Radioimmunoassay of urinary testosterone glucuronoside

285

RADIOIMMUNOASSAY John

F.

Hennam,

OF URINARY

William

P.

Department of Biochemical for Women, and Institute Dovehouse Street, London, Received:

TESTOSTERONE

Collins

and

Endocrinology, of Obstetrics SW3 6LT, U.K.

Ian

GLUCURONOSIDE F.

Sommerville.

Chelsea Hospital and Gynaecology,

11/U/72

ABSTRACT A simple method is described for the determination of testosterone glucuronoside in urine without prior hydrolysis or extraction. Appropriate amounts (5 ~1 male, 50 ~1 female urine) are dried at 100°C to eliminate nonspecific binding by urinary proteins. The residues are cooled, redissolved in buffer and equilibrated with antiserum to testosterone-17P-glucosiduronate-bovine serum albumin and tritiated testosterone glucuronoside. The unbound steroid is removed with dextran-coated charcoal. The total random theoretical percentage error was calculated as 7 5. The inter and intra assay precision were 18 and 9 $ respectively. Daily urine collections from 8 complete menstrual cycles have been analysed and the results related to the peak of urinary LH. In seven subjects, there was a visible peak of testosterone glucuronoside at mid cycle (LH peak + 1 day), in six a distinct peak in the luteal phase, and iz five a smaller peak during the follicular phase. The levels of testosterone glucuronoside in groups of healthy men and women were The corresponding range 273 + 169 and 19 + 10 pg/24 hrs. of values by a gag-liquid chromatographic method were 204 + 102 and 14 + 8. The values are discussed.

INTRODUCTION The is

level

frequently

of

testosterone

used

as

men and women.

The

biochemical

clinical

available

and methods

glucuronoside

an index

significance

reviewed

terms and

of

androgen of

has

(1)

urine

production

this

approach

been

evaluated,

preliminary

in

results

in in

both

the compared

21:2

STEROIDS

286

(2,

A new

3).

lite

was

method

suggested

prepared

and

by

for

the

the

work

described

the

determination of

of

Kellie

properties

of

testosterone-17P-glucosiduronate-bovine present in

report

a simple

method

glucuronoside The

mean

been

nickel-63

in and

compared

established

is

range with method,

electron

for urine

with

the

the

determination

without

of

values

those

in

al.

(4)

an

antiserum

of

this

of

testosterone

by gas-liquid

men a more

who to The

albumin.

hydrolysis

healthy

obtained

involving capture

prior

use

metabo-

et

serum

concerned

this

antiserum

or and

extraction.

women

have

time-consuming,

chromatography

detection. MATERIALS

Buffer Tricine (N-Tris(hydroxymethyl)methylglycine) buffered saline (TBS) containing 0.1 $ geltaine was prepared by dissolving 9 g sodium chloride, 1 g sodium azide and 1 g of gelatine in 1 litre of 0.1 M tricine in deionized distilled water (ddw). The pH was adjusted to 8.0 using 10 N sodium hydroxide and the solution stored at 4OC. Standards The glucuronosides of testosterone, androsterone, aetiocholanolone and dehydroepiandrosterone, oestradiol17P-glucuronoside and testosterone sulphate were donated as their sodium salts from the M.R.C. Steroid Reference Collection, Westfield College, London, N.W.3. A sample of epitestosterone glucuronoside was obtained from Professor A.E. Kellie, Courtauld Institute of Biochemistry, The Middlesex Hospital Medical School, London, WlP 5PR. Standard solutions were prepared in TBS or ddw.

3 H-glucuronoside Testosterone-1,2specific activity 42.3 mCi/mM was obtained from New England Nuclear Corp., Boston, Mass. A solution was prepared containing 10 @i/ml in ethanol. Before use, 100 ~1 are removed, thoroughly dried under N2 at 60Oc and redissolved in

with

Feb. 1973

10 ml 20,000

STEROIDS of TBS. One hundred ~1 containing dpm (100 pg) are used in the assay

287

approximately system.

