Radioimmunoassay for urinary metabolites of prostaglandin F2α

Antibodies against the main urinary metabolite of PGF_2α in the human, 5α,7α-dihydroxy-11-ketotetranorprosta-1,16-dioic acid, were raised in rabbits. The compound was coupled selectively in the ω position to bovine serum albumin prior to injection. The resulting antibodies did not distinguish between tetranor compounds varying only in structure at the ω carbon, and thus the assay could be used also for other metabolites of PGF_2α, e.g. the main urinary metabolite in the guinea pig, 5α,7α-dihydroxy-11-ketotetranorprostanoic acid. Labeled ligands for the assays were prepared either $\text{in vivo}$ by injection of |17,18-³H|-PGF_2α into humans after several days' treatment with indomethacin, or $\text{in vitro}$ by incubation of |17,18-³H|-15-keto-13,14-dihydro-PGF_2α with mitochondria from rat liver. The sensitivity of the assay was 10 pg or 4 pg with these two preparations, respectively.The assay was employed for a number of measurements: normal daily excretion in a number of humans; excretion of urinary metabolites during treatment with prostaglandin synthetase inhibitors in human subjects, or after intravenous injection of PGF_2α; excretion during human pregnancy; and prostaglandin production in the guinea pig during normal estrous cycles and pregnancies and after estrogen treatment.The results of these studies were in several cases compared to similar measurements earlier performed using mass spectrometric methods, and were found to agree well. Thus, this radioimmunoassay provides a simple and accurate method for estimating prostaglandin production, particularly suitable for long-term studies and for cases where repeated blood sampling must be avoided.     

Measurement of prostaglandin F2α metabolites by radioimmunoassay

Antibodies against 15 keto PGF_2α and 13,14 dihydro 15 keto PGF_2α were produced in goats and rabbits using the appropriate prostaglandin protein conjugate. Tritium labeled 15-keto, and 13,14 dihydro 15-keto PGF_2α were prepared from ³H-PGF_2α. These antibodies and ³H-labeled compounds were used to develop radioimmunoassays for the respective F_2α metabolites. The antibodies had relatively little cross-reactivity (≤0.1%) with the parent F_2α molecule. Infusion of PGF_2α in monkeys increased 15-keto-h₂ levels 10–20 fold higher than PGF_2α in peripheral plasma. The levels of this metabolite were not altered detectably during clotting, indicating relatively slow rates of PGF_2α metabolism in vitro. These assays should be useful to follow release rates of exogenous prostaglandins from various formulations and delivery systems, and in vivo tissue synthesis of PGF_2α, where low levels preclude measuring the parent compound.     

Synthesis of prostaglandin E2 and prostaglandin F2α by toadfish red blood cells

Saline washed red blood cells of the toadfish convert [1-¹⁴C] arachidonic acid to products that cochromatograph with prostaglandin E₂ and prostaglandin F_2α. This synthesis is inhibited by indomethacin (10 μg/ml). Conversion of arachidonic acid to prostaglandin E₂ was confirmed by mass spectrometry. When saline washed toadfish red blood cells were incubated with a mixture of [1-¹⁴C]-arachidonic acid and [5,6,8,9,11,12,14,15,-³H]-arachidonic acid, comparison of the isotope ratios of the radioactive products indicated that prostaglandin F_2α was produced by reduction of prostaglandin E₂. The capacity of toadfish red blood cells to reduce prostaglandin E₂ to prostaglandin F_2α was confirmed by incubation of the cells with [1-¹⁴C] prostaglandin E₂.     

A study of prostaglandin F2α as the luteolysin in swine: I. Effect of prostaglandin F2α in hysterectomized gilts

Experiments were conducted to determine if prostaglandin F_2α (PGF_2α) is luteolytic in swine. In Experiment 1, four bilaterally hysterectomized gilts were injected with PGF_2α at 0800 (10mg) and 2000 hours (10mg) and four gilts received .9% saline at the same times on day 17 after onset of estrus. Treatments were reversed in the two groups of gilts 21 days later. All eight PGF_2α treated gilts exhibited estrus an average of 88.0 ± 13.5 hours after treatment and average duration of estrus was 66.0 ± 16.4 hours. Saline treated controls did not exhibit estrus. Two additional gilts were hysterectomized bilaterally and the saphenous artery catheterized on day 7 after onset of estrus. PGF_2α injected on day 17 resulted in a precipitous decline in plasma progestin concentration and onset of estrus by 110 and 90 hours in gilts 1 and 2, respectively. Another bilaterally hysterectomized gilt, with CL marked with India ink, received PGF_2α on day 17. Estrus occurred 92 hours later and, on day 4, regression of marked CL to corpora albicantia and presence of newly formed CL was confirmed at laparotomy.In Experiment 2, 12 bilaterally hysterectomized gilts were treated with PGF_2α at 0800 (10mg) and 2000 hours (10mg) on either day 8, 11, 14 or 17 after onset of estrus. None of the gilts treated on days 8 and 11 exhibited estrus. Two of three gilts treated on day 14 and all three gilts treated on day 17 exhibited estrus at an average of 116.0 ± 9.8 hours post-treatment. Average duration of estrus was 49.6 ± 8.8 hours.     

