[Marine fungus Stilbella aciculosa as a potential producer of prostaglandins].

The amount and composition of fatty acids in the fungus Stilbella aciculosa associated with the marine macroorganism Apostichopus japonica (trepang) were determined by gas-liquid chromatography and gas chromatography-mass spectrometry. In the culture liquid of S. aciculosa, prostaglandins (PG) of groups E and F were revealed by UV spectroscopy. This finding was confirmed by the presence of direct precursors of PG, polyunsaturated eicosapentaenoic and docosahexaenoic acids, in the culture liquid. The biomass of this fungus contained PG of group B.

The biosynthesis of the 3-series prostaglandins in rat uterus after alpha-linolenic acid feeding: mass spectroscopy of prostaglandins E and F produced by rat uteri in tissue culture.

The abnormal uterine activity associated with dietary n-3 fatty acids may result from competitive inhibition of PG2 production. Uterine synthesis of 2- and 3-series prostaglandins F(PGF) and E(PGE) was studied using mass spectrophotometry in rats fed diets containing predominantly n-3 fatty acid, n-6 fatty acid, or control pelleted diet. Mass spectra of PGF (Me, TMS and Me, TBDMS derivatives) synthesised by uteri of n-3 fed rats were characterised by 8 ions containing the n-3 double bond, and m.i.d. of the 651/653 ions of PGF-Me, TBDMS indicated PGF3 alpha synthesis (44 +/- 8% and 13 +/- 2% of PGF release by uteri incubated + or -5 micrograms/ul calcium ionophore A23187 respectively). In uteri from the control diet group incubated with ionophore, PGF3 alpha ions were detected and PGF 3 alpha represented 9.5 +/- 1.0% of PGF alpha release. Similarly, analysis of PGE from uteri of n-3 fed rats indicated that PGE3 (16 +/- 6% of PGE) was released in the presence of ionophore A23187. Synthesis of 3-series PG by rat uteri was detected after only 3 weeks of n-3 diet. The capacity to synthesise 3-series PG increased at intracellular calcium concentrations which mimicked cell calcium during decidual autolysis at parturition. These experiments suggest that uterine synthesis of 3-series PG is regulated by the specifity of enzymes incorporating fatty acids, rather than by the cyclooxygenase enzyme.

A fast, nondestructive purification scheme for prostaglandin H2 using a nonaqueous, bonded-phase high-performance liquid chromatography system.

Arachidonic acid metabolism produces several biologically important compounds including the leukotrienes and prostaglandins. Prostaglandin H2 (PGH2) is the first metabolite in the arachidonic acid cascade leading to all other prostaglandins. Pivotal to our understanding of PGH2's biology is the ability to separate it in pure form from the numerous other arachidonic acid metabolites produced in a biological milieu. The extensive literature on PGH2 biology and metabolism has relied almost exclusively on the traditional method of separation using gravity flow silicic acid columns. In our hands, such PGH2 preparations were found to contain varying amounts of 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT), PGE2, PGF2 alpha and other minor impurities as determined by further chromatographic and mass spectral analyses. Analytical separation of PGH2 and other arachidonic acid metabolites has been accomplished using reversed-phase HPLC. However, the labile nature of this molecule in aqueous systems makes such techniques unacceptable for preparative isolation of high purity PGH2 and has necessitated the development of a totally nonaqueous separation. To this end, we attempted several stationary phases and found that the cyano-bonded phase showed the best selectivity for resolving PGH2 from its major contaminants. Separations were performed on self-packed columns using a hexane-isopropanol gradient. Peaks were detected both by liquid scintillation counting and uv spectrophotometry (214 nm). Structure assignments were made by chromatographic comparison with authentic standards (PGF2 alpha, PGE2), biological activity (PGH2--platelet aggregation), and by ammonia direct chemical ionization mass spectrometry (HHT, hydroxy-5,8,10,14-eicosatetraenoic acid, PGH2, PGE2, PGF2 alpha). The latter technique, which by its very nature volatilizes all organic material in the sample, was particularly useful in determining not only that the PGH2 preparations were free from the aforementioned side products, but that they were also free from lipid, protein, and other potential residues frequently found in biological preparations.