Antiserum Samples of antiserum produced by rabbits to testosterone-glucosiduronate-bovine serum albumin (batch nos. 50 and 51) were kindly donated by Professor A.E. Kellie. The serum was diluted 1:4 with TBS and stored at -15'C. Prior to use aliquots from each batch were diluted 1 to 250 with TBS and refrozen. The final dilution was made immediately before use. Dextran-coated charcoal Norit A charcoal from the Sigma Chemical Co., St.Louis, U.S.A. was washed with ddw until all fine particles MO., were removed. The purified material was thoroughly dried at 12OOC. Dextran T 70 from Pharmacia, Uppsala, Sweden, was dissolved in ddw (containing 0.1 $ sodium azide (w/v)) to a concentration of 100 mg/ml. The Norit A charcoal (5 g) and dextran T 70 (5 ml) were added to 1,000 ml of The solutions were stored at 4OC. TBS. Radioactivity measurement The scintillation fluid was prepared by dissolving 6.0 g of 2,5-diphenyloxazole (PPO) and 0.5 g 2-p-phenylenebis (5-phenyl-oxazole) (POPOP) in 1,000 ml of toluene and SubS8qU8ntly adding 500 ml of Triton X-100 (Koch-Light Laboratories Ltd., Colnbrook, Bucks, England). The solution was stored in the dark at 2OC for at least 2 hours before use. All samples were added to disposable plastic vials (Kautex Poly Vials, Packard Instruments Ltd.) containing 10 ml of scintillant and counted for a total of 4000 counts (+ 1.6 $) i n an automatic liquid scintillation system. The counting efficiencies were determined for each sample from standard curv s for an external standard channels ratio method using a a source. METHOD a)

Equilibration with antiserum The volume of urine from a 24-hour collection is adjusted to 2000 ml with ddw. If the initial amount ie then the total volume is recorded greater than 2000 ml, and th8 final answer corrected accordingly. Aliquots (50 ~1) are removed in triplicate with a Biopette (Schwarz Mann Bio Research, Orangeburg, New York 10962) which has a disposable tip. Seven hundred ~1 of ddw are added to

21:2

STEROIDS

the contents mixed on a Rotathe aliquots of male urine, mixer (Hook & Tucker Ltd., 301 Brixton Road, London, S.W.9) affecting a 1 in 15 dilution. The and 700 ~1 removed - thus glass 75 x 10 mm) containing 50 pl from tubes (disposable, both men and women, are transferred to a dry block heater and dried at 100°C for 15 min. After cooling to room temperature, the residue is redissolved in 100 ~1 of TBS. A standard solution of testosterone glucuronoside is serially diluted (1:l v/v) in TBS such that 100 ~1 contain 0.125 and 0.0625 ng. A standard curve is 2, 1, 0.5, 0.25, prepared by taking 100 ~1 of TBS for the zero point and 100 pl5of each standard solution in triplicate. One hundred H-testosterone glucuronoside solution in TBS is pl of added to both standards and unknowns. Finally, 100 pl of antiserum at an appropriate dilution (1:2,000) in TBS is added and the solutions mixed on a Rotamixer before placing in a refrigerator at 4OC. The time allowed for equilibration is standardised at 15 min, but for convenience may be allowed to continue overnight with a slight increase in initial percentage binding (5 $). b)

Separation of antibody bound steroid Dextran-coated charcoal, at 4OC, is maintained in suspension by a magnetic stirrer and 1 ml added to all tubes with an automatic pipetting syringe. oAfter mixing, the tubes are placed in a refrigerator at 4 C for 10 min, and then centrifuged (10 min, 7 C, 2,000 rpm). The supernatant, containing the bound fraction, is decanted into a counting vial and 10 ml of scintillation fluid added. A mixture of 100 pl of in TBS, 100 ~1 of antiserum to assay tubes in triplicate, that the initial percentage c)

Factors affecting binding The optimum dilution of percentage binding and overall checked at regular intervals. study, a dilution of 1:2,000

3 H-testosterone glucuronoside and 1. 1 ml of TBS was added decanted and counted so binding could be determined.

and displacement antiserum in terms of initial displacement of steroid is Throughout the present (v/v) was used.

