British Journal of Obstetrics and Gynaecology February 1975. Vol. 82. pp. 142-145
METABOLISM OF PROSTAGLANDIN FBGC WITHIN THE HUMAN UTERUS IN EARLY PREGNANCY BY
M. J. N. C . KEIRSE J. G. WILLIAMSON AND
A. C. TURNBULL Nufield Department of Obstetrics and Gynaecology, University of Oxford and Division of Obstetrics and Gynaecology, Oxfordshire Area Health Authority (Teaching), John Radclifle Hospital, Oxford OX3 9DU. Summary The potential for intra-uterine metabolism of prostaglandins in early pregnancy was studied by incubation of prostaglandin F,a (PGF,a) with tissues obtained by termination hysterectomy at 7 and 16 weeks gestation. Enzymes regulating the degradation of prostaglandins were demonstrated in placenta and membranes and to a lesser extent, in myometrium and decidua. It is believed that these enzymes may have a physiological r61e in maintaining the continuity of pregnancy, and that the high degradation of PGFza in placenta and membranes may be one of the reasons that, for termination of pregnancy, far higher doses are required intra-amniotically than extra-amniotically.
IT is now common knowledge that naturally occurring prostaglandins are metabolized by a chain of reactions which, in man, starts with oxidation at carbon atom 15 by the enzyme 15-hydroxy-prostaglandin dehydrogenase (Samuelsson et al., 1971). The recognition of the initial step in the degradation of prostaglandins has led to the synthesis of 15-methyl analogues of the natural prostaglandins which are resistant to degradation by 15-hydroxy-prostaglandin dehydrogenase (Bundy et al., 1971; Yankee and Bundy, 1972). Since natural prostaglandins are rapidly inactivated on perfusion through the lungs (Ferreira and Vane, 1967; Piper et al., 1970), it is perhaps not surprising that their 15-methyl analogues display a higher potency and a longer duration of action than the parent compounds
(Kirton and Forbes, 1972; Karim and Sharma, 1972). These analogues also proved to be far more active than the natural compounds, when administered locally (intra- or extra-amniotically) for the termination of pregnancy (Bygdeman et al., 1972; Karim and Sharma, 1972; Amy et a]., 1973). This could suggest that there is a potential for intra-uterine metabolism of natural prostaglandins in the second trimester of pregnancy, and that the large doses of natural prostaglandins required for the termination of pregnancy may be dictated partially by the need to overcome or compensate for their degradation. To test this hypothesis, we investigated, in vitro, the capacity of various intra-uterine tissues to metabolize a prostaglandin. 142
METABOLISM OF PGFza
METHODS
143
RESULTS
A complete uterus containing an intact gestational sac, was obtained by total hysterectomy from each of two patients, at 7 and 16 weeks gestation respectively. In both cases pregnancy was terminated on social grounds, and no pathology, other than fibroids, was found. At 16 weeks gestation, placental villous tissue membranes, umbilical cord, fetal skin, macroscopically normal myometrium and decidua were investigated whilst at the earlier gestation only normal myometrium and villous tissue were investigated. The method used was that described by Keirse and Turnbull (1974). All tissues were homogenized and the homogenates incubated with 3H-prostaglandin F p (’H-PGFZa; 0 .9 pg./g. of tissue) in 0.1M potassium phosphate buffer, pH 7.4, containing 2 mM. nicotinamide adenine dinucleotide (NAD) at 37 “C.for various lengths of time. A placental homogenate, previously inactivated by boiling, was used for the “control” incubation. After the reactions had been stopped by acidifying and addition of ethanol, PGFp was extracted and separated from its metabolites by thin layer chromatography. The plates were scanned for radioactivity using a radiochromatogram scanner, and the relevant zones were eluted for quantitative determination of radioactivity by liquid scintillation counting.
