The origin of circulating 13,14-dihydro-15-keto-prostaglandin F2α during delivery

All uterine tissues as well as the fetal membranes and the placenta can form prostaglandins from endogenous precursors but it is not clear which of the tissues is the main site for the increase in PGF_2α production during human parturition. To examine this question, we measured plasma prostaglandin levels before and at intervals after expulsion of the fetus, placenta, and membranes. The concentration of PGFM at the beginning of the second stage of labor was significantly higher than before the onset of labor. Five minutes after the birth of the infant, the concentration had doubled. Thirty minutes after the expulsion of placenta and membranes, plasma PGFM had fallen to the levels at full dilatation; two hours postpartum it was still significantly raised over levels before labor. Since the halflife of PGFM in the circulation is about 7 minutes, these findings indicate that the uterine tissues are important sources of PGFM during labor. In contrast, endogenous oxytocin levels, which were significantly raised over control levels at the second stage of labor, did not change during the third stage, and decline postpartum to control levels. Oxytocin infusion did not influence PGFM levels at 5 and at 30 minutes postpartum, but raised them at 2 hours.     

Synthesis of 6-keto-PGF1α by ram seminal vesicle microsomes

At low substrate/enzyme ratios, and in the absence of reduced glutathione (GSH), the major prostaglandin (PG) biosynthesised by the ram seminal vesicle cyclo-oxygenase from arachidonic acid was 6-keto-PGF1α. The addition of nanomolar amounts of reduced GSH suppressed biosynthesis of this product and stimulated the formation of PGE2; 1-epinephrine enhanced the conversion of the substrate but had not effect on the type of product formed. 15-Hydroperoxy arachidonic acid selectively inhibited formation of 6-keto-PGF1α (IC50 100 μM) but blocked synthesis of all cyclooxygenase products at concentations greater than 1 mM. At substrate concentrations of 30 μM or greater, synthesis of 6-keto-PGF1α was inhibited and PGE2 and PGF2α were the main products formed.     

A radioimmunoassay of 15 (S) 15 methyl prostaglandin F2α

A sensitive and relative specific radioimmunoassay for 15 (S) 15 methyl prostaglandin F_2α has been developed to enable the measurements of the concentrations of the drug in biological fluids after its administration for therapeutic abortion. The precision, accuracy and specificity of the assay are described.     

The stability of 13,14-dihydro-15 keto-PGE2

13,14-Dihydro-15 keto-PGE₂ decomposes by first order reaction kinetics, dependent on pH, temperature and albumin concentration. Under common experimental conditions at or near neutrality in the absence of albumin decomposition is suspended with the formation of 13,14-dihydro-15 keto-PGA₂. Cyclization into 11-deoxy-13,14-dihydro-15 keto-11,16-bicyclo-PGE₂ occurs at elevated pH in purely aqueous buffers, and also at or near neutrality in the presence of albumin. Albumin accelerates, quantitatively, the decomposition of 13,14-dihydro-15 keto-PGE₂ and promotes, qualitatively, the formation of the bicyclo rearrangement product. High performance liquid chromatographic analysis indicates that the cyclization product exists in at least three epimerically distinct forms. The two major epimers have been synthesized and isolated in pure form. They differ, mainly, by their optical rotation.     

Disappearance of prostaglandins F2α and 15-methyl F2α from amniotic fluid as measured by radioimmunoassay

A sensitive and relatively specific radioimmunoassay for 15 (S) 15 methyl prostaglandin F_2α was used to determine the levels of the drug in amniotic fluid after it had been injected intra-amniotically for termination of second trimester pregnancy. The disappearance of the free acid (tham salt) and methyl ester of the prostaglandin analogue were similar. The results of this preliminary study suggest that the drug rapidly equilibrates in the fluid and this is followed by a slow removal from the amniotic sac. A comparison with a similar study with PGF_2α, revealed that the analogue had a longer half-life in the amniotic fluid.     

