519
PATTERN OF ESTETROL CONJUGATION IN THE HUMAN (1) H. JIRKU, S. KADNER and M. LEVITZ (2) Department of Obstetrics and Gynecology, New York University School of Medicine, New York, N.Y. 10016 Received 10/23/71 ABSTRACT Following the intravenous injection of 3H-estetrol (E~) into an adult human subject the urinary excretion of tritium was virtually complete within 72 hours. The metabolites consisted almost exclusively of conjugates of estetrol. Each conjugate was purified by column chromatography, identified by the elution pattern and by enzyme hydrolysis and estimated on the basis of radioactivity recovered in the appropriate column fraction. The values are as follows: E4-ring-D-glucosiduronate , 89qo; E4-3 glucosiduronate, 2.8~0; E4-sulfoglucosiduronate , 0.4~; E4-Nacetylhexosaminide, O.5~; E~-sulfo-N-acetylhexosaminide, 0.5~. Some conjugates (4.%) were not identified. Uneonjugated E 4 was 2o1~. It appears that E 4 combines the conjugating patterns of estriol and 15ahydroxyestradiol. Estetrol (E4) (3) combines the structural features of estriol (E3) and 15~-hydroxyestradiol
(15~-OHE2).
interesting patterns of conjugation. glucosiduronates,
E 3 and 15a-ONE 2 display
E 3 is excreted as the 16 and 3
the 3 sulfate and as a double conjugate, estriol-3
s~fate-16 glueosiduronate.
15a-OHE~2 is excreted mainly as the 15-N-
acetylglucosaminide and the 3 sulfate-15 N-acetylglucosaminide. The metabolism of labeled E 4 in the man was studied by Fishman (4).
He found that virtually all the radioactivity was excreted with-
in 2 days and that 6 0 - 8 ~ siduronafie(s),
of the radioactivity was estetrol gluco-
in order to gain further insight into the nature of
the glucosiduronate fraction and to characterize some of the unknown conjugates, a urine sample previously analyzed by Fishman was investigated further.
520
ST ER O I D S
19:4
MATERIALS AND METHODS Subject and Collection. Twenty percent aliquots from the 3 day urine collections obtained from subject FN following the intravenous injection of 3H-E 4 were pooled and analyzed. This sample was generously donated by Dr. J. Fishman, after he completed his studies (4). Chromatography and Electrophoresis. ' Most separations were effected by gradient elution chromatography on 36 g of acid-washed Celite 545 in a 2X) x 45 em column (HBV = 55 ml) as described previously (5). Each column was eluted finally with methanol in order to detect highly polar conjugates. The solvent systems (1-15) are listed in Table 1 of the publication accompanying this report (6). Gradient elution chromatography on alumina was carried out with 220 g of neutral Woelm alumina deactivated with 9% water~ in a 2.5 x 55 cm column (5). The volumes used were as follows: 650 ml of 95% ethanol in the mixing chamber, 700 ml of water (pH 7.0 - 7.4) and lOO0 ml of 0.2% NH4OH. High voltage paper eleetrophoresis was carried out in pyridine-acetic acid buffer,
p~ 6.3 (5). Estrogens. Unlabeled E 4 and 6,7-3H-Eu were kindly donated by Dr. j. Fishman. 6,7-3H-E4-3 glucosiduronat~ was prepared by incubating labeled E 4 in guinea pig liver homogenate fortified with DDPGA (6,7). 6,7-~H-E4-3 sulfate was obtained as a by-product of the glucosiduronidation. E4-3 methyl ether and its 15,16-acetonide were prepared in the following way: 6,7-3H-E4 in 5 ml of dichloromethane was treated with excess diazomethane at 4 ° for 36 hr. The product was chromatographed on Celite in system 12. About 8 ~ of the tritium was eluted in holdback volume (HBV) 1-2, as compared to HBV 5-6 for E 4 (Table i). The material in HBV 1-2 was chromatographed in system 14. About 70~o of the tritium appeared as a single peak in HBV 13-14. Trihydroxyestrogens are eluted in HBV 12-14 in this system. The presumed E4-3methyl ether was converted to the 15,16-acetonide by treatment with perchloric acid in acetone (8). The acetonide was purified by chromatography in system ll. Enzyme ~drolysis. Glucosiduronates and sulfates were hydrolyzed respectively with ~-glucuronidase (Ketodase) and sulfatase (Mylase P) as described previously (9)- N-Acetylhexosaminides were hydrolyzed with ~-N-aeetylhexosaminidase extracted from jack bean meal (]O). The enzyme was generously donated by Dr. Yu-Teh Li. Usually the sample in 0.2 ml of 0.05 M citrate buffer was incubated with 4.5 units (ll) of G-N-acetylhexosaminidase for 2 days at 25 °. For each incubation a parallel control was run in which enzyme was omitted. In addition, parallel incubations with ~-glucuronidase were carried out in the presence of saecharolactone.