At 16 weeks gestation there was considerable metabolism of PGF,, by the membranes and placental homogenates ; PGF2a concentrations dropped to less than half their original concentration (0 a9 pg./g. of tissue) within three minutes, as shown in Figure 1. Myometrial and decidual homogenates were less active, metabolizing respectively 15 and 5 per cent of the PGF,, in 30 minutes. The fetal skin and umbilical cord, on the other hand, appeared to be devoid of enzymatic activity since the PGFp concentrations after incubation up to 60 minutes were not different from those in the control incubation with boiled homogenate. The results obtained with myometrium and villous tissue at 7 weeks gestation were similar to those observed at 16 weeks, showing that prostaglandin metabolizing enzymes are present in the placenta as early as the 7th week of pregnancy. Differentiation of the metabolites by thin layer chromatography showed that PGFp was metabolized to at least two compounds. Using an incubation of membranes as an example, the distribution of the total radioactivity between PGF2a and these metabolites (M, and M2) after various lengths of time, is shown in Figure 2. M, and M, showed a mobility on thin layer chromatography identical respectively to that
fetal skin & c o r d
I decidua 1
myometrium
placenta membranes t i m e (min.)
FIG.1 Metabolic degradation of PGF2a in homogenates of fetal skin, umbilical cord, decidua, rnyornetrium, placenta, and fetal membranes obtained at 16 weeks gestation.
144
KEIRSE, WILLIAMSON AND TURNBULL
30
60
time irnin.1 FIG.2
Distribution of the radioactivity between PGF,, and its metabolites (M, and M,) during incubation of 3H-PGF2a with a homogenate of membranes. obtained at 16 weeks gestation.
of 15-kcto PGF,, and 15-keto-13,14-dihydro PGF2,.
DISCUSSION The observations made in this study demonstrate the presence of enzymes regulating the degradation of prostaglandins, in placenta and membranes and to a lesser extent in myometrium and decidua, in early pregnancy. Since both E and F prostaglandins are substrates for 15hydroxy-prostaglandin dehydrogenase (AnggLd and Samuelsson, 1966), the present observations using PGF,, as a substrate can also be applied to PGE,. In fact, PGE is generally metabolized faster than PGF in the in vitro situation (Anggird and Samuelsson, 1966; Jarabak, 1972; Schlegel et al., 1974). The placenta has previously been shown to contain a high prostaglandin dehydrogenase activity in vitro (Jarabak, 1972; Schlegel et al., 1974). The present study, however, demonstrates that the enzymatic activity is present in villous tissue as early as the 7th week of pregnancy, and that this activity is not limited to placental tissue. The present findings certainly support the hypothesis that there is a high potential for intra-uterine metabolism of prostaglandins in the second trimester of pregnancy. The extent to which this potential, observed in vitro, applies in vivo is not known. It is interesting, however,
that the myometrium is divided from the amniotic cavity by two structures of fetal origin, membranes and placenta, which showed a remarkably high degradation of prostaglandins in vitro. Prostaglandins administered intraamniotically for termination of pregnancy have been shown to disappear steadily from the amniotic cavity (Pace-Asciak et al., 1972) and Karim (1972) has suggested that they diffuse through the membranes to reach their site of action in the myometrium. It is almost certain that a proportion of these prostaglandins must be inactivated before reaching the myometrial target and this may well be one of the reasons that, for termination of pregnancy, far higher doses of prostaglandins are required intraamniotically than extra-amniotically (Wiqvist et al., 1973). Prostaglandins have been implicated as causative agents in the process of spontaneous and habitual abortion (Karim and Hillier, 1970; Bodin, 1971). It is possible that the physiological r61e of the prostaglandin-degrading enzymes is to maintain the continuity of pregnancy. The fact that the highest enzyme activities were found in fetal tissues, membranes and placenta, may provide yet another argument for a fetal r61e in the maintenance of pregnancy. ACKNOWLEDGEMENTS The tissues were obtained by permission of
METABOLISM OF PGF,~
Mr. E. A. Williams and Mrs. B. R. Hicks gave expert technical assistance. The work was sponsored by Programme Grant G971/SO9 from the Medical Research Council. PGF,, was a gift from Professor D. A. van Dorp, Unilever Research, Vlaardingen, The Netherlands. REFERENCES Amy, J. J., Karim, S. M. M., and Sivasamboo, R. (1973): Journal of Obstetrics and Gynaecology of the British Commonwealth, 80, 1017. Anggird, E., and Samuelsson, B. (1966): Arkiv for Kemi, 25, 293. Bodin, M. (1971): British Medical Journal, 2, 587. Bundy, G., Lincoln, F., Nelson, N., Pike, J., and Schneider, W . (1971): Annals of the New York Academy of Sciences, 180, 76. Bygdeman, M., Btguin, F., Toppozada, M., Wiqvist, N., and Bergstrom, S. (1972): Lancet, 1, 1336. Ferreira, S. H., and Vane, J . R. (1967): Nature, 216,868. Jarabak, J. (1972): Proceedings of the National Academy of Sciences of the United States of America, 69, 533. Karim, S. M. M., and Hillier, K. (1970): Journal of Obstetrics and Gynaecology of the British Commonwealth, 77, 837.
