Immunoassay of unconjugated estriol in serum of pregnant women monitored by chemiluminescence

3138

123

IMMUNOASSAY PREGNANT

OF

WOMEN

W.Klingler,

Institut

UNCONJUGATED MONITORED

O.Haupt,

for B i o e h e m i s c h e

Hochschule

Received:

LObeck,

ESTRIOL

IN SERUM

OF

BY C H E M I L U M I N E S C E N C E

G.v.Postel

and

Endokrinologie

Ratzeburger

Allee

R.Knuppen

der M e d i z i n i s c h e n

160,

D-2400

LObeck

?-6-83

ABSTRACT

6-Oxoestriol-6-(O-carboxymethyl)oxime-aminobutylethyi-isolumino] conjugate was synthesized. This l u m i n o g e n i c estriol derivative enabled us to d e v e l o p a solid phase i m m u n o a s s a y method for the d e t e r m i n a t i o n of u n c o n j u g a t e d estriol in serum of p r e g n a n t w o m e n by the m e a s u r e m e n t of the bound estriol-isoluminol conjugate upon o x i d a t i o n with a h y d r o g e n peroxide/microperoxidase system. The s e n s i t i v i t y of the assay was 700 pmo]/l. R e s u l t s o b t a i n e d by r a d i o i m m u n o a s s a y and the described m e t h o d showed good a g r e e m e n t (r = 0.95). The chemiIuminescent method is a p p l i c a b l e in the routine measurement of u n c o n j u g a t e d estrio].

INTRODUCTION The blood being in

determination is

of the

serum

total

an

unconjugated directly

important fetus

steroid

without

problems

dioactive

the

any

after

extraction

especially

Volume 42, N u m b e r 2

function

have with

Alternative

S T

~ z o

in m a t e r n a l

for m o n i t o r i n g of the

the

placenta.

well-

Estriol

by r a d i o i m m u n o a s s a y ;

either

enzymatic

or the

is a n a l y z e d

procedures

waste.

concentrations

parameter

determined

is m e a s u r e d

dioimmunoassay some

and

is c o m m o n l y

estriol

of estriol

after

hydrolysis

ether

procedure. many

regard

Although

advantages, to the

methods

x x~

extraction

like

the

they

disposal enzyme

or ra-

pose

of raimmuno-

August, 1983

124

~

assays spin

(1,2),

different

of

In this for women

(5) the

too

been

radioactive

developed compounds.

including

could

be

an

(3,4)

ours

to avoid Experiments

have

alternative

or e l e c t r o n -

shown

the in that

to r a d i o a c t i v e

(6-11).

paper

quantitation without

x x~l

immunoassays

have

laboratories

chemiluminescence

labelling,

~m ~ o

fluorescence

immunoassays

disadvantages

~

we d e s c r i b e

a chemiluminescence

of c o n j u g a t e d

an e x t r a c t i o n

estriol

in serum

immunoassay of pregnant

procedure.

