2246 407

DIRECT RADIOIMMUNOASSAY OF SPECIFIC URINARY ESTROGEN GLUCOSIDURONATES IN NORMAL MEN AND NONPREGNANT WOMEN*

K. Wrightl, D.C. Collins2, P.I. Musey and J.R.K. Preedy

Departments of Medicine and Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30303

Received: 11-4-77

ABSTRACT

Direct radioimmunoassays are described for the measurement of each of three specific estrogen glucosiduronates: estrone gluco- siduronate, 17B-estradiol-17-glucosiduronate and estriol-16a-gluco- siduronate in urine. Each assay utilizes a specific antiserum prepared by complexing the carboxylic acid group of the appropriate glucosiduronate to the e-amino group of lysine in bovine serum albumin or bovine thyroglobulin. The antisera showed little or no cross reactivity toward other estrogens that might be present in significant amounts in urine. These antisera were used for the direct assay of the conjugates in urine from normal men and nonpreg- nant women without prior extraction or chromatography. The values were similar to those obtained after extraction, chromatographic purification on DEAE-Sephadex and subsequent immunoassay. The fol- lowing mean values + SE (ug/g creatinine) were obtained: estrone glucosiduronate, male 10.1 + 0.6, follicular phase female 17.3 f 1.6, luteal phase female 31.8 + 2.5; 17B-estradiol-17-glucosiduronate, male 1.7 f 0.3, follicular phase female 2.4 + 0.1, luteal phase female 4.2 t 0.4; estriol-16cr-glucosiduronate, male 1.8 + 0.2, follicular phase female 4.7 + 0.9, luteal phase female 10.0 f 1.6.

INTRODUCTION

Urinary estrogen conjugates classically have been quantitated by

chemical measurement of the unconjugated estrogen after hydrolysis

(3,4). There are several disadvantages to this procedure. Acid

hydrolysis is nonspecific and may lead to destruction of the molecule

(4), while enzyme hydrolysis, although more specific, is subject to

inhibition by endogenous urinary substances (3). Chromatographic

methods to separate hydrolyzed or unhydrolyzed estrogens are time

*A preliminary presentation of parts ofthesedata was made at the 61st Annual Meeting of the Federation of American Societies for Experimental Biology, April, 1977.

Volwne 31, Nwnber 3 S WBBOXDI March, 2978

408 S TmEOXDI

consuming. Fluorimetric, gas-liquid chromatographic and calorimetric

methods of quantitating hydrolyzed estrogens are relatively insensi-

tive. Finally, any form of hydrolysis may destroy the identity ofthe

starting compounds and therefore does not distinguish between a

single estrogen conjugated at different positions.

Recently, chromatographic methods have been reported which

isolate individual estrogen conjugates on Sephadex (5,6) or as the

trimethylammonium salt derivatives on LH-20 (7,8). The purified

estrogen conjugatewasthen hydrolyzed and the free estrogen measured

by radioimmunoassay (7,8

cific, these methods are

a limited number of samp

Development of spec

. Although reasonably sensitive and spe-

time consuming and can only be applied to

es.

fit antisera to various steroid conjugates

(9) and the development of a radioimmunoassay specific for testos-

terone-17-glucosiduronate (10,ll) indicated the feasibility of

developing a radioimmunoassay specific for individual estrogen

glucosiduronates. In this communication we describe the production

of antisera against steroid-protein conjugates of estrone gluco-

siduronate, 176-estradiol-17-glucosiduronate and estriol-16a-gluco-

siduronate. These antisera were used for the development of specific

radioimmunoassay procedures for these estrogen glucosiduronates

without extraction, chromatographic purification or hydrolysis.

Using these techniques, we report values for these three specific

estrogen conjugates in the urine of normal men and nonpregnant women.