The use of phosphate (pK 7.2, pH 7.0) and tricine (pK 8.1, pH 8.0) buffers were compared. At the same dilution of antiserum, the initial percentage of 3Htestosterone glucuronoside bound was similar (+ 5 $), but the displacement of radioactivity by 1 ng of nonlabelled testosterone glucuronoside was greater (lo-15 in the tricine buffer. In addition, the use of tricine

$)

Feb. 1973

STEROIDS

buffer gave a higher and efficiency (21 ,+ 3 $).

more

289

reproducible

counting

The effect of temperature on the parameters of equilibrium were investigated and found to be similar to that observed for other antisera (5, 6). However, only relatively slight changes in the initial percentage binding and displacement were found with equilibration time longer than 15 min. d)

Standard curves A standard curve (cold) prepared as described above is shown in Fig. 1. The amount of 3H-testosterone glucuronoside bound to antibody is expressed as dpm ordinate) and the non-labelled steroid covers the range ! O-2 ng).

In addition, standard curves (hot) have been prepared using varying amounts of 3H-testosterone glucuronoside with no additional non-labelled material. Solutions of 3Htestosterone glucuronoside were prepared in TBS containing times 20,000 dpm 8, 4, 3, 2, 1.5, 1.25, 1, 0.75 and 0.5 per 100 ~1. Tubes containing 100 ~1 of these solutions with 100 pl of TBS and 100 ~1 of antiserum were prepared in triplicate. The fraction of radioactivity bound, multiplied by the amount of radioactivity added to the unknowns (100 ~1 3H-testosterone glucuronoside solution), was plotted as the ordinate, against the ng of radioactive testosterone glucuronoside minus the ng of labelled testosterone glucuronoside added to the unknowns. This produced standard curves which were not significantly different from the *cold' ones. This method was devised to check the purity and accuracy of the testosterone glucuronoside solutions and to simplify the procedure. The 'hot' curve may also be continued on the negative side of the ordinate so that the zero point may be more precisely defined (Fig. 2). e)

Calculation of results A desk-top computer, the Olivetti Programma 101, is used to calculate the mean dpm for each point on the standard curve and the unknowns. The dpm's are calculated directly from the cpm and channels ratio using a quadratic equation derived from counting quenched standards. A standard curve is constructed with each batch of and the scale of the abscissa written as urine samples, pg from standard curve and pg testosterone glucuronoside/ 24 hrs (pg x 0.04 for female urine, pg x 0.6 for male urine) (Fig. 1).

290

STEROIDS

21:2

EVALUATION a)

Theoretical The

pipettes

assessment

precision

of

the

was

to

be

theoretical as

found percentage

calculated

errors, b)

from

was

the

found

to

be

of

the

error

constant ,+ 1.3

volume $.

The the

semi-automatic total

error

of

usual

laws

for

the

less

than

7.1

(k.

random

complete

procedure, combination

of

Accuracy The

three to

accuracy

approaches. show

The

whether

the

glucuronoside/24 taken

for

ted

that

the

limits,

of

the

calculated

from

men and

women,

amounts

of

10 replicate mean

was

was

within ~1.

to

to from

urine

were

the

testosterone mean

values

of

containing

healthy

assayed The

91

demonstra-

experimental

groups

shown

volume of

Authentic

urine

are

urine

6 women

glucuronoside.

determinations from

linear,

designed

analyses

6 men and

on urine

added

the

the

from

testosterone

of

similar

testosterone

recoveries

of

from

25-100

amounts

assessed were

amount

from

assays

been

independent

urine

range in

glucuronoside,

has

experiments

results

response

over

first

was

The

amounts

method

calculated

hrs assay.

varying

for

of

in

$ for

results

from

Table

1.

The

women

and

89 $

men. To

test

the

possible

interference

of

closely

related

Feb. 1973

steroid

glucuronosides

these

compounds

urine)

were

on (in

prepared

in

on the

value

determined.

