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This paper reports an online SPE-LC-MS/MS method for the simultaneous quantification of prostaglandins (PGE 2 and PGF 2α ) in menstrual fluid samples. To meet this goal human peripheral serum was used as surrogate matrix. The analytes were trapped on an OASIS HLB cartridge for 3 min, for sample cleanup and enrichment, and then transferred during only 42 s to an HSS T3 C18 analytical column, for separation and analysis. Prostaglandins (PGs) were detected by selected reaction monitoring in negative ion mode, PGE 2 (m/z 351 → 315) and PGF 2α (m/z 353 → 193) using isotope-labeled internal standard (PGE 2 -d 4 , m/z 355 → 319). The concentration linear range was of 10.34-1.034 ng mL -1 and the lower limit of quantification (LLOQ) was 10.34 ng mL -1 for both PGs. Validation parameters were successfully assessed according to the European Medicines Agency guideline (EMA), also comprising the FDA normative. The method showed no matrix effect and process efficiency around 100%, in addition to only 15 min of analysis time with lower solvent consumption. The method application was carried out using two menstrual fluid sample groups: control (n = 15) and treatment group (n = 7; samples from women that used Tahiti lemon juice). The PGF 2α levels were found to be higher in treated group than in control group (p ≤ 0.05), denoting an effect of the intake of Tahiti lemon juice on the menstrual inflammatory process. The on-line method herein reported could be useful for the analysis of PGs from large research studies., (Copyright © 2019 Elsevier B.V. All rights reserved.)

There is a clinical need for more relevant coverage of bioactive lipids using smaller sample volumes. Therefore, we have validated a tandem mass spectrometry method for combined solid phase extraction of 37 compounds in the oxylipin (OxL) and 14 in the endocannabinoid (eCB) metabolome, as well as prostamides. The limits of quantification (LOQ) for compounds in the eCB metabolome were in the range 0.5-1000 fg on column, intraday accuracy and precision ranges (%) were 83-125 and 0.3-17, respectively, and interday accuracy and precision ranges (%) were 80-119 and 1.2-20, respectively, dependent upon the compound and the concentration studied. Corresponding values for OxL were 0.5 fg-4.2 pg on column (LOQ), 85-115% (inter- and intraday accuracy) and <5% (precision). The combined extraction method was successfully applied to tissues, cell extracts, human plasma and milk samples. A deeper study of levels in elk, pig and cow brain, as well as cow heart and liver revealed tissue and species-specific elevation of eicosanoids: arachidonate diols, 20-HETE and 12(S)-HEPE (cow liver), LTB4 (cow brain), and monohydroxy metabolites (HETEs), epoxides and 5-oxo-ETE in elk brain, which might be caused by factors of stress and/or post-mortem reactions in the tissues., (Copyright © 2015 Elsevier Inc. All rights reserved.)

For the first time, the potential immunomodulators prostaglandin E2 and γ-aminobutyric acid (GABA) have been revealed in the plerocercoid Diphyllobothrium dendriticum, which is a parasite in the tissues and abdominal cavity of the Baikal omul Coregonus migratorius. The localization of immunomodulators in parasite tissues was compared with the location of typical markers of the nervous system (serotonin (5-HT) and FMRF-amide) and a marker of microtubules (α-tubulin). Prostaglandin E2 was revealed in the cells that are immunoreactive to α-tubulin and are situated in the cortical parenchyma outside the central nervous system (CNS). It is supposed that prostaglandin E2 is produced by the frontal glands and is carried out onto the tegument surface through specialized ducts. Immunoreaction to GABA was revealed in the central and peripheral nervous systems. GABA-ergic neurosomes, the neurites of which form a net on the surface of muscle layers and in the subtegument, were revealed in the cerebral ganglion and main nerve cords. The morphological characteristics for the identification of serotoninergic neurons in the CNS were described.