145
Karim, S. M. M., and Sharma, S. D. (1972): Journal of Obstetrics and Gynaecology of the British Commonwealth, 79, 737. Karim, S. M. M. (1972): Journal of Reproduction and Fertility, Supplement, 16, 105. Keirse, M. J. N. C., and Turnbull, A. C. (1974): Journal of Endocrinology. (In press.) Kirton, K. T., and Forbes, A. D. (1972): Prostaglandins, 1, 319. Pace-Asciak, C., Wolfe, L. S., Gillett, P. G . , and Kinch, R. A. (1972): Prostaglandins, 1, 469. Piper, P. J., Vane, J. R., and Wyllie, J. H. (1970): Nature 225, 600. Samuelsson, B., Granstrom, E., Green, K., and Hamberg, M. (1971): Annals of the New York Academy of Sciences, 180, 138. Schlegel, W., Demers, L. M., Hildebrandt-Stark, H. E., Behrman, H. R., and Greep, R. 0. (1974): Prostaglandins, 5, 417. Wiqvist, N., Bygdeman, M., and Toppozada, M. (1973): In Prostaglandins in Fertility Control 3. Edited by S. Bergstrom, WHO, Karolinska Institutet, Stockholm, p. 80. Yankee, E. W., and Bundy, G. L. (1972): Journal of the American Chemical Society, 94, 365 1.
METABOLISM OF PROSTAGLANDIN FBGC WITHIN THE HUMAN UTERUS IN EARLY PREGNANCY BY
M. J. N. C . KEIRSE J. G. WILLIAMSON AND
A. C. TURNBULL Nufield Department of Obstetrics and Gynaecology, University of Oxford and Division of Obstetrics and Gynaecology, Oxfordshire Area Health Authority (Teaching), John Radclifle Hospital, Oxford OX3 9DU. Summary The potential for intra-uterine metabolism of prostaglandins in early pregnancy was studied by incubation of prostaglandin F,a (PGF,a) with tissues obtained by termination hysterectomy at 7 and 16 weeks gestation. Enzymes regulating the degradation of prostaglandins were demonstrated in placenta and membranes and to a lesser extent, in myometrium and decidua. It is believed that these enzymes may have a physiological r61e in maintaining the continuity of pregnancy, and that the high degradation of PGFza in placenta and membranes may be one of the reasons that, for termination of pregnancy, far higher doses are required intra-amniotically than extra-amniotically.
