Separation and quantification of prostaglandins E1 and E2 as their panacyl derivatives using reverse phase high pressure liquid chromatography

Separation and quantification of prostaglandin E1 (PGE1) and prostaglandin E2 (PGE2) were achieved using reverse phase high performance liquid chromatography (HPLC). Panacyl bromide (p-(9-anthroyloxy)phenacyl bromide) (PAB) derivatives of PGE2 and PGE1 were prepared. Reverse phase HPLC using a linear gradient of 56% to 80% acetonitrile in water containing 0.10% acetic acid gave baseline resolution of the two derivatives. A 3 um diameter particle, C18 column provided good resolution and reproducible recoveries. Human synovial tissue cells were incubated with the precursor fatty acids for PGE1 or PGE2 and stimulated with a crude Interleukin 1 (IL-1) preparation. Cells grown in the presence of dihomogammalinolenic acid (DGLA), the precursor for PGE1, made significantly more PGE1 than cells grown in control medium or in the presence of arachidonic acid, precursor for PGE2. PGE2 synthesis was reduced when DGLA was added to cells (resting or IL-1-stimulated).     

Phosphatidylethanolamide derivatives of prostaglandins E1 and E2

Prostaglandins E₁ (PGE₁) and E₂ (PGE₂) have been coupled with the amine group of phosphatidylethanolamine (PE) by means of dicyclohexylcarbodiimide. These complexes basically mimic the relaxant and contractile effects of the corresponding free prostaglandins (PGs) on various smooth muscle preparations, but exhibit a delayed onset of action and a lower affinity for the PG receptors. The complexes are comparable with the free, parent PGs, in their intrinsic activities. The same holds true for the effects on blood pressure and on the motility of the uterus . The PGE₂-PE complex is hydrolysed to release obviously free PGE₂ by cell-free homogenates prepared from various tissues, but not by blood plasma. The PGE₂-PE complex is immunologically indistinguishable from the free PGE₂.     

Separation of prostaglandins E2 and F2α from their pulmonary metabolites by thin-layer chromatography

A simple thin-layer chromatographic system has been developed to separate PGE₂ and PGF_2α from 13,14-dihydro-PGF_2α and other pulmonary metabolites of prostaglandins. The separation is achieved on Kieselgel-60 F-254 plates impregnated with both silver nitrate and boric acid. The system should be of use in the purification and tentative identification of primary prostaglandins.     

Effect of prostaglandins E1, E2 and F2α on neutrophil aggregation

Recently we have found that chemotactic factors stimulate neutrophils in suspension to aggregate. Because of an obvious analogy to platelet aggregation, we examined the influence of three prostaglandins on this process. Prostaglandins E₁, E₂ and F_2α alone did not cause aggregation of the neutrophils but were able to partially inhibit the aggregation response induced by the synthetic chemotactic tripeptide, formly-methionyl-leucyl-phenylalanine. The minimal inhibitory concentrations for prostaglandins E₁, E₂ and F_2α were 10⁻⁷, 10⁻⁶ and 10⁻⁵M, respectively. These results are similar to those found for the prostaglandin-induced inhibition of platelet aggregation. It may be, therefore, that neutrophil aggregation, like platelet aggregation, is modulated by intracellular prostaglandins and other products of arachidonic acid metabolism.     

Failure of prostaglandins E1 and E2 to alter canine gastric mucosal cyclic nucleotides

The action of prostaglandins and indomethacin on gastric mucosal cyclic nucleotide concentrations was evaluated in 18 anesthetized mongrel dogs. Prostaglandins E₁ (PGE₁) and E₂ (PGE₂) (25 μg/kg bolus, then 2 μg/kg/min) were administered both intravenously (4 experiments; femoral vein) and directly into the gastric mucosal circulation (10 experiments; superior mesenteric artery). The possible synergistic effect of pre-treatment and continuous arterial infusion of indomethacin (5 mg/kg bolus for 5 min, then 5 mg/min), a prostaglandin synthetase inhibitor, with PGE₂ was studied in 4 experiments. Antral and fundic mucosa were biopsied and measured by radioimmunoassay for cyclic nucleotides. Doses of PGE₁ and PGE₂ which inhibited histamine-stimulated canine gastric acid secretion did not significantly alter antral or fundic mucosal cyclic nucleotide concentrations. Concomitant infusion of PGE₂ with indomethacin did not potentiate the mucosal nucleotide response compared to PGE₂ alone. These studies fail to implicate cyclic nucleotides as mediators of the inhibitory acid response induced by PGE₁ or PGE₂ in intact dog stomach.     

