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In vitro biosynthesis of estrogens in placentas from normal and toxemic pregnancies
In vitro biosynthesis of estrogens in placentas from normal and toxemic pregnancies KESHO
R.
PARVATI
K.
GOURI
HINGORANI, Delhi,
PH.D.
MALKANI,
S. KOSHTI,
VERA New
LAUMAS,
F.R.C.O.G., M.Sc.,
M.Sc.
F.R.C.O.G.,
F.A.C.S. (TECH.)
F.A.C.S.,
F.I.C.S.
India
In vitro biosynthesis of estrogens in microsomal fractions obtained from placentas from 7 normal and 7 toxemic cases has been investigated under identical experimental conditions. I9-Hydroxyandrost-4-ene-3, I7-dione, androst-4-ene-3, 17-dione, and testosterone were used as substrates and studied for their conversion into estrogens. The conversion of these substrates was found to be low in the toxemic placenta compared to the normal placenta. Kinetic studies with I9-hydroxyandrost-4-ene-3, 17-dione which has been considered to be an obligatory intermediate in the biosynthesis of estrogens showed that in both normal and toxemic placentas the maximum conversion was attained within one hour, and this remained unchanged when incubations were carried out even up to 4 hours. In kinetic studies also the toxemic placenta showed a lower conversion compared to the conversion found in normal cases. Human chorionic gonadotropin was found to have no eflect on the conversion of 19-hydroxyandrost-4-ene-3, 17-dione to estrogens in either normal or toxemic placentas. The present finding of low estrogen biosynthesis in toxemic cases is discussed.
1 N R E c E N T years considerable evidence has accumulated from both in vitro and in vivo studies which indicates that the placenta is the principal source of the greatly increased amounts of estrogens produced during human pregnancy. The studies of Ryan2 demonstrated the conversion of androgens to estrogens in high yield by a preparation of human placental microsomes. The exact biosynthetic pathways which in vivo lead to estrogen formation have not been quite clear. For a time it was assumed that human placenta, like the ovary, could synthesize estrogens from acetate, but extensive research in this area has shown that this may not be true. The concept of complete placental autonomy has ,
now been replaced by the theory that the placenta is an incomplete endocrine organ which is capable of synthesizing active hormones only from preformed steroidal precursors reaching it via the fetal and maternal bloodstreams. An active role3-5 has been ascribed to the fetus for the synthesis of steroidal precursors which later undergo transformation to estrogens in the placenta. The placenta is thus responsible for the final synthesis of large amounts of estriol formed during normal human pregnancy. Urinary estimation of estriol is regarded as a valuable test for placental function6 in the later stages of pregnancy. The level of estriol falls rapidly with placental death, and low urinary estriol values have been reported in cases of toxemia. The decline in urinary estriol levels in the later weeks of pregnancy in cases of toxemia indicates some impairment in the synthesis of estrogens by the fetal-placental unit.
From the Reproductive Biology Research Unit and the Department of Obstetrics and Gynaecologly, All-India Institute of
Medical
Sciences.
This work The Ford
was supported by grants from Foundation and the Indian of Medical Research.
Council
1062
In vitro placental biosynthesis of estrogens
tn view of the knowledge of estrogen biosynthesis in the normal human placenta, a study of estrogen biosynthesis in placentas from cases of toxemia of pregnancy seemed highly desirable. In the present paper, a study of the in vitro conversion of androgen precursors to estrogens in normal and toxemic placentas is presented. Material
and
methods
Tissue preparation. Human placenta was obtained from patients delivered in the AllIndia Institute of Medical Sciences Hospital. The placenta was immedately transported to the laboratory under ice in a thermal flask. It was dissected free of fetal membranes and blood vessels at Y’ C. The remaining placental tissue was weighed and homogenized in a Vertis 23 homogenizer for 1 minute in phosphate buffer according to the procedure of Ryan.’ The buffer contained 0.25M sucrose, 0.05M phosphate, and 0.04M nicotinamide at pH 7.0. One volume of buffer to three parts of placental tissue by weight was used for homogenization. The homogenate was filtered through cheesecloth and fractionated by differential centrifugation in the Beckman Spinco ultracentrifugation Model L. The nuclear fraction was separated by centrifugation at 6,000 x g for 10 minutes. The supernatant from the nuclear fraction was again centrifuged at 17,500 x g for 30 minutes and the residue containing the mitochondrial fraction was separated. The supernatant from the mitochondrial fraction was subjected to centrifugation at 105, 001) x ‘g for 60 minutes when the microsoulal fraction settled down in the bottom of the tube. The microsomal fraction which contained the active enzyme was stored in a deep freeze at -12’ C. until the experiment was carried out. Incubation. In all incubations a placental microsomal fraction in 1 ml. buffer obtained from 7.5 Gm. of placental tissue was used. It was incubated with 150 pg of various steroidal precursors in the presence of a TPNH-generating system consisting of 10 pmoles triphosphopyridine nucleotide (TPN) , 60 pmoles glucose-6-phosphate, and 1.5 K
1063