MATERIALS AND METHODS

Nonradioactive steroids and steroid conjugates were obtained from Sigma Chemical Co., St. Louis, MO., or Steraloids, Wilton, N.H., and purified by recrystallization. Purity was confirmed by thin-

S TmEOXDI 409

layer chromatogra Ci/mM), [2,4,6,7- Hl'estriol !

h Radioisotopes [2 4,6 7-3H] estrone (SA=84 (SA=84 Ci/mi) a;d [6,7-3H]-17B-estradiol-

17-glucosiduronate (SA=59 Ci/mM) were obtained from New England Nuclear Co., Boston, Ma.

Tritiated estrone glucosiduronate and estriol-16a-glucosiduronate of high specific activity were biosynthesized by incubation of the appropriate [3H] estrogen precursor with uridine -5'-diphosphoglucu- ronic acid. A microsomal preparation from rhesus monkey liver was used as an enzyme source (5). The [3H] conjugates were purified by chromatography on DEAE-Sephadex as previously described (5).

Unlabelled steroid standards were prepared and diluted in distilled undenatured ethanol. Phosphate buffered saline (PBS) (O.lM aqueous sodium phosphate, pH 6.8, containing (W/V) 0.9% NaCl, 0.1% ethylmercurithiosalicylate and 0.1% bovine serum albumin (Sigma, RIA grade)) was used as a diluent for the antisera and urine samples. The working solutions of [3H] estrogen conjugates were prepared fresh daily in PBS because of the increased rate of breakdown with prolonged storaae in PBS.

Radioactivity was measured using Packard liquid scintillation spectrometers, models 3320 and 3255, and ScintiverseTM (Emory Formu universal scintillation cocktail.

Antisera: Estrone glucosiduronate was coupled to bovine thyroglobu lin using the mixed anhydride procedure essentially as described by Erlanger ti ti (12). Antisera to estrone glucosiduronate were

la)

raised in adult male New Zealand white rabbits as previously described (13). The production of antisera to estriol-16a-glucosiduronate has already been reported (14,15). The antiserum to 17B-estradiol-17- glucosiduronate, a gift of Dr. A.E. Kellie, was prepared by a similar procedure (9).

The cross reactivities of other steroids and steroid conjugates for the antisera were determined as previously described and expressed as the percent equivalent of the appropriate standard (13).

Sample Collection and Preparation: Twenty-four-hour urine col- lections were made by normal men and women of reproductive age. Samples were collected without preservative and stored at 4"untilthe collection was complete. Aliquots were then stored at -20" until analyzed. No aliquot was used after having been thawed and refrozen three times.

Urine dilutions of 1:50, l:lOO, 1:500 and 1:lOOO were prepared in PBS and analyzed within 48 hr. Aliquots of 500 ul of at least 2 dilutions were assayed in triplicate.

Three different amounts of each estrogen glucosiduronate were added to a male urine sample. These samples were prepared and assayed as above.

Creatinines were measured on a Guilford automatic spectropho- tometer using a modified Jaffe reaction.

Radioinunoassay Procedures: Samples or standards were added to assay tubes in 500 ul PBS. The appropriate antiserum and tracer were added in a volume of 100 ~1 each. After thorough mixing, samples were incubated 60 min or overnight at 4". Bound and free

418 S -EmEOICDI

conjugates were separated in a 15 min incubation at 0" with 1 ml dextran coated charcoal (.5% Norit, .l% dextran in PBS), After centrifugation, the supernatant was decanted and counted after an equilibration period of 2 hr.

DEAE Chromatography: A 15 ml sample of urine containing 35,000 dpm of each of the estrogen glucosiduronates was extracted with 30 ml 95% ethanol by vigorous mixing and standing overnight at -20" (5). The sample was centrifuged and the supernatant removed and taken to dryness in a flash evaporator. cm DEAE-Sephadex column (5).

The residue was applied to a 1 x 60 The column was washed with 500 ml

deionized distilled water, and the estrogen glucosiduronates were eluted with a linear gradient of O-O-0.4M NaCl. An aliquot of each fraction was counted and each radioactive peak was pooled and assessed for recovery.

