Urinary estrogens I improved method for quantitation of urinary estrogens

285

URINARY ESTROGENS

I

IMPROVED METHOD FOR QUANTITATION OF URINARY ESTROGENS

Joseph C. Touchstone,

Taras Murawec,

Ofelia Brual and Meinert Breckwoldt Steroid Laboratory Department of Obstetrics and Gynecology and Harrison Department of Surgical Research University of Pennsylvania School of Medicine Philadelphia, Pennsylvania Received 7/30/70 Revised 10/5/70 ABSTRACT A specific method for quantitation of the three classical urinary estrogens, estrone, estradiol-178, and estriol is described. The method involves conjugate extraction, enzyme hydrolysis, phenolic separation, thin layer chromatography of the phenolic steroids in a 20% acetone-80% isopropylether system and finally separation and quantitation by gas chromatography. A formation of derivatives is not required. The precision, sensitivity, and specificity of the method are evaluated. Examples of the application of this method are evaluated. Examples of the application of this method are described.

Most methods

for quantitation of estrone,

17 8, and estriol have been modifications Brown

(i) and Bauld

chromatography

estradiol-

of methods of

(2) which are time consuming.

Gas

can overcome this disadvantage provided

286

S T E R O I D S

sufficient p r e l i m i n a r y p u r i f i c a t i o n However,

some methods

paration

of derivatives.

17:3

can be accomplished.

are tedious because

of the pre-

The present paper describes

development of a relatively

simple method for routine

quantitation

(El) , estradiol-17 B (E2)

and estriol

of the estrone

(E3) in the urine of n o n p r e g n a n t women.

Sufficient p u r i f i c a t i o n was matography

the

achieved by thin layer chro-

to render formation of derivatives

prior to gas chromatography.

unnecessary

A more reliable d e t e r m i n a -

tion was therefore possible.

EXPERIMENTAL

REAGENTS

Tritiated E 1 , E2, and E 3 were obtained from New England Nuclear, Boston, Massachusetts. Purity was assessed by paper and thin layer chromatography. Nonradioactive estrogens were obtained as a generous gift from Ayerst Limited, Montreal, Canada, or p u r c h a s e d from Mann Biochemical Co., New York, N.Y. All reagents were of analytical grade. Ether, isopropyl ether and ethyl acetate were kept over ferrous sulfate and redistilled. Hexane and toluene were w a s h e d with sulfuric acid, sodium bicarbonate and redistilled. Distilled water was redistilled in an all glass apparatus. Acetone was redistilled.

EQUIPMENT

Thin layer plates of 250p thickness made with silica gel G were obtained from Photo Scientific Research Co., Chester, Pa. and were activated at ll00C for i0 minutes just prior to use. The gas chromatograph was a Glowall model 210 gas chromatograph equipped with an argon ionizing

March 1971

ST ER O I D S

r a d i u m d e t e c t o r (22.5 ~C). A 6 ft glass column was p a c k e d w i t h a c o m b i n a t i o n of 10% QF-1 (fluorosilicone polymer, Dow Corning) and 5% L-45 (methylsilicone polymer, G e n e r a l Electric) on Gas C h r o m (80-100 mesh). This was c o n d i t i o n e d at 260 ° for one day. Priming with e s t r o g e n was c a r r i e d out e a c h day b e f o r e q u a n t i t a t i o n . The r e c o r d e r was a S p e e d o m a x H (Leeds and N o r t h r u p Co.) w i t h a s p e e d of 3 inch/min. For acetates a 5% L-45 c o l u m n was required. A P a c k a r d "Tricarb" s p e c t r o m e t e r M o d e l 3375 was used for l i q u i d s c i n t i l l a t i o n . All counts w e r e corr e c t e d to 100% e f f i c i e n c y by e x t e r n a l s t a n d a r d i z a t i o n . S u f f i c i e n t c o u n t i n g time was a l l o w e d to y i e l d m a x i m u m r e l a t i v e s t a n d a r d e r r o r of 2% for each sample. For counting, each sample was d i s s o l v e d in 1 ml of e t h a n o l and 15 ml of POP (2% b i s - M S B and 98% PPO) 0.4% in r e a g e n t grade t o l u e n e was added.