IT is now common knowledge that naturally occurring prostaglandins are metabolized by a chain of reactions which, in man, starts with oxidation at carbon atom 15 by the enzyme 15-hydroxy-prostaglandin dehydrogenase (Samuelsson et al., 1971). The recognition of the initial step in the degradation of prostaglandins has led to the synthesis of 15-methyl analogues of the natural prostaglandins which are resistant to degradation by 15-hydroxy-prostaglandin dehydrogenase (Bundy et al., 1971; Yankee and Bundy, 1972). Since natural prostaglandins are rapidly inactivated on perfusion through the lungs (Ferreira and Vane, 1967; Piper et al., 1970), it is perhaps not surprising that their 15-methyl analogues display a higher potency and a longer duration of action than the parent compounds
(Kirton and Forbes, 1972; Karim and Sharma, 1972). These analogues also proved to be far more active than the natural compounds, when administered locally (intra- or extra-amniotically) for the termination of pregnancy (Bygdeman et al., 1972; Karim and Sharma, 1972; Amy et a]., 1973). This could suggest that there is a potential for intra-uterine metabolism of natural prostaglandins in the second trimester of pregnancy, and that the large doses of natural prostaglandins required for the termination of pregnancy may be dictated partially by the need to overcome or compensate for their degradation. To test this hypothesis, we investigated, in vitro, the capacity of various intra-uterine tissues to metabolize a prostaglandin. 142
METABOLISM OF PGFza
METHODS
143
RESULTS
A complete uterus containing an intact gestational sac, was obtained by total hysterectomy from each of two patients, at 7 and 16 weeks gestation respectively. In both cases pregnancy was terminated on social grounds, and no pathology, other than fibroids, was found. At 16 weeks gestation, placental villous tissue membranes, umbilical cord, fetal skin, macroscopically normal myometrium and decidua were investigated whilst at the earlier gestation only normal myometrium and villous tissue were investigated. The method used was that described by Keirse and Turnbull (1974). All tissues were homogenized and the homogenates incubated with 3H-prostaglandin F p (’H-PGFZa; 0 .9 pg./g. of tissue) in 0.1M potassium phosphate buffer, pH 7.4, containing 2 mM. nicotinamide adenine dinucleotide (NAD) at 37 “C.for various lengths of time. A placental homogenate, previously inactivated by boiling, was used for the “control” incubation. After the reactions had been stopped by acidifying and addition of ethanol, PGFp was extracted and separated from its metabolites by thin layer chromatography. The plates were scanned for radioactivity using a radiochromatogram scanner, and the relevant zones were eluted for quantitative determination of radioactivity by liquid scintillation counting.
At 16 weeks gestation there was considerable metabolism of PGF,, by the membranes and placental homogenates ; PGF2a concentrations dropped to less than half their original concentration (0 a9 pg./g. of tissue) within three minutes, as shown in Figure 1. Myometrial and decidual homogenates were less active, metabolizing respectively 15 and 5 per cent of the PGF,, in 30 minutes. The fetal skin and umbilical cord, on the other hand, appeared to be devoid of enzymatic activity since the PGFp concentrations after incubation up to 60 minutes were not different from those in the control incubation with boiled homogenate. The results obtained with myometrium and villous tissue at 7 weeks gestation were similar to those observed at 16 weeks, showing that prostaglandin metabolizing enzymes are present in the placenta as early as the 7th week of pregnancy. Differentiation of the metabolites by thin layer chromatography showed that PGFp was metabolized to at least two compounds. Using an incubation of membranes as an example, the distribution of the total radioactivity between PGF2a and these metabolites (M, and M2) after various lengths of time, is shown in Figure 2. M, and M, showed a mobility on thin layer chromatography identical respectively to that
fetal skin & c o r d
I decidua 1
myometrium
placenta membranes t i m e (min.)
FIG.1 Metabolic degradation of PGF2a in homogenates of fetal skin, umbilical cord, decidua, rnyornetrium, placenta, and fetal membranes obtained at 16 weeks gestation.
144
KEIRSE, WILLIAMSON AND TURNBULL
30
60
time irnin.1 FIG.2
Distribution of the radioactivity between PGF,, and its metabolites (M, and M,) during incubation of 3H-PGF2a with a homogenate of membranes. obtained at 16 weeks gestation.
of 15-kcto PGF,, and 15-keto-13,14-dihydro PGF2,.