Levels of 13,14-dihydro-15-keto-PGE2 in some biological fluids as measured by radioimmunoassay

A rabbit antiserum directed toward the prostaglandin E₂ metabolite 13,14-dihydro-15-keto-prostaglandin E₂ (KH₂PGE₂) was produced by immunization with a human albumin-KH₂PGE₂ conjugate. The antiserum recognized the 15-keto-group (it cross reacts with 13,14-dihydro-prostaglandin E₂ 0.2%); the saturated 13,14-bond (it cross reacts with 15-keto-prostaglandin E₂ 7%); the 9-keto group (it cross reacts with 13,14-dihydro-15-keto-prostaglandin F_2α 5%); and the 11-hydroxy group (it cross reacts with 13,14-dihydro-15-keto-PGA₂ 0.4%).By subjecting the antiserum to preparative isoelectric electrofocusing, populations of antibodies that varied in their cross reaction with 13,14-dihydro-15-keto-prostaglandin F_2α (KH₂PGF_2α) from 20% to 1% were obtained. The levels of KH₂PGE₂ in plasma of rat and mouse as measured by radioimmunoassay of the unfractionated plasma were 0.39 ± 0.07 ng/ml and 0.41 ± 0.13 ng/ml, respectively. Recovery of exogenously added KH₂PGE₂ from human plasma was 100%. Radioimmunoassay with two antisera; an antiserum directed toward KH₂PGF_2α that cross reacts with KH₂PGE₂ 1% and the antiserum to KH₂PGE₂, demonstrated that KH₂PGE₂, not KH₂PGF_2α, was being measured with the anti-KH₂PGE₂. The levels of KH₂PGE₂ in rat plasma did not vary with sex. In rats, the levels of KH₂PGE₂ markedly increased after exercise stress.In mice carrying a spindle-cell sarcoma (SAI) and a fibrosarcoma (SaD2), the levels of KH₂PGE₂ in the plasma increased with time after transplantation. The increase was not observed in the plasma of mice carrying a transplantable anaplastic carcinoma (15091AK), a lymphatic leukemia (AW5147), two mammary adenocarcinomas (CADI, CAD2), a myeloid leukemia (C1498), and a hepatoma (BW7756).     

Determination of 6-keto-PGF1α, 2,3-dinor-6-keto-PGF1α, thromboxane B2, 2,3-dinor-thromboxane B2, PGE2, PGD2 and PGF2α in human urine by gas chromatography—negative ion chemical ionization mass spectrometry

A method for quantification of 6-keto-PGF_1α, 2,3-dinor-6-keto-PGF_1α, TXB₂, 2,3-dinor TXB₂, PGE₂, PGD₂ and PGF_2α in human urine samples, using gas chromatography—negative ion chemical ionization mass spectrometry, is described. Deuterated analogues were used as internal standards. Methoximation was carried out in urine samples which were subsequently applied to phenylboronic acid cartridges, reversed-phase cartridges and thin-layer chromatography. The eluents were further derivatized to pentafluorobenzyl ester trimethylsilyl ethers for final quantification by gas chromatography—mass spectrometry. The overall recovery was 77% for tritiated 6-keto-PGF_1α and 55% for tritiated TXB₂. Urinary levels of prostanoids were determined in a group of six volunteers before and after intake of the thromboxane synthase inhibitor Ridogrel, and related to creatinine clearance.     

Effects of dibuturyl cAMP, LHRH, and aromatase inhibitor on simultaneous outputs of prostaglandin F2α, and 13,14-dihydro-15-keto-prostaglandin F2α by term placental explants

Explants from term human placentas were maintained in culture with daily changes of medium. Daily output of PGF_2α and PGFM¹ decreased during the course of the incubation. Addition of 4 μg/ml DHEAS or 67 μg/ml LDL cholesterol had no effect on output of PGF_2α or PGFM. Addition of 1.6, 3.2, or 6.4 μg/ml of LHRH to the culture plates had no effect on output of PGFM or PGF_2α, but LHRH increased hCG output. Dibutyryl cAMP (1mM, 2mM, and 4mM) increased output of PGF_2α, PGFM, and hCG. Aromatase inhibitor decreased hCG output, but it was without effect on output of PGF_2α, or PGFM. Significant correlations were demonstrated between progesterone, PGFM, PGF_2α, and hCG, suggesting that PGF_2α originates in the syncytiotrophoblast cell. The ability of LHRH to stimulate output of hCG but not PGF_2α while dbcAMP stimulated both suggests that either PGF_2α and hCG arise in different cells or that LHRH does not act through cAMP.     

Plasma levels of 13,14-dihydro-15-keto prostaglandin F2α, estrogens, and progesterone during stretch-induced labor at term

The uterus of six healthy multiparous women at term was mechanically stretched by a rubber catheter and balloon. Apparent labor was inaugurated in all cases within 5 hours and increased progressively with time. Advanced cervical softening and dilatation were also evident after the stretch treatment. Significant increases in the levels of 13,14-dihydro-15-keto-prostaglandin F_2α (PGFM) were observed with the progress of treatment (P < 0.01). Plasma estrogens and progesterone levels did not change significantly during the treatment (P > 0.05). Stretching and/or resulting uterine contractions appear to induce the secretion of prostaglandin F_2α (PGF) from the organ, which in turn seems to be involved in both cervical softening, and the onset and progress of labor, under stable conditions of plasma estrogens and progesterone.     