METHOD

i.

Extraction

OF E X T R A C T I O N

of C o n j u g a t e s :

An entire 24 h o u r urine s p e c i m e n was a d j u s t e d to pH 1.5 w i t h 5 N HC1. A m m o n i u m sulfate (25% w/v) was added. The urine was then e x t r a c t e d 3 times w i t h ethyl acetate. E x t r a c t i o n was c a r r i e d out in a counterc u r r e n t m a n n e r by p a s s i n g 200 ml aliquots of the specimen t h r o u g h 3 s e p a r a t o r y funnels c o n t a i n i n g 250 ml of ethyl acetate. The o r g a n i c phases were c o m b i n e d and the e t h y l a c e t a t e was e v a p o r a t e d in vacuo. Last traces of e t h y l acetate w e r e b l o w n off w i t h a s t r e a m of n i t r o gen. 2.

Hydrolysis

of Conjugates:

F i f t y ml of 1.5 N HCI was added to the ethyl acetate e x t r a c t and the e x t r a c t was r e f l u x e d for 20 min. W h e n e n z y m e h y d r o l y s i s was used, 50 ml of m a l e a t e buffer s o l u t i o n of pH 6.8 c o n t a i n i n g 200 units of b a c t e r ial 8 - g l u c u r o n i d a s e / m l (Sigma C h e m i c a l Co., St. Louis, Mo.) w e r e added. A f t e r h y d r o l y s i s the s o l u t i o n was adj u s t e d to pH 4 w i t h 1.0 N HCl. 3.

Extraction

of Free

Steroids:

The h y d r o l y s a t e was e x t r a c t e d 3 times w i t h isop r o p y l e t h e r (30, 20, 20 ml) and the combined e t h e r w a s h e d once w i t h 5 ml each of 8% N a H C O 3 and water.

287

ST ER O I D S

288

4.

17:3

E x t r a c t i o n of Phenols:

An equal volume of hexane was added to the isopropyl ether extract. The phenolic component was extracted with 1 N NaOH, 15 ml 3 times and these alkaline fractions were combined and b a c k w a s h e d with 15 ml of toluene. The toluene fraction was discarded. The alkaline phase was adjusted to pH 1.0 with 5 N HCI and extracted 3 times with 15 ml of ether, w a s h e d with 5 ml of 8% NaHCO 3 and water and evaporated. 5.

Thin Layer Chromatography

(TLC):

The estrogen extract was separated by TLC using a solvent system of 15% acetone in isopropyl ether on a 20 x 20 cm plate. One lane was p r o v i d e d for the reference steroids. The zones corresponding to El, E 2 and E 3 (located by spraying the reference lane with ferric c~loride p o t a s s i u m ferricyanide reagent) were scraped into individual separatory funnels containing 15 ml of water of pH 4 (HCI) and extracted 3 times with ether (15, 15, 15 ml). The ether extract (E 1 and E 2) was w a s h e d once with 8% Na2CO 3, once with 8% NaHCO 3 and finally twice with water 5 ml each. The E 3 was w a s h e d only with 8% NaHCO 3 and water. 6.

Gas Chromatography

(GLC) :

The eluted zones were then subjected to GLC for further separation and quantitation. 7.

Recovery :

For assessment of recovery tritiated estrogens were added before hydrolysis. After the e x t r a c t i o n was completed aliquots were removed for counting as described in the equipment section. Solvents were e v a p o r a t e d with a stream of nitrogen before addition of the scintillation fluid.