Prostaglandins (PG) and isoprostanes (iso-PG) may be derived through cyclooxygenase or free radical pathways and are important signaling molecules that are also robust biomarkers of oxidative stress. Their quantification is important for understanding many biological processes where PG, iso-PG, or oxidative stress are involved. One of the common methods for PG and iso-PG quantifications is LC-MS/MS that allows a highly selective, sensitive, simultaneous analysis for prostanoids without derivatization. However, the currently used LC-MS/MS methods require a multi-step extraction and a long (within an hour) LC separation to achieve simultaneous separation and analysis of the major iso-PG. The developed and validated for brain tissue analysis one-step extraction protocol and UPLC-MS/MS method significantly increases the recovery of the PG extraction up to 95 %, and allows for a much faster (within 4 min) major iso-PGE2 and -PGD2 separation with 5 times narrower chromatographic peaks as compared to previously used methods. In addition, it decreases the time and cost of analysis due to the one-step extraction approach performed in disposable centrifuge tubes. All together, this significantly increases the sensitivity, and the time and cost efficiency of the PG and iso-PG analysis.

A simple extraction method for the analysis of PGE(2) and PGF(2α) in gonad samples from Atlantic cod and further quantification by using liquid chromatography-tandem mass spectrometry is proposed. The evaluation of the best solvent extraction conditions and the analytical performance parameters are reported. The method was highly selective for both prostaglandins and the calibration curves, based on the internal standard method, were linear between 5 and 1000 ng mL(-1) for PGE(2) and PGF(2α), with limits of detection of 1 ng mL(-1) and 1.5 ng mL(-1) and recovery values of 99.999±0.002 and 99.967±0.023 respectively. The homogenization of samples using liquid nitrogen combined with the developed extraction protocol can be implemented in different types of biological tissues., (Copyright © 2012 Elsevier B.V. All rights reserved.)

Culturing cells in three dimension (3D) provides an insight into their characteristics in vivo. We previously reported that human mesenchymal stem/stromal cells (hMSCs) cultured as 3D spheroids acquire enhanced anti-inflammatory properties. Here, we explored the effects of hMSC spheroids on macrophages that are critical cells in the regulation of inflammation. Conditioned medium (CM) from hMSC spheroids inhibited lipopolysaccharide-stimulated macrophages from secreting proinflammatory cytokines TNFα, CXCL2, IL6, IL12p40, and IL23. CM also increased the secretion of anti-inflammatory cytokines IL10 and IL1ra by the stimulated macrophages, and augmented expression of CD206, a marker of alternatively activated M2 macrophages. The principal anti-inflammatory activity in CM had a small molecular weight, and microarray data suggested that it was prostaglandin E2 (PGE2). This was confirmed by the observations that PGE2 levels were markedly elevated in hMSC spheroid-CM, and that the anti-inflammatory activity was abolished by an inhibitor of cyclooxygenase-2 (COX-2), a silencing RNA for COX-2, and an antibody to PGE2. The anti-inflammatory effects of the PGE2 on stimulated macrophages were mediated by the EP4 receptor. Spheroids formed by human adult dermal fibroblasts produced low levels of PGE2 and displayed negligible anti-inflammatory effects on stimulated macrophages, suggesting the features as unique to hMSCs. Moreover, production of PGE2 by hMSC spheroids was dependent on the activity of caspases and NFκB activation in the hMSCs. The results indicated that hMSCs in 3D-spheroid cultures are self-activated, in part by intracellular stress responses, to produce PGE2 that can change stimulated macrophages from a primarily proinflammatory M1 phenotype to a more anti-inflammatory M2 phenotype., (Copyright © 2012 AlphaMed Press.)

The primary prostaglandins PGE(2) and PGF(2 alpha) are metabolized in tissues by a series of enzymatic and non-enzymatic reactions. To measure metabolic rates and individual reaction rates it is necessary to extract the parent prostaglandins and metabolites before the separation and quantification of each compound is achieved. Here we have established and optimized a solid phase extraction (SPE) procedure to recover PGE(2), PGF(2 alpha) and their six enzymatic and non-enzymatic tissue metabolites from aqueous solutions including urine, plasma and tissue homogenate. We have used octadecyl-bonded silica gel as the stationary phase and methanol-water mixtures as binary mobile phases. The volumes and concentrations of the washing and elution solutions were optimized individually for each PG. Recoveries of all PG standards were quantitative except for PGEM, which was recovered at 80% efficiency. Biological matrix components interfered with the extraction in a PG- and matrix-specific fashion. Inclusion of 1% formic acid in the loading mixture raised recoveries from urine, plasma and tissue homogenate to >or=90%. This SPE method is the first that has been optimized by systematic elution studies for PGE(2), PGF(2 alpha) and the complement of their tissue metabolites. The procedure is simple, robust and can serve as an effective pre-purification step before downstream separation and quantification of each tissue metabolite of PGE(2) and PGF(2 alpha) from complex biological matrices.