Termination of early gestation with vaginal prostaglandin F2α tablets

The abortifacient activity of prostaglandin F_2α was investigated by placing one or two 50 mg tablets of prostaglandin F_2α in THAM salt into the vagina of nine women less than 4 weeks pregnant at intervals of 2 to 4 hours for a 24 hour period. Serum levels of HCG, estradiol (E₂), progesterone and 17α-hydroxyprogesterone were measured by radioimmunoassay prior to starting therapy and at frequent intervals thereafter for 48 hours. All but two patients had significant side-effects, mainly diarrhea and vomiting, indicating that systemic absorption took place. Although bleeding was induced in 8 of 9 women, only 3 had complete abortions. A D&C was performed on all patients 48 hours after starting therapy. A significant fall in HCG levels was noted only in the patients who aborted. Only 3 of the 9 women had significant changes in steroid levels. A fall in progesterone and 17α-hydroxyprogesterone occurred in the 3 women who aborted and took place following the fall in HCG. Estradiol levels remained in the same range in all subjects. These findings indicate that prostaglandin F_2α when administered in this vehicle and this dosage is relatively ineffective as an abortifacient. When effective, its action would appear to be due to contractions of uterine muscle and not secondarily to luteolysis.     

Sexual behavior of castrated boars treated with prostaglandin F2α

The objectives were to test the hypothesis that exogenous prostaglandin F_2α (PGF_2α) temporarily restores sexual behavior of castrated boars, and to evaluate effects of PGF_2α on serum hormone concentrations. At 35 d after castration, nine lean-type adult boars were randomly assigned to three treatments in a 3 × 3 latin square (with three replicates). Treatments were three doses of PGF_2α doses (0, 10, and 20 mg) and three periods of treatment, with 5 d between each period. Serum testosterone (T) concentrations were non-detectable at the start of the experiment. Serum concentrations of estradiol (E2), LH, prolactin (PRL), and cortisol were unaffected (P > 0.05) by PGF_2α treatment. The interval from treatment to ejaculation in boars treated with 10 mg (758 s) or 20 mg (660 s) PGF_2α did not differ, but were different (P < 0.05) from control boars (>1 800 s). Ejaculation duration and false mounts differed (P < 0.05) between control boars and boars treated with 10 or 20 mg PGF_2α. In conclusion, PGF_2α treatment did not change serum concentrations of T, E2, LH, PRL, or cortisol, but restored sexual behavior. This restoration may have been due to an effect of PGF_2α directly in specific areas of the brain, or indirectly via release of other hormones that stimulated areas in the brain that affected sexual behavior.     

A radioimmunoassay for 6-keto- prostaglandin F1α

A radioimmunoassay for 6-keto-prostaglandin F_1α has been developed. The assay is accurate and sensitive but since the antiserum cross-reacts 5–10% with prostaglandins (PGs) of the E and F series, solvent extraction and thin layer chromatography are required fo absolute specifity. The assay has been validated by comparison with a radiochemical assay and by the use of an inhibitor of 6-keto PGF_1α formation, 15-hydroperoxy arachidonic acid. 6-Keto PGF_1α was found to have a low cross reaction with antisera directed against PGE₂, PGF_2α and thromboxane B₂.     

Induction of abortion by intra- and extra-amniotic prostaglandin F2α administration

Legal abortion was induced by intrauterine administration of prostaglandin F_2α in 115 patients during the 11th – 20th week of pregnancy. An intra-amniotic method was used in 61 of the cases, an extra-amniotic one in 54 cases. The average total dose administered was 35.1 mg (range 5 – 65 mg) in the intra-amniotic group, and 6358 μg (range 1500 – 14000 μg) in the extra-amniotic group. Abortion rate was 92 % in the intra-amniotic material and 72 % in the extra-amniotic material, and side-effects, mainly gastrointestinal irritation, were noted in 74 % of the intra-amniotic cases and 54 % of the extra-amniotic ones. If total doses of 4750 μg or more were administered in the extra-amniotic cases, abortion rate went up to 80 %, but the frequency of side-effects simultaneously increased to 64 %. No serious complications occurred. Intrauterine prostaglandin induction is well suited therapeutic abortions in the second trimester, and the intra-amniotic technique is more practicable than the extra-amniotic one. The latter is applicable in cases where the puncture of the amniotic cavity is difficult to achieve, e.g. in cases of fetus mortuus and hydatiform mole.     

