6α-Hydroxylation in human placenta perfusion

307

6a-HYDROXYLATION IN HUMAN PLACENTA PERFUSION Lise C&lard and R. Knuppen Laboratoire de Chimie Hormonale de la Maternite de Port-Royal, Paris, France and Chemische Abteilung, Chirurgische Universitats-Klinik, Bonn-Venusberg, Germany Received

June

28, 1965

Abstract Perfusion -in vitro of human placenta with radioactive oestradiol-

178 or oestrone led to the formation of a fraction more 'polar' than

the starting material. In this fraction 6u-hydroxyoestradiol-170 has been identified by paperchromatography in various systems and by microchemical reactions as a major component. The same compound was also found after perfusion of 4-14C-testosterone, 4-14C-dehydro-epi-androsterone and 6,7-3H-dehydro-epi-androsterone-sulphate.

Introduction In previous experiments an increase of the 'oestriol-like' fraction was observed after perfusion of oestrone and oestradiol-17B through human placental. The 'oestriol' fraction thus obtained was very small in relation to the degree of aromatisation of 16a-hydroxylated Cl9-compounds 2 and reduction of 16a-hydroxyoestrone. More recently, Bolt& -et al. were unable to identify oestriol after perfusion -in situ of placenta without foetus. It seemed therefore of interest to investigate further the nature of the 'oestriol-like'product. The present experiments show conclusively that, in addition to as yet unidentified compounds, the 'oestriol-like' fraction contains 6a-hydroxyoestradiol-17'B as a major component, formed metabolically by 6a-hydroxylation after perfusion of neutral and phenolic steroids. Materials and methods Paperchromatographic solvents: Chloroform/formamide (system 1) ; chloroform-ethyl ; v/v)/formamide (system 2) ; monochloromonochlorobenzene-ethyl acetate (3:l;v/v) benzene/formamide /formamide (system 4 ; benzene-petroleum ether-methanol-water (4:6:7: 3) (system 5).

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9 human placentas were perfused with 6,7- 'H-oestradiol-170, 4-14Coestradiol-178, 4-14C-oestrone, 4-14C-testosterone, 1,2-3H-A4-androstenedione, 4-14C-dehydro-epi-androsterone or 6,7-3H-dehydro-epi-androsteat diff rent terms of pregnancy following the method rone-sulphate described previously 3,P. All radioactive material was purified by column- or thin layer chromatography before use. The quantitative assays were made in a Tricarb Packard Automatic Liquid Scintillator Spectrometer with two channels permitting simultaneous assays of 14C and 3X. The full term placentas were obtained from a natural confinement and the two early term placentas by hysterotomy for therapeutic abortion. The time between delivery and the beginning of the perfusion varied between 20 and 30 min. The perfusion was performed in a closed circuit with Krebs-Ringer solution and blood (0 :ih'). After 30 min. of perfusion the radioactive steroid, dissolved in 2 ml ethanol, was injected into the umbilical artery, and the perfusion was carried out for two hours. In most experiments, one hour after the injection of the steroid, 10000 I.U. of HCG (Laboratoire Kndopancrine, Paris) were added to the medium. In one experiment (expt no. 5, see table) a NADPH2-generating system was used, instead of HCG. Two different methods were used for the extraction and the purification of the radioactive material. Usually (method A) an aliquot of the perfusion fluid was extracted 3 times with an equal volume of ether. The combined ether extracts were washed with a saturated NaHCO3-solution and with water and then concentrated to 50 ml in vacua. This solution was then extracted 4 times with 20 ml of N-MaOr kfter adjustment to pH 8.5 the NaOH extract was reextracted 3 times with ether. After washing with water, the extract was evaporated to dryness, the residue dissolved in 1 ml ethanol and distributed twice between 80 ml of benzene/ petroleum ether (1:l; v/v) and 70 ml of water. The aqueous phase was washed back with 20 ml of benzene/petroleum ether (1:l; v/v), and then extracted 3 times with 100 ml of ether after addition of 8.5 ml 0.07 m phosphate buffer,'Phe organic phase was washed with water and evaporated to dryness. In some experiments the 'polar' fractions thus obtained were chromatographed on celite columns with ethylene dichloride-methanolwater (10:7:3) before paperchromatography. In two experiments (method B) the perfusion fluid was extracted 3 times with an equal volume of ether and twice with ethyl acetate. The combined extracts were washed with saturated NailCOz-solutions and evaporated to dryness -in vacua after washing with water. J

Results Identification of the 'polar' fraction. After perfusion of 4- l4Coestrone and 4- 14C-oestradiol-17B with full term placentas method B was used for the isolation of the 'polar' radioactive fraction. The residue thus obtained was subjected to chromatography on formamide-impregnated paper with chloroform as mobile phase5. The chromatograms were run for 6 hours. The radioactive zone corresponding to the position of Ga-hydroxy-

Sept.

