 ml ot benzene taking care that the columns never ran dry. The benzene extract was introduced directly into the column using a disposable Pasteur pipet. The eluate was discarded and the column was then washed with an additional 5 ml of benzene which was also discarded. Elution of androsterone was carried out with 5 ml of benzene containing 1% ethanol and was collected in a 5 ml volumetric flask made to the mark with the same solvent if necessary. Duplicate 1.0 ml aliquots were pipetted into counting vials, evaporated to dryness at 45OC under nitrogen and counted with scintillant A for the estimation of recovery which averaged 89.9 +5.2 (S.D.)%. Duplicate one milliliter aliquots were generally used for RIA. Howe& this volume could be varied depending upon androsterone levels to be expected. 2. Androsterone sulfate: For recovery, approximately 1000 cpm of 1,2-3Handrosterone-3-sulfate (60 cpm/pg) in ethyl alcohol was pipetted into each glass stoppered centrifuge tube and evaporated to dryness under nitrogen. Plasma samples (usually 0.05 ml, but variable according to the amount of androsterone sulfate expected) were measured intotre tubes, mixed on a “vortex” mixer, and kept at room temperature for 15 minutes and remixed. Saturated aqueous sodium chloride (0.95ml) was then added to each tube and mixed, followed by 5 ml of ethyl acetate. After agitating for one minute, the phases were separated by centrifuging at high speed and the bulk of the aqueous (lower) layer was removed by means of a disposable Pasteur pipette and discarded. Sufficient anhydrous sodium sulfate was added to the ethyl acetate extract so that it formed a solid cake with the residual aqueous layer,. After mixing, the ethyl acetate extract was decanted into a 50 ml Pyrex glassstoppered bottle and the residue was washed with an additional 2 ml of ethyl acetate which was combined with the extract. Two milliliters of glacial acetic acid were introduced and the stoppered bottles were incubated in a hot air oven at 70°C overnight. Weights were put on the stoppers in order to prevent them from blowing off. The solvent mixture was then evaporated to dryness at 45°C under nitrogen and the residue was extracted with 2 x 5 ml of benzene. These extracts were transferred directly into washed Al203 columns. The elution of androsterone was identical to the procedure described previously. For the estimation of recovery, duplicate 1.0 ml aliquots were pipetted into counting vials, dried, 0.5 ml of water added then 18 ml of scintillant B. The recovery of 1,2-3H-androsterone-3-sulfate (as 1,23H-anckosterone) averaged 76.2 + 6.5 (SD.)%. Duplicate 0.05 ml aliquots were taken for RIA but could be varieddepending upon androsterone-sulfate levels. Scintillant B was used for estimating recovery because labeled androsterone sulfate, which was introduced at the onset and which also has to be counted, is too polar to dissolve in scintillant A; thus a conversion factor in the recovery calculation was obviated. Tritium counting in the binding step after precipitation of the y -globulins required scintillant A (2 phase system) because of the presence of ammonium

S

732

sulfate

TDEOID=

in the supernatant.

Radioimmunoassay 1.

0.5 ml aliquots

(approximately 10 pc/ml)

6,000

are pipetted

tubes in sequence plasma

extracts

are prepared so that

are introduced (in duplicate).

by diluting

respectively

(0.5

blank

through

as well

of non-specific containing

2.

binding

the solvent.

the simultaneous in standard

estimation

of non-pecific

The tubes are agitated 5 minutes, incubated 3.

remixed at 4°C

utes. al

approximately

5%)

under a stream

and/or

pools.

androp

For the deter-

triplicate

tubes are

0.25 tube,

receive

0.25

for about

5 seconds,

of dry nitrogen

constructed

to each

mixer

in a

for this purpose

ml of antiserum except

ml of standard with

(diluted

the tubes for the

5 seconds,

covered

the free steroid

ml of an aqueous

tube while

room temperature

which

on a “vortex”

incubation,

0.25

to each

plasma

buffer

heated

parafilm,

only.

at 45°C

for

and finally

for 48 hours.

Following

form as follows:

Each run also includes

for androsterone

of 64 such tubes.

is then added

for an additional

per ml) picograms

and 0.5 ml of benzene.

to 45“C

binding

10 to 400

level).

