Estrogeg conjugates of hen's

RMESH S. MATHUR Department of Agricultura1 chemisday, Macdonald College of McGill University, Montreal, Quebec

Received December 23, 1968

MAT HUE^, R. S. Estrogen conjugates of hen's urine. Can. J. Biochem. 47, 535-538 (1969). Estrone-6,7-W and 0rnithine-5-~'C were injected concurrently into a hen and the steroid-conjugated

fractions of the urine subsequently excreted were examined for the presence of radioactive phenolic steroids. The results indicated that neither estrone-6,743 nor its phenolic in vivo conversion products were excreted as conjugates with ornithine. Possible biliary excretion of such conjugate(s), however, was not excluded by the experiment.

The fowl is known to excrete benzoic acid in conjugation with ornithine as dibenzoylornithine (1). This fact prompted us to investigate whether or not any of the steroid estrogens are also excreted in conjugation with ornithine.

Materials and IMethsds A Rhode Island Red hen was modified surgically (2)

so as to exteriorize the ureteral openings and permit the collection of wine.

Radioactive M6zterial Estrone-6,743 (specific activity, 500 mCi/mM) was

purchased from the Radiochemical Centre, Amersham, England. Its radiochemical purity was over 99% as judged by thin-layer chromatography, and hence it was used as received.

Brnit'hine-5-14C (specific activity, 8.3 mCilmM) was purchased from the New England Nuclear Corporation, Boston, Mass. and was used as received.

Steroids Reference estrogens were bought from various com-

mercial sources. Estradiol-17p-3-sulfate was a gift from Dr. W. Y. Kkdani, Woswell Park Memorial Institute, Buffalo, N.Y.

Sol%9ents A18 solvents except methanol were of analytical grade

and were redistilled before use. Methanol was 'spectrana- lyzed' grade (Fisher Scientific Co.) and was used as received.

Thin-Layer Chromatography Thin-layer chromatography was performed on 20 cm

x 20 cm glass plates coated with 0.25 mm thickness of siiica gel G (Merck). The solvents used are given in Table I.

lMacdonald College Joum~kl Series No. 585. T h e systematic and (in brackets) trivial names of the

steroid estrogens mentioned in the present communication are as follows: 1,3,5(10>estratrien-3-ol-17-one (estrone); 1,3,5(10)-es&atriene-3,1'S~-diol (estradiol-87D) ; 1,3,5(60)- estratriene-3, I7oc-diol (estradiol-$7~) ; and 5,3,5(10)- estratrien-3,168,17$-trio1 (1 6-epIestrioI).

Miomtiuity Measurements Measurements were performed in a Packard Tri-Carb

liquid-scintillation spectrometer, model 3003. The pro- cedure was that described by Rood et al. (6). Settings at the counter were adjusted so that only 14C was counted in the green channel and that mainly tritium along with a small proportion of 14C was counted in the red channel. Corrections for this overflow of 14C into the red charnel were made by using a calibration curve prepared by using standard 14C toluene samples. The counting efficiency of each isotope was ascertained by using I4C- and 3H-toluene standard solutions. Measure- ment of radioactivity was done in duplicate and the 14C- and 3M- standard solutions were added separately to the separate vials of the sample being assayed in duplicate. The maximum counting efficiency of "C was 54% and that of tritium was 28 "/,.

Experimental Procedure Estroned,7-W ((1.5 x lo9 d.p.m.) and ornithine

5-14C (2.22 x lo8 d.p.m.) were injected intramuscularly into a laying hen and the urine was collected over three successive 24-h periods. Each 24-h sample was separately filtered and aliquots thereof were assayed for radio- activity.

An aliquot of the urine for the first 24-h sample (day 1) was taken for solvent partition according to the procedure described in Fig. 1. Each fraction thus obtained was assayed for radioactivity. Fraction 4 (n-butanol fraction) was further chromatographed in system 1 and in system 2. Aliquots of fraction 3 and fraction 4 were subjected to enzymatic treatment. The following enzyme preparations were used: (a) Ketodase (Warner-Chilcott Co., Toronto, Canada) with 0.50 M acetate buffer, pH 5.0; (b) Ketodase in presence of 1,4-saccharolactone (Calbiochem), the saccharolactone being added at a concentration of 1 mg/ml; (c) phenol sulfatase (Sigma Chemical Co., St. Louis, Mo.,) with 0.15 M sodium acetate buffer, pH 5.0; and (6) hyaluronidase, type IV (Sigma Chemical Co.) with 0.10 M sodium citrate buffer, pH 4.3. A11 imuba- tions were carried out for 24 h at 37 "C. The amount of the added radioactivity which could then be extracted by chloroform (3 x 1 volume) from the incubation mixture was compared with that extracted by chloroform from a blank incubation without addition of enzyme.