1965

hp.

Term of

NO.

placenta

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309

Steroid perfused I

I ,

OO-“yWOxy-

I

oestrodiol178 fraction

Full term

Full term

I

Counts per minute

,I,#

6-0x0-

o-aenyarooestra-

oestrone 1

dioL17p 2

II.

oa-nydroxyoertro

dial-3-methyl ether 3

6,7-sH-oestradiol-17fi 9.7X 10s cpm

4728

2792

6,7-sH-oestradiol-17P

2860

1597

6.2X 10s cpm

4-‘4C-testosterone 0.96X106 cpm Full term

6,7-sH-oestradioL17p 2.3X106 cpm

412

840 4878

3090

201 1278

I-“C-oestrone 0.9XlOs cpm

1651

1449

6,?-JH-dehydro-epiandrosterone-sulphate 7.2X 10s cpm

940

612

168

456

4-IT-dehydro-epiandrosterone 3.3X 10s cpm

300

311

289

318

Full term

4-W-testosterone 1.3X10* cpm

160

24 Weeks

4-14C-oestradiol-176 0.6X 10s cpm

472

86

1,2-sH-androstenedione 2.5X 10s cpm

273

157

6,?-sH-oestradiol-l?fl 3SXlOs cpm

1720

965

0

0

Full term

9 week,

4-14C-testosterone 1.2XlOscpm

1 after oxidation of 6o-hydroxy-oestradiol-17@ 2 after elimination of the 6a-hydroxygroup 3 after methylation of 6a-hydroxy-oestradioLl7p

oestradiol-17I3 was eluted with methanol at room temperature. The residue was rechromatographed in system 2 for 15 hours. In this system the presence

of two radioactive zones (P-l and P-2) was observed. P-l show-

ed a mobility of 21.0 cm/15 hrs (that of P-2 was 22.9 cm/15 hrs) corresponding to the position of 6a-hydroxyoestradiol-178, whereas the distances from the starting line of oestriol and 6l3-hydroxyoestradiol-178 were 26 and 17.6 cm respectively.

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For further identification, P+l was eluted from the paperchromatograms with methanol after the addition of authentic 6a-hydroxyoestradiol-170. The solution was evaporated in vacua, and aliquots were subjected to microchemical reactions. Oxidation of P-l with chromic acid 6 in acetone yielded a substance which was indistinguishable from 6-0x0oestrone as judged by paperchromatography in the systems 3 and 5. The position of 6-oxo-oestrone detected by its U.V. light absorption corresponded to the maximum of the radioactive area. One third of the material (P-l ox), obtained after elution of the chromatograms, was treated with sodium borohydride in methanol at room temperature for 2 hoursT The product obtained had the same mobility as 6a-hydroxyoestradiol-17l3 in the systems 1 and 2. Catalytic reduction of P-l ox in ethyl acetate 8 with platinum oxide in an atmosphere of hydrogen led to the formation of two compounds which had the same mobility as 6Shydroxyoestrone 6B-hydroxyoestradiol-17I3 in the systems 1,2 and 4.

and

f/henP-l was trea-

ted under reflux with methanolic hydrochloric acid for one hour, a 'less polar' product was formed (P-l HCl), which had the same mobility as 6-dehydrooestradiol-17B as judged by the paperchromatographic system 3. Catalytic treatment of P-l HCl in an atmosphere of hydrogen resulted in the formation of oestradiol-170. In one experiment with a very young placenta (9 weeks) no 14C-6ahydroxyoestradiol-17B was found after perfusion of 4- 14 C-testosterone; 7 however, a small amount of 6,'(-3H-ba-hydroxyoestradiol-l'/Bwas formed from 6,7-'H-oestradiol-17l3, which was perfused simultanously with the same placenta. In 7 experiments, after preparation of the extracts following method A, quantitative estimations were carried out after elution of the areas containing 6a-hydroxyoestradiol-17B from paperchromatograms and counting the radioactivity in a Tricarb bcintillation Counter. Jhe amount of 6u-hydroxyoestradiol-l'i'l3 was compared to the total radioactivity of the ether soluble fraction, and constituted, 0.1-0.9 of this (see table). In some experiments, the 'oestriol-like' fraction was methylated with dimethyl sulphate according to Brown 9 and then subjected to chromatography on alumina. The radioactive material was eluted with 1.4/o ethanol in benzene, corresponding to authentic 6a-hydroxyoestra-