Peripheral plasma concentrations of 13,14-dihydro-15-keto-prostaglandin F2α and progesterone around luteolysis and during early pregnancy in the goat

Peripheral plasma concentrations of 13,14-dihdyro-15-keto-prostaglandin F_2α (PGFM) and progesterone were determined during both luteolysis in the oestrous cycle and early pregnancy in four goats. Luteal regression, characterised by decreasing progesterone concentrations, began on day 12 or 13. PGFM concentrations showed a pulsatile pattern around this time, with peak concentrations increasingly markedly as progesterone levels fell and oestrus approached. During early pregnancy progesterone concentrations did not fall after day 12 and no marked elevation of PGFM above basal values of 50–150 pg/ml was detected.     

The impermeability of the basic cell membrane to thromboxane-B2, prostacyclin and 6-keto-PGF1α

Washed rabbit red blood cells (RBCs) were suspended in electrolyte solution containing ³H-labeled prostacyclin (PGI₂), thromboxane (TxB₂) or 6-keto-PGF_1α and ¹⁴C-labeled sucrose or thiourea. Following 1 to 30 min incubation with ¹⁴C-sucrose, ³H-TxB₂ or ³H-6-keto-PGF_1α, the ¹⁴C or ³H space of packed RBCs remained essentially constant, yielding mean values (±S.E.) for all time periods of 6.1 ± 0.3, 9.5 ± 0.5 and 6.5 ± 0.4%, respectively. After 1 min of incubation at 4° or 23°C at a pH of 7.4 or 8.5 with trace amounts (10⁻⁹M) of ³H-PGI₂ or in the presence of added PGI₂ (10⁻⁵M) or ethacrynic acid (1.6 × 10⁻⁴M), the apparent PGI₂ space of packed RBCs ranged from 16 to 27%, decreasing to about 7% by 30 min. When RBCs were resuspended in fresh ³H-PGI₂ every 5 min, their ³H content increased very slowly (apparent PGI₂ space <40% at 30 min) as compared to thiourea (distribution space > 80% within 5 min). Over 90% of this ³H activity was lost from the RBCs in less than 2 min during elution at 4° or 23°C. It is concluded that RBC membranes and thus, presumably, the basic cell membrane in general, is not fundamentally permeable to PGI₂, 6-keto-PGF_1α or TxB₂. Hence, the effective entry of these cyclooxygenase products into some cells or their passage across tight-junctional capillaries or epithelial membranes must require facilitated or active transport processes as was shown to be the case for E, F and A PGs. This implies that the distribution, pharmacological action and metabolism of these and presumably all related cyclooxygenase products are selective rather than unrestricted.     

Production rates of prostaglandin F, 6-keto-PGF1α and thromboxane B2 by perifused human endometrium

Human endometrium obtained from fresh hysterectomy specimens was perifused for 7 hr in 95% O₂/5% CO₂ at 37°C. The phase of the menstrual cycle was determined by histological examination. The concentrations of PGF, 6-keto-PGF_1α and TxB₂ in 20 min fractions of the perifusion medium were measured by radioimmunoassay and production rates were calculated in terms of dry weight of tissue. Biphasic patterns of production were observed; high initial values fell to about 20% at 2 hr and then increased to relatively stable values at about 4 hr which were maintained for the next 2 hr. During this latter period, production rates in endometria taken at different phases of the cycle differed markedly from each other; the production rates of PGF in secretory and early proliferative endometria were low (15.8 ± 2.6, mean ± SEM and 67.2 ± 8.3 ng/min/g respectively) whereas they were high in late proliferative and premenstrual endometria (188.0 ± 16.7 and 196.4 ± 16.9 ng/min/g respectively). The patterns of production of 6-keto-PGF_1α and TxB₂ were similar to those of PGF but the absolute values were much lower (<10%). We conclude that the observed rates of production of prostaglandins by perifused human endometrium are consistent with synthesis being stimulated either by estrogen or withdrawal of hormonal support and being inhibited by progesterone.     