glucose-6-phosphate dehydrogenase. units The total volume of the incubation medium was 5 to 6 ml. The androgen steroidal prccursors were taken down in the bottom of the incubation flasks, The incubation was carried out in 25 ml. conical flask at 137”’ C. for one hour in Dubnoff shaker with air as the gas phase. Extraction. The incubations xvcre terminated by the addition of a small quantity of chloroform. At this stage carrier 6:i Westradiol (approximately 180,000 d.p.rn. in 0.2 ml. ethanol) was added for rc:co\ycry correction. The incubation mixture w:ts rxtracted three times with four volumes of chloroform. The chloroform extracts were pooled, made up to a known volume, and an aliquot taken out for radioactivity colinting. The remaining pooled chloroform extract was dried to a small quantity under nitrogen. Chromatography. The toluene-proi,ylene glycol system of Zaffaroni and Burton’ as modified by Savard8 was used for the pllrification and separation of estrogens formed after incubation. Standard clsteronc: and estradiol- 17,B were spotted along with the residue from the above chloroform extract. After running the paper in the t&en+propylene glycol system for 2 to 3 holrrs. the strips containing the standards were r~r~vecl and developed by spraying with freshly prepared ferric chloride-ferricyanidr rcaqent. The area corresponding to the standards on the strips containing the unknown r>stracts were cut and extracted with a mistnre of chloroform and methanol ( 1: 1). ‘t‘ht rhloroform-methanol extract was cotlcc-sntratcd under nitrqgen gas to a small xdurrlr~ in a small centrifuge tube. It was spottt*d on a Silica gel plate 10.25 mm.\ aloiig \\.ith rstrone and estradiol standards. ‘The plate was developed in a tank containinq hcmzenemethanol (9: 1). The area corresponds to the standards on the thin-layer plate %vhere the extracted estrone and estradiol were run, were separately scraped off, and were extracted separately with a mixture of chloroform-methanol ( 1 :I). The extracted estrone and estradiol were made up to a known volume and 5 per cent of this taken for
1064
August 15, lS68 Am. J. Obst. & Gym.
Laumas et al.
counting. The rest of the extracts were evaporated to dryness and estimated for estrone and estradiol. The recovery correction was carried out with the addition of 6:7 H3-estradiol in all cases. However, in separate experiments with 6:7 H3-estrone it was found that the recovery correction for estrone was almost the same as that for estradiol. Quantitation of estrone and estradiol by extraction of the Kober color. The content of estrone and estradiol in the unknown samples, puriiied by chromatographic procedure as mentioned above, was estimated by extraction of the Kober color. The Kober color reaction was carried out according to the procedure of Kober as modified Adlercrentez.e Four milliliters of chloroform containing p-nitrophenol (2 per cent) was added to the Kober reaction tube, shaken for 20 seconds, and centrifuged at -IO0 C. for 2 minutes. The chloroform layer was separated out and quantitated by measuring at 500, 538, and 576 rnp in a Zeiss spectrophotometer. Corrected optical density was obtained with Allen’s correction. Radioactivity counting. All radioactivity samples were counted in Packard Tricarb Model 3314 (Packard Instrument Company, La Grange, Illinois). The samples to be counted were taken down in the scintillation vials and 10 ml. of the scintillation liquid, 4.0 Gm. PPO (2, 5-diphenyloxazole) and 100 mg. dimethyl POPOP 1, 4-bis2- (4-methyl-5-phenyl-oxazolyl) -benzene dissolved in a liter of redistilled toluene, was added. The counting efficiency of 3H with this counter was about 33 per cent. Urinary estriol. Urinary estriol was estimated by the method of Laumas, Hingorani, and MalkanilO which is a modification of the procedure of Brown. I1 It consistedof dilution of the urine to 2 liters, acid hydrolysis, extraction of an aliquot with ether, column or thin-layer chromatography, Kober color reaction, extraction of the Kober color with chloroform containing 2 per cent p-nitrophenol, and quantitation with a Zeiss spectrophotometer. Kinetic studies. Incubations with the
microsomal fraction from the sameplacenta were carried out separately and terminated after different intervals of time. Each of the incubations was worked up according to the procedure given above. Results
The data on per cent conversion of androgen precursors to estrogens by placental microsomal fraction of 7 placentas from normal pregnancy casesare given in Table I. Similar data on placentas from 7 toxemic cases are given in Table II. The urinary estriol values determined at different times during the course of the pregnancy are also recorded (Tables I and II). The results given in these tables have been obtained after incubation of the microsomal fraction obtained from the same amount of placental tissue, namely, 7.5 Gm. in all the experiments. Three androgenic precursors, namely, 19-hydroxyandrost-4-ene-3, I’/-dione, androst-4-ene-3, 1‘I-dione, and testosterone, were used for the studies. It may be pointed out that the per cent conversion given in Tables I and II represents the conversion of the precursors to estrone and estradiol combined after making the recovery corrections. The mean conversion and range with various precursors are given in Table III. It would be seen from Table I that in placentas obtained from the normal pregnancies, the conversion of 19-hydroxyandrost-4-ene-3, 17-dione to estrogensis of the order of 45.4 to 62.4 per cent with a mean of 55.3 per cent. The mean conversion of androst-4-ene-3, 17-dione to estrogen was 19.5 per cent with a range from 12.0 to 3 1.5 per cent while the conversion of testosterone to estrogens was of the order of 3.5 to 19.1 per cent with a mean of 8.5 per cent. The toxemic casesshowed a conversion of 19-hydroxyandrost-4-ene-3, 17-dione to estrogen to the extent of 14.9 per cent with a range of 2.9 to 27.5 per cent which was quite low compared to the normal cases. Androst-4-ene-3, 17-dione conversion to estrogen was of the order of 6.9 per cent with a range of 3.5 to 13.3 per cent. Testosterone conversion to estrogen in toxemic
Volume Number
In vitro
101 8
placental
biosynthesis
of estrogens
!065
Table I. Conversion of androgen precursors to estrogensin placentas from normal pregnancies
Case No.