A glass manifold

treatment

buffer)

” 0”

from

from standard

(averaging

in benzene

(1 .O pg androsterone

described

prepared

and the chromatographed

contain

is used for the

the procedure

of

the same

of androsterone

solution

to the tubas

The tubes are then heated

lPO,OOO

added

of a stock

1,2-3H-androsterone

bath to remove

permits

solutions

as extracts

included

glass test tubes; into

(in triplicate)

portions

in benzene

the stock solution

standards

ml of benzene

mination

water

by diluting

Ten standard

when added

taken

terone-sulfate

of 1,2-3H-androsterone

into a series of disposable

0.5 ml aliquots

a water

of a solution

cpm, 45 pg, prepared

on a “vortex”

mixing

for 15 minutes

The supernates

and

are decanted

is separated

saturated

solution mixer.

centrifuged

into

from its antibody-bound

of ammonium

sulfate

is

The tubes are maintained

in the cold at 2500

fresh tubes and recentrifuged

rpm for

at

15 min-

for an addition-

15 minutes. 4.

scintillant set at

A 0.25

ml aliquot

A by shaking

150 excursions

per minute

for at least 2 hours before In order

to determine

cate

0.5 ml aliquots

0.25

ml of a solution

(NH4)2804 counted

(mixed

of each supernate

at room temperature and then

radioactive

made 1:l).

permitted

amount

tracer

from 0.5% After

fraction)

of 20,800

of lO.ml

10 ml of 6000)

in the counter

in each

in counting buffer,

with (Model

counts are taken.

of radioactivity

are dried

BSA in borate

addition

is mixed shaker

to equilibrate

assay; a total

the total

of the diluted

(free

on an Eberbach

vials

pH 8.0,

of scintillant,

tube,

tripli-

and mixed

with

and saturated

the vials

are

as usua I.

Figure

1, a plot of % 1,2-3H-androsterone

ical &se res nse curve obtained range of trip Pp ~cate determinations

bound vs. mass, represents

a typ-

over a range of O-400 pg. The mean and the of the % bound radioactivity are shown for each

s

TlllEOXDI

dose. In addition, 0.25 ml, 0.5 ml, and 1.0 ml aliquots of a chromatographed plasma pool extract were analyzed for androsterone; the quantities fall along the curve. Alternately, plotting the standard curve as a &t-log transformation, linearized it over a range of 10-400 pg.

Figure 1. Dose response curve for androsterone using rabbit antiserum to A - 17-BSA at a dilution of 1:20,000. #

mean and range of triplicate

v chrcmatographed

determinations.

plasma pool extract and aliquot analyzed.

733

S

734

TDROIDb

Calculations

P9

picograms

=

of androsterone

bound in all

analyses

as for the radioactivity

present

sequence

recovery

rection

of procedural can be neglected

R

=

procedura

I recovery

s

=

ml plasma

extracted

CA

=

factor

from dose response

when

for nonspecific

in the column

aliquots

determinations analyzing

curve.

binding (this

The % as well

as a conlatter

cor-

for AS).

fraction

to compensate

in androsterone

for column

aliquots

used and change

in units

analyses

corresponding

CAS=

taken

is corrected

compensating

A

=

ng/dl

=

E

(CA)

AS

=

pg/dl

=

E RS

(CAS)

factor

in androsterone

sulfate

analyses

RESULTS Sensitivity From the dose response detect

approximately

blanks

for the complete

8 + 2 (S.D.)

pg;

the presence (24); terone

and

androsterone-sulfate,

limit

= blank

values

of 7 ng/dl

for androsterone

sulfate.

these values

55 and 62 yrs),

and adrenalectomy,

was approximated

showed

x S.D.

each

Assuming

blanks

can

for the water

runs weresndrosterone,

pg.

The least detectable by the following

average

dose in

relationship

to be 13 pg for androp recoveries

to calculated

for androsterone-sulfate.

of whom had undergone

androsterone

of reagent

values

and was found

are equivalent

and 4 pg/dl

mean

selected

6 + 3 (S.D.)

(pg) +2.5

14 pg for androsterone respectively,

(42,

However

assay in 20 randomly

and 76.2%

women

the assay in the absence

5 pg of androsterone.

of these blank

detection

curves,

mean

blank

values

Plasma from three

mastectomy,

and androsterone-sulfate

of 89.9%

oophorectomy,

concentrations

S indistinguishable

735

?FBDOXDI

from the procedural water blanks.

Precision Androsterone:

The intra-assay precision was determined by 15 assays of a

normal male pool for androsterone. coefficient

of variation

(C.V.)

runs (assays in duplicate)

The mean was 42 22

of 4.8%;

ng/dl with a

the interassay precision in 5 different

showed a mean of 42 24

Androsterone-sulfate:

(SD.)

The intra-assay

(SD.)

ng/dl,

8.6%

C.V.

precision in each of 3 plasma pools

assayed 12 times within one run were as follows: (mean + S.D. in pg/dl and C.V. in %): 14 + 1.1, 7.9%; assayed in duplicate

25 + 1.7, 6.8%;

34 + 2.9, 8.5%.