Formation of 6-keto-PGF1α by collecting tubule cells isolated from rabbit renal papillae

Homogeneous populations of collecting tubule epithelial cells have been isolated from rabbit renal papillae by a sequence of procedures involving: (a) dissociation of the tissue by mincing and treatment with trypsin; (b) destruction of contaminating non-collecting tubule cells by differential lysis in hypotonic media and (c) collection and washing by repeated centrifugation. The isolated cells have been characterized as being derived from the collecting tubules on the basis of anatomical source, size and histological staining for both NADH diaphorase activity and cyclooxygenase antigenicity. The cells are judged to be viable by several criteria including their ability to exclude both trypsin and vital dyes, their capacity to metabolize glucose and leucine and their ability to retain distinctive morphology following 10–14 days in culture media. Homogenates of freshly isolated collecting tubule cells when incubated with [³H]-arachidonic acid yielded radioactive products identified by thin-layer chromatographic behavior in multiple solvent systems as 6-keto-PGF_1α, PGF_2α, PGE₂, PGD₂ and a monohydroxy acid, probably HHT. No lipoxygenase-like activity was detected. At arachidonate concentrations of 2 or less, the major product was 6-keto-PGF_1α; while at substrate concentrations of greater than 10 , PGE₂ was the major radioactive prostaglandin formed. Similar distributions of products were observed when homogenates of dissociated renal papillae enriched in medullary interstitial cells were incubated with arachidonic acid. Our results indicate that collecting tubule cells do contain significant prostacyclin synthetase activity and suggest that PGI₂ plays a role in the function of mammalian collecting tubules.     

A simple method for preparation of valency hybrid hemoglobins, (αβ)2 and (αβ)2

The improved methods for the preparation of valency hybrid hemoglobins, (α³⁺β²⁺)₂ and (α²⁺β³⁺)₂ were presented. The (α³⁺β²⁺)₂ valency hybrid was separated from the solutions of partially reduced methemoglobin with ascorbic acid, by using CM 32 column chromatography. The (α²⁺β³⁺)₂ valency hybrid was also isolated from hemoglobin solutions, which were partially oxidized with ferricyanide, by chromatography on CM 32 column. These valency hybrid hemoglobins were found to be single on isoelectric focusing electrophoresis. Present procedures are very simple and are suitable for the bulk preparation of (α³⁺β²⁺)₂ and (α²⁺β³⁺)₂ valency hybrids.     

A radioimmunoassay for 6-keto-prostaglandin F1α utilizing an antiserum against 6-methoxime-prostaglandin F1α: Application to study renal synthesis of prostacyclin

PGI₂ and 6-keto-PGF_1α were converted to 6-methoxime-PGF_1α (6-MeON-PGF_1α) by treatment with methoxyamine HCl in acetate buffer. The formed 6-MeON-PGF_1α was measured by radioimmunoassay. Antisera were raised in rabbits after immunization against 6-MeON-PGF_1α-BSA conjugate. Diluted 1:20.000 to bind 50% of the tracer (³H-6-MeON-PGF_1α, 100 Ci/mmol), the antiserum cross reacted 0.8% with PGE₂, 1% with PGF_2α and less than 0.2% with PGD₂, PGF_1α, PGF_2β and TXB₂. The radioimmunoassay was used to estimate release of PGI₂ and 6-keto-PGF_1α from chopped rabbit renal medulla and cortex incubated in Krebs-Ringer bicarbonate buffer (37°C, 30 min). The 6-keto-PGf_1α radioimmunoassay was validated in biological samples by mass fragmentography. The chopped medulla (n=5) released 38±9 ng/g/min and the cortex (n=5) 4.7±2.0 ng/g/min, while the release of immunoreactive PGE₂ (iPGE₂) and iPGF_2α was 171±26 and 74±13 ng/g/min from the medulla and 4.3±1.3 and 2.7±0.3 ng/g/min from the cortex, respectively. The results confirm previous findings, which indicate that in the renal medulla prostaglandin endoperoxides are mainly transformed to prostaglandins, while in the cortex transformation to PGI₂ seems to be of greater importance.     