---
Urinary (mg./24
fnitiads
1 2 3 4 5 6 7
v. V. P. K. N. K. K.
L. D. D. L. K. D. V.
Percentage
Time of estriol estimation (weeks’ gestation)
estriol hr.)
4.4
34
5.2
37 34,40 37,38,39,40
6.0, 6.8 10.1, 9.8, 8.9, 8.5
I9-Hydroxyandrost-4-ene-3, 17 dione
conversion
to estrogens I ’
Androst-4-ene3,17-dione
60.0 62.4 49.2 49.8 51.8 45.4 48.5
Testosterone
31.5 13.1
L9.1
18.6 22.3 12.0
5.1 3.0 6.8
Table II. Conversion of androgen precursors to estrogens in placentas from toxemic* pregnancies
Case No. 1 2 3 4 5 6 7 *Patients
Estriol (mg./24 hr.)
Initials
with
I. K.
Nil
34
;:JAc. s. s: Ii. D.t H. K. G. V.3
2.4, 2.2 4.3 4.7 4.4,4.0 5.8,5.9
36,37 31 34 32,37 30,31
blood
pressure
tDelivered of twins. $Case of toxemia. Estriol
above
values
140/90
at 30 weeks.
Percentage
Time of estriol estimation (weeks’ gestation)
and/or
conversion
were
at 40 weeks.
considered Placenta
to estrogens --.-_-
Androst-4-ene17-dione
2.9 27.5 19.2 a.5 17.3 16.2 12.5
albuminuria
Delivery
19-Hydroxyandrost-4-ene3,l f-dione
to have obtained
----
Testosterone
5.6
?.I
7.4 13.3 3.5 5.2
5.h 5.5 2.3 5.1
---
_ ._-_._-.
toxemia. 10 weeks
after
estriol
estimation.
Table III. Summary of the results on conversion of androgens precursors to estrogensin normal and toxemic pregnancies* Percentage Type
19-hydroxyandrost-4ene-3,17-dione
of pregnancy
Normal pregnancy with toxemia - Pregnancy *Figures
in parentheses
indicate
55.3 14.9 the
(45.4-62.4) ( 2.9-27.5)
conversion Androst-4-ene-3,17dione 19.5 6.9
(12.0-31.5) ( 3.5-13.3)
to estrogens Testosterone a.5 (3.0-19.1) 3.9 ( 1 .l-5.6) -..
- ..--_
range.
placentas ranged from 1.1 to 5.6 per cent with a mean of 3.9 per cent. From these results it becomes obvious that all three precursors studied, 19-hydroxyandrost-4-ene3, 17-dione, androst-4-ene-3, 17-dione, and testosterone, showed a decreased conversion to estrogens in toxemic placentas compared to normal placentas. Kinetic studies. Incubations for which results are presented in Tables I and II were
carried for 60 minutes in every case. It would be of interest to find out whether any increased conversion of androgen to estrogen could be obtained by increasing or decreasing the incubation time. For this purpose kinetic studies with normal and toxemic placental microsomal fractions were carried out using 19-hydroxyandrost-4-ene-3, 17dione as the substrate. The results are presented in Fig. 1. It can be seen that in
1066
Laumas et al.
August 15, 19citl Am. J. Obst. & Gpncc.
O----ONORMAL t
- - + TOXAEMIA
60
INCUBATION
Fig.
1. Kinetics
of
placental microsomal
the
conversion fractions from
TIME
(IN
mts)
of 19-hydroxyandrost-4-ene-3, normal and toxemic cases.
3 normal and the 4 toxemic cases maximum conversion was attained in 60 minutes. In the 3 normal cases, 6 to 10 per cent conversion of 19-hydroxyandrost-4-ene-3, 17-dione to estrogen could be found in 10 minutes, while in 3 cases of toxemia of pregnancy very little conversion of substrate could be detected at this time. In one case of toxemia (P. B.) about 7 per cent conversion was attained in 10 minutes. At 30 minutes, in normal cases the conversion had already reached more than 20 per cent while in 3 toxemic pregnancy cases it is less than 5 per cent. But in the fourth case of toxemia of pregnancy the conversion had gone to the extent of 15 per cent at this time. At 60 minutes in both the groups of normal and toxemic pregnancy cases maximum Conversion was attained. It was of the order of 45.4 to 62.4 per cent in normal cases while in toxemia pregnancy cases the maximum conversion in 60 minutes was of the order of 3.4 to 22.9 per cent. In either of the two groups, the conversion did not increase at 120 minutes or 240 minutes, as is evident from Fig. 1.
