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1.
A method is described for the quantitative analysis of eicosanoids (arachidonic acid metabolites, nee, prostaglandins) by reverse-phase high-pressure liquid chromatography following formation of the ester derivative with p-(9-anthroyloxy)phenacyl bromide. The lower limit of detection of the eicosanoid ester is 280 pg (ultraviolet—254 nm) and approximately 50 pg (fluorescence 249 emission, 413-nm cutoff). We separated the esters of seven common eicosanoids by reverse-phase chromatography with acetonitrile and water. Thromboxane B2 chromatographs as two species and coelutes with PGF. Separation of all others is adequate, including the three metabolites of prostacyclin (6-keto-PGF, 6-keto-PGE1, 13,14-dihydro-6,15-diketo-PGF). We obtained good correlation between radioimmunoassay and derivative analysis of standard 6-keto-PGF extracted from lactated Ringer's solution with standard technique, as well as 6-keto-PGF quantitation from tissue culture medium that had contained pulmonary endothelial cells. This method should be applicable to analysis of eicosanoids extracted from biological matrices.  相似文献   

2.
The transformation of 6-keto-PGF to two prostacyclin metabolites, 2,3-dinor-6-keto-PGF (I) and 2,3-dinor-6,15-diketo-13,14-dihydro-PGF (II) by Mycobacterium rhodochrous UC-6176 is described. The finding that the bacterium oxidized 6-keto-PGF to the 6,15-diketo metabolite II shows that it contains 15-hydroxy prostaglandin dehydrogenase and Δ13 reductase enzyme systems.  相似文献   

3.
Prostacyclin (PGX) strikingly increases cyclic AMP concentrations in human platelets. Prostacyclin is approximately 10 times more active than PGD2, 30 times more active than PGE1 and more than 1000 times more active than its stable end product, 6-oxo-PGF.These results correlate well with the anti-aggregating activity of prostacyclin, compared with PGE1 and PGD2.  相似文献   

4.
Western diets are enriched in omega-6 vs. omega-3 fatty acids, and a shift in this balance toward omega-3 fatty acids may have health benefits. There is limited information about the catabolism of 3-series prostaglandins (PG) formed from eicosapentaenoic acid (EPA), a fish oil omega-3 fatty acid that becomes elevated in tissues following fish oil consumption. Quantification of appropriate urinary 3-series PG metabolites could be used for noninvasive measurement of omega-3 fatty acid tone. Here we describe the preparation of tritium- and deuterium-labeled 6-keto-PGF and their use in identifying urinary metabolites in mice using LC-MS/MS. The major 6-keto-PGF urinary metabolites included dinor-6-keto-PGF (∼10%) and dinor-13,14-dihydro-6,15-diketo-PGF (∼10%). These metabolites can arise only from the enzymatic conversion of EPA to the 3-series PGH endoperoxide by cyclooxygenases, then PGI3 by prostacyclin synthase and, finally, nonenzymatic hydrolysis to 6-keto-PGF. The 6-keto-PGF derivatives are not formed by free radical mechanisms that generate isoprostanes, and thus, these metabolites provide an unbiased marker for utilization of EPA by cyclooxygenases.  相似文献   

5.
Granulosa cells isolated from mature Graafian follicles of swine produced significant quantities of immunoreactive 6-keto-PGF1α under chemically defined conditions in vitro. Luteinizing hormone elicited a dose-dependent stimulation of 6-keto-PGF1α accumulation, but follicle stimulating hormone, prolactin, L-epinephrine, estradiol-17B, or PGE2 were devoid of effect. The time-dependent in vitro production of 6-keto-PGF1α by ovarian cells was susceptible to inhibition by indomethacin, U-51506, cycloheximide, and actinomycin D. These observations implicate granulosa cells in the specific and hormonally regulated production of prostacyclin.  相似文献   