A new extraction method has been developed for the extraction of prostaglandin E(2) (PGE(2)) from human plasma of patients suffering chronic inflammatory disorders. The extraction solvents were optimised systematically and simultaneously by using a central composite design. The optimised method involves precipitation of the protein fraction, centrifugation, evaporation and dissolution of the supernatant in the mobile phase, screening to confirm the presence of the analyte, and quantification of the positive samples by liquid chromatography tandem ion-trap mass spectrometry. Tandem mass spectrometry in negative mode was performed by isolating and fragmenting the ion [PGE(2)-H](-) signal m/z 351. Identification and quantification was carried out by extracting the ion fragment chromatograms at 333, 315 and 271 m/z. The quantitative determination was linear for the low nanogram (1-50 ng/ml) and upper picogram (400-1000 pg/ml) range studied, using 15 and 0.5 ng/ml of internal standard, respectively. The lower limit of detection was 2.5 pg for an injection volume of 25 microl. The optimised extraction method showed high reproducibility (coefficients of variation<4%) and recovery values, estimated from standard addition experiments, ranging from 96 to 98%.

A number of immunomodulatory molecules are present in the placenta, including cytokines, prostaglandins, progesterone and indoleamine 2,3-dioxygenase. An undefined factor capable of down-regulating T-cell activity has recently been reported [1] as being produced by short-term cultures of placental fragments. By careful repetition of these studies we have confirmed that chorionic villi isolated from term placenta produce a low molecular weight, heat stable factor capable of inhibiting the IL-2-dependent proliferation of mouse CTLL-2 cells. This activity was not due, however, to a previously unknown immunosuppressive molecule, but rather to prostaglandin E2 (PGE2). Expression of cyclooxygenase (COX)-2 was detected in the syncytiotrophoblast of chorionic villi explants using immunohistochemistry. Culture of the explants in the presence of the COX-1/COX--2 inhibitors indomethacin and diclofenac, or with the COX-2-selective inhibitor DFP, blocked the production of the immunosuppressive factor. The immunosuppressive activity was restored by adding PGE2 to the supernatants obtained from diclofenac-inhibited explants. A number of different receptors are involved in mediating the biological effects of prostaglandins. By utilizing selective antagonists of individual receptors, we have established that the immunosuppressive effect of PGE2 on CTLL-2 cells is exerted via the EP4 receptor. Thus, addition of an EP4-selective antagonist, but not of EP1 or EP3 antagonists, abolished the immunosuppressive effect of PGE2 on CTLL-2 cells. This may have implications for attempts to selectively manipulate T-cell responses.

Bacterial hydroxy fatty acids and alpha-hydroxy fatty acids have been demonstrated in complex lipid extracts of subgingival plaque and gingival tissue. However, little is known about the relationship between these hydroxy fatty acids in plaque and gingival tissues or the significance of these complex lipids in promoting inflammatory periodontal disease. The present study determined the percentages of ester-linked and amide-linked hydroxy fatty acids in complex lipids recovered from plaque and gingival tissue samples and the relationship between bacterial hydroxy fatty acids and alpha-hydroxy fatty acids in the lipid extracts. To evaluate a potential role for these hydroxy fatty acids in inflammatory periodontal disease, gingival tissue samples were examined for a relationship between prostaglandin E2 (PGE2) and hydroxy fatty acids recovered in gingival lipid. This investigation demonstrated that alpha-hydroxy fatty acids are only ester linked in plaque lipids but are largely amide linked in gingival tissue lipids. Furthermore, the level of alpha-hydroxy fatty acid in gingival lipid is directly related to the level of the bacterial hydroxy fatty acid 3-OH iso-branched C17:0 (3-OH iC17:0) in the same lipid extract. However, the relationship between hydroxy fatty acids in gingival lipids does not parallel the fatty acid relationship observed in plaque lipids. Finally, alpha-hydroxy fatty acid levels in gingival tissue lipids correlate directly with the recovery of PGE2 in the same tissue samples. These results demonstrate that alpha-hydroxy fatty acid levels in gingival lipids are directly related to both 3-OH iC17:0 bacterial lipid levels and PGE2 levels. These results indicate that in periodontal tissues there are unusual host-parasite interactions involving penetration of bacterial lipid in association with an altered gingival lipid metabolism and prostaglandin synthesis.