Each purified estrogen glucosiduronate was quantitated by the direct radioimmunoassay of the intact conjugate as described above after serial dilutions of l:lO, 1:20, 1:50 and 1:lOO in PBS. The values were correlated with values obtained by direct analysis of the same sample.

RESULTS

Antisera: Antisera to estrone glucosiduronate with useful titers

were obtained within 4 months of beginning injection. The antisera

to be studied further were identified by testing each antiserum for

its cross reaction with estrone sulfate, and the antiserum with the

lowest cross reactivity for estrone sulfate was used in the assay.

The cross reactivities of each of the estrogen glucosiduronate

antisera with unconjugated steroids is shown in Table I. Each of the

antisera showed significant cross reaction with some of the unconju-

gated estrogens.

Table II shows the cross reactivities of the antisera with conju-

gates of estrogens and with nonestrogenic steroid conjugates. The

following conjugated estrogens had significant cross reactivity:

estrone sulfate with the estrone glucosiduronate antiserum (3.911,

estriol-17-glucosiduronate with the 17B-estradiol-17-glucosiduronate

antiserum (5.4%) and with the estriol-16o-glucosiduronate antiserum

411

Table I. Percent cross reaction of various unconjugated steroids with antisera against protein derivatives of estrone glucosiduronate, 17G-estradiol-17-glucosiduronate and estriol-l&-glucosiduronate.

Compound

estrone 176-estradiol estriol 17a-estradiol 2-hydroxyestrone 2-hydroxyestradiol 2-hydroxyestriol estrone-2-methyl ether estrone-3-methyl ether 16-ketoestrone 16-ketoestradiol 16a-hydroxyestrone 16-epiestriol 17-epiestriol 16,17-epiestriol estetrol progesterone testosterone cortisol ethinylestradiol mestranol diethystilbestrol

Antisera against:

EJG/J E316aGA E217GGA

10.2

:::

;*: 0:o 0.0

1K 4:8 1.1 0.2 0.3

;*: 0:o 0.0 0.1

::: 0.0 0.0

18.4 11.7 15.0

;*z 0:3

K 0:o

1z 1717 7.0

3.; 3:9 0.0

K! 4:2

:::

1i.Y 2:3

89.5 0.4 0.2

Kl 0:5

::;

;:':

;*: 0:9 0.6

:*: 3:g 0.4 1.2

(4.3%), and estriol-16-sulfate with the 17B-estradiol-17-glucosiduron-

ate antiserum (4.2%) and the estriol-16a-glucosiduronate antiserum

(13.9%). None of the antisera showed significant cross reacitvity

with the nonestrogenic steroid conjugates.

Radioimmunoassay: Standard curves were established for each of the

estrogen glucosiduronates. A typical logit-log standard curve for

estrone glucosiduronate is shown in Fig. 1. The least detectable

amount for estrone glucosiduronate was 2.6 pg. The useful range of

412 S W=ZIBOID6

Table II. Percent cross reaction of various conjugated steroids with antisera against protein derivatives of estrone glucosiduronate, 176-estradiol-17-glucosiduronate and estriol-16a-glucosiduronate.

Antisera against:

Compound

estrone glucosiduronate estrone sulfate 17B-estradiol-17- glucosiduronate estriol-16a- glucosiduronate estradiol-3- glucosiduronate estriol-3- glucosiduronate estriol-3-sulfate estriol-17- glucosiduronate estriol-16-sulfate androsterone-3- glucosiduronate etiocholanolone-3- glucosiduronate testosterone-17i3- glucosiduronate pregnanediol-3- glucosiduronate dehydroisoandrosterone- 3-sulfate

E E316cxGA

100.0 0.7 3.9 0.1

0.0 1.6 00.0

0.0

1.6

0.0 4.3 5.4 0.6 13.9 4.2

0.0

0.2

0.2

0.0

0.0

100.0

1.4

0.7 0.0

0.3

0.2

0.3

0.5

0.2

E217BGA

0.6 0.5

1.7

1.1

1.4 0.5

0.2

1.0

0.7

1.7

0.2

the standard curve was found to be 2.6 - 80.0 pg.