The

results

assay ratios

TBS. of

to

The

those

additive

testosterone of

mixtures

system,

found

of in

effect

of

these

glucuronoside

10 analyses

are

was

shown

in

Table

Precision Estimates

obtained

by

Snedecor

(7).

variation

of

(cv)

and

n = number On 60 with

coefficient

of

variation

d)

Sensitivity The

these

analyses

is

given

by

the

cv=

-

d2+ 2n

of

in was

lower

proposed

were by

coefficient

following

of

equation

of duplicates duplicate

performed

from was

two

:-

x

100

in

the

lo-350

9 46.

different

pg/24

In

hrs,

same

the

40 duplicate

assays

the

coefficient

18 $.

limit

can be determined measurement

the

assay)

determinations.

ranging

variation

inter

procedure

values value of duplicate

and

determinations

values

determinations

the

In

duplicate

of

(intra of

highest-lowest lowest

d =

assay,

precision

application

where

the

the

similar

compounds

c)

291

STEROIDS

of depends

of

this

testosterone upon value.

the

glucuronoside error

As the

which

associated standard

with

deviation

2.

21:2

STEROIDS

292

on the the of

first

point

standard the

curve

from

~1 of

the

in

ng on the lower

approx.

hrs

determinations)

0.0625

as

to

may be

resolving

value

power

which

different

by

limit

of

2 pg/24

hrs

from

men.

urine

obtained

the

as

on urine

samples

for

on the

mean standard volume,

and

15

pg/24

the

concentrations twice

using

in

up to

100

be

e)

Specificity

each

sample.

hrs

specificity

1 pg/24

for

samples from

of

this

value

were

performed

amounts

The from

of

was

the

the

hrs

for

urine men.

standard curve.

corrected samples

for from

Accordingly,

samples

varied

results

were

not

the

antiserum

in

terms

by

less

considered

of

women,

when

different.

of

dpm

mean

standard

masses,

l

testosterone

corresponding

the

from

significantly

about

of

different the

amounts

as

deviation

mass was read

were

significantly

The

standard

as

taken

analyses varying

be defined

may

estimate

deviations

these

to

method

Replicate

The

sample

the

An

containing

glucuronoside.

deviation

next.

follows.

calculated

of

may be confidently

from

obtained

than

of

taken

30 pg/24

overlaps

urine.

that

The

and

never

12 replicate

a reading

corresponds

women,

curve

$ on

safely

sensitivity

The

was

0,

This

standard

(4.0

may be

sensitivity.

Increased

the

deviation

corresponding

standard

urine

of

the

Feb. 1973

ability

of

closely

related

labelled

testosterone

has

been

reported

was

undertaken

activity an

of

authentic

equal

same

testosterone.

liquid

50 $ of

gluouronoside.

testosterone

competition

for

was

obtained

by

4 -

7 years

and

adults.

The

glucuronoside

in

the

defined

mean

and

range

been

of

compared

chromatographic

analysis

from

four

with

evidence urine

of

by

obtained This

from

below

sensitivity.

obtained

procedure.

epi-

were

of

those

with

ovariectomised,

subjects

values

obtained

in

values

limit

radio-

expected

of

calculated

lower

that

Indirect

the

all

of

sites

2).

potency

Analysis

ratio

binding

(Table

-

have

displacement

testosterone

adrenalectomised

assay

The

the

with

relative

of

aged

The

maximum

in

study

above,

mass

no

sites

A similar

was

mixtures,

with

described The

glucuronoside

hrs

binding

testosterone

specificity

2 pg/24

for (4).

procedure

results.

steroid

girls

al.

compete

of

testosterone

six

et

to

2 ng

showed

urine,

Kellie the

with by

by

of

by

the

obtained

steroids

glucuronoside

using

essentially was

293

STEROIDS

radioimmunoby

method

a gasinvolved

n

addition

each).

extracts fractions

at are

glucuronoside

testosterone

After

16 hrs

for

The

JH-testosterone

14 C labelled

and of

of

after

hydrolysis

37OC), fractionated containing

and

with

extraction

with

(2,000 (500

diethyl

thin-layer

testosterone

hydrolysis

P-glucuronidase

extraction by

before

located

units/ml,

ether,

chromatography are

dpm

the (TLC).

by

auto-

STEROIDS

294

radiography (THFB)

and

are

internal

eluted.

formed

and

standard

is

nickel

heptafluorobutyrate

rechromatographed added

heptafluorobutyrate with

The

21:2

and

63 electron

by

capture

A third

on TLC.

the

determined

derivatives

mass of

testosterone

gas-liquid

chromatography

detection

(8).

