00021-972X/78/4606-0947$02.00/0 Journal of Clinical Endocrinology and Metabolism Copyright © 1978 by The Endocrine Society
Vol. 46, No. 6 Printed in U.S.A.
Plasma Concentrations of Prostaglandins during Late Human Pregnancy: Influence of Normal and Preterm Labor* MURRAY D. MITCHELL,f ANTHONY P. FLINT,! JAY BIBBY, JANE BRUNT, JILL M. ARNOLD,§ ANNE B. M. ANDERSON, AND ALEXANDER C. TURNBULL Nuffield Department of Obstetrics and Gynecology, University of Oxford, John Radcliffe Hospital, Headington, Oxford 0X3 9DU, United Kingdom ABSTRACT. Highly sensitive and specific RIA procedures have been used to measure prostaglandin concentrations in the peripheral circulation of late pregnant and parturient women. The concentrations of prostaglandin E (PGE) and prostaglandin F (PGF) in plasma samples assayed within 4 weeks of collection were not significantly different among the groups studied, the levels (mean ± SRM, picograms per ml) were: late pregnancy (n = 13): PGE, 4.8 ± 1.0; PGF, 6.2 ± 0.5; early term labor (n = 5): PGE, 6.8 ± 1.5; PGF, 7.9 ± 0.7; late term labor (n = 5): PGE, 5.4 ± 2.2; PGF, 12.4 ± 3.5; and preterm labor (n = 7): PGE, 4.4 ± 0.4; PGF, 6.9 ± 1.4. The concentration of 13,14-dihydro-15-keto-prostaglandin F (PGFM) in late pregnancy was 59.0 ± 7.8 pg/ml. During spontaneous term labor, the concentration of PGFM was significantly elevated (P < 0.01) to 142.8 ± 32.3 pg/ml in early labor and 282.7 ± 55.3 pg/ml in late
labor. The concentration of PGFM in plasma from patients in preterm labor (62.7 ± 17.4 pg/ml) was not significantly different from that found during late pregnancy, but was significantly lower than levels found at term during early labor (P < 0.05). The concentration of PGE increased significantly in frozen plasma samples stored for more than 4 weeks in all groups studied; the concentration of PGF was significantly elevated after storage only in the late pregnancy group (P < 0.01). The plasma concentration of PGFM in all groups studied was unaffected by storage. It is concluded that measurement of PGFM concentrations is the most reliable method available of monitoring prostaglandins in the peripheral circulation and that great care must be exercised in the assay and interpretation of prostaglandin levels in human plasma. (J Clin Endocrinol Metab 46: 947, 1978)
P
ROSTAGLANDINS have been impli- this fluid must still be interpreted cautiously, cated in the mechanism(s) of parturition as the measured levels may be influenced by in many species (1, 2). In man, studies have the method of collection (10). been hampered by the low endogenous levels Previous investigations of prostaglandins in present, due to high rates of clearance (3), as peripheral plasma have described concentrawell as the problem of prostaglandin produc- tions greatly exceeding the calculated levels tion by platelets (4). Most useful data con- (11), although recent data are closer to the cerning the role of prostaglandins in human expected levels (12-14). Using recently develparturition have come from measurements in oped, highly sensitive and specific RIAs which amniotic fluid (5-8) which contains higher can measure peripheral plasma concentrations concentrations of prostaglandins than periph- of prostaglandins similar to the calculated leveral plasma and little prostaglandin-metabol- els, we have studied the changes in concentraising activity (9). Amniotic fluid is, however, tion of prostaglandin E (PGE), prostaglandin less available than blood and, indeed, the re- F (PGF), and 13,14-dihydro-15-keto-prostasults of measurements of prostaglandins in glandin F (PGFM) during late pregnancy and parturition. Received July 5, 1977. * This work was supported by a Medical Research Council (UK) Program grant awarded to A.C.T. f To whom requests for reprints should be addressed. X Present address: A.R.C. Institute of Animal Physiology, Babraham, Cambridge. § Present address: Royal United Hospital, Bath, England.