A typical standard curve for 176-estradiol-17-glucosiduronate

is shown in Fig. 2. The useful range for this standard curve was

found to be 5.7 - 80 pg with the least detectable amount of 5.7 pg.

A typical logit-log standard curve for estriol-16a-glucosiduron-

ate is shown in Fig. 3. The least detectable dose for this estrogen

conjugate was shown to be 2.5 pg with a useful range of 2.5 - 80.0

Pg.

S -l?6EOIDb 413

99

98 1 97

96 95 1 80

1

IO

3

I

3

2

Fig. 1. A typical logit-log standard curve for direct assay of estrone glucosiduronate.

The intra-assay and interassay variation for each of the

estrogen glucosiduronate radioimmunoassays are shown in Table III.

The intra-assay variation was calculated from samples reading at 3

different points on the standard curve. The coefficients of

variation were less than 10% at each of the points. The interassay

variation was calculated from repeated analysis of a urine sample

from a normal woman (sample 1) and man (sample 2) in a number of

different assays.

414 s ‘SIIEOXD-

99

98

97 1 96 95 1

pg E,l7GA

Fig. 2. A typical logit-log standard curve for direct assay of 17B-estradiol-17-glucosiduronate.

Recovery experiments were carried out on a sample of urine

from a normal man. Three different amounts of each estrogen gluco-

siduronate were added to aliquots of the urine and the urine was

assayed. The results of this experiment are shown in Table IV.

In each case, the amount of each estrogen glucosiduronate assayed

was not significantly different from the amount added (~~0.05).

The validity of the direct assay for estrogen glucosiduronates

S TmEOflD- 415

I] I I , , , , , , I I I 1 I 2 3 45

rrlrrl

K) 2o 40 60 8OlOO 200 pg E,IGGA

Fig. 3. A typical logit-log standard curve for direct assay of estriol-16a-glucosiduronate.

was tested by comparing values obtained for urine samples assayed

directly with those obtained after purification by chromatography

on DEAE-Sephadex. The correlation of values obtained for estrone

glucosiduronate from 9 urine samples with and without chromatography

is shown in Fig. 4. The values showed a correlation coefficient of

0.98. The regression coefficient was 0.96 r 0.07 with an intercept

of 2.55 _C 1.94 ug/l.

416 S ‘B?DEOfD=

Table III. The intra-assay and interassay variation for the direct assay of estrone glucosiduronate, 17B-estradiol-17- glucosiduronate and estriol-16a-glucosiduronate in urine. See text for a description of the method for calculation.

INTRA-ASSAY VARIATION

(n=3)

SAMPLE 1 SAMPLE 2 SAMPLE 3

mean f SD CV mean k SD CV mean + SD CV (pg) (pg) (pg)

estrone glucosiduronate 11.7kl.l 9.32 51.21-0.5 1.02 79.3k2.2 2.73

176-estradiol-17- glucosiduronate 6.3kO.6 9.09 23.550.6 2.43 46.OkO.9 1.90

estriol-16a- glucosiduronate 9.4+0.3 3.09 20.4*0.3 1.42 49.6i2.3 4.68

INTERASSAY VARIATION

SAMPLE 1 SAMPLE 2

Number of mean + SD mean + SD Assays (PSI cv (pg) cv

estrone glucosiduronate

175-estradiol-17- glucosiduronate

estriol-16a- glucosiduronate

10 38.5k2.3 5.90 30.3k4.1 13.40

8 32.755.7 17.34 29.2k2.9 9.94

9 44.7k3.8 8.58 22.7k3.1 13.71

The correlation of values obtained from 6 urine samples for 178-

estradiol-17-glucosiduronate with and without chromatography is shown

in Fig. 5. The correlation coefficient was 0;97 with a regression

S TDEOIDI 417

Table IV. The mean values * SE measured in a sample of male urine after addition of known amounts of estrone glucosiduronate, 178-estradiol-17-glucosiduronate and estriol-16a-glucosid- uronate. The number of samples analyzed is shown in parentheses.