The latter pool, was

in 12 runs to give the interassay precision of 33 + 4.4,

13.3%.

Recovery Increasing amounts of androsterone and androsterone sulfate were added to portions of two plasma pools which were then analyzed procedure.

for A and AS by the RIA

Each measurement was performed in quadruplicate;

the data are

shown in Table I. Table I. Recovery of Added Steroid to Plasma Androsterone (ng/dl)* Pool A Added 0 25 50 100 200

Recovered 21 53 106 203

Androsterone Sulfate (pg/dl) Pool 6 Added 0 14 27 55

* Endogenous androsterone, 7 ng/dl; endogenous androsterone-sulfate, The amounts recovered have been corrected for endogenous steroid.

Recovered 13 25 53

14 pg/dl.

S

736

TDROIDCII

Specificity The specificity steroids

of the antiserum

of the Cl*,

significant

and C2,

cross-reactivity

the steroids in high

Cl9,

which

enough

remain Table

levels

Only

the antiserum

which

shows the greatest

Cross-Reactivity

during

of Various

with

of the Cl9

would

to be present

would,

Steroids

with

Antiserum

(androsterone)

37

3o-Hydroxy-4-androsten-17-one

12 9-l

/?-Dihydroxy-5cr-androstan-l-/-one

3/3-Hydroxy-So-androstan-17-one

7

3o-Hydroxy-5_androsten-17-one

5 (etiocholanolone) A-203

3&Hydroxy-5&androstan-17-one 3&Hydroxy-5_androsten-17-one

(dehydroisoandrosterone)

< 0:01

17/3-Hydroxy-4-androsten-3-one

(testosterone)

< 0.01

17P_Hydroxy-5~androsten-3-one

(dihydrotestosterone)

< 0.001

5-Androstene-3@,17&diol

< 0.01

(estradiol)

0.3

3a-Hydroxy-So-pregnan-2O-one

0.03

3o-Hydroxy-5Bpregnan-20-one 11 -Keto-5a-pregnane-3o,

< 0.01 < 0.01

4 -Androstene-3,17-dione 1,3,5-Estratriene-3,17/l-diol

17a,20&2

0.03

1 -tetrol

3P_Hydroxy-5o-pregnan-20-one

< 0.01

20-Keto-5o-pregnane-3a,11/3,17cu,21-tetrol

< 0.01

3bHydroxy-5-pregnen-20-one

< 0.01

Cortisol

co.01

Progesterone

< 0.01

*Cross-reactivity competing antibody

steroid (X

100).

*

100

5cY-Androstane-3cr,17Bdiol

3cr-Hydroxyd&androstan-17-one

if

chromatography.

% Cross-Reactivity

3a-Hydroxy-Sa-androstan-17-one

showed

of the assay.

cross-reactivity

represents

the ratio

required

to displace

of the mass of androsterone 50%

of the 3H-androsterone

of

II. None of

be expected

the specificity

Substance

3o,ll

a number

series

as can be seen in Table

to influence

columns

was tested

members

cross-reactivity

in the circulation

on the Al203

II.

series.

show significant

So-Androstan-3@,17&diol present,

with

for androsterone

to the mass of bound

to the

This paper

Rosenfeld and Hellman (27)

17-60 14-82 38-61 pre-menopaUSal

M F M F

Gas-Liquid chr~atogra~y

RIA

2”2

24 31

41 51

52.6 (mean) 45.0 (mean)

M F

Gas-Liquid

ch roma togra phy

Wang et al (26)

55 2 28 52231

68 23

41.4

40.5

AndrosteroneSulfate (pg/dl)

24 21

57: 442

27 58

1) SD 80 30

24-76 21-76

M F

RIA

This paper

(wI’d Mean 2 160 !: 70 2

hdrosterone

542 32 46’28

20-45 18-30

M F

RIA

Youuefnejadian et al (15)

17 7

No.

in Normal Human Subjects

40 40

20-45 20-40

M F

Age (yr)

Sex

Double isotope derivative

Method

of Androsterone and Androsterone-3-Sulfate

Gandy and Peterson (25)

Author

Plasma Concentration

Table 111

10-l 14 16-138

21-130 6- 68

4-104 5-129

35-135 18- 98

24-l 17 12-w

60-290 30-l 10

Range

S

738

T=PIROIDIB

Plasma concentrations Table III lists normal levels of plasma andosterone in male and female subjects. Although there are significant isotope derivative is excellent

and androsteronesulfate

Plasma samples were obtained in the morning. differences

between the RIA data and the double-

values for androsterone obtained by Gandy and Peterson (25) there

agreement between our values and those obtained by Youssefnejadian

al (15) by RIA.