EXPERIMENTAL i

Materials, s o l u t i o n s and m i s c e l l a n e o u s . Estriol,,estrone, estradiol-178, cortisol and p r o g e s t e r o n e were p u r c h a s e d from Merck, Darmstadt, FRG; estriol-]-sulphate, estriol-3g l u c u r o n i d e and e s t r i o l - 1 6 ~ - g l u c u r o n i d e from Sigma, MOnchen, FRG; 16-epi-estriol and 17-epi-estriol from Paesei, Frankfurt, FRG; 1 7 - h y d r o x y p r o g e s t e r o n e from Fluka, Ulm, FRG; microperoxidase (MPll) from Sigma, M~nchen, FRG; c a r b o x y methoxylamine hemihydrochloride from EGA-Chemie, Steinheim, FRG; precoated silica gel thin layer c h r o m a t o g r a p h y plates (Polygram Sil G/UV 254) from M a c h e r y and Nagel, D~ren, FRG; p r e c o a t e d silica gel thin layer c h r o m a t o g r a p h y plates (silica gel 60 F254) and all other c h e m i c a l s from Merck, Darmstadt, FRG. Anti-estriol antibodies raised in rabbits against 6-o xoestriol-6-(O-carboxymethyl)oxime-bovine serum albumin and fixed to solid p a r t i c l e s ("Amerlex") were kindly p r o v i d e d by Amersham International, Amersham, UK. The c o n c e n t r a t i o n of this p a r t i c l e s u s p e n s i o n was identical to that of the Amer]ex Radioimmunoassay kit but the s u s p e n s i o n was not co]ourcoded. Stock s o l u t i o n s of steroids were p r e p a r e d in ethanol, and just prior to use, d i l u t e d to the desired c o n c e n t r a t i o n s in assay buffer. Microperoxidase was d i s s o l v e d at 1 mg/ml in Tris-HC] buffer (0.01 M, pH 7.4)7 this stock s o l u t i o n could be u s e d w i t h i n 6 months. Just prior to use, 0.04 ml of this s o l u t i o n was d i l u t e d with 8 ml of borate buffer (0.075 M, pH 8.6) y i e l d i n g a 2.5 pM enzyme solution. The oxidant s o l u t i o n was p r e p a r e d by adding 1 m] of 55 % h y d r o g e n p e r o x i d e solution to 114 mi Tris-HCl buffer (0.01 M, pH 7.4). Melting points were d e t e r m i n e d with a m i c r o s c o p e h o E - s t a g e and are uncorrected. Infrared spectra using p r e s s e d KBr

•1 " 1 -

] ~ o

x

x)

m,

125

discs were r e c o r d e d with a B e c k m a n A c c u l a b 4 s p e c t r o m e t e r . Light e m i s s i o n s were m e a s u r e d with a B i o l u m a t LB 9500 (Laboratorium Prof. B e r t h o l d , W i l d b a d , FRG). The pump s y s t e m of the a u t o m a t i c i n j e c t i o n of the l u m i n o m e t e r w a s e q u i p p e d with teflon tubes to avoid c o n t a c t b e t w e e n o x i d a n t s o l u t i o n and metal. Synthesis of 6-oxoestriol-6-(O-earboxymethyl)oxime. 6O x o e s t r i o l (0.725 g) (15) was d i s s o l v e d in 50 ml of ethanol. Fifty-five ml of 1 M s o d i u m a c e t a t e s o l u t i o n and I g of carboxymethoxylamine hemihydrochloride were added and refluxed for 4 h. E t h a n o l was e v a p o r a t e d in vacuo and the remaining aqueous phase was a d j u s t e d to pH 2 with l M HCI. This acidified phase was e x t r a c t e d three times with ethyl acetate. The c o m b i n e d o r g a n i c layers were w a s h e d with water until neutral and evaporated in vacuo. The r e s i d u e was r e d i s s o l v e d in s a t u r a t e d s o d i u m b i c a r b o n a t e s o l u t i o n and extracted three t i m e s with ether to remove r e s i d u a l 6 - o x o e s triol. The r e m a i n i n g a q u e o u s phase was a d j u s t e d to pH 2 and extracted three times with ethyl acetate. The ethyl a c e t a t e was w a s h e d with w a t e r and e v a p o r a t e d in vacuo. The s e m i c r y s ratline residue was c r y s t a l l i z e d from m e t h a n o l / w a t e r yielding w h i t e n e e d l e s (0.9 g, mp. i G g - 1 7 0 ° C ) . Analytical data were i d e n t i c a l to those p u b l i s h e d by Kuss et al. (14). S y n t h e s i s of 6 - o x o e s t r i o l - 6 - ( O - c a r b o x y m e t h y l ) o x i m e - a m i n o butyleth~1-isoluminol (E3-6-CMO-ABEI). 6[N-(6-aminobutyl) -N-ethylJamino-2,3-dihydrophthalazine-l,4-dione ( " A B E l " ) was synthesized according to the method of S c h r o e d e r et al. (12). 6-Oxoestriol-6-(O-carboxymethyl)oxime (250 mg~ was suspended in 7 ml of dry dioxane and 0.5 ml of tri-nb u t y l a m i n e was added. The s u s p e n s i o n was c o o l e d to ii ° a~d 0.22 ml of i s o b u t y l c h l o r o f o r m a t e was added. The s o l u t i o n turned clear and it was stirred for 60 min at iI °. Aminobutylethyl-isoluminol (280 mg) was dissolved in a mixture of 31 ml of d i o x a n e and 31 ml of water and the pH was a d j u s t e d to 8.5 at O°C. The m i x e d a n h y d r i d e was added d r o p w i s e to the s t i r r e d ABEl s o l u t i o n . D u r i n g this p r o c e d u r e the pH was kept b e t w e e n 8.3 and 8.5 with s o d i u m h y d r o x i d e solution (0.3 M). The solution was s t i r r e d for 3.5 h at O°C. After adjusting the pH to 7.0 the s o l u t i o n was diluted with 200 ml of w a t e r and the solid m a t e r i a l was f i l t e r e d off and was d r i e d in vacuo. Yield: 420 mg of solid material m e l t i n g atl145-149°C-~. The p r o d u c t was i d e n t i f i e d by IR (3360 cm , vO-H; 1765, vC=O) and e l e m e n t a l a n a l y s i s . The p r o p o s e d s t r u c t u r e of the s y n t h e s i z e d m o l e c u l e is shown in Fig. I. Before use, 3 pg of this tracer s u b s t a n c e was p u r i f i e d by thin-layer c h r o m a t o g r a p h y on s i l i c a gel using t o l u e n e / c h l o roform/methanol (40j40:20) as solvent system. E - 6 - C M O ABEl (Rf = 0.15) ~ o e x t r a c t e d with m e t h a n o l from the plate and diluted with p h o s p h a t e b u f f e r (O.O1 M, pH 6.2) to the final d i l u t i o n of 40 p g / O . l ml.

