John A. Salmon

The chemical structure of prostaglandin X (prostacyclin)

The chemical structure of prostaglandin X, the anti-aggregatory substance derived from prostaglandin endoperoxides, is 9-deoxy-6, 6alpha-epoxy-delta5-PGF1alpha. The stable compound formed when prostaglandin X undergoes a chemical transformation in biological systems in 6-keto-PGF1alpha. Prostaglandin X is stabilized in aqueous preparations by raising the pH to 8.5 or higher. The trivial name prostacyclin is proposed for 9-deoxy-6, 9alpha-epoxy-delta5-PGF1alpha.

Further studies on the enzymatic conversion of prostaglandin endoperoxide into prostacyclin by porcine aorta microsomes.

A simple, rapid radiochemical assay for prostacyclin synthesis has been used to characterize the enzyme in arterial walls which converts prostaglandin endoperoxides to prostacyclin. The enzyme displays a broad pH optimum, and catalyses a rapid conversion of saturating concentrations of the endoperoxide at 37 degrees C. Hydroperoxides of several unsaturated fatty acids are potent inhibitors of the enzyme, and act in a time dependent manner. The isomerase which converts prostaglandin endoperoxides to prostaglandin E2 or D2 was not detected in the arterial wall.

Biosynthesis and biological activity of leukotriene B5

Several studies indicate that increased intake of eicosapentaenoic acid (EPA) in the diet may lead to decreased incidence of thrombotic events. Most investigators agree that this is achieved by competitively inhibiting the conversion of arachidonic acid (AA) to thromboxane A2 in the platelets. The effect of high EPA-intake on the formation of prostacyclin is less clear. However, EPA is a good substrate for lipoxygenase enzymes which results in formation of hydroperoxy- and hydroxy-acids, and, in some cases, leukotrienes. The biological activities of the leukotrienes derived from arachidonic acid suggest that they mediate or modulate some symptoms associated with inflammatory and hypersensitivity reactions. In order to clarify the possible effect of dietary manipulation on inflammatory processes, leukotriene B5 (LTB5) was prepared and its biological activities assessed. LTB5 was biosynthesized by incubation EPA with glycogen-elicited polymorphonuclear neutrophils (PMN) from rabbits in the presence of the divalent cation ionophore, A23187. The LTB5 was extracted from the incubate using mini-reverse phase extraction columns (Sep-pak) and purified by reverse-phase high pressure liquid chromatography (RP-HPLC). The purity of the product assessed by repeat RP-HPLC and straight phase (SP) HPLC was greater than 95%. Ultra-violet spectrophotometry of the product confirmed its purity and also provided assessment of the yield. The biological activity of LTB5 was assessed and compared with that of LTB4 in the following tests: aggregation of rat neutrophils, chemokinesis of human PMN, lysosomal enzyme release from human PMN and potentiation of bradykinin-induced plasma exudation. In all these tests, LTB5 was considerably less active (at least 30 times) than LTB4.

A radioimmunoassay for leukotriene B4

A radioimmunoassay for leukotriene B4 has been developed. The assay is sensitive; 5 pg LTB4 caused significant inhibition of binding of [3H]-LTB4 and 50% displacement occurred with 30 pg. The specificity of the assay has been critically examined; prostaglandins, thromboxane B2 and arachidonic acid do not exhibit detectable cross-reactions (less than 0.03%). However, some non-cyclic dihydroxy- and monohydroxy-eicosatetraenoic acids do cross-react slightly (e.g. diastereomers of 5,12-dihydroxy-6,8,10-trans-14-cis-eicosatetraenoic and 12-hydroxy-5,8,10,14-eicosatetraenoic acids cross-react 3.3% and 2.0% respectively). The assay has been used to monitor the release of LTB4 from human neutrophils in response to the divalent cation ionophore, A23187. The immunoreactive material released during these incubations was confirmed as LTB4 by reverse phase high pressure liquid chromatography following solvent extraction and silicic acid chromatography.

The release of leukotriene B4 during experimental inflammation

Leukotriene B4 (LTB4) has been detected by radioimmunoassay in inflammatory exudates obtained following the implantation of saline- or carrageenan-soaked polyester sponges in rats. The immunoreactive material was confirmed as LTB4 after extraction and purification by high pressure liquid chromatography. The peak concentration (6.9 +/- 0.5 ng/ml) was detected 6 hr after implantation of sponges soaked in 0.5% carrageenan; thereafter the level declined and was undetectable after 16-24 hr. The concentration of LTB4 during the early phase of the inflammatory response (4-8 hr) is sufficient to induce leukocyte aggregation, chemotaxis and degranulation of polymorphonuclear leukocytes (PMN) in vitro. Therefore, LTB4 may mediate, at least in part, the influx of PMN and contribute to other events which characterise the inflammatory response. The level of thromboxane B2 (TXB2) in the inflammatory exudate followed a similar time-course to that of LTB4 although the maximum concentration was higher (15-30 ng/ml). However, prostaglandin E2 (PGE2) exhibited a different time-course; the maximum level (20-30 ng/ml) was also reached 6-8 hr after implantation but remained elevated at 24 hr. The PMN count in the sponges and the concentrations of both LTB4 and TXB2, but not PGE2, were significantly reduced by prior treatment of the animals with colchicine. This suggests that PMN are the major source of LTB4 and TXB2 in the inflammatory exudate whereas PGE2 is produced in significant amounts by other tissues.