Methods and Materials Patients and sampling Peripheral venous blood samples were obtained from patients in late pregnancy before the onset of labor (beyond 34 completed weeks) and during
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948
MITCHELL ET AL.
labor of spontaneous onset within 2 weeks of term. The pregnancies were uncomplicated, with no major fetal or maternal pathology. Blood was also collected from women admitted in preterm labor (defined as labor occurring spontaneously for no apparent reason before 37 completed weeks of pregnancy; in the present group of women, 28-36 weeks of pregnancy). Samples were obtained before any treatment was instituted. Cervical dilatation was less than 4 cm in all cases. No blood samples were collected within 1 h after vaginal examination. When samples were obtained during active labor, cervical assessment always followed sampling. Samples in "early" labor were obtained when the cervix was 4 cm dilated or less; those in "late" labor were taken at a cervical dilatation between 5-8 cm. Blood samples were collected into ice-cold tubes containing 0.1 ml solution of acetylsalicylic acid (5 mg/ml) and 0.1 ml solution of ethylenediaminetetraacetic acid (70 mg/ml). After centrifugation at 10 C and 1,500 X g plasma was separated and stored at —20 C until analysis.
JCK&M • 1978 Vol46 • No 6
(specific activity > 130 Ci/mmol; New England Nuclear, Boston, and Radiochemical Centre, Amersham) and an iodinated tracer was used for PGFM synthesized by previously described methods (15). Prostaglandin standards were in the range 0-50 pg. Bound and free prostaglandins were separated by using a suspension of dextran-coated charcoal. The mean mass of added prostaglandin required to displace zero point binding by 50% during these studies for each assay was: PGE, 5.3 pg; PGF, 10.1 pg; and PGFM, 13.1 pg. The mean least detectable mass (mass resulting in a response 2 SD away from the zero dose response) in each assay during these studies was: PGE, 0.54 pg; PGF, 0.83 pg; and PGFM, 1.61 pg. Statistical analysis The Mann-Whitney rank sum test was used to test for significant differences between groups.
Results Validation of assays
Assay details The assays utilized antisera kindly donated by Doctor F. Dray (Pasteur Institute, Paris) and the procedures employed were based on his published methodology (13, 15), with some modifications. All solvents were Distol reagent grade (Fisons Scientific Apparatus, Leics, UK) and silicic acid was 100 mesh (Mallinckrodt Co., MA). After adding approximately 1200 cpm tritiated PGFM to monitor recoveries, plasma samples (4 ml) were extracted twice with an acidified mixture of cyclohexane and ethyl acetate (1:1, vol/vol). Samples were applied to microcolumns containing silicic acid (0.5 g), in cyclohexane-ethyl acetatemethanol (60:40:2, vol/vol). After a wash procedure using cyclohexane-ethyl acetate (60:40, vol/vol; 5 ml), prostaglandins were eluted in a 5-ml fraction of cyclohexane-ethyl acetate-methanol (60:40:20, vol/vol). All procedures were carried out under nitrogen pressure and all of the glassware was siliconized. The column fractions were evaporated under nitrogen and then dissolved in 1 ml buffer (1 M K2HPO4, 80 ml; 1 M KH2PO4, 20 ml; 0.9% NaCl, 900 ml; NaN3, 1 g; and gelatin, 1 g). A known volume (0.2 ml) was taken for the determination of radioactivity to estimate recoveries. Duplicate samples (0.1 ml) were taken for RIA of PGE, PGF, and PGFM. Samples were assayed at three dilutions and good agreement was observed between results calculated from different dilutions. Final antibody dilutions were 1:45,000 in all assays, tritiated tracers were used for PGE and PGF assays
The major modification to the previously published methodology was the elution of all three prostaglandins in one fraction. Comparison of this procedure with group separation procedures is shown in Table 1. As might be expected from the cross reactivities of the antisera (Table 2), the modification employed did not alter the measured level of prostaglandins. Both methods were used during the period of this study. Inter- and intraassay coefficients of variation were determined on plasma pools of mixed umbilical cord and maternal plasma and are shown in Table 3. Concentrations of PGE and PGF Analysis of plasma concentrations of prostaglandins revealed significantly higher levels TABLE 1. Prostaglandin concentrations in freshly collected plasma from nonpregnant subjects Prostaglandin concentration (pg/ml; mean ± SD; n = 10) PGE 1.7 ± 0.9
PGF 3.3 ± 0.8
PGFM 52.1 ± 5.4
A. Using group separation technique B. Using single 1.3 + 0.4 3.6 ± 0.7 56.9 ± 4.3 fraction elution The differences between A and B are not statistically significant.
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PROSTAGLANDINS AND PARTURITION TABLE 2. Cross reactivities of the antisera used in these studies % Cross reaction with antisera raised against PGE2 PGE2 PGF a , PGFM 15-Keto-PGE2 13,14-Dihydro-PGE2 15-Keto-PGF2ll 13,14-Dihydro-PGF2l, PGA2 PGB2
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Vol. 46, No. 6 Printed in U.S.A.