Steroid pg added pg recovered mean + SE (n)

estrone glucosiduronate

10.4 10.3 f 0.7 (10)

26.1 24.7 f 0.6 (10)

36.5 33.8 + 0.6 (10)

17f3-estradiol-17- glucosiduronate

11.5 11.4 + 0.6 (8)

28.7 28.3 + 1.1 (8)

40.1 39.4 + 1.5 (7)

estriol-16a- glucosiduronate

10.0 8.7 + 0.4 (8)

25.1 21.3 + 0.8 (7)

35.2 32.8 + 1.3 (4)

coefficientof 0.86 ? 0.10 and an intercept of 0.48 + 0.54 ug/l.

Fig. 6 shows the correlation of values obtained for estriol-16a-

glucosiduronate from 6 urine samples with and without chromatography.

The correlation coefficient was 0.99 with a regression coefficient of

1.01 i 0.04 and an intercept Of -0.31 f 0.44 Ug/l.

In the case of each estrogen glucosiduronate analyzed with and

without chromatography, the correlation coefficient was significantly

different from 0 (~~0.05). In all cases, the coefficient regres-

sionwas not significantly different from 1 (~~0.05) and the intercept

was not significantly different from 0 (~~0.05).

418 S =EmEOXDI

y = 0.96x + 2.55

r = .98

I I I I I

RldOwith 20 CHROMATOGRAPHY 30 40 50 (pg/l)

Fig. 4. Correlation of values for estrone glucosiduronate in 9 samples of urine from males and nonpregnant females estimated by direct radioimnunoassay and by radioimmunoassay after chromatographic separation on DEAE-Sephadex. The line relating the values is the calculated regression line.

Urinary Values: Urine samples from normal men and women in the mid-

follicular (day 8) and midluteal (day 22) phases of the cycle were

analyzed by direct radioimnunoassay and expressed as F-g/g creatinine.

The mean values and ranges are summarized in Table V.

419

IO-

22 g- \ - ;;3

8-

7;’ E 6-

b =- W a 4-

3 3-

2- y = 0.86x + 0.48

1 4 I I I I I I I I I I

1234561891011

RIA after CH~~ATOGRAPHY (pg/l) Fig. 5. Correlation of values for 176-estradiol-17-glucosiduronate in

6 samples of urine from males and nonpregnant females esti- mated by direct radioimmunoassay and by radioimmunoassay after chromatographic separation on DEAE-Sephadex. The line relating the values is the calculated regression line.

Of the three conjugates measured, estrone glucosiduronate was the

major estrogen glucosiduronate excreted in both men and women. The

mean value + SE obtained in men was 10.1 + 0.6 ug/g creatinine,

whereas women in the follicular and luteal phases had mean values

of 17.3 f 1.6 and 31.8 * 2.5 ug/g creatinine respectively. The

estriol-16a-glucosiduronate values were considerably lower than the

420 S TBEOXDl

257

5-

1 I I I 1

5 IO 15 20 25

RIA with CHROMATOGRAPHY (&I)

Fig. 6. Correlation of values for estriol-16a-glucosiduronate in 6 samples of urine from males and nonpregnant females estimated by radioimmunoassay after chromatographic separation on DEAE-Sephadex. The line relating the values is the calcu- lated regression line.

estrone glucosiduronate values in both men and women. The 176-

estradiol-17-glucosiduronate values were less than the estriol-16cl-

glucosiduronate values in women.

Statistical analysis of the excretion patterns (Table V) indicates

that the excretion of estrone glucosiduronate, 176-estradiol-17-

TABL

E V.