April 1972
Table i.
ST E R O ID S
521
Elution behavior of estetrol and its derivatives on Celite columns
Solvent System*
Steroid ii
12
13
14
15
Holdback Volume
E4**
-
5-6
5
-
9
E4-3raethyl ether
-
1-2
-
13-I~
-
Eh-3 methyl ether ~5,16-acetonide
2
.
.
.
.
E4-15,16-acetonide
5-6
.
.
.
.
4
1
-
7-8
1
-
5-6
-
iO-ii
i-2
-
12
-
E3
-
3-4
3
l~
4
15~-O~ 2
-
4-5
4
-
6
E3-3 methyl ether
8
15~-OHE2-3 methyl ether
9
E1
16-epiE 3
4
1
* See (6) for solvent systems. * * S e e Reference Section for abbreviations.
-
-
9-10
-
522
ST ER O I D S
19:4
Isolation and Identification of Metabolites. The urine was passed through a 5.2 x 94 cm column charged with IOOO g of Amberlite XAD-2 as described by 8radlow (12), and the metabolites were eluted with methanol. The solvent was flash evaporated and the residue dissolved in 95~ ethanol. Gradient elution chromatography on alumina was then carried out yielding two zones, A and B. Zone A (elution volume OI O O O m l ) was fractionated by partition chromatography on Celite in system iO (see Table 2 for the elution Characteristics of estetrol conjugates). Three peaks were eluted. Peak A-I (HBV i) was rechromatographed in system !2. Peak A-!I (HBV 2-3) was incubated twice, first with 4.4, then with 2.9 units of ~-N-acetylhexosaminidase. Between incubations the material was passed through a i x 20 cm XAD-2 column (Ii). The resulting aglycone was extracted 5 times with ecual volumes of ether. The ether extract was subsequently purified in system 12. Peak A-Ill (HBV 5) appeared to be a sulfo-N-acetylglucosaminide. Following incubation with 4.4 units of ~-N-acetylhexosaminidase for 48 hr at 25 ° the hydrolysate was saturated with NaCI and extracted with n-butyl alcohol. An aliquot was checked for completeness of hydrolysis by thin layer chromatography on a silica gel G plate with the solvent system methanol-ethyl acetate l:l. In this system the following approximate Rf values were observed: E4, 0.73; E4-3S, 0.78; E4-HNAc , 0.65; and E4-SHNAc , 0.58. The residue from the butanol extract was chromatographed in system 7. A single compound was eluted in HBV 1-2 and incubated with 20 mg of M~lase P in 5 ml of O.1 M acetate buffer, pH 6.0, for 14 hr at 37 °. Following extraction with ether the sample was chromatographed in system 12. Since the aglycones from A-II and A-III had the same polarity (HBV 6), they were pooled and chromatographed in system 13 together with ~C-15~-OHE 2. The elution rate relative to ! 5 ~ - 0 ~ 2 was determined. Authentic E 4 (20 ~g) was added to the tritiated material eluted in HBV 5 and the acetonide was prepared. It was purified in system Ii and its elution rate relative to E l was determined.