Plasma Concentrations of Prostaglandins during Late Human Pregnancy: Influence of Normal and Preterm Labor* MURRAY D. MITCHELL,f ANTHONY P. FLINT,! JAY BIBBY, JANE BRUNT, JILL M. ARNOLD,§ ANNE B. M. ANDERSON, AND ALEXANDER C. TURNBULL Nuffield Department of Obstetrics and Gynecology, University of Oxford, John Radcliffe Hospital, Headington, Oxford 0X3 9DU, United Kingdom ABSTRACT. Highly sensitive and specific RIA procedures have been used to measure prostaglandin concentrations in the peripheral circulation of late pregnant and parturient women. The concentrations of prostaglandin E (PGE) and prostaglandin F (PGF) in plasma samples assayed within 4 weeks of collection were not significantly different among the groups studied, the levels (mean ± SRM, picograms per ml) were: late pregnancy (n = 13): PGE, 4.8 ± 1.0; PGF, 6.2 ± 0.5; early term labor (n = 5): PGE, 6.8 ± 1.5; PGF, 7.9 ± 0.7; late term labor (n = 5): PGE, 5.4 ± 2.2; PGF, 12.4 ± 3.5; and preterm labor (n = 7): PGE, 4.4 ± 0.4; PGF, 6.9 ± 1.4. The concentration of 13,14-dihydro-15-keto-prostaglandin F (PGFM) in late pregnancy was 59.0 ± 7.8 pg/ml. During spontaneous term labor, the concentration of PGFM was significantly elevated (P < 0.01) to 142.8 ± 32.3 pg/ml in early labor and 282.7 ± 55.3 pg/ml in late
labor. The concentration of PGFM in plasma from patients in preterm labor (62.7 ± 17.4 pg/ml) was not significantly different from that found during late pregnancy, but was significantly lower than levels found at term during early labor (P < 0.05). The concentration of PGE increased significantly in frozen plasma samples stored for more than 4 weeks in all groups studied; the concentration of PGF was significantly elevated after storage only in the late pregnancy group (P < 0.01). The plasma concentration of PGFM in all groups studied was unaffected by storage. It is concluded that measurement of PGFM concentrations is the most reliable method available of monitoring prostaglandins in the peripheral circulation and that great care must be exercised in the assay and interpretation of prostaglandin levels in human plasma. (J Clin Endocrinol Metab 46: 947, 1978)
P
ROSTAGLANDINS have been impli- this fluid must still be interpreted cautiously, cated in the mechanism(s) of parturition as the measured levels may be influenced by in many species (1, 2). In man, studies have the method of collection (10). been hampered by the low endogenous levels Previous investigations of prostaglandins in present, due to high rates of clearance (3), as peripheral plasma have described concentrawell as the problem of prostaglandin produc- tions greatly exceeding the calculated levels tion by platelets (4). Most useful data con- (11), although recent data are closer to the cerning the role of prostaglandins in human expected levels (12-14). Using recently develparturition have come from measurements in oped, highly sensitive and specific RIAs which amniotic fluid (5-8) which contains higher can measure peripheral plasma concentrations concentrations of prostaglandins than periph- of prostaglandins similar to the calculated leveral plasma and little prostaglandin-metabol- els, we have studied the changes in concentraising activity (9). Amniotic fluid is, however, tion of prostaglandin E (PGE), prostaglandin less available than blood and, indeed, the re- F (PGF), and 13,14-dihydro-15-keto-prostasults of measurements of prostaglandins in glandin F (PGFM) during late pregnancy and parturition. Received July 5, 1977. * This work was supported by a Medical Research Council (UK) Program grant awarded to A.C.T. f To whom requests for reprints should be addressed. X Present address: A.R.C. Institute of Animal Physiology, Babraham, Cambridge. § Present address: Royal United Hospital, Bath, England.