RESULTS

a)

Hydrolysis There is no universally

hydrolysis results

accepted m e t h o d

of steroid conjugates.

for

Table I gives

of a comparison of enzymatic

and acid

March 1971

ST ER O I D S

TABLE COMPARISON

OF M E T H O D S

289

I FOR HYDROLYSIS

OF

ESTRIOL-~H-GLUCURONIDE * ADDED TO URINE

Experiment 2

Enzyme % Recovery

Acid Hydrolysis % Recovery

1

94

80

2

87

90

3

92

75

4

80

20

5

80

62

6

80

75

7

90

52

AVERAGE

IGenerous

86+6% 3

g i f t of Dr. M o r t i m e r

65+23%

s

Levitz

2 U r i n e s a m p l e s d i v i d e d and d i f f e r e n t p r o c e d u r e s c a r r i e d out: E n z y m e as d e s c r i b e d in t e x t A c i d h y d r o l y s i s = u r i n e m a d e to 15% HCI and r e f l u x e d 30 m i n u t e s 3Mean + s t a n d a r d

deviation

290

ST ER O I D S

hydrolysis.

Enzyme hydrolysis

and was found to provide for gas chromatography lower and higher hydrolized b)

samples

(Extraction)

Different

solvents

indicated

for extraction water

were

Recovery removed ever,

of various

clearer

of a number of sol-

ether

showed potential

from urine.

solvents

The

was used as an

studies

indicated

that isopropyl

separation

resulted

three estrogens

during

shown in Figure

I less than i:i isopropyl

The add-

in good recoveries

of all

this step of the procedure.

Further

How-

with 1 N NaOH the

of E1 and E 2 was not satisfactory.

gave poorer recovery.

ether

from water quantitatively.

ition of hexane

c)

extracts.

of the solvents.

the estrogens

not improve

was

to determine

in providing

of free estrogens

during phenolic

recovery

studied

that isopropyl

index of polarity

Background

were found in the enzyme

of the polarity

solubility

suitable

than after acid hydrolysis.

which was most effective

vents

the most

after TLC.

Partition

An investigation

was more reproducible

extracts

recoveries

17:3

addition

As

ether-hexane

of hexane did

the recovery.

TLC Contemporary

rogen extracts

systems

used for TLC of urinary

gave inadequate

zone after elution

revealed

resolution.

substances

est-

The E 1

with a retention

March 1971

ST ER O I D S

291

f~ 0 to

_0

H

0 - (D

m I".-

to

Z

I

~.o

_ ot--

0 to

_0 0 r-

_o 0 u cl o r-

q~ tO X q~

T

o

Z 0

Z

Z

0

1"4

0 H

Z

to

r..) Z 0 r.j

rD H 0

Z tn

o.9_ N

Z H

I

I

I

1

0

0

0

0

0

D

o

if)

rj

~n I H

~9 M

292

ST ER O I DS

time different chromatographic

17:3

from that of E 1 as seen in the gas pattern.

The same u n c e r t a i n t y was

shown for the E 2 fraction. acetone or 7% ethanol specific separations

A system containing

in isopropyl ether provided more of the estrogens

substance which travels with estrone systems d)

15%

on TLC

(6).

A

in conventional

is separated well in this new system.

GLC GLC of the extracts

described,

yields

as prepared by the method

separation

and quantitation.

packing used in the column provides tion available

the only separa-

for E 1 and E 2 w i t h o u t derivative

ation because of fluorosilicone The QF-I substrate, terol from estrone.

however,

(QF-I) present

form(7).

did not separate choles-

The substrate L-45 will do this

but does not separate E1 and E 2. two substrates

The

By combining

in a single column,

these

both purposes were

served and a much more reliable column was obtained. This separation was desirable

since the aim of the

methods was to avoid derivative

preparation.

The high

percentage of substrate used has the advantage of providing sequently,

a longer retention better

time for estrogens.

Con-

separation between the estrogens

extraneous material

has been achieved than can be

obtained at shorter retention times of columns with low substrate concentration.