RESULTS The from

method

a group

throughout of

has

of the

been

healthy

as

in

the

Table

first

histogram

day

the

the

peak

is

in

Fig.

3.

-1 the

are

7 after

the

peak

7 days of

preceding individually,

daily

1 of

of

on or within

LH,

7 had

a visible

and

5 had

a small with

-14

first

to

If a visible

rise variable the

in

-8 of

the rise

mean and

are

1 to

(LH)

from

mid the

after

days

cycle, peak

phase

in

follicular of

to

considered

luteal

range

-7

7 days

the

urine from

days

are

the

in

collections

14 are

at

defined

+ S.D.)

7 the

cycles

is

are

A composite

bleeding;

peak

+ 24 hrs

cycle

(mean

8 to

the

values

occurred.

days

Days

of

hormone

day and

menstruation.

range

variation

samples

women - a total

menstrual

bleeding

Days

LH.

7 had

the

urine collections

eight

luteinising

prior,

urinary

from

mean and

daily

the

occurring

A comparison

from

on which

to

1 to

men and cycle

Day

relative

days

24-hour

The

3.

showing

shown

to

menstrual

235 determinations.

shown

applied

values

i.e. of

of

urinary the

cycle,

phase. obtained

Feb. 1973

by

STEROIDS

GLC

and as

on urine

women pg

are

shown

T

from

in

testosterone

wt

mol

samples

295

small

Table

groups

4.

All

mol

TH.FB

wt

healthy

results

glucuronoside/24

288;

of

hrs

are

(mol

wt

men expressed

TG 465;

484).

DISCUSSION This based

method

has

the

principles

upon

gas-liquid plasma

noside

is

by

various

is

quick

as

hydrolytic

same

day.

the

capacity up

to

the

results

The

a large

required

is

a liquid

samples

to

resolves

the

urinary

proteins.

obviates in

efficiency

and

and

only

the

at of

precision.

procedure

on

perform

samples

is

that

day

the and

is

high to

day

equipment

Heating

for

100°C

15 min

the

use

due

leads

to

The

preparation

improved of

is

buffer of

one

salts

counting standard

glucuronoside there

to

tricine

precipitation

and

that

of

the

effectively

binding

testosterone in

of

method

obtained to

features.

with

fluid

be

expensive

non-specific

associated

the

counter.

unique

some

tritiated the

advantage

Furthermore,

scintillation

simplifies

number

Another

dryness

problems

using

are

scintillation

problem

the

curves

has

released

Furthermore,

analyse

low

glucuro-

testosterone

simple

are

method

as

steps

costs

The

not

individual

run.

binding

testosterone

may

per

on

protein

12 urines

to

100

and

procedures dilution,

competitive

example,

such

over

isotope

procedures.

running

urine

For

measured

double

and

proteins.

-

advantages of

chromatography

using

-

distinct,

only, less

standard

solution

to

then

material

the

method

labelled of

removes

unbound

fraction The be

(95

accuracy

by

of

of The

on

the

same

from

as

analyses

error of

errors practical

each

gave

in and

the

in usual

calculated

by

the

The

for that

replicate

determi-

coefficients

within

and

of random be

7.0 and

The

similarity is

of between

analyses

procedure

error

taken

authentic

duplicate

to

way.

urine

appears

experimental

gave

total

calculated

hrs

within

coefficients The

bound counting.

indicates

women

by

step

antibody

assayed

assessed

determined

was

effectively equilibration

of of

well

12 $.