~
Zone B. Zone B %~s purified by chromatography in system ~ . In this instance, a 2.5 x 55 cm column (HBV = 125 ml) was employed. Three zones of radioactivity were obtained. The least polar, B-1 (~BV l) was rechromatographed in system 6o A single peak was eluted in HBV 6-8 and treated with 1 ml of Ketodase in 4 ml of O.1 M acetate buffer, pH 4°65, for 18 hr at 37 ° . Extraction of the hydrolysate with ether and subsequent chromatography in system 12 resulted in a single peak in HBV 5-6. The elution volumes of the steroid and its acetonide were compared, respectively, with that of 15~-OHE 2 in system 13 and with that of estrone in system ll. The second zone, B-II (HBV 2-4) was chromatographed in system 2. Two major distinct zones, B-II-1 (HBV ll-12) and B-II-2 (}{BV 13-15) were eluted. Zone B-II-1 gave a single peak in system 5 (HBV 4-5)
April 1972
Table
ST ER O ID S
2.
Elution
behavior
of estetrol
Conjugate
Solvent
1
2
4
GA+*
conjngates
on C e l i t e
columns
Systems*
5 Holdback
E4-ring-D
523
6
7
9
i0
Volume
2
-
4-5
1-2
6-8
3-4
I
E4-3C_~%
-
11-12
-
4-5
-
-
-
E4-HNAc
.
.
.
.
.
.
2-3
E4-SHNAc
.
.
.
.
.
.
5
E4-3S
i-2
.
E4-SGA
-
13-15
-
-
X-GA
-
13-15
-
-
X
.
E3-16GA
i
-
-
-
3
E3-3GA
5
-.
8
1-2
6-8
4-5
.
.
.
.
.
.
15~-OHEISGNAc
.
.
I~-O~2SG~o
*
See
(6) f o r s o l v e n t
**
See
Reference
.
.
.
-
-
3 4
.
2-3
.
.
systems.
Section
I-2
for a b b r e v i a t i o n s .
I
I
4-5 4
6
3-4 6-7
1-2
3
524
ST ER O I D S
19:4
corresponding in mobility t o E 4 - 3 glucosiduronate which was prepared by incubation of E h with guinea pig liver homogenate fortified with UDPGA (6). Its mo~ility on high voltage paper electrophoresis was slightly greater than that of Eq-16 glucosiduronate. Following hydrolysis with 8-glucuronidase~ extraction with ether yielded a steroid with the elution characteristics of E 4 in system 12. Zone B-!I-2 was purified by high voltage paper electrophoresis at 4000 volts for 2.5 hr. Two components were separated: B-II-2G with the mobility of a glucosiduronate, and B-II-26 migrating like a sulfoglucosiduronate. Following chromatography in system 9, fraction 8-1I-2Gwas eluted in HBV 4. Upon treatment with 6-glucuronidase a less polar compound (X) was formed and eluted in HBV 1 in the same system. This compound did not migrate on high voltage paper electrophoresis. Solvolysis with O.1 N HC104 in tetrahydrofuran (13) was carried out. The material extracted with ether from aqueous solution was chromatographed in system 12. Treatment of Fraction B-II-2~ with phenolsulfatase resulted in the formation of a compound with the mobility of an estetrol-ring-D glucosiduronate in system 1. Subsequent treatment of the monoconjugate with 6-glucuronidase yielded an ether-extractable steroid with the mobility of E 4 in system 13. Quantitation. An aliquot of each coltn~n eluate was assayed. The recovery of radioactivity ranged between 90 and 95%. On the assumption that the losses were uniform the counts in each radioactive zone were corrected to 100~/o recovery. Thus, the values presented are semiquantitative. On the Position of Attachment of Glucosiduronic Acid. Zone B-l: A single peak was obtained upon chromatography in system 5 (HBV 1-2). It was subjected to the methylation procedure essentially as described by Brown (14) except that only one 1 ml portion of dimethyl sulfate was used. After one hour of incubation at 37 ° the reaction mixture was neutralized with dilute acetic acid at O ° and then passed through a 1 x 50 cm Amberlite XAD-2 column to remove the salts. An aliquot of the methanolic eluate from the Amberlite column was dissolved in 4 ml of O.1 M acetate buffer, pH 4.65, and extracted once (v/v) with ether. The pM was readjusted and 1 ml of Ketodase was added. Following incubation for 18 hr at 37 ° the sample was again extracted (5 x 5 ml) with ether and the organic extract chromatographed in system 12 to check for completeness of methylation. The rest of the eluate from Amberlite was then treated with 2 x 1 ml of dimethyl sulfate and the procedure described above was repeated. The fractions eluted in HBV 1-2 in system 12 were combined and chromatographed in system lh together with standard 16-epiE~ and E 3. A single peak of radioactivity was obtained in HBV 13-14. Following treatment with perchloric acid in acetone, chromatography in system ll resulted in a single peak in HBV 2, presumably the 3-methyl ether of estetrol-15, 16-acetonide.