[59] Extraction and thin-layer chromatography of arachidonic acid metabolites

Publisher Summary This chapter discusses the extraction and thin-layer chromatography of arachidonic acid metabolites. In some cases prostaglandins may be determined directly in aqueous samples (e.g., radioimmunoassay or bioassay of tissue perfusates and cell culture fluids) without recourse to extraction, but more usually, extraction into an organic solvent is a prerequisite to separation and/or quantitation. The advantages of extraction are that it eliminates protein, imparts some specificity to the assay and, by concentrating material, improves the sensitivity of the analysis. The choice of any particular extraction technique is governed by its efficiency, specificity, reproducibility, and practicability. The extraction procedures efficiently remove all fatty acids, prostaglandins, and other hydroxy acids, but obviously if specific analysis of a particular compound is required some further purification is necessary. Many chromatographic procedures can be employed for analysis of prostaglandins, but thin layer chromatography has much to commend it in terms of efficiency, simplicity, and economy of money and effort.

The effects of BW755C and other anti-inflammatory drugs on eicosanoid concentrations and leukocyte accumulation in experimentally-induced acute inflammation

BW755C (3‐amino‐1‐[m‐trifluoromethyl)phenyl]‐2‐pyrazoline HCl) reduced the concentration of leukotriene B4 (LTB4), thromboxane B2 (TXB2) and prostaglandin E2 (PGE2) in exudate derived from the subcutaneous implantation in rats of 0˙5% carrageenanimpregnated polyester sponges. Polymorphonuclear leukocyte (PMN) migration into the inflammatory exudate was also decreased. The inhibition of LTB4 may, in part, account for the lower number of cells in the exudate since LTB4 is a potent leukotactic agent. Inhibition of LTB4‐formation and cell migration by BW755C was dose‐related, but the two dose‐response curves were not parallel. Cell influx still occurred at doses of BW755C that completely inhibited formation of LTB4: this indicates that, although LTB4 may have a chemotactic role in‐vivo, other factors must also contribute to cell migration into the inflammatory exudate. Treatment of rats with dexamethasone also caused a reduction in leukocytes and eicosanoids in the exudate. As with BW755C, there was a differential effect on PMN and LTB4: dexamethasone (1 mg kg−1) reduced PMN accumulation by 40% but LTB4 formation was inhibited by 70%. Leukocyte accumulation was also inhibited by the non‐steroidal anti‐inflammatory drugs (NSAIDs), indomethacin and flurbiprofen. These drugs reduced the concentration of both PGE2 and TXB2 in exudate but that of LTB4 was unchanged. This suggests that reduction of PMN accumulation by indomethacin and flurbiprofen is mediated by a mechanism other than inhibition of LTB4‐synthesis. Aspirin also reduced the levels of PGE2 and TXB2 in the exudate but did not consistently affect PMN influx, thereby confirming that inhibition of cyclo‐oxygenase does not reduce cell migration in inflammation.

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

Abstract 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.

Effect of orally administered eicosapentaenoic acid (EPA) on the formation of leukotriene B4 and leukotriene B5 by rat leukocytes

Eicosapentaenoic acid (EPA) is a poor substrate for the fatty acid cyclo-oxygenase but is a good substrate for lipoxygenase enzymes which catalyse the biosynthesis of hydroperoxy-acids, hydroxy-acids and leukotrienes. Recently, we reported that leukotriene B5 (LTB5) was at least 30 times less potent than LTB4 in causing aggregation, chemokinesis and degranulation of polymorphonuclear leukocytes in vitro. In this paper, the effect of oral administration of EPA on LTB4 and LTB5 production by rat leukocytes stimulated with the calcium ionophore, A23187, was assessed. The concentration of LTB was determined by radioimmunoassay and also by reverse-phase high pressure liquid chromatography using PGB3 as internal standard. Supplementation of a normal rat diet with EPA (240 mg/kg per day) for 4 weeks caused a significant increase in the formation of LTB5 and a decrease in the synthesis of LTB4 by stimulated leukocytes. The EPA-rich diet significantly increased the EPA content of leukocyte phospholipids without altering the content of arachidonic acid (AA) or linoleic acid. The ratio of EPA/AA in leukocytes correlated (r = 0.795, P less than 0.001) with the LTB5/LTB4 ratio produced after stimulation of leukocytes. If LTB4 has a chemotactic role during inflammation, the present data suggest that an EPA rich diet could decrease the accumulation of leukocytes at sites of inflammation.

Chemotactic response to some arachidonic acid lipoxygenase products in the rabbit eye.

The effects of arachidonic acid, its cyclo-oxygenase and lipoxygenase products and the synthetic chemotactic peptide, formyl-methionyl-leucyl-phenylalanine (FMLP) on leukocyte accumulation in the aqueous humour and intraocular pressure in the rabbit were studied in vivo. Substances were injected into the anterior chamber of the eyes of anaesthetised rabbits using a closed circuit perfusion system. Injection of arachidonic acid, prostaglandins E1 and E2, and the monohydroperoxy and hydroxy acids of the lipoxygenase pathway did not result in any significant accumulation of leukocytes in the anterior chamber. In contrast, FMLP and 5,12-diHETE (Leukotrine B4) resulted in significant dose dependent accumulation of leukocytes into the aqueous humour. Leukocytes appeared in the aqueous humour between 2 and 3 h after the injection of either FMLP or LTB4 and the response was maximal at 4 h. None of the lipoxygenase products tested had any effect on intraocular pressure in contrast to the profound effects observed with arachidonic acid and the E type prostaglandins. FMLP had a small but significant effect on intraocular pressure at the highest dose tested for leukocyte accumulation. These results indicate that the effects of the cyclo-oxygenase products of arachidonate metabolism are mainly vascular in the rabbit eye in contrast to the predominantly cellular effects of lipoxygenase products. Thus in the eye, the interaction of cyclo-oxygenase and lipoxygenase products of arachidonate metabolism may be important in the development of both acute and chronic ocular inflammation.