Corpus luteum regression induced by ultra-low pulses of prostaglandin F2α

In view of the pulsatile nature of PGF_2α secretion from the ovine uterus at the time of luteolysis, experiments were designed to examine the effect of pulsed infusions of PGF_2α on luteal function and to re-examine the minimal effective levels of PGF_2α required to induce luteolysis. To mimic physiological conditions, hour-long infusions of PGF_2α in increasing concentrations were given either 4 times in 19 h or 5 times in 25 h into the arterial supply of the autotransplanted ovary in conscious sheep on day 12 of an induced cycle. Blood flow and progesterone secretion rate from the ovary were used to monitor directly the luteolytic effect of administered PGF_2α. The concentration of LH in peripheral plasma was measured throughout each infusion experiment and the presence of a preovulatory peak of LH was used as an indicator of the permanence of luteal regression. Four pulses of PGF_2α in 19 h caused complete corpus luteum regression in only 1 of 4 animals whereas the addition of a fifth pulse (5 pulses in 25 h) caused permanent regression in 4 out of 4 animals. Infusion of 5 hour-long pulses of saline or PGF_2α at a rate of <0.04 μg/h did not induce permanent suppression of progesterone secretion. The average total effective dose of PGF_2α required to induced luteal regression when given as 5 pulses was 1/40th of the amount currently regarded as the minimal effective one when given by constant infusion into the ovarian artery. In another series of experiments the luteolytic effect of a single hour-long pulse of 0.1 μg/h PGF_2α given daily for either 3 or 4 days was investigated. A significant fall (ANOVA, F_0.01) in progesterone secretion rate, which reached a nadir at $\text{5.3 ± 2.2}\text{h (}\text{x}\text{±}\text{S.D.}\text{, n=15)}$, was followed by a recovery of progesterone secretion rate. Permanent luteal regression did not occur with this protracted regimen, suggesting that a relatively short pulse frequency of PGF_2α over a minimal period of 24 h is a necessary condition for physiological regression of the corpus luteum in sheep.     

Inhibition of prostaglandin F2α-induced reflex bradycardia and hypotension by meclofenamic acid

Intravenous injection of prostaglandin F_2α (4–15 μg/kg, i.v.) produces an increase in pulmonary arterial pressure in conjunction with reflex bradycardia and hypotension in the anesthetized cat. Meclofenamic acid (30 mg/kg, i.v.) inhibited the bradycardia and the reflex contribution to the systemic hypotension. Neither the PGF_2α-induced pulmonary vasoconstriction nor the direct systemic vasodilator actions of PGF_2α were blocked by meclofenamate. In addition, the reflex responses caused by i.v. veratrine and 5-HT were not inhibited by meclofenamate. These results suggest that meclofenamic acid selectively blocks the afferent mechanism by which PGF_2α induces reflex bradycardia and hypotension in the cat.     

Levels of prostaglandin F2α in amniotic fluid during pregnancy and labour

Levels of prostaglandin F_2α (PGF_2α) in the amniotic fluid were determined by radioimmunoassay. Concentrations of the prostaglandin were relatively constant between 15 and 35 weeks' gestation, but an increase was observed after 36 weeks. The rise was continued up to 44 weeks. A still greater elevation of PGF_2α levels was recorded during labour, when the levels were related to the amount of cervical dilatation.Amniotic fluid PGF_2α levels in toxaemia of pregnancy did not significantly differ from those found in normal pregnancy.     

Radioimmunoassay of main urinary metabolite of prostaglandin F2α in normal subjects

Radioimmunoassay of 5α,7α-dihydroxy-11-keto-tetranorprosta-1, 16-dioic acid, main urinary metabolite of prostaglandin F F_2α (PGE_2α-MUM), was performed in normal subjects. Twenty-four hours secretion of PGF_2α-MUM were 29.04 ± 9.73 μg in males and 18.22 ± 5.88 μg in females on an average. And an oral administration of aspirin resulted in the remarkable decrease of PGF_2α-MUM in both sexes.     