The columns described

and

March 1971

ST ER O I D S

h a v e b e e n in use

for as l o n g as 3 months.

EVALUATION

Table estrone,

II shows

OF THE M E T H O D

the r e s u l t s

estradiol-178

These

unlabeled

conjugates

estrogen,

case was

was c o m p a r a b l e .

satisfactory

for r o u t i n e

noted within

the d e t e r m i n a t i o n s .

culating

~g);

in each

can be c o n s i d e r e d

of the m e t h o d was d e t e r m i n e d by cal-

of a s i n g l e n o r m a l

(mean 2.4

under

L i t t l e v a r i a t i o n was

the s t a n d a r d d e v i a t i o n

for 5 r e p l i c a t e

tritiat-

The r e c o v e r y

The r e c o v e r y

in the o r d e r of 60%, w h i c h

Precision

obtained with

a d d e d to w a t e r ,

urine.

use.

s t u d y of

added before hydrol-

results were

a d d e d to n o r m a l

both conditions

of r e c o v e r y

and e s t r i o l

y s i s of the urine.

ed e s t r o g e n s

293

in a r e p l i c a t e

u r i n e pool.

determinations

The s t a n d a r d d e v i a t i o n was

for e s t r a d i o l - 1 7 8

~g) ; and 0.50 ~ g for e s t r i o l

assay

0.25

it was

for e s t r o n e 0.15

(mean 2.18

(mean 5.3 ~g), w i t h

a coef-

f i c i e n t of v a r i a t i o n of 8, 7, 9%, r e s p e c t i v e l y . The s e n s i t i v i t y

of the m e t h o d

is l i m i t e d by the

r a n g e of l i n e a r i t y of the c a l i b r a t i o n

curve

detector

is r e a d i l y

used.

tained with steroid.

A measurable

the d e t e c t o r

u s i n g as low as 0.05

The r e c o r d e r w i l l

b u t the m e a s u r e m e n t

response

for the

at this

ob-

~g of

show a p e a k at 0.02 ~g, level is not r e l i a b l e .

294

ST ER O I D S

17:3

cxl

r-I

,~

+1 0"~

+1 CO

+1 CO

r~

r--

rr)

r-+l CO

P+t (~1

L~ +! CO

+1

+l

+1

,<

0 -,-I

Z H

0 E-, I~1

'0 '0 '0

F.I.1

IJ u} Z +1

H H

m

0 E-t 0 H

E-I 0

H m E-,

0 U

Z

Z

0

r..) r--

I

Z L~ 0

F-T4

0 H

o E~ [/}

March 1971

Thus,

ST ER O I D S

0.25

ug can be quantitated

295

in a 24 hour urine

specimen. The specificity

of the procedure

the purification

technique,

steps employed.

The improved

thin layer system as

packing

graphy

specificity.

results

obtained

in

and the chromatographic

well as the combination tend to increase

is inherent

used for gas chromato-

for the acetates

The quantitative

were

similar

to those

of the free steroid. Figure rogen

II shows

fractions

clearly

the gas chromatograms

after TLC.

The estrogen

of estrogen

of no great advantage,

derivatives

Tables ermination

during

The most abundant

Estradiol-178

in very low amounts. the results

gnanediol

the results

the menstrual

On many days the estrone

that of estriol.

results

in

done

estrogen was estthan

III shows graph-

estrogen,

a complete

The method has been successfully urine of rhesus monkeys.

cycle

was consistently

Figure

during

of det-

level was higher

of temperature,

determinations

to be

and separation

along with the pregnanediol

at the same time.

ically

are

of the free steroids.

III and IV demonstrate of estrogens

three women

appears

as quantitation

can be done with chromatograms

present

peaks

separated.