Our androsterone~ulfate

concentrations

are in good agreement with

the gas-liquid

chromatography values obtained by Wang et al (26) and Rosenfeld

and Hellman

(27) although the data of the latter authors demonstrated a sex differ

ence which might be related

to the age differences

et

in the female subject chosen

for analysis, post menopausal values tending to be lower (26). ACKNOWLEDGEMENTS We are grateful to Mrs. Hilda Lederer, Mr. Andy Grigorian and Miss Anne Thalarsinos for their valuable assistance. This work has been supported by Grants from the Nationai Heart and Lung Institute (HL 147341, the National Cancer Institute (CA 07304), and the General Clinical Research Centers Branch (RR 53) of the National Institutes of Health. REFERENC ES

1.

2. 3. 4. 5. 6. 7. 8.

Abbreviations: A, androsterone, 3a-hydroxy-%-androstan-17-one; AS, androsterone sulfate, DHA, dehydroisoandrosterone, 3&hydroxy+androstenI7-one; DHAS, dehydroisoandrosterone sulfate; RIA, radioimmunoassay; BSA,, bovine serum albumin; Hgg, human gamma globulin; A-17-BSA, So-hydroxy%-androstane-17 (O-carbox~ethyl) oxime BSA. Hellman,L,, Ekadlow, H-L., Zumoff, B., Fukushima,D.K. and Gallagher, T.F, J Clin Endocr 12, 936 (1959). Hellman, L. and Rosenfeld,R.S., J Clin Endocrinol Metab 5, 424 (1974). Bulbrook, R.D., Hayward, J.L. and Spicer, CC., Lancetz, 395 (1971). Rao, L.G.S., Nature 235, 220 (1972). Marmorston, J., GellerP.J., Weiner, J.M., Allain, C.C., Pare, J.H., Bush, I.E. and Roberts, J.B., Physiol Chem and Physics_2, 337 (1970). Rao, L.G.S., Lancetz, 390 (1970). Dey, AX., Abbott, EC., Rust4I.E. and Senciall, I.R., Can J Biochem 50, I273 (1972).

S 9. 10. 11. 12. 13. 14. 15. 16. 17. 18. 19. 20. 21. 22. 23.

24.

25. 26. 27.

WDEOXDI

Nieschlag, E., Loriaux, D.L. and Lipsett, M.B., Steroids 19,669 (1972). Buster, J.E. and Abraham, G .E., Anal Letters?, 203 (19’32). Sekihara, H., Ohsawa, N. and Ibayashi, H., Steroids 20, 813 (1972). Sekihara, H. and Ohsawa, N., Steroids 24,317 (1974r Rosenfeld, R.S., Rosenberg, B.J. and HelGan, L., Steroidsz, 799 (1975). Smith, M.R., Rudd, B.T., Shirley, A., Rainer, P.H.W., Williams, J.W. Duignan, N.M. and Bertrand, P.V., Clin Chim Acta 5, 5 (1975), Youssefnejadian, E., Collins, W. P. and Sommerville, I-F., Steroids 22, 63 (1973). Rosenfeld, R. S., Rosenberg, B. J., Kream, J. and Hellman, L., Steroids 21, 723 (1973). Kream, J., Rosenfeld, R.S., Rosenberg, B.J. and Hellman, L.,Clin Chem 12, 713 (1972). Dusza, J.P., Joseph, J.P. and Bernstein, S., Steroids 12 49 (1968). Mayes, D.M. and Nugent, C.A., Steroids 15, 389 (1976). Furuyama, S., Mayes, D.M. and Nugent, Cx., Steroids l& 415 (1970). Erlanger, B.F., Borek, F., Beiser, SM. and Lieberman, S., J Biol Chem 228, 713 (1957). Etchard, G., Ann NY Acad Sci, 51,660 (1949). Odell, W.D., Abraham, G,A., Skow&, W.R., Hescox, MAA. and Fisher, D.A. in Principles of Competi~tive Protein Binding Assays (Odell, W.D..and Daughaday, W.H. eds.), pp. 5/-/6, Lippincott, Philadelphia, 1971. Ekins, R. and Newman, B., Theoretical Aspects of Saturation Analysis. in Steroid Assay by Protein Binding, E. Diczfalusy, Ed, Transactions of the Second Symposium, Karolinska Symposia on Research Methods in Reproductive Endocrinology, Stockholm, 1970. Gandy, H.M. and Peterson, R.E., J Clin Endocrinol Metab 28, 949 (1968). Wang, D.Y., Bulbrook, R.D., Thomas, B.S. and Friednan, K, J Endocrinology c, 567 (1968). Rosenfeld, R.S. and Hellman, L., Steroids fi, 675 (1969).

739