D2/E2-isoprostanes are prostaglandin D2/E2-like compounds that are produced in vivo as non-enzymatic products of free radical catalyzed peroxidation of arachidonic acid. One E2-isoprostane that should be formed is 15-E2t-isoprostane (8-iso-prostaglandin E2). 15-E2t-isoprostane has been shown to exert potent biological activity but proof that it is formed in vivo is lacking. Evidence is now presented that 15-E2t-isoprostane is formed in vivo by demonstrating that an endogenous E2-isoprostane with a retention time on capillary GC identical with that of 15-E2t-isoprostane co-chromatographs through four high resolving HPLC purification procedures with authentic radiolabeled 15-E2t-isoprostane.

Prostaglandin E2 is observed at elevated levels during human immunodeficiency virus (HIV) infection and thus may contribute to the HIV-dependent immunosuppression. The mechanisms responsible for this increase are not understood. Evidence indicates that the viral envelope proteins perturb membrane signaling mediated by the CD4 receptor, suggesting that the free envelope protein and/or the intact virus may be responsible for the increase in prostaglandin E2 levels. In this study, we have used THP-1 human monocytes and THP-1 cells differentiated by 12-O-tetradecanoylphorbol-13-acetate treatment into macrophages to determine if the HIV envelope protein, gp120, or an anti-CD4 receptor antibody stimulates prostaglandin formation by interacting with the CD4 receptor. Incubation of THP-1 cells with OKT4A antibody greatly stimulated the CD4-p56lck receptor complex as estimated by enhanced p56lck autophosphorylation, while the gp120 gave small but significant responses. Monocytic THP-1 cells poorly metabolized arachidonic acid to prostaglandin E2 and thromboxane B2 as measured by high-pressure liquid chromatography analysis. Western blot (immunoblot) and Northern (RNA) blot analyses revealed that unstimulated monocytes expressed little prostaglandin H synthase 1 and 2 (PGHS-1 and -2). Incubation of the monocytes with lipopolysaccharide, OKT4A, or gp120 did not increase the formation of prostaglandins. The expression of PGHS-1 or PGHS-2 was also not increased. Differentiation of the monocytes to macrophages by 12-O-tetradecanoylphorbol-13-acetate treatment resulted in increased expression of PGHS-1 and increased formation of prostaglandins compared with that for the monocytes. Lipopolysaccharide stimulation of the macrophages increased the formation of prostaglandins and increased the expression of PGHS-2 in the macrophages. However, OKT4A or gp120 preparation, at concentrations that stimulated p56lck autophosphorylation, did not enhance the formation of prostaglandins or the expression of PGHS-1 or PGHS-2. OKT4A and gp120 also did not stimulate the release of arachidonic acid, indicating that phospholipase A2 was not activated by the CD4 receptor in either the THP-1 monocytes or macrophages. These results indicate that activation of the CD4-p56lck receptor signal transduction pathway by the HIV envelope protein does not increase prostaglandin formation.

Our earlier studies have shown that mammalian pancreatic group I phospholipase A2 (PLA2-I) has its specific receptor (PLA2 receptor) on a wide range of mammalian cells and that the receptor-binding capability of PLA2-I is a property of this molecule separable from its enzymatic activity. To clarify whether PLA2 activity is required for eliciting a biological response via the receptor or not, we examined the enzymatic activity of PLA2-I/PLA2 receptor complex and the inducibility of prostaglandin (PG) E2 production in rat mesangial cells by mutant PLA2s-I. Using a recombinant soluble PLA2 receptor, we first found that PLA2-I could not hydrolyze a phospholipid substrate when complexed with the receptor. In the next experiment using various mutant porcine PLA2s-I, we found that PGE2 production in rat mesangial cells could be induced by a mutant PLA2-I which retained the receptor-binding activity but had almost completely lost its enzymatic activity. These findings indicate that the enzyme action of PLA2-I is not required for a PLA2-I-induced biological response, i.e., the augmentation of PGE2 production in rat mesangial cells.