Formation

rone.

of the est-

The monkey

and pre-

cycle.

used on the urine

showed

296

ST ER O I D S

17:3

Acetyloted Steroids

Free Steroids

Aliquot from El TLC Froctlon

Estrone TLC Fraction

Allquot from E2TLC Froctlon

Estrodiol-17~ TLC Froction

Aliquot from E3 TLC Froctlon Reference

Reference •0.25 ~9

i

o so.°

\ '

FIGURE

6

II

'

i~

'

J~

Time in minutes

- ESTROGEN PREGNANT ACETYLATED

0.25 4~g EttCo.25 Aa.9 Jl (2 &c Reference II I J no, so,~g

2'4mi..

FRACTIONS WOMEN

FROM

COMPARING

STEROID.

URINE

OF

NON-

THE

FREE

TO

THE

March 1971

ST ER O I D S

TABLE ESTROGEN

AND

PATIENT

PREGNANDIOL THROUGHOUT

Day of Cycle

297

III OUTPUT

THE

OF

MENSTRUAL

AN

INFERTILITY CYCLE

El*

E2*

E3*

5

0.6

-

-

2.8

7

1.0

0.4

0.3

2.0

9

1.2

0.4

-

4.2

i0

.

12

0.3

-

-

2.1

14

0.4

-

-

2.3

16

0.5

-

-

1.4

18

1.9

0.4

-

0.8

20

0.3

-

-

i.i

22

2.2

-

-

3.1

24

2.9

-

-

0.2

26

1.4

i.i

3.7

1.4

28

1.0

0.5

-

0.7

30

0.6

-

-

0.4

32

-

-

-

i.i

-

=

not

*

=

ug/24

**

=

.

detectable

mg/24

hours hours

.

Pregnandiol**

.

298

ST ER O I D S

TABLE ESTROGEN

AND

PATIENT

Day of Cycle

THROUGHOUT

IV OUTPUT

THE

OF

AN

MENSTRUAL

INFERTILITY CYCLE

El*

E2*

E3*

1

2.0

0.8

2.5

1.3

2

2.6

i.i

2.8

1.5

3

1.7

3.0

2.2

1.2

4

1.2

0.22

2.5

i.I

5

1.6

0.9

3.4

0.8

6

1.8

1.4

2.1

0.6

7

3.9

1.2

4.0

0.9

i0

0.8

-

0.6

0.9

12

1.6

1.6

1.3

0.8

14

0.5

-

-

0.9

17

1.2

0.7

-

1.0

18

i.i

0.7

-

1.3

19

0.7

1.4

-

1.2

20

i.i

-

1.8

1.2

21

i.i

0.2

3.2

1.3

- = not * = **

PREGNANDIOL

17:3

detectable

ug/24 = mg/24

hours hours

Pregnandiol**

M a r c h 1971

S T E R O I D S

qD

299

•6t 9~

2.0 LO

E

s'r°n.a

i~

i

i

i

~

1

e, \

,

I

,

m

•

•

•

,

w

,

i

o

i

,

i

1

£stradiol . . . . . . . . .

,o~1

9

,

Estriol

5)~¢Menses i

z

,

6

i

,

,o

i

,2

,,

i

,6

,8 20 22

2426

Cycle Day

FIGURE

III-

URINARY

ESTROGENS

THROUGHOUT TEMPERATURE

AND PREGNANDIOL

A NORMAL = BASAL

MENSTRUAL BODY

CYCLE.

TEMPERATURE

300

ST ER O I DS

predominantly

estrone as seen in Figure

were no detectable The estrone

estrogens

IV.

There

in the anovulatory monkey.

levels went as high as 50 ug/day following

FSH/LH stimulation. detail

17:3

The results are reported

in

in the following paper of this issue.

DISCUSSION

Kellie

(3) reported almost q u a n t i t a t i v e

of steroid conjugates mixtures.

from urine by ether-alcohol

Varon et al,

steroid conjugates

removal

(4) and Kornel

(5) removed

by ethyl acetate after addition of

ammonium sulfate to urine. pH before extraction.