April 1972
ST ER O l D S
525
Zone B-II-I. An aliquot of this sample ~ s treated with 1 ml of dimethyl sulfate for 1 hr at 37 ° , the mixture was neutralized at 0 ° 8nd extracted with ether. Treatment with Ketodase and subsequent chromatography of the aglycone in system 12 resulted in essentially one peak which exhibited the elution behavior of E 4 in system 13.
RESULTS The excretion of radioactivity in urine by the subject studied following the intravenous injection of 3H-6,7-E 4 was found by Fishman to be virtually complete within 72 hours (4).
Furthermore, he found
that less than 2% of the tritium was extracted from the urine prior to enzyme treatment and that 8-glucuronidase hydrolyzed 82% of the radioactive material. drolysis.
An additional l ~
was liberated by acid hy-
In our study, chromatography on alumina resulted in the
separation of compounds containing glucuronic acid (Zone B,
95.7~)
from those devoid of it (Zone A, 3-7~). i~-Aeetylhexosaminid3s.
Zone A was fractionated in system 10 (system
numbers refer to the Celite partition columns listed in Tables 1 and 2).
Over 7C~/0 of the activity was eluted in HBV l, the major portion
of which (87%) had the mobility of E 4 in system 12.
The methanolic
eluate from system 12 was checked for E4-3 sulfate in system 1.
Less
than 4~ of Zone A had a mobility comparable to E4-3 sulfate, but because of low counts its identity could not be established. About 14¢ of Zone A was eluted in HBV 2-3 in system 10 (fraction A-II).
In two successive incubations with 8-N-acetylhexosaminidase,
6(~i'~and 76% of the activity were released into ether, as compared to 3¢ in a control sample.
This result strongly suggests the presence
STEROI
526
DS
19:4
of a conjugate containing N-acetylhexosamine, probably N-acetylglucosamine. Fraction A-III, comprising about 14~ of Zone A, was converted by ~-N-acetylhexosaminidase to a monoconjugate in 99~0 yield (against !~o in a control sample).
The degree of hydrolysis ~ms judged by thin-
layer chromatography on silica gel G.
The monoconjugate eluted in
HBV I-2 in system I exhibited the same mobility as the sulfate obtained whem E4-6,7-3H was incubated with fresh guinea pig liver homogenate (6).
Phenolsulfatase rendered 9(~ of the radioactivity (3~ in a
control) ether soluble.
The data suggest that fraction A-III repre-
sents a double conjugate containing both sulfuric acid and N-acetylhexosamine.
The steroid moiety in fractions A-II and A-Ill was
characterized as E 4 by its elution volume in systems 12 and 13.
Its
relative elution volume against I ~ - O H E 2 was 1.33 (standard E4=1.33 ). The acetonide had a relative elution volume of i . ~
(standard E 4-
acetonide=l.48) Compared to estrone in system ii. Glucosiduronates.
1~wo major radioactive zones were obtained follow-
ing chromatography of Zone B in system 9A. (94.~/0) was eluted in zone B-I. was ~
The bulk of the tritium
It was purified in system 6.
~Hydrolysis
upon treatment with ~-glucuronidase, with inhibition by sac'
charolactone.
The aglycone was characterized as E 4 by virtue of its
elution volume relative to I ~ O H E 2 in systems 12 and 13 and the elution volume of its acetonide relative to E 1 (1.44) in system I!. This conjugate accounted for 89% of the metabolites excreted in the urine.