Effect of human chorionic gonadotropin on the conversion of 19-hydroxyandrost-4ene-3, 17-dione. The effect of 100 T.U. and
IT-dione
to estrogens
in
1,000 I.U. of HCG on the conversion of 19hydroxyandrost-4-ene-3, 17-dione to estrogen was studied with the above incubation procedure and microsomal fractions from normal and toxemic placentas. There was no significant increase in the conversion of 19hydroxyandrost-4-ene-3, 17-dione to estrogen with HCG with either of the microsomal preparations. Comment The present work on the biosynthesis of estrogens in placentas from normal women and from cases of toxemia of pregnancy showed some remarkable differences in the rate of biosynthesis of estrogens. The results indicate that in the placenta in toxemia of pregnancy the conversion of 1 g-hydroxyandrost-4-ene-3, 17-dione to estrogen was quite low compared to that in normal placentas when incubations were carried out for one hour under the same experimental conditions. Other precursors studied, i.e., androst4-ene-3, 17-dione and testosterone, also showed a similar pattern of lower conversion in toxemic pregnancy cases. The kinetic studies (Fig. 1) Ient further support to the above results when it became clear that maximum
In vitro
conversion of 19-hydroxyandrost-4-ene-3, 17dione was attained in one hour both in normal and toxemic placentas. The kinetic studies also showed that the toxemic placentas could be clearly distinguished from normal placentas in that the former showed decreased biosynthesis of estrogen compared to the later cases. In the present study no effort was made to correlate the estriol excretion values with the degree of toxemia. Separate studies on this aspect of the work are in progress. The toxemia cases, however, showed lowered excretion of estriol except in Case No. 5 (B. D.), a case of twin pregnancy. In Case No. 7, urinary estriol determination was done at 30 weeks’ gestation whereas the placenta was obtained 10 weeks later. This explains why the androgen conversion rates may be decreased but the cstriol value not lowered. Hayano, Longchampt, and Dorfmanlz and Morato and associates13 have shown that 19-hydroxyandrost-4-ene-3, 17-dione is con\.erted to estrogen much more rapidly than androst-4-ene-3, 17-dione. Wilcox and Engel14 have provided kinetic evidence which shows that 19-hydroxyandrost-4-ene-3, t7&one is an obligatory intermediate between androst-4-ene-3, 17-dione and estrogens. The c,xperiments of Wilcox and EngeI14 further give support to the hypothesis that 19-hyclroxylation is the rate-limiting step in the aromatization of ring A of the steroid nucleus. The present results on the conversion of 19-hydroxyandrost-4-ene-3, If-dione to estrogens by the microsomal enzyme from
placental
biosynthesis
of estrogem
1067
the normal placenta are in accordance with the results of Hayano, Longchampt, and Dorfmanl” and Morato and associatrs.‘” However, this important precursor, i.e.. 1% hydroxyandrost-4-ene-3, 1 ‘I-dione which is an obligatory intermediate in the biosynthcbsis of estrogens, showed a reduced convex sion to estrogen when placental microsomal enzymes from cases of toxemia were used. ‘I‘his indicated the possibility that besides other defects, the toxemic placenta may be d(*frctive in the enzymes which bring about the aromatization reaction. It is likely that tile reduced conversion in the toxemic placenta may be due to inadequate availabiiity cbt‘ cofactors or precursors or a defect in the enzyme which converts androgen to cstro~~r~. However? in the present studies, wfTicicr~t excess of precursors were added to the incubations and it appears unlikely that ;L tcxemit placenta may be lacking the rC(luired precursors. Human chorionic gonadotropin had no effect on the conversion of androgrn pr’erursors in normal or toxemic placentas. Ryan’ also did not find any effect of HCG (in the conversion of androgens by normal pla.t:ental microsomes. The present results taken together indicate that there probably is .t lack of the aromatizing enzymes or thst the enzymes have been inactivated in the ttrxcmic placenta. This could result in dccrcasc,d biosynthesis and also be a possible cause for the lower excretion of estrogens in sr1c:11 curses and resultant placental insufficiency.
REFERENCES
1. Laumas, K. R.: Preliminary 2. 3.
4. 5. 6. 7.
report presented
at the Symposium on Placenta held at G. S. Medical College, Bombay, Oct. 1-2, 1966. Ryan, K. J.: J. Biol. Chem. 234: 268, 1959. Simmer, Hans H., Easterling, W. E., Pion, R. J., and Dignam, W. J.: Steroid 4: 125, 1964. Siiteri, P. K., and MacDonald, P. C.: J. Clin. Endocrinol. 26: 751, 1966. Levitz, M.: J. Clin. Endocrinol. 26: 773, 1966. Frandsen, V. A., and Stakemann, G.: Acta cndocrinol. 44: 183, 1963. Zaffaroni, A., and Burton, R. B.: J. Biol. Chem. 193: 749, 1951.
8. Savard, K.: J. Biol. Chem. 202: 457. i953. 9. 10. 11. 12.
13.
1-i.
Adlercrentez, H.: Acta endocrinol. Suppl.) 72: 71, 1962. Laumas, K. R., Hingorani, V., and Malkani, P. K.: Enzymol. biol. et clin. 6: 0~7, 1966. Brown, J. B.: Lancet 1: 704. 1956. Hayano, M.. Longchampt, J. E., and Dorfman, R. I.: Acta endocrinol. (Suppl.) 50: 699, 1960. Morato, T., Hayano, M., Dorfman. R. I., and Axelrod, L. R.: Biochem. Biophvs. Rcs. Comm. 6: 334, 1961. Wilcox, R. B., and Engcl. L. I,. Steroid (Suppl.) 1: 49, 1965.