Measurement of prostaglandin E3 and other eicosanoids in biologic samples using high pressure liquid chromatography and radioimmunoassay

A method to measure PGE₃ in biologic samples is described. Complete resolution of PGE₃ from PGE₁ and PGE₂ is achieved by reverse- phase high pressure liquid chromatography. Quantification is carried out by radioimmunoassay using an antibody directed against PGE₂ that has high cross-reactivity with PGE₃. Using this method, a marked increase in PGE₃ production by mouse kidney tissue and in rat urine was demonstrated after supplemental feeding of ω-3 fatty acids. This method can also be applied to measurement of 6-keto-PGF_1α and TXB₂ in the same samples.     

Effects of prostaglandins E1 and E2 on activity in laryngeal and pharyngeal afferent fibers

Prostaglandins (PG) E₁ and E₂ were applied topically to the receptive fields of feline laryngeal and pharyngeal sensory receptors, while action potentials were recorded from single - or few-fiber preparations of the superior laryngeal nerve. When initially dissolved in ethanol, PGs stimulated these sensory receptors. If ethanol was not used as a solvent for the PGs, they did not stimulate the sensory receptors. Similarly, local application of dilute (0.025%, v/v) solutions of ethanol alone excited the receptors, whereas phosphate buffer alone did not. Thus PGE₁ and PGE₂ do not themselves stimulate sensory receptors in the larynx and pharynx. These findings suggest that irritant properties of PGEs on upper airways are attributable to the ethanol used as a solvent.     

Neurological and electroencephalographic changes in newborns treated with prostaglandins E2 and E2

Six newborns with obstructive right heart lesions were examined neurologically and electroencephalographically during treatment with prostaglandin (PG) E₁ or E₂ given to maintain patency of the ductus arteriosus and to increase pulmonary blood flow. PG was administered intravenously or intraarterially in the aortic isthmus proximal to the ductus arteriosus. Besides a rise in arterial oxygen saturation, all patients had some sign of central nervous system involvement. The electroencephalogram showed minor changes suggestive of sedation. In addition, three patients in whom PG given intravenously presented various combinations of neurological abnormalities (“myoclonic jerks”, apnoeic spells, hiccup) of subcortical origin. Side-effects subsided after stopping the treatment anf posed no problem in the management of the patients. These findings confirm the usefulness and safety of the PG therapy and indicate that the intraaortic route of administration is preferable.     

Interactions of prostaglandins with subpopulations of ovine luteal cells. I. Stimulatory effects of prostaglandins E1, E2 and I2

Preparations of small and large steroidogenic cells from enzymatically dispersed ovine corpora lutea were utilized to study the effects of luteinizing hormone (LH) and prostaglandins (PG) E₁, E₂ and I₂. Cells were allowed to attach to culture dishes overnight and were incubated with either LH (100 ng/ml), PGE₂, PGE₂, or PGI₂ (250 ng/ml each). The secretion of progesterone by large cells was stimulated by all prostaglandins tested (P < 0.05) while the moderate stimulation observed after LH treatment was attributable to contamination of the large cell population with small cells. Prostaglandins E₁ and E₂ had no effect on progesterone secretion by small cells, while LH was stimulatory at all times (0.5 to 4 hr) and PGI₂ was stimulatory by 4 hr. Additional studies were conducted to determine if the effects of PGE₂ upon steroidogenesis in large cells were correlated with stimulated activity of adenylate cyclase. In both plated and suspended cells PGE₂ caused an increase (P < 0.05) in the rate of progesterone secretion but had no effect upon the activity of adenylate cyclase or cAMP concentrations within cells or in the incubation media. Exposure of luteal cells to forskolin, a nonhormonal stimulator of adenylate cyclase, resulted in marked increases in all parameters of cyclase activity but had no effect on progesterone secretion. These data suggest that the actions of prostaglandins E₁, E₂ and I₂ are directed primarily toward the large cells of the ovine corpus luteum and cast doubt upon the role of adenylate cyclase as the sole intermediary in regulation of progesterone secretion in this cell type.     

Prostaglandin E1 and E2 in bovine semen: Quantification by gas chromatography

Prostaglandins PGE₁ and PGE₂ extracted from bovine semen, purified via silicic column chromatography were quantified by gas chromatography as their methoxime methyl ester trimethylsilyl ether derivatives. The PGE₁ and PGE₂ concentrations of 19 bovine semen samples ranged from 395 ± 225 and 487 ± 407 ng/ml, respectively. A constant 1:1 ratio between PGE₁ and PGE₂ was observed. There was no relationship between PGE and sperm motility, but high sperm counts were generally associated with decreased PGE levels. The direct precursors of PGE₁ and PGE₂, i.e. 20:3n6 and 20:4n6, occurred in low concentrations compared to other related unsaturated fatty acids, i.e. 18:2n6 and 22:5n6 of the n-6 family.     