Me

an co

ncen

trat

ion

k SE

(w

ith ra

nges

in

par

enth

eses

) of

es

tron

e gl

ucos

idur

onat

e,

176-

estr

adio

l-17

-glu

cosi

duro

nate

an

d es

trio

l-16

a-gl

ucos

idur

onat

e in

th

e ur

ine

of men

an

d wo

men

in th

e mi

dfol

licu

lar

(day

8)

and

midl

utea

l (d

ay 22)

ph

ases

of

the me

nstr

ual

cycl

e de

term

ined

by

dir

ect

radi

oinm

unoa

ssay

. Va

lues

ar

e ex

pres

sed

as p

g/g

crea

tini

ne.

p fo

r di

ff.

p fo

r di

ff.

foll

icul

ar fe

male

lu

teal

fe

male

be

twee

n ma

le

betw

een

male

me

an t

SE (n

) me

an +

SE (n

) fo

llic

ular

me

an f

SE (n

) &

foll

icul

ar

rang

e ra

nge

& lu

teal

ra

nge

phas

e

17.3

rt

1.

6 (1

8)

estr

one

gluc

osid

uron

ate

(6.8

_ 31

.5)

31.8

+

2.5

(15)

10

.1 +

0.6

(6)

(9.8

- 4

7.9)

c.

01

(7.6

- 11

.3)

c.05

17B-

estr

adio

l-17

- 2.

4 I

0.1

(78)

4.

2 f

0.4

(15)

1.

7 ?r

0.3

(6)

gluc

osid

uron

ate

(0.9

- 3.4

) (2

.2 -

7.

5)

1.01

(1

.0 -

2.9)

c.

05

estr

iol-

16a-

gl

ucos

idur

onat

e

4.7

+ 0.

9 (1

8)

10.0

*

1.6

(15)

1.

8 k

0.2

(6)

(1.3

- 16

.5)

(3.2

- 23

.4)

<.Ol

(1

.0 -

2.5

) c.

05

422 S TlOICOXDIll

glucosiduronate and estriol-16a-glucosiduronate was significantly

lower in men than in women during the follicular phase. The

excretion of all three estrogen glucosiduronates was significantly

elevated in the luteal phase of the cycle when compared with the

follicular phase.

DISCUSSION

The antisera against each of the estrogen glucosiduronates was

prepared by linking the protein to the carboxylic group of the gluco-

siduronic acid rather than introducing a carboxylic acid group into

the estrogen molecule at another point. This procedure has been used

by several laboratories to prepare antisera against steroid glucosid-

uronates (9,11,13). Based on our previous experience with the devel-

opment of antisera against free estrogens (13), we expected the

greatest amount of cross reactivity to occur in the region where the

glucosiduronate molecule is attached to the estrogen. A review of

the cross reactivities in Tables I and II indicate that this was

generally true.

The significant cross reaction of the estrogen glucosiduronate

antisera with the unconjugated estrogens does not present a problem

in the urinary assay because of the extremely low concentration of

the latter in urine. None of the steroid conjugates showed any

significant cross reaction with the antibody to estrone glucosid-

uronate. None of the major estriol or 176-estradiol conjugates

present in significant concentration in urine showed significant

cross reaction with the estriol-16a-glucosiduronate or 176-estradiol-

17-glucosiduronate antisera. The only ring-D conjugates showing

significant cross reaction with either antiserum were estriol-16-

sulfate and estriol-17-glucosiduronate. These two estriol conjugates

are not thought to be present in significant amounts in urine.

The values for estrone glucosiduronate in normal men and women

are the first report of direct analysis of estrone glucosiduronate in

urine. The data presented in Table V indicate that the excretion of

estrone glucosiduronate is significantly lower in men than in women

during the follicular phase of the cycle. Estrone glucosiduronate

excretion is significantly increased in the luteal phase of the cycle.

Comparison of the values obtained by our direct method with other

methods requiring hydrolysis (3,4) suggest that estrone glucosid-

uronate is a major conjugate of estrone in the urine.