Methods and Materials Patients and sampling Peripheral venous blood samples were obtained from patients in late pregnancy before the onset of labor (beyond 34 completed weeks) and during
947
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948
MITCHELL ET AL.
labor of spontaneous onset within 2 weeks of term. The pregnancies were uncomplicated, with no major fetal or maternal pathology. Blood was also collected from women admitted in preterm labor (defined as labor occurring spontaneously for no apparent reason before 37 completed weeks of pregnancy; in the present group of women, 28-36 weeks of pregnancy). Samples were obtained before any treatment was instituted. Cervical dilatation was less than 4 cm in all cases. No blood samples were collected within 1 h after vaginal examination. When samples were obtained during active labor, cervical assessment always followed sampling. Samples in "early" labor were obtained when the cervix was 4 cm dilated or less; those in "late" labor were taken at a cervical dilatation between 5-8 cm. Blood samples were collected into ice-cold tubes containing 0.1 ml solution of acetylsalicylic acid (5 mg/ml) and 0.1 ml solution of ethylenediaminetetraacetic acid (70 mg/ml). After centrifugation at 10 C and 1,500 X g plasma was separated and stored at —20 C until analysis.
JCK&M • 1978 Vol46 • No 6
(specific activity > 130 Ci/mmol; New England Nuclear, Boston, and Radiochemical Centre, Amersham) and an iodinated tracer was used for PGFM synthesized by previously described methods (15). Prostaglandin standards were in the range 0-50 pg. Bound and free prostaglandins were separated by using a suspension of dextran-coated charcoal. The mean mass of added prostaglandin required to displace zero point binding by 50% during these studies for each assay was: PGE, 5.3 pg; PGF, 10.1 pg; and PGFM, 13.1 pg. The mean least detectable mass (mass resulting in a response 2 SD away from the zero dose response) in each assay during these studies was: PGE, 0.54 pg; PGF, 0.83 pg; and PGFM, 1.61 pg. Statistical analysis The Mann-Whitney rank sum test was used to test for significant differences between groups.
Results Validation of assays
Assay details The assays utilized antisera kindly donated by Doctor F. Dray (Pasteur Institute, Paris) and the procedures employed were based on his published methodology (13, 15), with some modifications. All solvents were Distol reagent grade (Fisons Scientific Apparatus, Leics, UK) and silicic acid was 100 mesh (Mallinckrodt Co., MA). After adding approximately 1200 cpm tritiated PGFM to monitor recoveries, plasma samples (4 ml) were extracted twice with an acidified mixture of cyclohexane and ethyl acetate (1:1, vol/vol). Samples were applied to microcolumns containing silicic acid (0.5 g), in cyclohexane-ethyl acetatemethanol (60:40:2, vol/vol). After a wash procedure using cyclohexane-ethyl acetate (60:40, vol/vol; 5 ml), prostaglandins were eluted in a 5-ml fraction of cyclohexane-ethyl acetate-methanol (60:40:20, vol/vol). All procedures were carried out under nitrogen pressure and all of the glassware was siliconized. The column fractions were evaporated under nitrogen and then dissolved in 1 ml buffer (1 M K2HPO4, 80 ml; 1 M KH2PO4, 20 ml; 0.9% NaCl, 900 ml; NaN3, 1 g; and gelatin, 1 g). A known volume (0.2 ml) was taken for the determination of radioactivity to estimate recoveries. Duplicate samples (0.1 ml) were taken for RIA of PGE, PGF, and PGFM. Samples were assayed at three dilutions and good agreement was observed between results calculated from different dilutions. Final antibody dilutions were 1:45,000 in all assays, tritiated tracers were used for PGE and PGF assays
The major modification to the previously published methodology was the elution of all three prostaglandins in one fraction. Comparison of this procedure with group separation procedures is shown in Table 1. As might be expected from the cross reactivities of the antisera (Table 2), the modification employed did not alter the measured level of prostaglandins. Both methods were used during the period of this study. Inter- and intraassay coefficients of variation were determined on plasma pools of mixed umbilical cord and maternal plasma and are shown in Table 3. Concentrations of PGE and PGF Analysis of plasma concentrations of prostaglandins revealed significantly higher levels TABLE 1. Prostaglandin concentrations in freshly collected plasma from nonpregnant subjects Prostaglandin concentration (pg/ml; mean ± SD; n = 10) PGE 1.7 ± 0.9
PGF 3.3 ± 0.8
PGFM 52.1 ± 5.4
A. Using group separation technique B. Using single 1.3 + 0.4 3.6 ± 0.7 56.9 ± 4.3 fraction elution The differences between A and B are not statistically significant.
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PROSTAGLANDINS AND PARTURITION TABLE 2. Cross reactivities of the antisera used in these studies % Cross reaction with antisera raised against PGE2 PGE2 PGF a , PGFM 15-Keto-PGE2 13,14-Dihydro-PGE2 15-Keto-PGF2ll 13,14-Dihydro-PGF2l, PGA2 PGB2
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