Prostaglandin E1 and E2 in bovine semen: Quantification by gas chromatography

Prostaglandins PGE₁ and PGE₂ extracted from bovine semen, purified via silicic column chromatography were quantified by gas chromatography as their methoxime methyl ester trimethylsilyl ether derivatives. The PGE₁ and PGE₂ concentrations of 19 bovine semen samples ranged from 395 ± 225 and 487 ± 407 ng/ml, respectively. A constant 1:1 ratio between PGE₁ and PGE₂ was observed. There was no relationship between PGE and sperm motility, but high sperm counts were apparently associated with decreased PGE levels. The direct precursors of PGE₁ and PGE₂, i.e. 20:3n6 and 20:4n6, occurred in low concentrations compared to other related unsaturated fatty acids, i.e. 18:2n6 and 22:5n6 of the n-6 family.     

Presence of prostaglandins E2 and A2 in canine gastric secretions

The presence of prostaglandins (PGs) was determined in gastric juice obtained from 3 conscious dogs, provided with a chronic gastric fistula. Outputs of acid (mequiv min^?1) and PGs (pg min^?1) were measured in gastric secretions stimulated by pentagastrin (100 or 200 ng kg^?1min^?1). Prostaglandin activity was estimated, after extraction and thin layer chromatography, by radioimmuno-assay of the PGB formed by treatment with alkali. Tritiated PGs were added to gastric juice for the purpose of correcting for PGs recovery. Using this method, the minimum mass of PGB which could be satisfactorily distinguished from zero was 25 pg. Prostaglandins A₂ and E₂ were present in pentagastrin-activated gastric secretions and averaged (mean ± SE, n = 8) 200.7 ± 18.1 and 260.1 ± 18.0 pg min^?1 respectively. The identity of PGA₂ and PGE₂ was confirmed by gas liquid chromatography combined with mass spectrometry. The amount of PGE₂ converted to PGA₂ during extraction, separation and conversion procedures was estimated from the amount of [³H] PGA₂ found when only [³H] PGE₂ had been added to a sample of gastric juice and averaged 14.5% ± 2.0. Our preliminary results support the possibility that PGE₂ and PGA₂ may be of physiological importance in the regulation of canine gastric secretions.     

Radioimmunoassay of prostaglandins E1, E2, and F2α in unextracted plasma, serum and myocardium

A radioimmunoassay procedure for the determination of PGE₁, PGE₂, and PGF₂α is presented. The procedure involves the pre-precipitation of each prostaglandin specific antiserum with the precipitating antisera (ARGG), and the use of these antisera mixtures in assaying for PGE₁, PGE₂, and PGF₂α. Applicability of the methods to unextracted plasma, serum and myocardial homogenate has been demonstrated through tests of specificity, recovery, reproducability and parallelism. A mathematical correction for cross-reactivity between PGE₁ and PGE₂, and their opposing antisera is given. To demonstrate the utility of the methodology in differentiation of experimental variables, prostaglandin concentrations in unincubated serum, incubated serum, and the rate of prostaglandin production in serum of dogs are given.     

Triphasic effect of prostaglandins E1, E2 and F2α on the fluid transport of isolated gall-bladder of guinea-pigs

Prostaglandins (PGs) F_2α, E₁ and E₂ exerted a triphasic influence on the fluid transport of isolated guinea-pig gall-bladders, when applied to the serosal side. PGE₁ and PGE₂ produced these effects in lower concentrations than F_2α. Directly after PG addition to the serosal side a short stimulation of fluid transport to between 200 and 400% was observed. The stimulatory effect of PGs was most distinct in gall-bladders from female guinea-pigs, less pronounced in male and nearly absent in pregnant animals. Since PGs increased intraluminal hydrostatic pressure in gall-bladders by contraction of the smooth muscle, experiments were performed in which hydrostatic pressure was increased by different procedures. These included the addition of imidazole (10⁻² M), raising of K⁺ in the bathing solution and an increase in intraluminal pressure by addition of Ringer's solution into the lumen. All three procedures stimulated fluid reabsorption temporarily in the same way as PGs, hence increase of intraluminal pressure is thought to be the reason for the observed temporary stimulation of fluid transport. Direct evidence for this thesis was obtained when the gall-bladder was mounted as a flat sheet over a chamber; in this preparation no stimulation of fluid transport was obtained. The second phase of the PG influence was characterized by a concentration-related inhibition of fluid reabsorption followed by a significant but small reverse of fluid transport (secretion of fluid). When PGs were applied to the mucosal side, only an inhibition of fluid transport was observed, which was much weaker compared to the addition to the serosal side.     