Mechanism of the inhibition of platelet aggregation produced by prostaglandin F2α

The inhibition of human platelet aggregation produced by PGF_2α is not specific for thromboxane A₂ mimetics. Aggregation waves induced by PAF and thrombin are also inhibited by PGF_2α (8 μM); ADP is unaffected. These effects are still seen in platelets from aspirin-treated donors and platelets desensitized to thromboxane-like agonists (e.g. 11,9-epoxymethano PGH₂). In contrast the thromboxane receptor antagonist EP 045 (up to 20 μM) had no effect on primary aggregation induced by PAF, thrombin and ADP. We have previously shown that EP 045 (IC₅₀ = 0.5 μM), displaces the specific binding of [³H] 9,11-epoxymethano PGH₂ to washed human platelets.PGF_2α produces small increases in cAMP levels, and both this effect and the anti-aggregation are diminished by the adenyl cyclase inhibitor SQ 22536. The rise in cAMP induced by PGF_2α is inhibited to a greater extent by the presence of ADP than by thrombin, PAF or a thromboxane mimetic. The ability of aggregating agents to inhibit this increase correlates inversely with their sensitivity to inhibition by PGF_2α.We suggest that the very weak effect of PGF_2α on cyclic AMP_ production is sufficient to account for its inhibitory activity, and it is unlikely to be a competitive antagonist at the platelet thromboxane receptor as suggested by others.     

Mass fragmentographic determination of prostaglandin F2α in human and rabbit urine

Analysis of prostaglandin F_2α (PGF_2α) in urine is a useful indicator of renal prostaglandin synthesis. A mass fragmentographic method for PGF_2α analysis in human urine was developed using [3,3,4,4-²H₄]PGF_2α as an internal standard and carrier. PGF_2α was extracted from urine (20 ml) with chloroform, purified by preparative thin-layer chromatography and converted to the methyl ester trimethylsilyl ether before analysis by gas chromatograph—mass spectrometry. The specificity of the urine analysis was demonstrated by retention time and the use of two pairs of fragments m/e 494/498 and 513/517 with the same results. The coefficient of variation for duplicate analysis averaged 12.6%, n = 17. Urine from recumbent women contained 4.9 ± 2.6 (S.D.) ng/ml or 4.1 ± 1.0 ng PGF_2α per mg creatinine (n = 10) with little diurnal variation. Male urine contained 5.0 ± 2.7 (S.D.) ng/ml or 3.7 ± 2.1 ng/mg creatinine (n = 10). Similar concentrations were found in boys and in girls. These observations indicate that urinary PGF_2α originates from the kidneys with little contribution from the male accessory sexual glands. This method can also be applied to analysis of PGF_2α in rabbit urine.     

Studies on prostaglandin F2α formation caused by pentametylenetetrazol-induced convulsions in rat brain

Prostaglandin F_2α formation caused by pentametylenetetrazol convulsions was studied as a function of the duration, the doses of the convulsant and the intensity of the seizures. It was shown by the statistical analysis of the results in the case of clonic convulsions that the amount of synthetized PGF_2α did not depend on the doses of convulsant, while close relation existed between the duration and the PGF_2α production. At the same time, during tonic convulsions lasting longer than 50 sec, no more increase in the PGF_2α content of the brain was observed. An experimental model is suggested to study $\text{in vivo}$ the mechanisms regulating the brain's prostaglandin biosynthesis.Pretreatment of the animals with reserpine did not affect the rate of convulsion-induced PGF_2α-formation.     

Failure of prostaglandin F2α to affect LH secretion in the ovariectomized ewe

Prostaglandin F_2α (PGF_2α) when administered to ovariectomized ewes by intra-carotid infusion did not alter either the pattern of tonic LH secretion or the LH surge evoked by estradiol, indicating that, in the sheep, the luteolytic action of PGF_2α does not involve alteration of LH secretion by the pituitary gland.     

Effects of repeated daily injections of prostaglandin F2α on ovaries in mares

In experiment 1, seven groups of pony mares (2 or 3/group) were given either no injections (controls), or 5(5X) or 10(10X) daily subcutaneous (SC) injections of 1.25 mg PGF_2α beginning on days 1, 7 or 13 post-ovulation. Compared to controls (24.5 days), the interovulatory interval was longer (P<.05) for day 7, 10X (33.5 days) and day 13, 10X mares (49.0 days) but was not different for the remaining groups. In experiment 2, nine groups of pony mares (4/group) were given either no injections (controls) or 1(1X) or 10(10X) daily SC injections of 1.25 mg PGF_2α beginning on day 2 of estrus or on days 1, 7 or 13 post-ovulation. Compared to controls (25.0 days), the interovulatory interval was longer (P<.05) for day 13 post-ovulation, 10X mares (40.0 days) and shorter (P<.05) for day 1 post-ovulation, 10X mares (14.5 days). The interovulatory interval for the remaining groups was not different (P>.05) from that for controls. In day 13 post-ovulation, 10X mares, the longer interovulatory interval did not appear to be related to a depression in either peripheral LH concentration (no effect of treatment on LH) or on follicular development (no effect of treatment on diameter of largest follicle). This suggests that circulating levels of gonadotropins were adequate for ovarian follicular development and ovulation and the effect of repeated daily injections of PGF_2α in preventing ovulation was likely exerted at the ovarian level directly on the follicle.