Both procedures

used low

Since pure estrogen glucuroni-

des were not available,

recovery studies were made

using pregnancy urine of known estrogen content added to water.

This pseudo-urine was first adjusted to pH

1.5, ammonium sulfate

(25% w/v)

was then added,

and

ethyl acetate extraction carried out before hydrolysis. Results of 5 determinations

indicated that ethyl ace-

tate extraction removed the conjugates tatively

(average 94.6%,

range

92-96).

almost quantiThe results

concurred with those of Kellie and indicated that pH of the extraction was not critical as long as it was below 6.

An ammonium sulfate concentration

not increase the recovery.

of 50% did

March 1971

ST ER O ID S

Refltencm Chromuloqrom EI and E2 0 S u g

3 01

Onl,eolecl £m F,oc~,on ~/10 ot 24h,s E2 El

E2

Triolld EI FroClion I,,'~Oo@ 24 hrs

E2

FIGURE

IV

- ESTROGEN BEFORE

FRACTIONS AND

AFTER

FROM

MONKEY

GONADOTROPIN

URINES TREATMENT

302

ST E R O I DS

The use of NaCI,

17:3

NH4CI and Na2SO 4 instead of

(NH4)2SO 4 was also investigated.

The results obtained

with these salts used for saturation of the urine were not satisfactory.

E t h e r - a l c o h o ~ butanol or c h l o r o f o r m

for extraction of the conjugates yielded extracts much less satisfactory tate.

for p u r i f i c a t i o n

than did ethyl ace-

These results were assessed by evaluation of

the gas chromatograms. The determining

factor in this m e t h o d

is the use

of isopropyl ether both in the extraction process well as the thin layer chromatography. ether has a lower polarity water

than ether

0.2 g/100 vs 7.8 for ether).

isopropyl material

ether probably removes from the urine,

as

Isopropyl (solutility in

For this reason

less of the polar

resulting

in cleaner extracts.

The use of the ethyl acetate extraction procedure to remove conjugates

has the advantage of reduction of

the solvent volume since a 24 hour urine collection reduced to 50 ml for the hydrolysis cedure is time saving and reduces

step.

is

This pro-

the amount of sol-

vents required during extraction. Since labeled steroid conjugates were not available,

it was not possible

hydrolysis.

to assess

losses during

Since this work was completed,

been shown that there is no difference labelled estriol g l u c o s i d u r o n a t e

it has

in recovery of

added before hydrolysis

March 1971

ST ER O I D S

to pregnancy plasma.

303

The m e t h o d of Levitz and Bassett

(9) has in our hands consistently

given a recovery

of 70% whether the conjugate or free estriol was added to the plasma or urine. The method described provides on gas chromatography the three classical

an extract which

yields chromatograms

estrogens

from which

can be quantitated.

The m e t h o d is simple enough for performance by a well trained technician. ments point out, to day.

As the replicate

recovery experi-

the results can be repeated from day

The application of the method to routine

clinical problems

is at present under investigation.

REFERENCES

Brown,

J. B.

B i o c h e m J.

2.

Bauld,

W. S.

Biochem.

3.

Kellie,

4.

Varon, H. H., Darnold, H. A., Forsythe, J. L. and Bishop, J. K. Fed. Proc. 26, 779, 1967.

.

.

6.

.

.

9.

Kornel,

A. E.

L.

Biochem.

J. Clin.

60,

185,

J. 63,

488,

J. 100,

Endocrin.

1955.

1956.

63, 1966.

23, 1192,

Touchstone, J. C., Murawec, T. and Brual, J. Chromatography 37, 359, 1968. Touchstone, J. C., Nikolski, Steroids 3, 569, 1964. Eberlein,

W.

R.

Steroids

Levitz, M. and Bassett, 1067, 1968.

M.

1963. O.

A. and Murawec,

14, 553,

T.

1969.

J. Clin.

Endocr.

28,