126

~

,,x- "m : ~ , o

x -nm

OH

HO~

-O 'H

N I

0 l

0:c-N-CCHzlc -

H

Fig.

",.H

~ " 1 1 fNH 0

i: P r o p o s e d s t r u c t u r e for e s t r i o l - 6 - c a r b o x y m e t ~ y l o x i m e aminobutylethyl-isoluminol (E3-6-CMO-ABFI)

Assay procedure. Standard s o l u t i o n s were O, 8.7, 17.4, 34.7, 69.4 and 138.8 nmol/l. The i m m u n o a s s a y was p e r f o r m e d by mixing serum (0.02 ml) and p h o s p h a t e buffer (0.02 ml, 0.01 M, pH 6.2) or estriol s t a n d a r d m a t e r i a l in the same buffer (0.02 ml) and e s t r i o l - f r e e serum (0.02 ml). Tracer s o l u t i o n (O.1 ml) and 0.5 ml of a s u s p e n s i o n of a n t i - e s t r i o l antibodies a t t a c h e d to the solid p a r t i c l e s ("Amerlex") were added. After i n c u b a t i o n time, c e n t r i f u g a t i o n and w a s h i n g of the precipitate, the c h e m i l u m i n e s c e n c e of the p r e c i p i t a t e was measured. The d e t a i l e d i m m u n o a s s a y p r o c e d u r e is shown in the flow scheme (Fig. 2). Light measurements. M e a s u r e m e n t s of light e m i s s i o n were performed by adding 0.I ml of borate buffer (0.075 M, pH 8.6), 0.05 ml sodium h y d r o x i d e s o l u t i o n (2M) and m i c r o p e r oxidase s o l u t i o n to the p r e c i p i t a t e . Just prior to m e a s u r e ment, 0.I ml of h y d r o g e n p e r o x i d e s o l u t i o n was added. Light measurements were started during this a d d i t i o n and c a l c u l a ted as integrals over a period of 30 seconds.