R.
PARVATI
K.
GOURI
HINGORANI, Delhi,
PH.D.
MALKANI,
S. KOSHTI,
VERA New
LAUMAS,
F.R.C.O.G., M.Sc.,
M.Sc.
F.R.C.O.G.,
F.A.C.S. (TECH.)
F.A.C.S.,
F.I.C.S.
India
In vitro biosynthesis of estrogens in microsomal fractions obtained from placentas from 7 normal and 7 toxemic cases has been investigated under identical experimental conditions. I9-Hydroxyandrost-4-ene-3, I7-dione, androst-4-ene-3, 17-dione, and testosterone were used as substrates and studied for their conversion into estrogens. The conversion of these substrates was found to be low in the toxemic placenta compared to the normal placenta. Kinetic studies with I9-hydroxyandrost-4-ene-3, 17-dione which has been considered to be an obligatory intermediate in the biosynthesis of estrogens showed that in both normal and toxemic placentas the maximum conversion was attained within one hour, and this remained unchanged when incubations were carried out even up to 4 hours. In kinetic studies also the toxemic placenta showed a lower conversion compared to the conversion found in normal cases. Human chorionic gonadotropin was found to have no eflect on the conversion of 19-hydroxyandrost-4-ene-3, 17-dione to estrogens in either normal or toxemic placentas. The present finding of low estrogen biosynthesis in toxemic cases is discussed.
1 N R E c E N T years considerable evidence has accumulated from both in vitro and in vivo studies which indicates that the placenta is the principal source of the greatly increased amounts of estrogens produced during human pregnancy. The studies of Ryan2 demonstrated the conversion of androgens to estrogens in high yield by a preparation of human placental microsomes. The exact biosynthetic pathways which in vivo lead to estrogen formation have not been quite clear. For a time it was assumed that human placenta, like the ovary, could synthesize estrogens from acetate, but extensive research in this area has shown that this may not be true. The concept of complete placental autonomy has ,
now been replaced by the theory that the placenta is an incomplete endocrine organ which is capable of synthesizing active hormones only from preformed steroidal precursors reaching it via the fetal and maternal bloodstreams. An active role3-5 has been ascribed to the fetus for the synthesis of steroidal precursors which later undergo transformation to estrogens in the placenta. The placenta is thus responsible for the final synthesis of large amounts of estriol formed during normal human pregnancy. Urinary estimation of estriol is regarded as a valuable test for placental function6 in the later stages of pregnancy. The level of estriol falls rapidly with placental death, and low urinary estriol values have been reported in cases of toxemia. The decline in urinary estriol levels in the later weeks of pregnancy in cases of toxemia indicates some impairment in the synthesis of estrogens by the fetal-placental unit.
From the Reproductive Biology Research Unit and the Department of Obstetrics and Gynaecologly, All-India Institute of
Medical
Sciences.
This work The Ford
was supported by grants from Foundation and the Indian of Medical Research.
Council
1062
In vitro placental biosynthesis of estrogens
tn view of the knowledge of estrogen biosynthesis in the normal human placenta, a study of estrogen biosynthesis in placentas from cases of toxemia of pregnancy seemed highly desirable. In the present paper, a study of the in vitro conversion of androgen precursors to estrogens in normal and toxemic placentas is presented. Material
and
methods
Tissue preparation. Human placenta was obtained from patients delivered in the AllIndia Institute of Medical Sciences Hospital. The placenta was immedately transported to the laboratory under ice in a thermal flask. It was dissected free of fetal membranes and blood vessels at Y’ C. The remaining placental tissue was weighed and homogenized in a Vertis 23 homogenizer for 1 minute in phosphate buffer according to the procedure of Ryan.’ The buffer contained 0.25M sucrose, 0.05M phosphate, and 0.04M nicotinamide at pH 7.0. One volume of buffer to three parts of placental tissue by weight was used for homogenization. The homogenate was filtered through cheesecloth and fractionated by differential centrifugation in the Beckman Spinco ultracentrifugation Model L. The nuclear fraction was separated by centrifugation at 6,000 x g for 10 minutes. The supernatant from the nuclear fraction was again centrifuged at 17,500 x g for 30 minutes and the residue containing the mitochondrial fraction was separated. The supernatant from the mitochondrial fraction was subjected to centrifugation at 105, 001) x ‘g for 60 minutes when the microsoulal fraction settled down in the bottom of the tube. The microsomal fraction which contained the active enzyme was stored in a deep freeze at -12’ C. until the experiment was carried out. Incubation. In all incubations a placental microsomal fraction in 1 ml. buffer obtained from 7.5 Gm. of placental tissue was used. It was incubated with 150 pg of various steroidal precursors in the presence of a TPNH-generating system consisting of 10 pmoles triphosphopyridine nucleotide (TPN) , 60 pmoles glucose-6-phosphate, and 1.5 K
1063