Zone B-If yielded two distinct peaks comprising 3~/~ of the
April 1972
ST ER O ID S
527
counts following chromatography in system 2. The major portion (52.4¢~ of Zone B-II), Fraction B-If-I, was hydro!yzed by 8-glucuronidase in 95~ yield.
The aglycone ~as characterized as E 4 by virtue of its
mobility in system 12.
The conjugate as such ~as characterized in
part by its mobility, which corresponded to that of E4-3-glucosiduronate (6). This conjugate was quantitatively the second most important metabolite in tbe urine (2.8q0). Fraction B-II-2, comprising about one-fourth of the activity in zone B-Ii, was separated into components ~ and ~ on high voltage paper electrophoresis.
At
4000 volts, fraction B-II-2(~ (72~) moved about 7 cm/hr, similar to estriol-16 glucosiduronate.
Fraction B-II-2~B (28~) had a mobility
comparable to that of estriol-3 sulfate-16 glucosiduronate (about 14 cm/hr). Ketodase.
The g!ucosiduronate-like material (X-CA) was treated with Subsequent chromatography in system 9 revealed that 78~
had been converted to a less polar substance which was non-extractable with ether (monoconjugate
? ) . In the presence of saccharolactone only
hydrolysis was observed.
The "monoconjugate" (X) was eluted in
HBV 1 in system lO, thus exhibiting less polarity than E4-HNAc (see Table 2 for comparison).
Compound X contained no ionizable group as
indicated by its lack of mobility on high voltage paper electrophoresis. Treatment with enzymes such as Mylase P, almond emulsin, Glusulase and jack bean meal ~-N-acetylhexosaminidase released at m o s t 6 % of the radioactivity into ether (34 in a control).
The ether extract obtain-
ed from the incubate of presumed ~ouble conjugate X-GA with Ketodase also contained about 6~ of the radioactivity.
This material had the
528
ST ER O I D S
mobility of E 4 in syste m 12.
19:4
Following solvolysis of X in tetra-
hydrofuran about 65% of the tritium became ether-soluble.
However,
chromatography of the extract in system 12 did not yield a distinct peak and the limited amount of material precluded further investigation. The sulfoglucosiduronate-like compound was converted in 69~0 yield to a monoconjugate by treatment with phenolsulfatase. monoconjugate so obtained was eluted in HBV 2 of system 1. mobility is characteristic of E4-ring-D glucosiduronate.
The This
Both the
un~wdrolyzed portion and a control sample containing no phenosulfatase retained the mobility of a sulfoglucosiduronate on high voltage paper electrophoresis.
Virtually complete hydrolysis witb inhibition by
saccharolactone was obtained by subsequently treating the monoconjugate with ~-glucuronidase.
The elution volume of the aglycone
relative to 15aOHE 2 (1.33) was again characteristic of E 4 in system 13. The composition of the urinary conjugates of estetrol expressed as per cent of total radioactivity excreted is presented in Table 3. Differentiation of ring$A glucosiduronate from ring-D-glucosiduronat__~e. The methylation procedure according to Brown (14) was employed to differentiate ring-D from ring-A glucosiduronates, as described previously.
An aliquot of fraction B-I was reacted once with one
milliliter of dime~hyl sulfate and extracted with ether to eliminate material hydrolyzed during methylation.
About 80~o of the radio-
activity was ether-extractable following treatment with ~-glucuronidase.
Of that, 47~ still had the polarity of E 4 in system 12 indicat-
April 1972
S T E R O 1D S
ing incomplete methylation.
529
When the methylation procedure was re-
peated twice with the partially methylated glucuronide, less than 2~ of the counts were released into ether before and 32~ after treatment with ~-glucuronidase.
The aglycone in the second ether
extract contained virtually no estetrol.
Over 80~0 as compared to
53~ after the first methylation had the polarity of standard estetrol3 methyl ether in systems 12 and 14.
It was converted in 90% yield
to an acetonide with a characteristic elution volume in system ll.