RESULTS Yield light

of

emission

light of

of

ABE1 and e s t r i o l - 6 - E M O - A B E l .

increasing

quantities

of

When t h e

ABE1 and e s t r i o l -

B

"I" |

20lalserum +201albuffer

'I*-0

127

201atstandard-solution *201alO-serum

~

L

X ~D!

1

100 ma(tracer solution 500pl antibody suspensmn

incubation, 2 hrs, 3'/°C centrifugation, 25min,2000g

supernatant

precipitate

i

50Opt buffer, pH 6.2 centrifugation, 25 rain,2000g

supernatant

predpitate

i

measurement

Fig.

2:

F l o w s c h e m e of the c h e m i l u m i n e s c e n c e unconjugated estriol.

6-CMO-ABEI shown

were

(Fig.

3).

fmol

(SD,

(SD,

p < 0.05).

Table

l:

measured, The

p < 0.05),

linearities

detection of

limit

a wide

of A B E I

estriol-6-CMO-ABEI

was

was

range 0.4

for

were

+ 0.15

I + 0.38

fmol

Influence of s e r u m on l i g h t e m i s s i o n . E 3 - 6 - C M O ABEI (150 fmol) w e r e d i s s o l v e d in O.1 ml p h o s p h a t e buffered s a l i n e ( 0 . 0 6 M, pH 7.4) or five d i f f e r e n t sera, respectively. Source

tracer tracer tracer tracer tracer tracer

over

immunoassay

in p r o t e i n - f r e e in s e r u m 1 in s e r u m 2 in s e r u m 3 in s e r u m 4 in s e r u m 5

Photoncounts buffer

(pH

7.4)

51273 52339 51911 50997 51772 52196

128

~

-Je ~

~.o

x x~ ~

Arbifrary

lighfunifs

100

/

/

e///"

/

50

10

1 Fig.

2

10

20

fmot/fube

3: L i n e a r i t y of light m e a s u r e m e n t s of i n c r e a s i n g quantities of ABEI and E 3 - 6 - C M O - A B E I , r e s p e c t i v e l y .

Influence

of

serum

components

on l u m i n e s c e n c e

measure-

ment. Light ing

measurements steroid

table

i.

aere

antibody the tity ces

found.

of solid

of

light

the

marker

other

emission.

of the

are

]ight

decrease

particles

results

five

influences

On the

suspension

original

the

serum

between

not

m]

chemiluminescent

The

differences

of 0.i

hand,

assays.

minimal

tube,

of

and

containshown

the

five

sera

of the

to a third

identical

does

in

significant

particles

signal

this

are

of the

solid

light

Because

sera

conjugate

emissions

the

in each

different

not

of

quan-

influen-

~-x~oxDm Performance with

varying

phosphate

of

assay.

quantities

buffer

With

increasing

sion

decreases.

to 138.8

the

as d e s c r i b e d

in the

curves

2 shows

were

estriol

carried

were

estriol

in

section.

light

established

a representative

out

dissolved

experimental

of u n c o n j u g a t e d

Calibration Table

reactions

of u n c o n j u g a t e d

amounts

nmol/].

The

129

emis-

from

dose

8.7

respon-

se curve. Table

2:

T y p i c a l estriol c h e m i l u m i n e s c e n c e i m m u n o a s s a y data. Light emission units represents photon counting. B l a n k s are p r e p a r e d in the same m a n n e r as the maximal b i n d i n g samples, but i n s t e a d of a n t i s e r u m buFFer was used. For f u r t h e r d e t a i l s see E x p e r i m e n t a l section.