6.
The metabolism of endogenous PGI2 (released by angiotensin II or bradykinin) and exogenous PGI2 by 15-hydroxy-PG-dehydrogenase and Δ13-reductase was studied in five different vascular beds of the anaesthetized cat. Plasma concentrations of 6-keto-PGF (the product of spontaneous hydrolysis of PGI2) and 6,15-diketo-13,14-dihydro-PGF (the metabolite formed from PGI2 by 15-hydroxy-PG-dehydrogenase and Δ13-reductase) were determined in the efferent vessels of the respective vascular beds by specific radioimmunoassays.No major metabolism of PGI2 by 15-hydroxy-PG-dehydrogenase and Δ13-reductase was detected in the head and the hindlimbs of the cat. In the lung exogenous (circulating) PGI2 was not metabolized, whereas PGI2 synthetized in the lung itself was converted to 6,15-diketo-13,14-dihydor-PGF. No significant amounts of 6,15-diketo-13,14-dihydro-PGF-immunoreactivity were detected in hepatic venous blood after infusion of PGI2 into the portal vein. However as also no 6-keto-PGF was found, the liver seems to efficiently extract PGI2 from the circulation. The cat kidney had the highest capacity of all vascular beds investigated to release endogenous and exogenous PGI2 as 6-15-diketo-13,14-dihydro-PGF. In other organs (vascular beds) investigated PGI2 is either metabolized less efficiently by the 15-hydroxy-PG-dehydrogenase or further transformed to other metabolites.  相似文献   

7.
Biosynthesis of prostaglandins in ob17 preadipose cells was studied in culture. Dihomo-γ-linolenic acid is exclusively converted to PGE1. Arachidonic acid behaves quantitatively as a more potent precursor, leading to the synthesis of PGE2 and 6-keto-PGF (stable product of prostacyclin). In all cases prostaglandin synthesis was confirmed directly by radioimmunoassay. This synthesis is maximal during the growth phase and decreases dramatically after confluence at a time where adipose conversion occurs, suggesting a possible relationship between both events.  相似文献   

8.
An NADP-linked 15-hydroxyprostaglandin dehydrogenase specific for prostacyclin was purified 1,300-fold from rabbit kidney. Prostaglandins E2, F, and 6-Keto PGF and thromboxane B2 were oxidized by the purified enzyme with rates of reaction less than 4% that of PGI2. Unlike other rabbit kidney NADP-linked 15-hydroxyprostaglandin dehydrogenases, this enzyme catalyzes oxido reduction more rapidly at the 15- position than at the 9- position and does not utilise NAD as a cofactor. It has a molecular weight of 62,000 and migrates on polyacrylamide disc gel electrophoresis as a single diffuse band. The reaction product was identified by thin-layer chromatography as 6,15-diketo PGF. Prostacyclin dhydrogenase is the first 15-hydroxyprostaglandin dehydrogenase described which is specific for the metabolism of prostacyclin.  相似文献   

9.
(5E)-6a-Carbaprostaglandin I2 (carbacyclin) was oxidized to (5E)-15-dehydro-6a-carbaprostaglandin I2 (15-dehydrocarbacyclin) by partially purified rhesus monkey lung prostaglandin dehydrogenase (PGDH). The (5E)-15-dehydro-6a-carbaprostaglandin I2 was isolated by preparative thin-layer chromatography and identified by gas chromatography-mass spectrometry. A Lineweaver-Burke plot gave an apparent Km value of 2.9 μM and a Vmax of 35.7 nmoles carbacyclin oxidized/mg protein/min. These values are similar to previously reported Km and Kmax values for PGI2 and PGE1.  相似文献   

10.
The activity of prostacyclin (PGI2), PGE1 or PGD2 as inhibitors of platelet aggregation in plasma from human, dog, rabbit, rat, sheep and horse was investigated. Prostacyclin was the most potent inhibitor in all species. PGD2 was a weak inhibitor in dog, rabbit and rat plasma whereas PGE1 and prostacyclin were highly active. Theophylline or dipyridamole potentiated the inhibition of human platelet aggregation by prostacyclin, PGE1 or PGD2. Compound N-0164 abolished the inhibition by PGD2 of human platelet aggregation but did not inhibit the effects of PGE1 or prostacyclin. The results suggest that prostacyclin and PGE1 act on similar sites on platelets which are distinct from those for PGD2.  相似文献   