TABLE 3 Urinary conjugates of estetrol Compound
Estimated percent of total radioactivity excreted *
E4
2.1
E4-N-acetylhexosaminide
0.5
E4-sulfo-N-acetylhexosaminide
0.5
E4-ring-D-glucosiduronate
89
E4-3-glucosiduronate
2.8
E4-sulfoglucosiduronate
0.4
X-glucosiduronate**
1.1
Not identified
3.8
*
See text for assumptions involved in quantitation
** See text for characteristics of this unknown conjugate The glucosiduronate from Zone B-II-1 was treated once with dimethyl sulfate. radioactivity.
Extraction with ether removed about 1% of the
Subsequent enzyme treatment released about 50°/o into
53o
STEROIDS ether.
19:4
Contrary to the pattern observed with Zone B-I virtually all
the material recovered following chromatography in system 12 (84~ of the ether extract) had the polarity of estetrol, as confirmed by chromatography in system 13.
No E4-3 methyl ether-like material
could be detected. DISCUSS ION Fis~man has shown that following the intravenous injection of 6'7-3H.E4 into a human subject virtually all the radioactivity is excreted as conjugates of E 4 and that glucosiduronates predominate (4).
The present report provides more information on the nature of
the glucosiduronates and partial structures of most of the other conjugates.
Furthermore, some analogies with the metabolism of E 3
and 15~-0~] 2 are indicated. The appropriate reference compounds were not available for use as internal standard.
Thus, identification was made on the basis of
elution characteristics of the conjugate as such on one or two Celite systems, enzyme hydrolysis and chromatography of the released steroid. In the ease of a double conjugate sequential enzyme hydrolysis and chromatography of theintermediate single conjugate was employed. This methodology does not preclude the presence of some radioactive impurities in the metabolite under observation. The monoglucosiduronates were separated by chromatography.
The
less polar glucosiduronate (89~) was methylated under conditions by which phenols react, and then treated with ~-glucuronidase affording E4-3 methyl ether. This sequence establishes the conjugate as a ringD glucosiduronate.
Unavailability of referencecompound precluded
April 1972
531
ST ER O ID S
further identification.
It is of interest to note that the ring-D
glucosiduronate E3-16GA is the predominant urinary conjugate of E 3 (15, 16).
The second glucosiduronate of E 4 was identified as the
3 glucosiduronate.
The compound could not be methylated with dimethyl
sulfate, presumably because the phenolic position was blocked.
Fur-
thermore, the conjugate had the chromatographic properties of E4-3GA synthesized in guinea pig liver homogenates.
Interestingly, E 3 is
also excreted in the urine as the 3 glucosiduronate in amounts less than that of the 16 glucosiduronate (16).
A further analogy with E 3
metabolism is the presence of E4-sulfoglucosiduronate in the urine. Partial hydrolysis of this double conjugate by phenolsulfatase places the sulfate moiety at carbon 3.
The e!ution characteristics of the
glucosiduronate so obtained support evidence for glucuronidation in ring-D without revealing the exact position of the glucuronic acid. E3-3S-16GA has been identified in several human body fluids including urine, (17,18,19).
E4-su!foglucosiduronate has been reported
in small amounts in the bile and urine of subjects receiving estrone
(20). Two other metabolites of interest were partially characterized. The first, a single conjugate, exhibited elution characteristics of N-acetylglucosaminides on chromatography on alumina.
Furthermore,
it was hydrolyzed by a pure preparation of 8-N-acetylhexosaminidase. Based on our recent studies which indicate that in the human this type of conjugation is specific for the 19x-position (5,21), it seems most likely that this conjugate is the
E4-15~-B-D-N-acetyl-
532
STER
glucosaminide.
OIDS
19:4
The second conjugate was converted to E4-3S follow-
ing treatment with B-N-acetylhexosaminidase.
Again by analogy with
the metabolism of 15G-OHE 2 (21) this double conjugate is probably the E4-B-sulfate-15G-B-D-N-acetylglucosaminide. Thus qualitatively the conjugates of E 4 appear to combine the structures exhibited by E 3 and 15G-OHE 2.