Assay

tube

Photon

Total

ii0 109 iii

Blank

Maximal

8.7

nmol/l

17.4

nmol/l

34.7

nmol/l

69.4

nmol/l

158.8

nmol/l

Sensitivity. could

be

27 26 27 20 20 20 15 16 15 12 12 12 8 8 8 6 6 7

counts

271 390 176 870 902 870 307 408 723 160 053 658 930 457 561 574 380 243 832 744 944 651 552 007

The

Mean

Mean-Blank

Binding (%)

27146

26265

i00

20290

19409

73.9

15983

15102

57.5

12399

11518

43.9

8840

7959

30.3

6737

5856

22.3

881

minimum

significantly

concentration

distinguished

from

of e s t r i o l

zero

was

that

calcula-

130

~

ted

from

SD)

three

pmol/l

of

of the

related

concentrations The

results

immunoassay

".~ i , o

response

i

",n,m

curves.

The

value

was

antibody.

compounds

was

obtained and with

The

and

determined with

by the

a commercial

("Amerlex

unconjugated

U.K.)

are

shown

Table

3:

estrial

in Table

percentage

of s t e r o i d s the m e t h o d described

with

high

serum

of A b r a h a m

(13).

chemiluminescence

available

RIA",

cross-reactiv-

radioimmunoasssy

Amersham

International,

3.

The cross r e a c t i o n s of d i f f e r e n t s t e r o i d s e x p r e s s e d as % c o n c e n t r a t i o n s g i v i n g 50 % i n h i b i t i o n of lab e l l e d a n t i g e n bound to a n t i b o d i e s .

Steroid

Chemiluminescence immunoassay

Radioimmunosssay

estriol estrone estradiol

lO0.O < O.1 < O.1

lO0.O < O.1 < O.l

cortisol 17-hydroxyprogesterone progesterone estriol-3-sulphate estriol-3-glucuronide estriol-16~-glucuronide 6-oxo-estriol 17-epi-estrio] 16-epi-estriol

< < < <

< 0.i < 0.i < O.l 0.3 0.5 < O.1 188.2 0.2 4.0

Precision. sults

are

700+340

(p
Specifity ity

dose

~

Within

shown

0.i O.1 O.1 O.1 0.7 < 0.I 229.0 < 0.i l.B

assay

in table

4.

and

between

assay

precision

re-

T

Table

4:

Within studies

~ ' ~ o x

~ m

131

a s s a y and b e t w e e n a s s a y two p l a s m a p o o l s (A and Mean + SD (nmolTl)

Withinassay A Withinassay B Betweenassay A Betweenassay B

was

One

diluted

regression trations

of was

8.4

I0

73.9+7.8

10.6

10

21.5+1.8

8.4

5

74.3+9.6

12.9

5

serum with

the

both

Coefficient of v a r i a t i o n (%)

20.2+1.7

Accuracy. week

p r e c i s i o n . For B) w e r e used.

from

different

measured

linear

a patient

and

volumes

and

the

of

the

the

of

a male

expected

correlation

40th

gestation serum.

estriol

The

concen-

coefficient

was

r =

serum.

The

0.94. Different recovery

amounts is

shown

Clinical women

assay two

by

at

same

the

patients le.

Thirty-one

Fig.

values

pregnancies placenta with

well:

in

the and

are

added

to

a male

5. serum

gestation

samples weeks

immunoassay

antibody

agreed

radioimmunoassay)

of

table

different

the

methods

normal

in

were

chemiluminescence

using

Estrio]

estriol

samples.

taken

estriol

of

suspension.

r = 0.95,

from

were and

The

pregnant

assayed

radioimmuno-

results

y = 5.17

for

of

+ 0.74x

the

( x =

5).

serum one

shown

intrauterine

of

five

patient in

with

figure

death

patients

4.

with

improved In

estrio]

the was

deficiency

serum not

obvious

of

two

detectab-

132

S

5:

Table

Serum Serum Serum

Recovery

~

~. x~o

of estriol

x x~m

in serum.

Value (nmoi/l)

Found (nmol/l)

17.4 34.7 52.i

18.1 31.2 48.0

I II Ill

Recovery

104.0 89.9 92.1

86~, eslrto&

34~7

17k

....

Fig.

4:

.