Isolation and identification of prostaglandins of the E and F series in testes and semen of lowland-black-white breed of bulls.

The prostaglandins of E and F series were obtained from testes and semen of sexually mature bulls of lowland-black-white breed. From 1 g of fresh testes tissue we obtained 7.01 X 10(-9) M prostaglandin of the F series (PGF) and 20.65 X 10(-9) M prostaglandin of the E series (PGE): from 11 of semen 3.28 X 10(-6) M of PGF and 10.58 X 10(-6) M of PGE were obtained as well. The prostaglandins thus obtained displayed biological activity in experiments on the isolated small intestine of the rabbit.

Hepatic transformation of prostaglandin D2 to a new prostanoid, 9 alpha,11 beta-prostaglandin F2, that inhibits platelet aggregation and constricts blood vessels.

The metabolic transformation of tritium-labeled prostaglandin D2 ([3H]PGD2) was investigated in the isolated Tyrode's-perfused rabbit liver. One major product was isolated and identified in the perfusate as a new prostanoid. The structure of this metabolite was further confirmed by gas chromatography-mass spectrometry and chemical methods to be 9 alpha,11 beta,15-L-trihydroxyprosta-5-cis, 13-trans-dienoic acid, namely (9 alpha,11 beta-PGF2). This new prostanoid was found to be an inhibitor of platelet aggregation and to cause constriction of canine coronary artery strips. These results suggested that on passage through the hepatic circulation exogenous PGD2 is converted to 9 alpha,11 beta-PGF2, the latter having a biological profile which differs from that of PGD2 and PGF2 alpha.

[Effect of the chromatographic purification method on the isolation of prostaglandins E and F from a natural lipid mixture and their stability]. / Vliianie sposoba khromatograficheskoi ochistki na vydelenie prostaglandinov E i F iz prirodnoi smesi lipidov i ikh ustoichivost'.

Separation of PGE2 and PGF2 alpha and their ethyl ethers on silica gel from a natural lipid mixture permits the attainment of the 97-100% purity of the substances. However, the losses for PGE2 amount to 20-25, those for PGF2 alpha to 35-40%. Thus chromatography on silica gel can be used only for analytical purposes. As regards the preparative purposes the best method of PG isolation from a natural lipid mixture is gel filtration with a preliminary separation of phospholipids from the mixture over the oleophilic sulfacationite KO-I. After separation on sephadex the preparations can be stored for up to 4-6 months at 5-8 degrees C.

Effect of exogenous gonadal steroids and pregnancy on uterine luminal prostaglandin F in mares.

Two experiments were conducted to assess the effect of exogenous hormone treatment on uterine luminal prostaglandin F (PGF). In the first experiment ovariectomized pony mares received either corn oil (21 days, n = 3), estradiol valerate (21 days, n = 3), progesterone (21 days, n = 3) or estradiol valerate (7 days) followed by progesterone (14 days, n = 4). Progesterone treated mares had higher (P less than .01) uterine luminal PGF compared with all other groups, and no differences were detected between other treatment comparisons. In Experiment II, uterine fluid was collected from 4 ovariectomized horse mares before and after treatment with estradiol valerate (7 days) followed by progesterone (50 days). Pretreatment uterine luminal PGF levels were lower (P less than .001) than post-treatment levels (.03 vs 76.80 ng/ml). In a third experiment PGF was measured in uterine fluid of pony mares on days 8, 12, 14, 16, 18 and 20 of the estrous cycle and pregnancy. In nonpregnant mares a day effect (P less than .03) was observed in which uterine fluid PGF increased during the late luteal phase and declined thereafter. In contrast, no day effect was observed in pregnant animals and uterine luminal PGF was lower (P less than .001) than in cycling animals. These studies indicate that exogenous progesterone administration results in a large increase in uterine luminal PGF, whereas, pregnancy results in suppression. Taken collectively with previous work from our laboratory, these results suggest that while the endometrium of pregnant mares is capable of producing large amounts of PGF, the presence of a conceptus impedes its synthesis and/or release which allows for luteal maintenance.