The values for estriol-16a-glucosiduronate reported here are

similar to those reported by Lehtinen and Adlercreutz (16) using a

direct radioimmunoassay. The values indicate that there is a signif-

icant rise in the.excretion of estriol-16a-glucosiduronate in urine

during the luteal phase of the cycle. The values reported here for

the excretion of estriol-16a-glucosiduronate by men were signifi-

cantly lower than those for women in the follicular phase. The

levels of estriol-16a-glucosiduronate are slightly lower than those

reported for total estriol in both men and women (3,4). This

suggests that while estriol-16a-glucosiduronate is a major form of

estriol in the urine, other metabolites are also present. Previous

work with radioactive metabolites of estriol in urine of nonpregnant

women suggests that significant amounts of estriol-3-glucosiduronate

and estriol-3-sulfate-16a-glucosiduronate are also excreted (17,18).

424 S TmEOIDm

This is also the first report of direct analysis of 17&estradiof-

17-glucosiduronate in urine of men or women. The values reported here

suggest that the excretion of 176-estradiol-17-glucosiduronate is

considerably lower than either estrone glucosiduronate or estriof-

?6o-glucosiduronate in women. The excretion of 176-estradiol-17-

glucosiduronate was also significantly increased during the luteal

phase.

When the values for 17~-estradiol-17-glucosiduronate were

compared to the total estradiol levels in urine from previous work

(3,4), it is clear that 17B-estradiol-17"glucosiduronate is a major

urinary form of estradiol-176 in women.

Thus, we have reported the development of specific radioimmuno-

assay procedures for the analysis of estrone glucosiduronate, estriol-

16a-glucosiduronate and 17B-estradiol-17-glucosiduronate in urine.

These procedures are specific and sensitive enough to measure these

estrogen glucosiduronates in men and nonpregnant women. They will

enable us to measure the levels of these specific estrogen gluco-

siduronates throughout the menstrual cycle and during various

disease states where an alteration in the metabolism of estrogens

might be expected.

ACKNOWLEDGEMENTS

This research was supported by NIH Grants ROl-HL-16394 and ROl- AM-13468. Dr. Collins is a recipient of U.S. Public Health Service Career Research Development Award l-K04-AM-70381 from the National Institute of Arthritis, Digestive and Metabolic Diseases.

REFERENCES

1. Present address: Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, Ct. 06510.

S tl?FEIEOXDb 425

2.

3.

4. 5.

6. 7.

a.

9.

10.

Address reprint requests to: Dr. D.C. Collins, Department of Medicine, Emory University School of Medicine, 69 Butler St., S.E., Atlanta, Georgia 30303. Brown, J.B., (1958).

A. Klopper and J.A. Loraine, J ENDOCRINOL 17:401 -

Preedy, J.R.K. and E.H. Aitken, J BIOL CHEM 236:1300 (1961). Musey, P.I., D.C. Collins and J.R.K. Preedy, STEROIDS 29:93 (1977).

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Hahnel,R., ANAL BIOCHEM lo:184 (1965). Young, B.K., H. Ji rku, STKadner and M. Levitz, AM J OBSTET GYNECOL 126:38 (1976). Levitz, M., H. Jirku, S. Kadner and B.K. Young, J STEROID BIOCHEM 6:663 (1975). Nellie, A.E., V.K. Samuel, W.J. Riley and D.M. Robertson, J STEROID BIOCHEM 6:91 (1975). The following trrvial names are used: androsterone-3-glucosiduronate=17-oxo-5c-androstan-3~-yl-~-D- glucopyranosiduronate; dehydroisoandrosterone-3-sulfate=17-oxoandrost-5-en-3~-yl- sulfate; 16-epiestriol=estra-1,3,5(lO)-triene-3,16B,l78-triol; 16,17-epiestriol=estra-1,3,5 lO)-triene-3,1@,17a-triol; 17-epiestriol=estra-1,3,5(10 -triene-3-16a,l7a-triol; I estetro1=estra-1,3,5(10)-triene-3,15~,16~,17~=tetro1; 17~-estradiol-3-glucosiduronate=l7~-hydroxyestra-l,3,5(lO 3-yl-B-D-glucopyranosiduronate; 178-estradiol-17-glucosiduronate=3-hydroxyestra-l,3,5(10) 17B-yl-B-D-glucopyranosiduronate; estrio1-3-g1ucosiduronate=16~,178-dihydroxyestra-1,3,5(10 ) 3-yl-B-D-glucopyranosiduronate; estriol-16a-glucosiduronate=3,17~-dihydroxyestra-l,3,5(10