Estriol values obvious normal with improved ta.

~

~

9estat~

in the serum of five p a t i e n t s with pregnancies and one patient (S-S) s u l p h a t a s e d e f i c i e n c y of the p l a c e n -

DISCUSSION The first CMO

synthesis time.

with

of e s t r i o l - 6 - C M O - A B E I

It was

chemically

prepared

aminobutylethylisoluminol.

The

is d e s c r i b e d by c o u p l i n g steroid-ABEl

for

the

E3-6eonju-

g

gate

proved

lo

be

with

a detection

ons

used.

In

ABEl

can

stored

without

be

~

]El I 1 0

I I)|

an e x c e l l e n t

limit

of

l fmol

ethanolic

stock

at 4Oc

significant

loss

133

tool

foc

chemiluminescence

under

the

reaction

solutions

in the

dark

the

for

at

~

C

conditi-

estrioJ-6-CMOleast

2 years

of a c t i v i t y .

nmot/L

"

•

52.1 •

a J

•

•

34,7

• co ee~ J

• • o

oo / ~

y(CIA)--0,74x(RIA]*5,17 r-0,9535

I?,4

n --31

17,~, Fig.5:

The se

52,1

69~4

nmoL/!

Comparison of s e r u m e s t r i o l levels d e t e r m i n e d by d i o i m m u n o a s s a y and c h e m i J u m i n e s c e n c e immunoassay.

estriol-6-CMO-ABEl

immunoassay

miluminescence. incubation serum

34,7

or

for

serum

The

(ii)

us

to d e v e l o p

unconjugated

assay

of a n t i b o d i e s standards,

enabled

method

with

the

estriol

requires marker

separation

ra-

a solid-phabased

three

on che-

steps:

conjugate

(i)

and w i t h

by c e n t r i f u g a t i o n ,

and

134

~

(iii)

subsequent

bound

label

oxidase

by

measurement the

separation

of

measurement

is not

the

conjugated

sitivity suits

the The

slighty

fact

is

the

2.5-40

ng/ml)

high

found

precision

of the to

due

using

step

the

the

luminescence

curves

for

of 0 . 2 2 - 3 . 4 7

were

obtained

to be 0.7

un-

pmo]/tube

and

nmol/l.

comparable

of the

use

after

components.

standard

are

of the

microper-

the

sen-

These

re-

to c o m m o n

RIA

chemiluminescence

that

to the

emission

a washing

range

specificity

inferior

H202

by s e r u m

covering

was

light

antigens,

conditions,

assay

~robably

free

affected

nmol/l;

and

with

introducing and

described

of the

methods. is

bound

estriol

(8.7-138.8

By

1- z ~

of the

oxidation

as c a t a l y s t .

Under

.z- ~-, ~ o

immunoassay

radioimmunoa§say;

of m i c r o p e r o x i d a s e

this

as cata-

lyst.

There one

is a high

clinical

assay

and

found

in

thod

quality

by

that

study.

but

by

values

assay

than

using

The

have those

the

results

estriol

with

is not

we

the

of t h i r t y

chemiluminescence

women

this

programs,

higher

estrioi

determined

of p r e g n a n t

control

gives

between

radioimmunoassay.

lower,

thod

An

samples

serum

are

correlation

the

concentrations

non-isotopic

surprising.

observed of other

immuno-

that

In e x t e r n a l our

RIA

laboratories

chemiluminescence

me-

method

mein

can

~ - ~

easily

be performed

recording

the

time consuming to

the

production

but meanwhile counters

should make to numerous

automatic

equipments

are available as well

laboratories.

hours.

The step of

was done manually.

This is

comparable

(Berthold,

Wildbad,

as the lack of radioac-

the c h e m i l u m i n e s c e n c e

field of steroids,

ble alternative

135

x ~m

three working

Such kind of apparatus

tivity tive

within

light

radioactivity

FRG).

la-~o

It seems

chemiluminesoence

immunoassay that,

attrac-

especially

in

will be a reasona-

to radioimmunoassay.