Fractions 3 and 4 were subjected to solvoBysis as described by Burstein and Lieberman (7). The estrogens liberated after 24-h incubation were chromatographed

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CANADIAN JOURNAL OF BIOCHEMISTRY. VOk. 47, 1968

TABLE I Thin-layer chromatographic systems

System No. Composition by volume Reference

1 Benzene - ethyl methyl ketone - ethanol - water (3:3 :3:1)

2 2-propanol~hloroform-methanol- (3)

10 N ammonia (10: 10:5 :2) 3 Cyclohexane - ethyl acetate - ethanol

(3)

(4.5:4.5 : 1) 4 Cyclohexane - ethyl acetate (1 : 1)

(4)

5 Chloroform - diethyl ether (3 :2) (4) (5)

Urine aliquot I

Results and Discussion 1 The total urinary recovery of "H and -C

extract with CWCIs (3 x 1 volume) was 17.9 % for 3~ and 36.1 % for 14C.

The proportions of the urinary radioactivity

CH@13 extract

extract with ethyl acetate (3 x 1 volume)

Eth extract A

adjust to pH 2 and extract with ethyl acetate (3 x 1 volume)

Eth extract B

extract with n-butanol (3 x 1 volume)

n-Butanol extract ~ ~ u e o i s phase (residue)

FIG. 1. Flowsheet of the method for solvent partition of the radioactive urine sample.

on thin-layer chromatoplates in system 5. Reference estrone, estradiol-l7p, estradiol-1 7a9 and 16-epiestriol were also spotted on the lateral strips. Following develop- ment, the reference estrogens were stained by the reagent of FoHin and Ciocalteu, and zones corresponding in chromatographic mobilities with the reference estrogens were removed and eluted. Each of the four presumptive estrogen fractions was chromatographed in three diflerent solvent systems (systems 3,4, and 5) for identification of the presumptive estrogen fractions.

Fraction 5 (aqueous residue) was subjected to acid hydrolysis (15% volume WCl) and the hydrolyzed material extracted with diethyl ether, the ether extract dried under vacuum, the residue dissolved in a small volume of methnoI, and chromatographed in system 5.

obtained in various fractions by the procedure outlined in Fig. I are shown in Table IT.

TABLE 11 Partition of the urinary radioactivity in the various

fractions obtained by the procedure outlined in Fig. 1 *

Solvent Percentage fraction

No. Composition 'H

1 Chloroform 7.1 1.2 2 Ethyl acetate (pH 6.5) 5.8 0.5 3 Ethyl acetate (pH 2.0) 28.9 1.7 4 n-Butanol (pH 2.0) 62.0 7.1 5 Aqueous residue. 15.8 72.5 Total 118.8 83.8

*Values expressed as percentage of total urinary radioactivity.

Thin-layer chromatography of fraction 4 in system B gave the following results (Table 111). (a) Almost all of the 14C activity remained at the line of application. (b) Tritium activity was located largely (35.0 %) in the region correspond- ing in mobility to reference estrone sulfate and estradiol-l7p-3-sulfate. The solvent front carried 27.7 % of the radioactivity.

Similar results were obtained when fraction 4 was chromatographed in system 2. This showed that the and '"6 activities were not together and hence estrone or its metabolites were not conjugated with ornithine. Presence of a large proportion of 3H activity at the solvent front showed that at least an appreciable proportion of the conjugated material present was acid- labile.