The role of glutathione in protecting prostaglandin (PG) generation after exposure of fibroblasts to oxidant stress was investigated. Exposure of 3T3 fibroblasts to H2O2, followed by washing and then 20 microM arachidonic acid, caused a dose-dependent decrease in PG synthesis as assessed by radioimmunoassay. PGE2 production decreased from 3.7 +/- 1.1 to 0.15 +/- 0.04 pmol/microgram protein, and prostacyclin (PGI2) formation decreased from 0.56 +/- 0.03 to 0.06 +/- 0.03 pmol/microgram protein after exposure to 200 microM H2O2. Decreasing intracellular glutathione with 50 micrograms/ml 1,3-bis(chloroethyl)-1-nitrosourea (BCNU) enhanced the H2O2-induced decrease in PGE2 synthesis. Another glutathione-depleting agent, 1-chloro-2,4-dinitrobenzene (CDNB), also potentiated the H2O2-induced decrease in PGE2 formation. However, although PGI2 production was decreased by H2O2, neither BCNU nor CDNB potentiated this decrease. Without oxidant stress, extreme glutathione depletion decreased PGE2 synthesis and caused PGI2 synthesis to exceed PGE2. In summary, oxidant stress decreases both PGE2 and PGI2 formation. However, the primary effect of decreasing cell glutathione during oxidant stress is a reduction in PGE2 formation, not PGI2. This implies that the predominant effect of glutathione depletion during oxidant stress is on the PGE2 isomerase(s) and not PGH synthase or PGI2 synthase.

Objective: The effects of a phorbol ester activator of protein kinase C and the glucocorticoids cortisol and dexamethasone on the enzyme activity of prostaglandin H synthase (cyclooxygenase) in confluent cultures of human amnion epithelial cells were tested., Study Design: Amnion epithelial cells from spontaneously delivered placentas at term were isolated and grown in culture until confluent. The activity of the enzyme prostaglandin H synthase was determined in these cells with a well-characterized enzyme assay monitoring the conversion of arachidonic acid to prostaglandin E2. The Michaelis-Menten constant and maximum velocity were determined from the substrate velocity data by means of Lineweaver-Burk plots., Results: Amnion cells lost most of their prostaglandin H synthase activity within 2 days of culturing. This activity could be restored when cells were treated with 12-O-tetradecanoyl phorbol-13-acetate, a phorbol ester activator of protein kinase C, or with the glucocorticoids cortisol and dexamethasone. Epidermal growth factor also increased the specific activity, whereas 17 beta-estradiol had no effect on the specific activity of the enzyme. There was a direct correlation between the specific activity of prostaglandin H synthase and the output of prostaglandin E2 by cells treated with these agonists., Conclusion: Our data support the view that increases in prostaglandin H synthase specific activity in intrauterine tissues can be caused by stimulation by specific agonists, and this in turn is responsible for enhanced prostaglandin output by these tissues.

When human synovial fluid as such was subjected to radioimmunoassays of prostaglandin E2 (PGE2) and leukotriene C4 (LTC4), there was no linear increase in PGE2 and LTC4 as the amount of synovial fluid was raised. For removal of substances thus disturbing the assay we developed a method of immunoaffinity purification of PGE2 and LTC4. A monoclonal antibody against PGE2 or LTC4 was coupled to BrCN-activated Sepharose 4B. When synovial fluid mixed with radiolabelled PGE2 or LTC4 was applied to the column of immobilized antibody, the ligand was adsorbed to the column and eluted with a mixture of methanol/water in a recovery of about 80%. The purified material showed a linearity between the amount of the sample and the value of radioimmunoassay. The one-step method was applied to synovial fluid from patients with rheumatoid arthritis, osteoarthritis and other joint diseases.