-trien-

trien-

-trien-

-trien- 1 16a-yl-B-D-glucopyranosiduronate; estriol-17-glucosiduronate=3,l6~-dihydroxyestra-l,3,5(lO)-trien- 17B-yl-B-D-glucopyranosiduronate; estriol-3-sulfate=16a,l7~-dihydroxyestra-l,3,5(lO)-trien-3-yl- sulfate; estriol-16-sulfate=3,l7~-dihydroxyestra-l,3,5(lO)-trien-l6~-yl- sulfate; estrone glucosiduronate=17-oxoestra-1,3,5(lO)-trien-3-yl-~-D- glucopyranosiduronate; estrone sulfate=17-oxoestra-1,3,5(lO)-trien-3-yl-sulfate; ethinylestradiol=17a-ethinylestra-1,3,5(lO)-triene-3,l7~-diol; etiocholanolone-3-glucosiduronate=17-oxo-5~-androstan-3~-yl-~-D- glucopyranosiduronate; 2-hydroxyestradiol=estra-1,3,5(lO)-triene-2,3,17B-triol; Z-hydroxyestrone=2,3-dihydroxyestra-1,3,5(lO)-trien-l7-one; 16a-hydroxyestrone=3,16cL-dihydroxyestra-1,3,5(lO)-trien-17-one; 16-ketoestradiol=3,178-dihydroxyestra~,3,5(lO)-trien-l6-one; 16-ketoestrone=3-hydroxyestra-1,3,5(lO)-trien-16,17-dione; mestranol= 3-methoxy-17-ethinylestr~-1,3,5(1O)-trien-l7~-ol; estrone-Z-methyl ether= 2-methoxy-3-hydroxyestra-1,3,5(10)-trien- 17-one.

426 S TmEOIDI

11.

12.

13.

14.

15.

16. 17. 18.

estrone-3-methyl ether= 3-methoxyestra-1,3,5(10)-trien-17- one; pregnanediol-3-glucosiduronate=2O~-hydroxy-5%-pregnan-3~-yl-~-D- glucopyranosiduronate; testosterone-17B-glucosiduronate=3-oxoandrost-4-en-l7~-yl-~-D- glucopyranosiduronate; Hennan, J.F., W.P. Collins and I.F. Sommerville, STEROIDS 21:285 (1973).

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Erlanqer, B.F., S.M. Beiser, F. Borek, F. Edel and S. Lieberman, Methods in Immunology and Immunochemistry (Eds., Williams, C.A. 2, Vol 1, p. 148 (1967). an M.W. ase , Wright, K., (1973).

D.C. Collins and J.R.K. Preedy, STEROIDS 21:755 -

Wright, K., D.C. Collins and J.R.K. Preedy, J STEROID BIOCHEM 5:303 (1974). Uright, K., D.C. Collins, M. Virkler, P.I. Musey, E. Dale and J.R.K. Preedy, AM J OBSTET GYNECOL, In Press (1978). Lehtinen, T. and H. Adlercreutz, J STEROID BIOCHEM 8:99 (1977). Stoa, K.F. and M. Levitz, ACTA ENDOCRINOL 57:657 (lg68). Levitz, M. and J. Katz, J CLIN ENDOCRINOL STAB 28:862 (1968). -