11.
Prostacyclin alternatively called prostaglandin (PG) I2 is an unstable metabolite synthesized by the arachidonate cyclooxygenase pathway. Earlier studies have suggested that prostacyclin analogues can act as a potent effector of adipose differentiation. However, biosynthesis of PGI2 has not been determined comprehensively at different life stages of adipocytes. PGI2 is rapidly hydrolyzed to the stable product, 6-keto-PGF, in biological fluids. Therefore, the generation of PGI2 can be quantified as the amount of 6-keto-PGF. In this study, we attempted to develop a solid-phase enzyme-linked immunosorbent assay (ELISA) using a mouse antiserum specific for 6-keto-PGF. According to the typical calibration curve of our ELISA, 6-keto-PGF can be quantified from 0.8 pg to 7.7 ng in an assay. The evaluation of our ELISA revealed the higher specificity of our antiserum without the cross-reaction with other related prostanoids while it exhibited only the cross-reaction of 1.5 % with PGF. The resulting ELISA was applied to the quantification of 6-keto-PGF generated endogenously by cultured 3T3-L1 cells at different stages. The cultured cells showed the highest capability to generate 6-keto-PGF during the maturation phase of 4–6 days, which was consistent with the coordinated changes in the gene expression of PGI synthase and the IP receptor for PGI2. Following these events, the accumulation of fats was continuously promoted up to 14 days. Thus, our immunological assay specific for 6-keto-PGF is useful for monitoring the endogenous levels of the unstable parent PGI2 at different life stages of adipogenesis and for further studies on the potential association with the up-regulation of adipogenesis in cultured adipocytes.  相似文献   

12.
Granulosa cells isolated from mature Graafian follicles of swine produced significant quantities of immunoreactive 6-keto-PGF1α under chemically defined conditions in vitro. Luteinizing hormone elicited a dose-dependent stimulation of 6-keto-PGF1α accumulation, but follicle stimulating hormone, prolactin, L-epinephrine, estradiol-17B, or PGE2 were devoid of effect. The time-dependent in vitro production of 6-keto-PGF1α by ovarian cells was susceptible to inhibition by indomethacin, U-51506, cycloheximide, and actinomycin D. These observations implicate granulosa cells in the specific and hormonally regulated production of prostacyclin.  相似文献   

13.
Several lines of investigation have suggested that exposure to ethanol may lead to alterations in both the synthesis and degradation of the E and F series of prostaglandins (PG). It has been suggested that these changes in PG metabolism underlie cartain of the pathophysiological consequences of chronic alcoholism but few data re availalbe as to the mechanism resonsible for these changes. We now report that chronic exposure to ethanol in moderate doses (17% total dietary calories as ethanol) and high doses (35% total dietary calories as ethanol) results in a concentration dependent loss of renal 15-hydroxy-prostaglandin dehydrogenase for the NAD mediated reactioins. Soluble fractions of kidney homogenates in teh presnet of > 10 Km concentratons of NAd exhibited dose dependent loss of specific and total organ PG dehydrogenase activity toward PGE2 and F. A similar dose dependent decrease in the Vmax of the NAD mediated reaction was measured for the oxidation of PGE1, E2, and F. Moderate doses of ethanol resulted in an increase in the Km for PGE2 and F. Km values for the NADP mediated reactions were not significantly influenced by exposure to high doses of ethanol other than for PGE1. These data suggest that chronic ethanol consumption results in a dose dependent, selective inhibition of the metabolism of PGs of the “2” series by the renal PGDH enzyme which utilizes NAD.  相似文献   

14.
A method is described for measurement of the cyclooxygenase products, thromboxane,prostacyclin, and prostaglandins (PG), and several prostaglandin metabolites. The procedure involves separation of the compounds by high-pressure liquid chromatography combined with identification and estimation by serologic analysis. These combined procedures have been used to identify and estimate five such products, PGE2, PGE1 PGF2α, PGF, and 6-keto-PGF, in the culture fluids of dog kidney cells stimulated by a tumor-promoting phorbol diester. The prostaglandin metabolites, 13,14-dihydro-15-keto-PGE2, 13,14-dihydro-15-keto-PF2, 13,14-dihydro-PGE2, and 13,14-dihydro-PGF, were not found in these culture fluids.  相似文献   