Prostaglandin E1 (PGE1) and prostaglandin E2 (PGE2), derived by enzymatic oxidation of cellular dihomogammalinolenic acid (DHLA) and arachidonic acid (AA), respectively, have diverse and, at times, distinct biological actions. It has been suggested that PGE1 specifically inhibits a variety of inflammatory processes, and, in light of the potential therapeutic benefit of PGE1 and its fatty acid precursor in inflammatory disorders, there is growing interest in the biochemical mechanisms which determine the balance between PGE1 and PGE2 synthesis. Metabolic studies in this area have been hampered by the difficulties in measuring the extremely small masses of these prostaglandins which are generated in cell culture systems. We studied the regulation of PGE1 versus PGE2 synthesis using an essential fatty acid-deficient, PGE-producing, mouse fibrosarcoma cell line, EFD-1. Because EFD-1 cells contain no endogenous AA or DHLA, we were able to replete the cells with AA and DHLA of known specific activities; thus, the mass of both cellular AA and DHLA, and synthesized PGE1 and PGE2, could be accurately determined. The major finding of this study is that production of PGE2 was highly favored over production of PGE1 due to preferential incorporation of AA versus DHLA into, and release from, the total cellular phospholipid pool. Further, we correlated the selective release of AA versus DHLA from total cellular phospholipids with the selective incorporation of AA versus DHLA into specific phospholipid pools. In addition, we showed that conversion of DHLA to AA by delta 5 desaturase was enhanced by increasing the cellular mass of n-6 fatty acids and by increasing the cell proliferative activity. Together, these results indicate that the relative abundance of PGE2 versus PGE1 in vivo is not merely a function of the relative abundance of AA versus DHLA in tissues, but also relates to markedly different cellular metabolism of these two fatty acids.

The profiling of eicosanoids, including prostaglandin E2 (PGE2), prostaglandin F2 alpha (PGF2 alpha), 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha), thromboxane B2 (TXB2) and leukotriene B4 (LTB4), in dog and human gall bladders was carried out by a combination of an effective and convenient clean-up procedure and gas chromatography with selected-ion monitoring. The clean-up procedure was based on the stepwise elution of their methyl ester derivatives from a silica gel column with n-hexane-ethyl acetate and ethyl acetate-methanol in various ratios. The LTB4 methyl ester was eluted with an n-hexane-ethyl acetate (2:1, v/v) fraction because LTB4 is more lipophilic than the other eicosanoids. The present method permitted the quantitation of trace amounts of eicosanoids, including LTB4, present in tissues in the order of pg/mg of protein, without interference from other endogenous substances. In experimental acalculous cholecystitis produced in dog, the levels of eicosanoids (except LTB4) were significantly changed. Of these eicosanoids, the level of 6-keto-PGF1 alpha was significantly higher in the seromuscular layer and correlated with the observed severe morphological changes. In human chronic cholecystitis with gallstones, the level of 6-keto-PGF1 alpha in the mucosal layer was significantly higher than that in the seromuscular layer. These data suggest that prostaglandin I2 may play an important pathophysiological role in the course of cholecystitis.

After the parenteral administration of the 3H-labeled prostaglandin E2-analogue (PGE2-analogue) to three healthy women, a number of metabolites were observed in the plasma, some of them still potentially pharmacologically active. The metabolite pattern in human plasma was very similar to the one observed in the urine of female guinea pigs, which received a total dose of 0.21 mg [3H]sulprostone by repeated sc administration over a period of 5 days. In vitro perfusion of an isolated guinea pig liver with 50 mg of [3H]sulprostone, dissolved in Tyrode's solution, yielded another source for the isolation of those metabolites, which were also present in human plasma. Both urine and perfusion medium were submitted to repeated HPLC-separations and the recovery during the purification procedure was calculated for each metabolite on the basis of recovered radioactivity after each step of purification. Chromatographically pure metabolites were submitted to GC/MS analysis, 1H-NMR and IR spectroscopy for structural elucidation. Besides the unchanged parent compound, four metabolites of sulprostone could be identified in human plasma by co-chromatography with the isolated compounds. One of two major metabolites was the PGA2-analogue of the parent drug, the other was a cyclization product of the beta-side chain of sulprostone with the cyclopentenone ring, preceded by delta 13 reduction. The two minor metabolites were the free acids of sulprostone and the PGA2-analogue.