It is emphasized that
only one urine sample was analyzed, that from a subject who excreted some 90~ of the urinary radioactivity in the glucosiduronate form. Another subject studied by Dr. Fishman excreted only 60~ of E 4 as glucosiduronates
(4).
Unfortunately the latter sample which may
have yielded higher percentages of the more unusual conjugates, was not available to us for analysis.
REFERENCES
1.
This investigation was supported by a grant from the National Cancer Institute, National Institutes of Health (Grant #5 RO1 CA 2071-18).
2.
Research Career Development Awardee, 5 K3-HD-18,422-O9, of the National Institute of Child Health and Human Development. To whom correspondence should be addressed.
.
The following trivial names and abbreviations are used: Estetrol (Eh) = estra-l,3,5 (iO)-triene-3 ,l~,16~,lT~-tetrol; estrone (E17 = estra-l,3,5(lO)-trien-17-one; estradi01 (E2) = estra-l~3,~(lO)-triene-3,17B-diol; estriol (E~) = estra-l,3,5 (lO)-triene-3,16~, 178-triol ; l~.hydroxyeston4 (I~-OKE I) = 3, l~-dihydroxyestra-i, 3,5 (lO) -trien-17-one ; l~f~-hydrox~estra' diol (I~-OHE2) = estra-l,3,5(lO)-triene-3,1~,lTB-triol;
STEROIDS
April 1972
533
16-epiestriel (16-epiE.~) = estra-l,3,5(iO)-triene-3,168,17Btriol; estetrol-N-acet#lhexosaminide (E4-HNAc); estetrol-sulfoN-acetylhexosaminide (E4-SHNAc); estetrol-sulfo-glucosiduronate (E4-SGA); estetrol-ring-D-glucosiduronate (~4-ring-D GA); estetrol-3-glucosiduronate (E4-3GA) = 15~,16d~,178-trihydroxyestra-l,3,5(lO)-trien-3-yl 8-D-glucopyranosiduronate; estetrol3-sulfate (E4-3S) = l~,16c~,17~-trihydroxyestra-l,3,5(iO)trien-3-yl sulfate; estriol-3-glucosiduronate (E3-3C~) = 16~, 17~-dihydroxyestra-l,3,5(lO)-trien-3-yl ~-D-glucopyranosiduronates; estriol-16-glucosiduronate (E3-16GA) = 3,17~-dihydroxyestra-l,3,5(lO)-trien-l~-yl ~-D-glueopyranosiduronate; estriol3-sulfate-16-glucosiduronate (E3-3S,16GA) = 3-sulfato-17~-hydroxyestra-l,3,5(lO)-trien-16~-yl ~-D-glueopyranosiduronate. 15~-hydroxyestrone-sulfo-N-acetylglucosaminide (15~-OKEISGNAc) = 3,sulfato-17-oxoestra-l,3,5(lO)-trien-15~-yl 2'-acetamido-2'deoxy-~-D-glucopyranoside; 15~-hydroxyestradiol-sulfo-N-acetyl glucosaminide (15~-OH~SGNAc) = 3-sulfato-17~-hydroxyestra-l,3, 5(10)-trien-15~-yl 2'-acetamido-2'-deoxy-~-D-glucopyranoside; HBV, holdback volume; TEAMS, triethylammonium sulfate. ,
Fishman, J., J. CLIN. ENDOCRIN. 31, 436 (1970).
5.
Jirku, H., and Levitz, M., J. CLIN. ENDOCRIN. 29, 615 (1969).
6.
Jirku, H., Kadner, S., and Levitz, M., STEROIDS, publication accompanying this paper.
.
.
Goebelsmann, U., and Diczfa!usy, E., Katz, J., and Levitz, M., STEROIDS 6, 859 (1965).
Dixon, R., STE O S 14, 714 (1969).
9.
Levitz, M., Twombly, G.H., and Katz, J., STEROIDS 6, 553 (1965).
I0.
Li, S-C., and Li, Y-T., J. BIOL. CHEM. 245 , 5153 (1970).
ll.
Cable, R.G., Jirku, H., and Levitz, M., BIOCHEMISTRY 9, 4587
12.
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