15.
The metabolism of endogenous PGI2 (released by angiotensin II or bradykinin) and exogenous PGI2 by 15-hydroxy-PG-dehydrogenase and Δ13-reductase was studied in five different vascular beds of the anaesthetized cat. Plasma concentrations of 6-keto-PGF (the product of spontaneous hydrolysis of PGI2) and 6,15-diketo-13,14-dihydro-PGF (the metabolite formed from PGI2 by 15-hydroxy-PG-dehydrogenase and Δ13-reductase) were determined in the efferent vessels of the respective vascular beds by specific radioimmunoassays.No major metabolism of PGI2 by 15-hydroxy-PG-dehydrogenase and Δ13-reductase was detected in the head and the hindlimbs of the cat. In the lung exogenous (circulating) PGI2 was not metabolized, whereas PGI2 synthetized in the lung itself was converted to 6,15-diketo-13,14-dihydor-PGF. No significant amounts of 6,15-diketo-13,14-dihydro-PGF-immunoreactivity were detected in hepatic venous blood after infusion of PGI2 into the portal vein. However as also no 6-keto-PGF was found, the liver seems to efficiently extract PGI2 from the circulation. The cat kidney had the highest capacity of all vascular beds investigated to release endogenous and exogenous PGI2 as 6-15-diketo-13,14-dihydro-PGF. In other organs (vascular beds) investigated PGI2 is either metabolized less efficiently by the 15-hydroxy-PG-dehydrogenase or further transformed to other metabolites.  相似文献   

16.
The ability of aortae from young and mature swine to produce prostacyclin (PGI2) has been determined. PGI2 was measured as its hydration product, 6-keto-PGF and assayed by stable isotope dilution GC-MS. There was no significant difference in 6-keto-PGF production between intimal strips from young and mature aortae in the basal state. In the presence of saturating concentrations of arachidonic acid, however, intimal strips from young aortae synthesized twice as much 6-keto-PGF as did older tissues. Fatty acid compositions of young and mature aortae were virtually identical, making dietary differences an unlikely explanation for the age-related decrease in PGI2 synthesis. Both young and mature vascular tissues produced essentially only PGI2; insignificant amounts of PGE2 and PGF were found.  相似文献   

17.
Prostaglandin E2 (PGE2) has previously been shown to inhibit sympathetic neurotransmission in different organs and species. Based on this inhibitory effect and on its reversal by cyclo-oxygenase inhibitors, PGE2 has been claimed to be a physiological modulator of in vivo release of norepinephrine (NE) from sympathetic nerves. It is now recognized that prostacyclin (PGI2) is the main cyclo-oxygenase product in the heart. We therefore addressed the question whether PGI2, within the same preparation, is formed in increased amounts during sympathetic nerve stimulation and has neuromodulatory activity.The effluent from isolated rabbit hearts subjected to sympathetic nerve stimulation or to infusion of NE or adenosine (ADO) was collected, and its content of PGE2 and 6-keto-PGF (dehydration product of PGI2) was analyzed using gas chromatography/mass spectrometry, operated in the negative ion/chemical ionization mode. Other hearts were infused with PGI2 and nerve stimulation induced outflow of endogenous NE into the effluent was analyzed using HPLC with electrochemical detection. Nerve stimulation at 5 or 10 Hz (before but not after adrenergic receptor blockade), as well as infusion of NE (10−6–10−5M) or ADO (10−4M) increased the cardiac outflow of 6-keto-PGF1α. Basal and nerve stimulation induced efflux of 6-keto-PGF1α was approximately 5 times higher than the corresponding efflux of PGE2. PGI2 dose-dependently inhibited the outflow of NE from sympathetically stimulated hearts, the inhibition at 10−6M being approximately 40%.On the basis of these observations we propose that PGI2 is a more likely candidate than PGE2 as a potential modulator of neurotransmission in cardiac tissue in vivo.  相似文献   

18.
The oxidation of 12-hydroxylauric acid methyl ester (12-OH-L-Me) and of ω-hydroxy-prostaglandins (ω-OH-PGs) such as 20-OH-PGB1 and 20-OH-PGE1, was demonstrated with liver cytosol from rat, rabbit, and guinea pig in the presence of NAD; however, NADP did not support this oxidation. (ω-1)-Hydroxy-compounds (11-OH-laurate and 19-OH-PGB1) and PGE1, PGF, and PGB1, all lacking the terminal (ω)-hydroxyl, did not reduce NAD. However, at pH 10, PGE1 slightly enhanced NAD reduction, suggesting that at this pH PGE1, could be a substrate for 15-hydroxy-PG dehydrogenase (PGDH). The oxidation products from incubations of 12-OH-L-Me, 20-OH-PGB1-Me, and 20-OH-PGE1 with guinea pig liver cytosol were isolated and identified by gas chromatography/mass fragmentation spectrometry as being the corresponding dicarboxylic acids. In contrast to the liver cytosol, guinea pig kidney cytosol had only a minimal effect on NAD reduction by 12-OH-L-Me but nevertheless did support the stimulation of NAD reduction by PGE1, and PGF, but not by PGB1, indicating the participation of kidney cytosolic PGDH in PGE1 and PGF oxidation and demonstrating that the oxidation of ω-OH to the carboxylic acid is not mediated by PGDH. Though the in vivo rate of oxidation of ω-OH-PGs has not been established, these results suggest that the urinary dicarboxylic-PG metabolites involve a multiple sequentialstep oxidation of PGs involving ω-hydroxylation by an NADPH-cytochrome P-450 system in the endoplasmic reticulum and the subsequent oxidation of the ω-OH by an NAD-dependent dehydrogenase in the cytosol.  相似文献   