glucose-6-phosphate dehydrogenase. units The total volume of the incubation medium was 5 to 6 ml. The androgen steroidal prccursors were taken down in the bottom of the incubation flasks, The incubation was carried out in 25 ml. conical flask at 137”’ C. for one hour in Dubnoff shaker with air as the gas phase. Extraction. The incubations xvcre terminated by the addition of a small quantity of chloroform. At this stage carrier 6:i Westradiol (approximately 180,000 d.p.rn. in 0.2 ml. ethanol) was added for rc:co\ycry correction. The incubation mixture w:ts rxtracted three times with four volumes of chloroform. The chloroform extracts were pooled, made up to a known volume, and an aliquot taken out for radioactivity colinting. The remaining pooled chloroform extract was dried to a small quantity under nitrogen. Chromatography. The toluene-proi,ylene glycol system of Zaffaroni and Burton’ as modified by Savard8 was used for the pllrification and separation of estrogens formed after incubation. Standard clsteronc: and estradiol- 17,B were spotted along with the residue from the above chloroform extract. After running the paper in the t&en+propylene glycol system for 2 to 3 holrrs. the strips containing the standards were r~r~vecl and developed by spraying with freshly prepared ferric chloride-ferricyanidr rcaqent. The area corresponding to the standards on the strips containing the unknown r>stracts were cut and extracted with a mistnre of chloroform and methanol ( 1: 1). ‘t‘ht rhloroform-methanol extract was cotlcc-sntratcd under nitrqgen gas to a small xdurrlr~ in a small centrifuge tube. It was spottt*d on a Silica gel plate 10.25 mm.\ aloiig \\.ith rstrone and estradiol standards. ‘The plate was developed in a tank containinq hcmzenemethanol (9: 1). The area corresponds to the standards on the thin-layer plate %vhere the extracted estrone and estradiol were run, were separately scraped off, and were extracted separately with a mixture of chloroform-methanol ( 1 :I). The extracted estrone and estradiol were made up to a known volume and 5 per cent of this taken for
1064
August 15, lS68 Am. J. Obst. & Gym.
Laumas et al.
counting. The rest of the extracts were evaporated to dryness and estimated for estrone and estradiol. The recovery correction was carried out with the addition of 6:7 H3-estradiol in all cases. However, in separate experiments with 6:7 H3-estrone it was found that the recovery correction for estrone was almost the same as that for estradiol. Quantitation of estrone and estradiol by extraction of the Kober color. The content of estrone and estradiol in the unknown samples, puriiied by chromatographic procedure as mentioned above, was estimated by extraction of the Kober color. The Kober color reaction was carried out according to the procedure of Kober as modified Adlercrentez.e Four milliliters of chloroform containing p-nitrophenol (2 per cent) was added to the Kober reaction tube, shaken for 20 seconds, and centrifuged at -IO0 C. for 2 minutes. The chloroform layer was separated out and quantitated by measuring at 500, 538, and 576 rnp in a Zeiss spectrophotometer. Corrected optical density was obtained with Allen’s correction. Radioactivity counting. All radioactivity samples were counted in Packard Tricarb Model 3314 (Packard Instrument Company, La Grange, Illinois). The samples to be counted were taken down in the scintillation vials and 10 ml. of the scintillation liquid, 4.0 Gm. PPO (2, 5-diphenyloxazole) and 100 mg. dimethyl POPOP 1, 4-bis2- (4-methyl-5-phenyl-oxazolyl) -benzene dissolved in a liter of redistilled toluene, was added. The counting efficiency of 3H with this counter was about 33 per cent. Urinary estriol. Urinary estriol was estimated by the method of Laumas, Hingorani, and MalkanilO which is a modification of the procedure of Brown. I1 It consistedof dilution of the urine to 2 liters, acid hydrolysis, extraction of an aliquot with ether, column or thin-layer chromatography, Kober color reaction, extraction of the Kober color with chloroform containing 2 per cent p-nitrophenol, and quantitation with a Zeiss spectrophotometer. Kinetic studies. Incubations with the
microsomal fraction from the sameplacenta were carried out separately and terminated after different intervals of time. Each of the incubations was worked up according to the procedure given above. Results
The data on per cent conversion of androgen precursors to estrogens by placental microsomal fraction of 7 placentas from normal pregnancy casesare given in Table I. Similar data on placentas from 7 toxemic cases are given in Table II. The urinary estriol values determined at different times during the course of the pregnancy are also recorded (Tables I and II). The results given in these tables have been obtained after incubation of the microsomal fraction obtained from the same amount of placental tissue, namely, 7.5 Gm. in all the experiments. Three androgenic precursors, namely, 19-hydroxyandrost-4-ene-3, I’/-dione, androst-4-ene-3, 1‘I-dione, and testosterone, were used for the studies. It may be pointed out that the per cent conversion given in Tables I and II represents the conversion of the precursors to estrone and estradiol combined after making the recovery corrections. The mean conversion and range with various precursors are given in Table III. It would be seen from Table I that in placentas obtained from the normal pregnancies, the conversion of 19-hydroxyandrost-4-ene-3, 17-dione to estrogensis of the order of 45.4 to 62.4 per cent with a mean of 55.3 per cent. The mean conversion of androst-4-ene-3, 17-dione to estrogen was 19.5 per cent with a range from 12.0 to 3 1.5 per cent while the conversion of testosterone to estrogens was of the order of 3.5 to 19.1 per cent with a mean of 8.5 per cent. The toxemic casesshowed a conversion of 19-hydroxyandrost-4-ene-3, 17-dione to estrogen to the extent of 14.9 per cent with a range of 2.9 to 27.5 per cent which was quite low compared to the normal cases. Androst-4-ene-3, 17-dione conversion to estrogen was of the order of 6.9 per cent with a range of 3.5 to 13.3 per cent. Testosterone conversion to estrogen in toxemic
Volume Number
In vitro
101 8
placental
biosynthesis
of estrogens
!065
Table I. Conversion of androgen precursors to estrogensin placentas from normal pregnancies
Case No.