18 $ respectively.

percentage

volume

8 -

samples

(q),

the

previously

men and

from

and

scintillation

glucuronoside

as

urine

is

recovery

is

preferred, only

glucuronoside/24

urine

precision

variation

it

initial

method

ranging

al. after

liquid

the

and

is

of

dextran-coated et

Hence for

the

urine

of

Hotchkiss

2 4 5).

testosterone

ddw

use

way

glucuronosides

of

accuracy

to The

testosterone

variation

for

the

Furthermore,

limits.

these

check

level

from

9 and

using

removed

endogenous

assay

that

is

assay.

nations

with

which

independent

steroids

the

compared

steroid

antiserum

use

conventional

order

described

additional

the

solutions.

as

for

be in

the

charcoal,

the

may

the

however,

in

material,

purity

with

If,

prepare.

non-labelled

to

21:2

STEROIDS

296

of

variation

of

theoretical $ from

replicate

combination

reassuring

in

of values

with

STEROIDS

Feb. 1973 regard

to

the

The

as

standard

that

male always

power

of

between mean

reading

below

30

and

resolution

i.e.

the

curve the

hrs may

value.

The

ability

to

been

be

of

samples

2 pg/24

adults

discriminate taking detercurve

It

method

in

by

standard

the

urine

men. by

resolving

values.

hrs

from

function

replicate

on the

that

obtained

urine

evaluated

mass

urine

the deviation

hypopituitary

positions

approach

in

where

for

deviations

of

be

standard

values

has

corresponding this

confidently

ovariectomised

this

different

pg/24

may

standard

the

values

a difference

and

as

standard

from

detect

of

the

the

concluded

the

suspected

method,

at

method

overlaps

with

similar

of

minations and

in

method.

adrenalectomised,

fallen

two

the

0,

girls,

the

the

never

patients

have

the

point

corresponding young

and

of

deviation

the

from

of

sensitivity

defined

of

validity

is can

safely

from

Increased analysing

women sensitivity

larger

volumes

urine. Investigations

indicate

into

that

closely

epitestosterone sites with

on

interference

related

glucuronoside the

antibody.

testosterone.

amounts

the

(0.5

The As

by

1.3 this

pg/24

this hrs),

compound

specificity

of

the

method

glucuronosides

including

do not

for

compete

principal

cross

steroid an is

is

reaction

present

more

than

is

in

overestimation no

binding

small due

10

‘$

to in

298

219

STEROIDS

women. A comparison urinary and by

gas-liquid

men

of

values

obtained

there

an

is

butyrates would

is and

by any

GLC

is

Although shown

to

to

then

dihydrotestosterone by

comparison

in

plasma

possible

of from

of the

of

a composite

by

Ismail

et

the

standards

internal the an

relative

cycle al.

(10)

is cross

assay,

excretion

is

v.ery

would

a GLC

in of and

these

compounds

provide

a in

the

glucuronoside. as

similar

if

testosterone

overestimation

of

been

present

of

testosterone

using

urine.

reaction

to

this

some

urinary

as

have the

levels

women,

obtained

overestimate

with

the

for

pattern

values

untreated

antisera

men and

explanation

determination However,

the

the

heptafluoro-

interfere

with

the

testosterone

glucuronosides

with

be

underestimate

virtually

analogy

may

urine

an

glucuronoside

by

in

represent

on

radically

higher

value

example,

are

steroid

mean

For

probably

5a-dihydrotestosterone urine,

times

Conversely,

performed

for

factors.

which

for.

the

difference

of

GLC,

available not

could

hydrolysis

correct

assay

This

values

radioimmunoassay

that

1.3

of

of

by

approx.

women.

radioimmunoassay

the

range

show

accumulation

prior not

and

glucuronoside

radioimmunoassay both

mean

chromatography

result

by

the

testosterone

from

if

of

expressed to

procedure.

that

in reported

terms

STEROIDS

Feb. 1973

299

REFERENCES 1.

Testosterone glucuronoside (androst-4-en-3-one-l7B-y1B-D-glucopyranosiduronic acid); androsterone glucuronoside (5a-androstan -17-one-3a-yl-B-D-glucop;pranosiduronic acid); aetiocholanolone glucuronoside -androstan-l7--one-3a-yl-P-D-glucopyranosiduronic acid); dehydroepiandrosterone glucuronoside (androst-5-ene-17one-3P-yl-b-D-glucopyranosiduronic acid); epitestosterone glucuronoside (androst-4-en-3-one-l7a-yl-B-D-glucoand dihydrotestosterone pyranosiduronic acid); glucuronoside (5a-androstan-3-one-l7p-yl+-D-glucopyranosiduronic acid).