Cross-Reactivity of Δ17-6-Keto-PGF1α with 6-Keto-PGF1α antibodies

The cross-reactivity of the PGI₃ metabolite, Δ17-6-keto-PGF_1α, with antibodies against 6-keto-PGF_1α for radioimmunoassays (RIA) has been investigated. Δ17-6-keto-PGF_1α was obtained either from commercial sources or after its purification from endothelial cells. In the latter case, primary cultured bovine aortic endothelial cells were incubated for 20 min at 37°C with 10 μM eicosapentaenoic acid (EPA) in the presence of 2 μM 13-hydroperoxy-octadecadienoic acid, an activator of the EPA cyclooxygenation, and the 6-keto-PGF_1α and Δ17-6keto-PGF_1α produced were separated by RP-HPLC. Then, cross-reactivities of the commercial and purified Δ17-6-keto-PGF_1α with 6-keto-PGF_1α antibodies were determined and found not to exceed 10%. In addition, the amounts of prostacyclin-related compounds detected by direct measurements in media of cells loaded with EPA were compared with those obtained after purification of 6-keto-PGF_1α. In accordance with the cross-reactivity data, we found that RIA in media mainly measured 6-keto-PGF_1α, the Δ17-6-keto-PGF_1α formed being undetected at 90%. It is concluded that 6-keto-PGF_1α antibodies generally used for RIA of 6-keto-PGF_1α are highly specific since they can discriminate a metabolite bearing an additional double bond such as the PGI₃ metabolite Δ17-6-keto-PGF_1α.     

Induction of second trimester abortion: Comparison between vaginal 15-methyl-PGF2α methyl ester and intra-amniotic PGF2α

The efficiency and acceptability of a single-dose, long-acting vaginal suppository containing 3.0 mg of 15-methyl PGF_2α methyl ester was compared with intra-amniotic administration of 50 mg of PGF_2α in 100 patients with a second trimester pregnancy termination. Within 24 hours, 78 per cent of the patients in the vaginal group and 92 per cent in the intra-amniotic group had aborted. The mean induction-abortion interval was 17.9 hours in the vaginal group and 15.8 hours in the intra-amniotic group.Gastrointestinal side-effects were more frequent, but the procedure was less painful, with vaginal 15-methyl PGF_2α methyl ester than with intra-amniotic PGF_2α.The vaginal route is technically simple for adaptation to large-scale use, but the high frequency of gastrointestinal side-effects still limits the acceptability of 15-methyl PGF_2α methyl ester in vaginal administration.     

Cardiovascular actions of prostaglandins F2α; 15-me-F2α; F1α; F2β and F1β in the cat

Prostaglandin F_2α (5μg/kg, i.v.) causes an increase in pulmonary arterial pressure, decrease in systemic arterial pressure, and reflex bradycardia in the anesthetized cat. The same dose of the 15-methyl analogue of PGF_2α produces the same triad of effects but of greater magnitude and duration. Although prostaglandins F_1α, F_2β and F_1β also cause the same cardiovascular effects as F_2α, there is a decrease in potency for all parameters measured, with PGF_2α>PGF_1α>PGF_2β>PGF_1β. When compared to the actions of PGF_2α in producing an increase in pulmonary arterial pressure, PGs F_1α, F_2β and F_1β were less potent by approximately 10, 100, and 1000 fold respectively.     

Prostaglandin F2α (PGF2α) and prolactin secretion in rats

Plasma prolactin and F-prostaglandins (PGF) were measured in anesthetized male Sprague-Dawley rats before and at 15, 30, 45 and 60 minutes following i.v. injection of either PGF_2α (4 mg/kg), chlorpromazine, 1 mg/kg or chlorpromazine (1 mg/kg) after pretreatment with i.p. indomethacin (2 mg/kg). Following PGF_2α administration, plasma prolactin levels increased significantly only at 15 and 30 minutes in spite of extremely high PGF levels throughout 60 minutes. Besides the expected rise in plasma prolactin, chlorpromazine caused a transient but statistically significant increase in PGF. Indomethacin blocked the chlorpromazine-induced PGF rise but not prolactin increase. Animals stressed with ether anesthesia showed elevation of plasma prolactin, which was not blocked by indomethacin although PGF concentration fell. These results indicate that PGF_2α can stimulate prolactin release. This effect does not appear to be physiologic since very high PGF levels are required. Furthermore, blockade of prostaglandin synthesis by indomethacin does not prevent the release of prolactin in response to chlorpromazine or stress. Our findings do not support a possible role of PGFs as intermediaries in prolactin release. However, it is possible that PGFs may work through other mechanisms not investigated in our study.     

Determination of prostaglandin F2α, E2, D2 and 6-keto-F1α in human cerebrospinal fluid

Prostaglandin (PG)F_2α, E₂, D₂ and 6-keto-F_1α were determined in human cerebrospinal fluid by a mass spectrometric technique. The samples were obtained from 12 patients with suspected intracranial disease. A 64 fold variation in PG levels was observed. The major PG was 6-keto-F_1α (0.12–15 ng/ml). PGF_2α and PGE₂ were present in lower concentrations PGD₂ was below the level of detection (0.05 ng/ml) except in one patient with extremely high total levels of PGs.