Free fatty acids in isolated pancreatic islets have been quantified by gas chromatography-mass spectrometry after stimulation with insulin secretagogues. The fuel secretagogue D-glucose has been found to induce little change in islet palmitate levels but does induce the accumulation of sufficient unesterified arachidonate by mass to achieve an increment in cellular levels of 38-75 microM. Little of this free arachidonate is released into the perifusion medium, and most remains associated with the islets. Glucose-induced hydrolysis of arachidonate from islet cell phospholipids is reflected by release of the arachidonate metabolite prostaglandin E2 (PGE2) from perifused islets. Both the depolarizing insulin secretagogue tolbutamide (which is thought to act by inducing closure of beta-cell ATP-sensitive K+ channels and the influx of extracellular Ca2+ through voltage-dependent channels) and the calcium ionophore A23187 have also been found to induce free arachidonate accumulation within and PGE2 release from islets. Surprisingly, a major fraction of glucose-induced eicosanoid release was found not to require Ca2+ influx and occurred even in Ca(2+)-free medium, in the presence of the Ca(2+)-chelating agent EGTA, and in the presence of the Ca2+ channel blockers verapamil and nifedipine. Exogenous arachidonic acid was found to amplify the insulin secretory response of perifused islets to submaximally depolarizing concentrations of KCl, and the maximally effective concentration of arachidonate was 30-40 microM. These observations suggest that glucose-induced phospholipid hydrolysis and free arachidonate accumulation in pancreatic islets are not simply epiphenomena associated with Ca2+ influx and that arachidonate accumulation may play a role in the signaling process which leads to insulin secretion.

The effects of histamine on endogenous prostaglandins and on the damage caused by 0.6 N HCl were tested in rat gastric mucosa. The damage was assessed both macroscopically and microscopically. Histamine increased the prostaglandin E2 level in vivo and stimulated the synthesis of prostaglandin E2 and 6-keto-prostaglandin F1 alpha in vitro. These effects of histamine were inhibited by treatment with cimetidine beforehand. Histamine inhibited both the gross visible mucosal damage and deep necrosis caused by the intragastric instillation of 0.6 N HCl. This effect was overcome by cimetidine or indomethacin, but not by diphenhydramine. These results suggest that histamine stimulates prostaglandin synthesis and induces cytoprotection in rat gastric mucosa, perhaps with the involvement of the H2 receptor.

18-Hydroxy-PGE1 and 18-hydroxy-PGE2 were identified in human seminal fluid by capillary gas-liquid chromatography-mass spectrometry. The levels of these prostaglandins was 1-2% of the corresponding 19-hydroxy-PGE compounds in human semen. 18-Hydroxy-PGE1 and 18-hydroxy-PGE2 are likely formed by cytochrome P-450 in seminal vesicles in analogy with the 19-hydroxy-PGE compounds. This was supported by the finding that microsomes of seminal vesicles of the cynomolgus monkey, Macaca fascicularis, supplemented with 1 mM NADPH, metabolized PGE1 to both 19-hydroxy-PGE1 (92%) and 18-hydroxy-PGE1 (8%). The hydroxylation of prostaglandins in seminal vesicles of primates may thus show a high but not absolute specificity for the penultimate carbon of prostaglandins.

A procedure using a solid-phase support has been developed for the isolation and derivatization of prostaglandins from biological matrices. The styrene-divinylbenzene cross-linked copolymeric macroreticular resin, XAD-2, was used as an adsorbent for prostaglandin E2 from biological samples, as a support for the oximation of the carbonyl group and as a catalyst for pentafluorobenzylation. The reactor bed was then linked to a Florisil column for a final chromatographic clean-up. Matrix effects were found to affect the yield, but recovery of the desired electrophoric products was comparable with methods reported in the literature. The ease of sample preparation suggests that this technique may be a viable approach to automating the processes for preparing prostaglandins from biological matrices for gas chromatographic analysis.

The content of prostaglandins in Yoshida tumor ascites fluid was evaluated by extraction of the arachidonic acid metabolites by means of octadecylsilyl-silica chromatography (ODS-extracts). The pattern of association of the icosanoids to ascites fluid colloids was determined by sequential precipitation with polyethylene glycol and extraction of the precipitates with methanol (M-extracts). Thin layer chromatographic analysis of these extracts demonstrated in the ODS-extract the presence of chromatographic fractions corresponding to prostaglandin E2 (4.6 micrograms/mL) and prostaglandin A2 (3.0 micrograms/mL), while in the M-extract only the fraction corresponding to prostaglandin A2 (9.2 micrograms/mL) was detected. This fraction was as the growth inhibitory activity reduced by approximately 60% in M-extracts from animals treated with Indomethacin. These results support previous indications that arachidonic acid metabolites are involved in the cytostatic activity of ascites fluid extracts. The preferential association of prostaglandin E2 to the ascites macroglobulin fraction could be a consequence of the presence of an unidentified tumor-dependent amphiphilic polymer in the Yoshida ascites fluid.