Separation of major prostaglandins, leukotrienes, and monoHETEs by high performance liquid chromatography.

A procedure using high performance liquid chromatography (HPLC) is described for the separation of major primary cyclooxygenase metabolites (prostacyclin metabolite-6ketoPGF1 alpha, thromboxane B2, and prostaglandins F2 alpha, E2, and D2), leukotrienes (C4, B4, and D4), monohydroxyeicosatetraenoic acids (15-, 11-, 12-, and 5HETEs), and free arachidonic acid. It is therefore possible to quantitate major arachidonic acid metabolites by a single chromatographic procedure. Using this technique we have determined that a major arachidonic acid metabolite of human lung macrophages co-elutes with leukotriene B4.

Separation and quantitative determination of radiolabeled prostaglandins, thromboxanes, 6-keto-prostaglandin F1 alpha and other arachidonic acid metabolites produced in biological material.

Evaluation of several thin-layer chromatographic procedures for the separation of various labeled arachidonic acid metabolites (including 6-keto-prostaglandin F1 alpha) produced in the biological system is described. Manual scanning and autoradiography of the plates developed by two-dimensional thin-layer chromatography was also done for locating the radioactivities due to arachidonic acid metabolites other than thromboxane B2 and the classical prostaglandins (PGF2 alpha, PGE2, and PGD2).

Effect of pregnancy and collection technique on prostaglandin F in the uterine lumen of Pony mares.

Uterine flushings were obtained through the cervix (Method A) and through the wall of the uterus after hysterectomy (Method B) of ovariectomized Pony mares after s.c. injection of oestrogen for 1 week and progesterone for 2 weeks (Exp. 1). Non-pregnant and pregnant mares were flushed by Method A on Day 14 after ovulation and the flushings compared with those of non-pregnant mares injected i.v. with flunixen meglumine, a prostaglandin synthetase inhibitor, shortly before flushing (Exp. 3). Uterine flushings were also collected by Methods A and B from non-pregnant and pregnant Pony mares on Day 14. Endometrial and embryonic tissues from these mares were incubated with and without flunixen meglumine (Exp. 3). In all experiments, pregnancy had a significant effect on PGF content of uterine flushings or incubation media. Flushings from pregnant mares had reduced levels of PGF and were not influenced by collection technique (Exps 1 & 3). Non-pregnant Pony mares treated with progesterone responded to cervical stimulation (Method A) with an increase in intrauterine PGF over levels measured after hysterectomy (Method B) (Exps 1, 2 & 3). There was no effect on endometrial production of PGF in vitro by any tissue combination in a 2 h incubation in Krebs-Ringer-bicarbonate buffer but after 12 h incubation in Minimum Essential Medium endometrial PGF production was significantly higher when the endometria were from pregnant mares than from non-pregnant mares. PGF production in vitro was significantly suppressed by flunixen meglumine, by yolk sac membranes, and yolk sac and trophoblast, but not by trophoblast alone. The low intrauterine PGF levels in pregnant mares and the low in-vitro PGF production in the presence of the conceptus membranes may reflect inhibition of PGF synthesis and/or release by the embryo.

Prostaglandin synthesis by glomeruli isolated from rats with glycerol-induced acute renal failure.