---
Urinary (mg./24
fnitiads
1 2 3 4 5 6 7
v. V. P. K. N. K. K.
L. D. D. L. K. D. V.
Percentage
Time of estriol estimation (weeks’ gestation)
estriol hr.)
4.4
34
5.2
37 34,40 37,38,39,40
6.0, 6.8 10.1, 9.8, 8.9, 8.5
I9-Hydroxyandrost-4-ene-3, 17 dione
conversion
to estrogens I ’
Androst-4-ene3,17-dione
60.0 62.4 49.2 49.8 51.8 45.4 48.5
Testosterone
31.5 13.1
L9.1
18.6 22.3 12.0
5.1 3.0 6.8
Table II. Conversion of androgen precursors to estrogens in placentas from toxemic* pregnancies
Case No. 1 2 3 4 5 6 7 *Patients
Estriol (mg./24 hr.)
Initials
with
I. K.
Nil
34
;:JAc. s. s: Ii. D.t H. K. G. V.3
2.4, 2.2 4.3 4.7 4.4,4.0 5.8,5.9
36,37 31 34 32,37 30,31
blood
pressure
tDelivered of twins. $Case of toxemia. Estriol
above
values
140/90
at 30 weeks.
Percentage
Time of estriol estimation (weeks’ gestation)
and/or
conversion
were
at 40 weeks.
considered Placenta
to estrogens --.-_-
Androst-4-ene17-dione
2.9 27.5 19.2 a.5 17.3 16.2 12.5
albuminuria
Delivery
19-Hydroxyandrost-4-ene3,l f-dione
to have obtained
----
Testosterone
5.6
?.I
7.4 13.3 3.5 5.2
5.h 5.5 2.3 5.1
---
_ ._-_._-.
toxemia. 10 weeks
after
estriol
estimation.
Table III. Summary of the results on conversion of androgens precursors to estrogensin normal and toxemic pregnancies* Percentage Type
19-hydroxyandrost-4ene-3,17-dione
of pregnancy
Normal pregnancy with toxemia - Pregnancy *Figures
in parentheses
indicate
55.3 14.9 the
(45.4-62.4) ( 2.9-27.5)
conversion Androst-4-ene-3,17dione 19.5 6.9
(12.0-31.5) ( 3.5-13.3)
to estrogens Testosterone a.5 (3.0-19.1) 3.9 ( 1 .l-5.6) -..
- ..--_
range.
placentas ranged from 1.1 to 5.6 per cent with a mean of 3.9 per cent. From these results it becomes obvious that all three precursors studied, 19-hydroxyandrost-4-ene3, 17-dione, androst-4-ene-3, 17-dione, and testosterone, showed a decreased conversion to estrogens in toxemic placentas compared to normal placentas. Kinetic studies. Incubations for which results are presented in Tables I and II were
carried for 60 minutes in every case. It would be of interest to find out whether any increased conversion of androgen to estrogen could be obtained by increasing or decreasing the incubation time. For this purpose kinetic studies with normal and toxemic placental microsomal fractions were carried out using 19-hydroxyandrost-4-ene-3, 17dione as the substrate. The results are presented in Fig. 1. It can be seen that in
1066
Laumas et al.
August 15, 19citl Am. J. Obst. & Gpncc.
O----ONORMAL t
- - + TOXAEMIA
60
INCUBATION
Fig.
1. Kinetics
of
placental microsomal
the
conversion fractions from
TIME
(IN
mts)
of 19-hydroxyandrost-4-ene-3, normal and toxemic cases.
3 normal and the 4 toxemic cases maximum conversion was attained in 60 minutes. In the 3 normal cases, 6 to 10 per cent conversion of 19-hydroxyandrost-4-ene-3, 17-dione to estrogen could be found in 10 minutes, while in 3 cases of toxemia of pregnancy very little conversion of substrate could be detected at this time. In one case of toxemia (P. B.) about 7 per cent conversion was attained in 10 minutes. At 30 minutes, in normal cases the conversion had already reached more than 20 per cent while in 3 toxemic pregnancy cases it is less than 5 per cent. But in the fourth case of toxemia of pregnancy the conversion had gone to the extent of 15 per cent at this time. At 60 minutes in both the groups of normal and toxemic pregnancy cases maximum Conversion was attained. It was of the order of 45.4 to 62.4 per cent in normal cases while in toxemia pregnancy cases the maximum conversion in 60 minutes was of the order of 3.4 to 22.9 per cent. In either of the two groups, the conversion did not increase at 120 minutes or 240 minutes, as is evident from Fig. 1.