Incubation of fractions 3 and 4 with Ketodase and hyaluronidase did not result in liberation of

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MATHUR: ESTROGEN CONJUGATES OF HEWS URINE 537

Thin-layer chromatography of n-bubanol extract (fraction 4) in system 1

Radioactivity

d.p.m. Fraction

No. I4C

1 2 3 4 5 6 7 8 9

10 11

Total

Percentage of total

I4C

6 .3 94.5 10.3 5.2 16.8 0 .3 2.7' - 1 .o - - -

TABLE IV Fraction 5 (aqueous residue): distkibution of the JH-radisactivity on chromatoplate following acid hydrolysis and subsequent extrwtircbn by dkthyl

ether (chromatopaphed in system 5)

Radioactivity

Fraction Percen tap Position of No. d.g.m. of total reference estrogens

*Location of reference dieonjugate (~tradio1-11~-3,17-disulfate). a conjugate(s) cannot be completely ruled ?Location of reference monoconjugate (estrone-3-sulfate). $Location of free phenolic steroids (atrone, atrerdio8-17B9 and

estradiol-1%). If estrogen-ornithine conjugates do exist, it

any significant amount of free estrogens. Phenol sulfatase on the other hand liberated 34.4% of the chloroform-extractable radioactivity from fraction 3 and 36.2% f om fraction 4. These results indicated absence of glucosiduronates and presence of sulfates.

Identification of the estrogens in fractions 3 and 4 following solvolysis of each of these fractions indicated the presence of estrone, estradiol- 17p, estradiol- 17a, and f 6epiestriol.

Fraction 5 (aqueous residue) was acid- hydrolyzed and the ether extract subjected to thin-layer chromatography. The distribution of the 'H activity on the chromatoplate is shown in Table IV. There was practically no 14C activity in the ether extract, suggesting that the I4C- labelled compounds were highly polar and that very small proportions of the radioactivity were present in locations corresponding in chromato- graphic mobility with the reference 16-epiestrio1, estradiol- 133, estradiol- l7a, or estrone.

From these results, it is clear that there was no appreciable in vivo conjugation and subse- quent excretion in urine of estrone and ornithine when the two substances were injected together into the hen. This observation does not, of itself, exclude completely the possibility that such a conjugate could, in fact be formed in vivo. Excretion in bile of conjugates which are entirely absent from urine is by no means impossible and therefore biliary excretion of

is likely that some active form of ornithine would be involved in such conjugation, and it is quite possible that either ornithine itself may not be the active form required for conjugation and/ or significant labelling of this active form in vivo might only take place if enough 14C-ornithine were injected to label the entire body pool of this amino acid.

It may be mentioned, however, that the hen excretes estrogens in the urine mainly as their sulfuric acid esters and that monosulfates and disulfates of steroid estrogens have been identi- fied in hen's urine following administration of estradi01-17f4-4-'~C (8). Hence if ornithine conjugates of the estrogens occur in the hen's urine they must be present as relatively very minor components.

Acknowledgments This work was supported in part by grant

AM-86130-END, National Institute of Arthritic and Metabolic Diseases, United States Depart- ment of Health, and in part by the National Research Council of Canada,. grant A-56. The author thanks Professor W. H. Common of MacDonald College, and Professor D. S. Layne of the University of Ottawa for helpful criticisms.

1. JAFFE, M.: Fber. dyt . chem. Ges. 10, 1925 (18771. Cited fmni Betoxication mechanisms. 2nd d. Williams, R. T. John Wiley and Sons Pnc., New York. 1959. p. 286.

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538 CANADIAN JOURNAL OF BIOCHEMISTRY. VOL. 47, 1969

2. APNSWORTH, L.: Poultry Sci. 44, 1541 (1965). 6 . FLOOD, C., LAYNE, D. S., RAMCHARAM, S., ROSSIPAL, 3. WUSEMAN, F. S., DODGSON, K. S., LLOYD, A. G., E., TAR, J. F., and TAIT, S. A. S.: Acta Endocrinol. 36,

ROSE, F. A., T ~ B A L L , Pd.: 9. Chromatog. 16, 237 (1961). 334 (8964). 7. & J R ~ ~ E ~ N , S., and LIEBERMAN, S.: J. Biol. Chem. 2.33,

4, LISISBBA, El. P., and DICZFALUSY, E.: Act8 EndocKin01. 331 (8958). 40, 60 (1962). 8. MATHUR, R. S., COMMON, R. H., COLLINS, D. C., and

5. SOBREVILLA, L., HAGERMANN, D., and VILLEE, 6 . : LAYNE,D. S.:Bischirn. Biophys,Acta,176,394(1969). Biochim. Biophys. Acta, 93, 665 (1964).

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