2.

Sommerville, I.F., and Collins, BIOCHEMISTRY AND PHARMACOLOGY, Academic Press, 2, 267, (1970).

W.P., Editor

3.

Loraine, J.A., THEIR CLINICAL Edinburgh and

HORMONE ASSAYS & S. Livingstone, 501.

4.

Kellie, D.M.,

J.

and Bell, E.T., APPLICATION, E. London, 1971, p.

A.E., Samuel, V.K., STEROID BIOCHEM.,

Riley, 2, 275,

ADVANCES IN M.H. Briggs,

.W.J., and (1972).

5.

Emment, Y., Collins, W.P., and ACTA ENDOCR. 2, 567, (1972).

6.

Collins, W.P., Mansfield, M.D., Alladina, Sommerville, I.F., J. STEROID BIOCHEM.

7.

Snedecor,

8.

Collins, W.P., Sisterson, J.M., Mansfield, M.D., and Sommerville, J. CHROMATOG. 2, 33, (1968).

9.

Hotchkiss, J., Atkinson, ENDOCRINOLOGY 3, 177,

10.

G.W.,

BIOMETRICS

8,

L.E., (1971).

Ismail, A.A.A., Harkness, R.A., ACTA ENDOCR. 58, 685, (1968).

Sommerville,

85,

2,

AND

Robertson, I.F.,

N.S., 333,

and (1972).

(1952).

Koullapis, I.F., and and

Knobil, Loraine,

STEROID

E.N.,

E., J.A.,

21:2

STEROIDS

300

STANDARD (Antibody

5000

I 500

Standard

1:20001

I pg. TG.

20

0 0

1 -

dilution

0

Fig.

CURVE

kq.124

300

curve

rq./24hrr.

for

hrs.’ Women’ ‘Men’

testosterone

I 1500

2(

W

60

80

900

1200

glucuronoside.

Feb. 1973

STEROIDS

E

6000

301

-

8

-500

0 Picograms

Fig.

500 Testosterone

1000 1500 Glucuronoside

2000

'Cold' prepared using of standard curves. 2 - A comparison labelled and non-labelled testosterone glucuronoside,. 'hot' prepared using only labelled material.

STEROIDS

302

21:2

Ij

-12

-10

-(I

l-l lllblr,,,., ml -6

-4

-J

0

2

4

i:

6

e

10

12

14

t

Urine

Fig.

3 - The level throughout

of testosterone 8 menstrual

LH

peak

glucuronoside cycles.

(mean

2 S.D.)

Feb. 1973

STEROIDS

TABLE

1 - THE RECOVERY OF TESTOSTERONE ADDED TO URINE.

URINE

of

NO.

@g/24

hrs

(mean of

Amount Amount

SOURCE

_+ S.D.)

variation

MAN

10

10

added

(pg)

recovered of

variation

+ 0.7

8.1

($4)

(pg)

330

+ 29

8.6

8.9

200

500

i84+ (qb)

GLUCURONOSIDE

WOMAN

determinations

Coeffic.

Coeffic.

303

12.4

23

446 10.9

+ 49

304

STEROIDS

8 0 d 0 in

21:2

Feb. 1973

STEROIDS

305

306

21:2

STEROIDS

4 - A COMPARISON GLUCURONOSIDE AND GAS-LIQUID

TABLE

OF THE VALUES FOR URINARY TESTOSTERONE BY METHODS INVOLVING RADIOIMMUNOASSAY CHROMATOGRAPHY.

Men (aged 20-40 yrs) Radioimmunoassay No. pg/24

Gas-liquid

of hrs

(RIA)

determinations

224

(mean

19 + 10

_+ S.D.)

chromatography No.

&g/24

of hrs Ratio

RIA/GLC

273

+ 1 6.4

(GLC)

determinations ( mean

20

+ S.D.)

22 14

+ 8 1.36

20 204

+ 102 1.34