In vitro PG synthesis by glomeruli isolated from rats with glycerol-induced acute renal failure (ARF) was measured by radiometric high performance liquid chromatography after incubation with [14C]arachidonic acid and radioimmunoassay (RIA). The four PGs, 6-keto-PGF1 alpha, TXB2, PGF2 alpha, and PGE2 were each synthesized by glomeruli from both control and treated rats but the synthesis rates were greater after glycerol. This increase was not apparent 1 hour after injection but, at 24 hours, all PGs were produced in greater amounts by glomeruli of treated rats. Thus, we studied PGE2, PGE2 alpha, and TXB2 synthesis by glomeruli at various time intervals after induction of ARF using direct RIA, PGF 2 alpha and TXB2 synthesis were greater only at 24 hours and only in the presence of arachidonic acid, whereas PGE2 synthesis was greater at 24 hours, irrespective of arachidonic acid, but at 48 hours only with arachidonic acid. The stimulatory effect of arachidonic acid was always greater in glycerol-treated than in control rats for these three PGs in the later period, whereas a significant decrease for PGE2 was observed at 1 hour. The late increase in PG synthesis may be due to stimulation of the renin-angiotensin system since it was abolished in rats pretreated for 48 hours with captopril. A late increase in PG synthesis by the papilla of the treated rats also was observed. We concluded that any increase in the glomerular production of vasoconstrictor PGs could contribute to the maintenance of acute renal failure, whereas the early fall in the stimulatory effect of arachidonic acid on PGE2 synthesis could play a role in its initiation.

Rapid extraction of oxygenated metabolites of arachidonic acid from biological samples using octadecylsilyl silica.

A rapid procedure for the efficient extraction of prostaglandins, thromboxanes and hydroxy fatty acids from urine, plasma and tissue homogenates has been developed. Fractions containing these substances are acidified and passed through a column of octadecylsilyl silica, which retains oxygenated metabolites of arachidonic acid. Phospholipids, proteins and very polar materials either are not retained or can be eluted with dilute aqueous ethanol. Nonpolar lipids and monohydroxy fatty acids are then eluted with petroleum ether or benzene. Subsequent elution of the column with methyl formate gives a fraction containing prostaglandins and thromboxanes which is much less contaminated with extraneous material than that obtained by conventional extraction of aqueous media with organic solvents. The methyl formate can be removed rapidly under a stream of nitrogen and the components of the sample purified directly by high pressure liquid chromatography (HPLC). An improved method for the purification of prostaglandins and TXB2 by HPLC on silica columns is reported.

Separation of prostaglandins, thromboxane, hydroxy fatty acids and arachidonic acid by high pressure liquid chromatography.

Separation of the major metabolites of arachidonic acid (AA) produced by the cyclo-oxygenase and the lipoxygenases was achieved by using reverse phase high-pressure liquid chromatography. Prostaglandins (PGs), thromboxane B2 (TXB2), and AA were separated on a C-18 radial compression column. An initial isocratic elution resolved the PGs and TXB2 which was followed by a linear gradient in order to elute AA. Variations of the gradient elution shape were required to permit the separation of 12-L-hydroxy-5,8,10-heptadecatrienoic acid, 5-12 and 15-hydroxy-5,8,11,14-eicosatetraenoic acid. The recovery of the labeled AA and its metabolites was investigated. Use of these separation methods and radiolabeled substrates should permit investigators to obtain reproducibly in one chromatographic run adequate separation and quantitation of both PGs and hydroxy fatty acid systems.

Quantitative determination of 6-keto-PGF1 alpha released by rat aorta: comparison of mass fragmentography and high resolution gas chromatography with electron capture detection.

Mass fragmentography (MF) and high resolution gas chromatography with electron capture detection (HRGC-ECD) were used for measuring 6-keto-PGF1 alpha, the stable hydrolysis product of prostacyclin (PGI2) released by fresh rings of rat aorta, incubated in the absence of the precursors arachidonic acid or prostaglandin endoperoxide (PGH2). The incubation medium was acidified, extracted, chromatographed on silicic acid column and derivatized. Comparable results were obtained analyzing each sample by MF and HRGC-ECD. Both methods proved to be suitable in terms of sensitivity and specificity for the measurement of 6-keto-PGF1 alpha produce by individual rat aortae.