DISCUSSION The observations made in this study demonstrate the presence of enzymes regulating the degradation of prostaglandins, in placenta and membranes and to a lesser extent in myometrium and decidua, in early pregnancy. Since both E and F prostaglandins are substrates for 15hydroxy-prostaglandin dehydrogenase (AnggLd and Samuelsson, 1966), the present observations using PGF,, as a substrate can also be applied to PGE,. In fact, PGE is generally metabolized faster than PGF in the in vitro situation (Anggird and Samuelsson, 1966; Jarabak, 1972; Schlegel et al., 1974). The placenta has previously been shown to contain a high prostaglandin dehydrogenase activity in vitro (Jarabak, 1972; Schlegel et al., 1974). The present study, however, demonstrates that the enzymatic activity is present in villous tissue as early as the 7th week of pregnancy, and that this activity is not limited to placental tissue. The present findings certainly support the hypothesis that there is a high potential for intra-uterine metabolism of prostaglandins in the second trimester of pregnancy. The extent to which this potential, observed in vitro, applies in vivo is not known. It is interesting, however,
that the myometrium is divided from the amniotic cavity by two structures of fetal origin, membranes and placenta, which showed a remarkably high degradation of prostaglandins in vitro. Prostaglandins administered intraamniotically for termination of pregnancy have been shown to disappear steadily from the amniotic cavity (Pace-Asciak et al., 1972) and Karim (1972) has suggested that they diffuse through the membranes to reach their site of action in the myometrium. It is almost certain that a proportion of these prostaglandins must be inactivated before reaching the myometrial target and this may well be one of the reasons that, for termination of pregnancy, far higher doses of prostaglandins are required intraamniotically than extra-amniotically (Wiqvist et al., 1973). Prostaglandins have been implicated as causative agents in the process of spontaneous and habitual abortion (Karim and Hillier, 1970; Bodin, 1971). It is possible that the physiological r61e of the prostaglandin-degrading enzymes is to maintain the continuity of pregnancy. The fact that the highest enzyme activities were found in fetal tissues, membranes and placenta, may provide yet another argument for a fetal r61e in the maintenance of pregnancy. ACKNOWLEDGEMENTS The tissues were obtained by permission of
METABOLISM OF PGF,~
Mr. E. A. Williams and Mrs. B. R. Hicks gave expert technical assistance. The work was sponsored by Programme Grant G971/SO9 from the Medical Research Council. PGF,, was a gift from Professor D. A. van Dorp, Unilever Research, Vlaardingen, The Netherlands. REFERENCES Amy, J. J., Karim, S. M. M., and Sivasamboo, R. (1973): Journal of Obstetrics and Gynaecology of the British Commonwealth, 80, 1017. Anggird, E., and Samuelsson, B. (1966): Arkiv for Kemi, 25, 293. Bodin, M. (1971): British Medical Journal, 2, 587. Bundy, G., Lincoln, F., Nelson, N., Pike, J., and Schneider, W . (1971): Annals of the New York Academy of Sciences, 180, 76. Bygdeman, M., Btguin, F., Toppozada, M., Wiqvist, N., and Bergstrom, S. (1972): Lancet, 1, 1336. Ferreira, S. H., and Vane, J . R. (1967): Nature, 216,868. Jarabak, J. (1972): Proceedings of the National Academy of Sciences of the United States of America, 69, 533. Karim, S. M. M., and Hillier, K. (1970): Journal of Obstetrics and Gynaecology of the British Commonwealth, 77, 837.
145
Karim, S. M. M., and Sharma, S. D. (1972): Journal of Obstetrics and Gynaecology of the British Commonwealth, 79, 737. Karim, S. M. M. (1972): Journal of Reproduction and Fertility, Supplement, 16, 105. Keirse, M. J. N. C., and Turnbull, A. C. (1974): Journal of Endocrinology. (In press.) Kirton, K. T., and Forbes, A. D. (1972): Prostaglandins, 1, 319. Pace-Asciak, C., Wolfe, L. S., Gillett, P. G . , and Kinch, R. A. (1972): Prostaglandins, 1, 469. Piper, P. J., Vane, J. R., and Wyllie, J. H. (1970): Nature 225, 600. Samuelsson, B., Granstrom, E., Green, K., and Hamberg, M. (1971): Annals of the New York Academy of Sciences, 180, 138. Schlegel, W., Demers, L. M., Hildebrandt-Stark, H. E., Behrman, H. R., and Greep, R. 0. (1974): Prostaglandins, 5, 417. Wiqvist, N., Bygdeman, M., and Toppozada, M. (1973): In Prostaglandins in Fertility Control 3. Edited by S. Bergstrom, WHO, Karolinska Institutet, Stockholm, p. 80. Yankee, E. W., and Bundy, G. L. (1972): Journal of the American Chemical Society, 94, 365 1.