Quantitative separation of estrogens,androgens and progestogens using reverse phase high pressure liquid chromatography

Dual UV wavelength scanning at 206 and 254 nm was used to develop a sensitive, separation and quantitation procedure for estrone, estradiol-17β, testosterone, 4-androstene-3,17,-dione, progesterone and 17-hydroxyprogesterone using reverse phase high pressure liquid chromatography. The difference in UV absorption of the estrogens from the androgens and progestogens allows for correction of the co-elution of testosterone with estradiol-17β and 4-androsten-3,17,-dione with estrone. Incorporation of an isocratic step-gradient provides improved separation while maintaining shortened elution times for the less polar steroids.     

Prostanoids and hemostasis in chickens: Anti-aggregating activity of the prostaglandins E1 and E2, but not of prostacyclin and prostaglandin D2

Aggregation of chicken thrombocytes was studied in whole blood using an electronic aggregometer. Serotonin (5-hydroxytryptamine, 5HT), arachidonic acid (AA) and collagen, but not adenosinediphosphate (ADP) induced aggregation. Prostaglandin (PG) endoperoxides were essential for arachidonic acid-induced aggregation, but were not involved in 5HT-induced aggregation, as indicated by inhibitory studies with indomethacin. Similar experiments indicated that biosynthesis of endogenous PG endoperoxides contributed to the aggregation induced by low concentrations of collagen, but was of little importance when high collagen doses were employed. PGE₁ and PGE₂ could abolish all types of aggregation studied, whereas prostacyclin (PGI₂) and PGD₂ were without any anti-aggregatory activity at 1 μg/ml. Between 1 and 100 ng/ml PGE₁ and PGE₂ inhibited arachidonic acid- and 5HT-induced aggregation dose-dependently.The lack of any hemostatic function of PGI₂ in chickens was also indicated by the absence of biosynthesis of endogenous PGI₂ in chicken aorta. PGI₂ was assessed as anti-aggregating activity, released by aortic fragments stirred in rabbit platelet rich plasma. Still, the presence of chicken aortic tissue i chicken whole blood inhibited 5HT-, but not arachidonic acid-induced aggregation. This inhibition was not affected by pretreatment of the aortic fragments with indomethacin or pargyline.     

Quantitative determination of prostaglandins E1 and F1α by multiple ion analysis

Quantitative assays for prostaglandins (PG) E₁ and PGF_1α are described using [3,3,4,4,5,6-²H₆]labeled prostaglandins as carriers and methyl ester-O-methyloxime-acetate (PGE₁) and methyl ester-acetate (PGF_1α) derivatives for gas - liquid chromatography/mass spectrometric analysis. Thin-layer argentation chromatography was used to separate PGE₁ from PGE₂ and 13, 14-dihydro-PGE₂. These latter compounds, which do not separate from PGE₁ using conventional thin-layer chromatography or under the gas - liquid chromatographic conditions used, can significantly interfere with the quantitative analysis of PGE₁. The method described prevents this interference and is therefore suitable for the accurate analysis of PGE₁ in biological samples containing a high concentration of PGE₂ and/or 13, 14-dihydro-PGE₂.     

Effects of intracerebroventricular administration of prostaglandins I2, E2, F2α and indomethacin on blood pressure in the rat

The effects of intraventricularly administered prostaglandins I₂ (PGI2), E₂ (PGE2), F_2α (PGF2α) and indomethacin on systemic blood pressure were investigated in conscious rats. PGI2 (1.25 – 10 g/kg) decreased blood pressure in a dose-related manner, whereas PGE2 (100 – 1000 ng/kg) dose-dependently increased blood pressure. Both PGF2α (0.31 – 20 μg/kg) and indomethacin (0.625 – 40 μg/kg) had no effects on blood pressure. These results indicate that intraventricular injection of PGI2 or PGE2 can induce significant changes in blood pressure, while endogenous prostaglandins synthesized in the brain seem to play a minor role in direct regulation of systemic blood pressure in the rat.     

Prostaglandins E1 and E2 stimulate release of intestinal brush border enzymes

Rat small bowel was perfused and in the absence of biliary and pancreatic secretion. Intraluminal release of sucrase, alkaline phosphatase, aminopeptidase and enterokinase was significantly increased after administration of PG E₁ and E₂ 1 and 5 μg/kg. This suggests a direct stimulation of the intestinal mucosa, which might be mediated through cyclic AMP ; dibutyryl cAMP significantly stimulates intraluminal release of proteins, sucrase and enterokinase.