Effect of human chorionic gonadotropin on the conversion of 19-hydroxyandrost-4ene-3, 17-dione. The effect of 100 T.U. and
IT-dione
to estrogens
in
1,000 I.U. of HCG on the conversion of 19hydroxyandrost-4-ene-3, 17-dione to estrogen was studied with the above incubation procedure and microsomal fractions from normal and toxemic placentas. There was no significant increase in the conversion of 19hydroxyandrost-4-ene-3, 17-dione to estrogen with HCG with either of the microsomal preparations. Comment The present work on the biosynthesis of estrogens in placentas from normal women and from cases of toxemia of pregnancy showed some remarkable differences in the rate of biosynthesis of estrogens. The results indicate that in the placenta in toxemia of pregnancy the conversion of 1 g-hydroxyandrost-4-ene-3, 17-dione to estrogen was quite low compared to that in normal placentas when incubations were carried out for one hour under the same experimental conditions. Other precursors studied, i.e., androst4-ene-3, 17-dione and testosterone, also showed a similar pattern of lower conversion in toxemic pregnancy cases. The kinetic studies (Fig. 1) Ient further support to the above results when it became clear that maximum
In vitro
conversion of 19-hydroxyandrost-4-ene-3, 17dione was attained in one hour both in normal and toxemic placentas. The kinetic studies also showed that the toxemic placentas could be clearly distinguished from normal placentas in that the former showed decreased biosynthesis of estrogen compared to the later cases. In the present study no effort was made to correlate the estriol excretion values with the degree of toxemia. Separate studies on this aspect of the work are in progress. The toxemia cases, however, showed lowered excretion of estriol except in Case No. 5 (B. D.), a case of twin pregnancy. In Case No. 7, urinary estriol determination was done at 30 weeks’ gestation whereas the placenta was obtained 10 weeks later. This explains why the androgen conversion rates may be decreased but the cstriol value not lowered. Hayano, Longchampt, and Dorfmanlz and Morato and associates13 have shown that 19-hydroxyandrost-4-ene-3, 17-dione is con\.erted to estrogen much more rapidly than androst-4-ene-3, 17-dione. Wilcox and Engel14 have provided kinetic evidence which shows that 19-hydroxyandrost-4-ene-3, t7&one is an obligatory intermediate between androst-4-ene-3, 17-dione and estrogens. The c,xperiments of Wilcox and EngeI14 further give support to the hypothesis that 19-hyclroxylation is the rate-limiting step in the aromatization of ring A of the steroid nucleus. The present results on the conversion of 19-hydroxyandrost-4-ene-3, If-dione to estrogens by the microsomal enzyme from
placental
biosynthesis
of estrogem
1067
the normal placenta are in accordance with the results of Hayano, Longchampt, and Dorfmanl” and Morato and associatrs.‘” However, this important precursor, i.e.. 1% hydroxyandrost-4-ene-3, 1 ‘I-dione which is an obligatory intermediate in the biosynthcbsis of estrogens, showed a reduced convex sion to estrogen when placental microsomal enzymes from cases of toxemia were used. ‘I‘his indicated the possibility that besides other defects, the toxemic placenta may be d(*frctive in the enzymes which bring about the aromatization reaction. It is likely that tile reduced conversion in the toxemic placenta may be due to inadequate availabiiity cbt‘ cofactors or precursors or a defect in the enzyme which converts androgen to cstro~~r~. However? in the present studies, wfTicicr~t excess of precursors were added to the incubations and it appears unlikely that ;L tcxemit placenta may be lacking the rC(luired precursors. Human chorionic gonadotropin had no effect on the conversion of androgrn pr’erursors in normal or toxemic placentas. Ryan’ also did not find any effect of HCG (in the conversion of androgens by normal pla.t:ental microsomes. The present results taken together indicate that there probably is .t lack of the aromatizing enzymes or thst the enzymes have been inactivated in the ttrxcmic placenta. This could result in dccrcasc,d biosynthesis and also be a possible cause for the lower excretion of estrogens in sr1c:11 curses and resultant placental insufficiency.
REFERENCES
1. Laumas, K. R.: Preliminary 2. 3.
4. 5. 6. 7.
report presented
at the Symposium on Placenta held at G. S. Medical College, Bombay, Oct. 1-2, 1966. Ryan, K. J.: J. Biol. Chem. 234: 268, 1959. Simmer, Hans H., Easterling, W. E., Pion, R. J., and Dignam, W. J.: Steroid 4: 125, 1964. Siiteri, P. K., and MacDonald, P. C.: J. Clin. Endocrinol. 26: 751, 1966. Levitz, M.: J. Clin. Endocrinol. 26: 773, 1966. Frandsen, V. A., and Stakemann, G.: Acta cndocrinol. 44: 183, 1963. Zaffaroni, A., and Burton, R. B.: J. Biol. Chem. 193: 749, 1951.
8. Savard, K.: J. Biol. Chem. 202: 457. i953. 9. 10. 11. 12.
13.
1-i.
Adlercrentez, H.: Acta endocrinol. Suppl.) 72: 71, 1962. Laumas, K. R., Hingorani, V., and Malkani, P. K.: Enzymol. biol. et clin. 6: 0~7, 1966. Brown, J. B.: Lancet 1: 704. 1956. Hayano, M.. Longchampt, J. E., and Dorfman, R. I.: Acta endocrinol. (Suppl.) 50: 699, 1960. Morato, T., Hayano, M., Dorfman. R. I., and Axelrod, L. R.: Biochem. Biophvs. Rcs. Comm. 6: 334, 1961. Wilcox, R. B., and Engcl. L. I,. Steroid (Suppl.) 1: 49, 1965.