19.
Prostaglandin (PG) synthesis and degradation were examined in different regions (epithelial versus non-epithelial structures) of the rat distal colon by both HPLC analysis of [14C] arachidonate (AA) metabolites and by specific radioimmunoassays. Intact isolated colonic epithelial cells synthesized mainly PGF2α and TXA2, as monitored from the formation of its stable degradation product TXB2 (PGF2α > TXB2 > 6-keto-PGF1α, the stable degradation product of PGI2=PGD2=PGE2=13,14-dihydro-15-keto-PGF2α). The profile of PG products of isolated surface epithelial cells was identical to that of proliferative epithelial cells. However, generation of PGs by surface epithelium was 2 to 3-fold higher than by proliferative cells both basally and in the presence of a maximal stimulating concentration (0.1 mM) of AA. The latter implied a greater synthetic capacity of surface epithelium, rather than differences due to endogenous AA availability. The major sites of PG synthesis in colon clearly resided in submucosal structures; the residual colon devoid of epithelial cells accounted for at least 99% of the total PGs produced by intact distal colon. The profile of AA metabolites formed by submucosal structures also differed markedly from that of the epithelium. The dominant submucosal product was PGE2. PGE2 and its degradation product 13,14-dihydro-15-keto-PGE2 accounted for 63% of the PG products formed by submucosal structures (PGE2 PGD2 > 13,14-dihydro-15-keto-PGE2 > PGF2α=TXB2=6-keto-PGF1α > 13,14-dihydro- 15-keto-PGF2α). By contrast, epithelial cells, and particularly surface epithelium, contributed disproportionately to the PG degradative capacity of colon, as assessed from the metabolism of either PGE2 or PGF. When expressed as a percentage, epithelial cells accounted for 71% of total colonic PGE2 degradative capacity but only 23% of total colonic protein. Approximately 15% of [3H] PGE2 added to the serosal side of everted colonic loops crossed to the mucosal side intact. Thus, at least a portion of the PGE2 formed in the submucosa reaches, and thereby can potentially influence functions of the epithelium.  相似文献   

20.
The aim of this study was to investigate circadian variation in concentrations of arachidonic acid(AA) metabolites in relation to the circadian pattern in bronchial patency. Blood samples were obtained at 4-hr intervals from 2000 of 1 day until 1400 of the next from 12 diurnally active asthmatic and six diurnally active non-asthmatic patients. Bloods were analyzed for the prostanoids thromboxane A2 (measured as stable metabolite 6-keto-PGF1a), PGE2, and PGF2a. Airways patency was assessed by self-measurement of peak expiratory flow (PEF). In asthmatics, circadian variation was detected in PEF as well as PGE2 and TXB2. The circadian trough of the PEF rhythm closely coincided with the circadian peak of the PGE2 and TXB2, rhythms. In the controls, the PEF was not circadian rhythmic. Of the AA metabolites only 6-keto-PGF1a exhibited 24-hr bioperiodicity in the controls. The controls exhibited a significantly higher circadian mean of PEF (P < 0.001), while the asthmatics had a lower 24-hr average PGE2 but greater mean TXB2/PGE2 ratio. The obstructive effect caused by the overall 24-hr deficiency of PGE2 in asthmatics is possibly amplified by the increased of TXB2 during the early morning hours. This dissociation of the temporal patterns in TXB, and PGE, levels over the 24 hr is discussed as a characteristic finding for asthmatics.  相似文献   

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