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1965

311

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diol-j-monomethyl ether. Ron-radioactive oestriol-j-monomethyl ether, normally found in the perfusion fluid, was eluted with 2.577 ethanol in benzene. Discussion The experiments reported here leave no doubt that the metabolite P-l is identical with ba-hydroxyoestradiol-1i'B. This compound is formed as a metabolite after perfusion of human placenta with phenolic and neutral steroids. On the basis of the foregoing results it is likely that the 'oestriol-like' fraction, previously obtained after the perfusion of oestradiol-17B, consisted partially of ba-hydroxyoestradial-17l3. No evidence was obtained for 6B- or 16a-hydroxylation. The constant presence of radioactive material (p-2) with a mobility of 22,9 cm/15 hours on the paperchromatograms in the system 2 suggests that 15a-hydroxyoestradio1-178 might also be present in placenta perfusion 10 isolated and identified 15a-hyfluid. Recently, Knuppen and Breuer droxyoestrone as main metabolite when oestrone was incubated with adrenal tissue. It appears likely that placenta tissue is also capable of hydroxylating oestrogens at C-15. 6-hydroxylation of phenolic compounds has been described after in11,12 . The occurrence of 6-hydroxylacubation with liver preparations tion of cortisol is well known during pregnancy".

More recently, Fo-

14 observed that the excretion of 6-oxygenated metabolites therby -et al. increased steadily during pregnancy. On the basis of the present results with placenta perfusion and of the finding by Hagopian -et al. 15 that 6-oxo-progesterone is formed after placenta perfusion with progesterone it seems reasonable to assume that the placenta may be active in the process of 6-hydroxylation during pregnancy. There are two possible metabolic pathways by which 6-oxygenated oestrogens could arise. One involves the direct hydroxylation of oestrone and oestradiol17~ in position 6, the second pathway involves the aromatisation of 6substituted neutral Clg-steroids. The result reported here confirm the first hypothesis, whereas previous experiments 16 showed that the second pathway is also possible in the placenta. Acknowledgements The authors wish to thank IVIms. E.Alsat and C.Ego for their skilled technical assistance and Prof.E.E.Baulieu for permission to carry out

312

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radioactive assays in his laboratory. We are also greatly indebted to Prof.H.Breuer for his interest in the progress of this work, and to Dr. E.R.Smith, London, for reading the manuscript.

References 1. Varangot,J., CQdard,L and Yanotti,S., Excerpta Xedica International Congress Series 51, Abstr. 396, Milan (1962). 2. Boltk,E., Xancuso,S., Eriksson,G., Wiqvist,N. and Diczfalusy,E., Acta endocr. (Kbh.) U_, 535 (1964). 3. C&dard,L., Varangot,J., Yanotti,S., C.R.Acad.Sci.(Paris) 154,1870 (1962) 4. Varangot,J., C&dard,L. and Yanotti,S., Amer.J.Obstet. Gynec.(in press). 3.

Knuppen,R., Z.Vitamin-Hormon- u. Fermentforsch. l2, 355 (1962).

6. Bowden,K., Beilbron,I.M., Jones,E.R.J. and Weedon,B.C.L., J.chem. Sot. 39 (1946). 7. Knuppen,R., Behm,M. and Breuer,H., Hoppe-Seylers Z. physiolWChem. z,

145 (1964).

8. Qintersteiner,G. and koore,M., J.Amer.chem.Soc. 8l, 442 (1959). 9. Brown,J.B., Bi0chem.J. 60, 185 (1955). 10. Knuppen,R. and Breuer,H., Hoppe-Seylers Z. physiol.Chem., m,

159

(1964) 11. Xueller,G.C. and Humney,G., J.Amer.chem.Soc. 1p, 1004 (1957). 12. Breuer,H., Knuppen,R. and Pangels,G., Biochim,biophys.Acta (Amst.) a,

1 (1962).

13. Frantz,A.G., Katz,F.H. and Jailer,J.X., Proc.Soc.exp.Biol.(N.Y.) 105, 41 (1960). 149 Fotherby,K., James,F., Kamyab,S., Klopper,A. and Wilson,G.R., J..&ndocr. 2, XXV (1965). 15. Hagopian,m.,Pincus,G., Carlos,J. and Romanoff,E.B., Endocrinology 58, 38 (1956). 16. Breuer,B. and Knuppen,R., 7.Symposium der Deutschen Gesellschaft fiir Endokrinologie, domburg/Saar 1960, Springer, Beidelberg,p.273(1961).