ACKNOWLEDGEMENTS We thank Amersham International plc, U.K. and Amersham Buchler, FRG for providing us with solid-phase antiserum suspension. We thank L a b o r a t o r i u m Prof. Dr.Berthold, FRG for providing us with a Biolumat LB 9500 luminometer.

REFERENCES i. Sch~neshofer, M., ARZTL. LAB. 24, 94 (1978). 2. Klingler, W., NUCL.-MED. 21, 161 (1982). 3. Briggs, J., Elings, V.B. and Nicoli, D.F., SCIENCE 212, 1266 (1981). 4. Ullmann, E.F., Schwarzberg, M. and Rubenstein, K.E., d. BIOL. CHEM. 251, 4172 (1976). 5. Sayo, H. and Hosokawa, M., YAKUGAKU ZASSHI I00, 56 (1980). 6. Kohen, F., Kim, J.B., Lindner, H.R. and Collins, W.P.,

STEROIDS 38, 73 ( 1 9 8 1 ) . P a z z a g l i , M., Kim, J . B . , M e s s e r i , G., M a r t i n a z z o , G., Kohen, F . , F r a n c e s c h e t t i , F . , Tommasi, A . , S a l e r n o , R. and S e r i o , M., CLIN. CHIM. ACTA i 1 5 , 287 ( 1 9 8 1 ) . 8. S t r a s b u r g e r , C . J . , F r i c k e , H. and Wood, W.G., FRESENIUS Z. ANAL. CHEM. 311, 351 ( 1 9 8 2 ) . 7.

9. Schroeder, H.R., Yeager, F.M., Boguslaski, R.C. and Vogelhut, P.O., J. IMMUNOL. METHODS 25, 275 (1979). lO. Arakawa, H., Maeda, M. and Tsuli, A., CHEM. PHARM. BULL. 30, 3O36 (1982). ii. Klingler, W., Haupt, 0., von Postel, G. and Knuppen, R., ACTA ENDOCRINOL. (Kbh) Suppl. 246, 22 (1982). 12. Schroeder, H.R., Boguslaski, R.C., Carrico, R.J. and Buckler, R.T., METHODS ENZYMOL. 57, 424 (1978).

136

~

~

"t~ I t O

I

I } !

Abraham, G.E. ACTA ENDOCRINOL. (Kbh) 183, 1 ( 1 9 7 4 ) . Kuss, E., G o e b e l , R. STEROIDS 19, 509---~i-972). 0 . , J. BIOL. CHEM. 133, 219 Longwe]l, B., W i n t e r s t e i n e r , (1940). 16. The f o l l o w i n g trivial names are used: e s t r i o l = E = 1,3,5(lO)-estratriene- 3,16~,17G-triol; estriol sulphate = 1,3,5(lO)estratriene -3,16~,17G-triol-3-sulphate; estriol-3-glucuronide : 1,3,5(lO)estratriene-3,16~, 178t r i o l - 3 - D - g l u c o p y r a n o s i d u r o n i c acid; e s t r i o l - 1 6 ~ - g l u c u r onide = 1 , 3 , 5 ( l O ) e s t r a t r i e n e - 3 , 1 6 ~ , 1 7 0 - t r i o l - 1 6 ~ - D - g l u c o p y r a n o s i d u r o n i e acid ; e s t r o n e = 3 - h y d r o x y - l , 3 , 5 ( l O ) e s t r a t r i e n - 1 7 - o n e ; est radiol = 1 , 3 , 5 ( l O ) e s t r a t r i e n e 3,17S-diol; cortisol = llB,17,21-trihydro×y-4-pregnene3,20-dione; progesterone = 4-pregnene-3,20-dione; 16epi-estriol = 1,3,5(lO)-estratriene-3,16B,170-triol; 17epi-estriol = 1,3,5(10)-estratriene-3,16a,17o-triol. 13. 14. 15.