Quantitative analysis of 5-oxo-6,8,11,14-eicosatetraenoic acid by electrospray mass spectrometry using a deuterium-labeled internal standard.

5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), a metabolite of arachidonic acid formed by the 5-lipoxygenase pathway, is a potent eosinophil chemoattractant that may be an important mediator in asthma. To further investigate the physiological and pathological roles of 5-oxo-ETE we have developed a mass spectrometric assay employing a tetradeuterated analog (5-oxo-[11,12,14,15-(2)H]ETE) as an internal standard. Collision-induced dissociation of the quasimolecular anion of 5-oxo-[11,12,14,15-(2)H]ETE (m/z 321) resulted in the formation of a major ion at m/z 207 that retained all four deuterium atoms. Measurement of the ratio of ions at m/z 203 (endogenous 5-oxo-ETE) and m/z 207 permitted quantitation of this compound by liquid chromatography-mass spectrometry-mass spectrometry using multiple reaction monitoring. The resulting assay was highly sensitive (< or =20 pg/sample) and selective, enabling detection of the amount of 5-oxo-ETE produced by as few as 10,000 neutrophils. This assay should permit measurement of 5-oxo-ETE in biological fluids, enabling evaluation of its role in asthma and other inflammatory diseases.

Biological inactivation of 5-oxo-6,8,11,14-eicosatetraenoic acid by human platelets.

Neutrophil-derived 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is a potent activator of neutrophils and eosinophils. In the present study we examined the biosynthesis and metabolism of this substance by platelets. Although platelets contain an abundant amount of 5-hydroxyeicosanoid dehydrogenase, the enzyme responsible for the formation of 5-oxo-ETE, they synthesize only very small amounts of this substance from exogenous 5-hydroxyeicosatetraenoic acid (5-HETE) unless endogenous NADPH is converted to NADP+ by addition of phenazine methosulfate. Similarly, relatively small amounts of 5-oxo-ETE were formed by A23187-stimulated mixtures of platelets and neutrophils, which instead formed substantial amounts of two 12-hydroxy metabolites of this substance, 5-oxo-12-HETE and 8-trans-5-oxo-12-HETE, which were identified by comparison with authentic chemically synthesized compounds. These metabolites were also formed from 5-oxo-ETE by platelets stimulated with thrombin or A23187. In contrast, unstimulated platelets converted 5-oxo-ETE principally to 5-HETE. Neither 5-oxo-12-HETE nor 8-trans-5-oxo-12-HETE had appreciable effects on neutrophil calcium levels or platelet aggregation at concentrations as high as 10 micromol/L, but both blocked 5-oxo-ETE-induced calcium mobilization in neutrophils with IC50 values of 0.5 and 2.5 micromol/L, respectively. We conclude that platelets can biologically inactivate 5-oxo-ETE. Unstimulated platelets convert 5-oxo-ETE to 5-HETE, with a 99% loss of biological potency, whereas stimulated platelets convert this substance to 12-hydroxy metabolites, which possess antagonist properties.

5-oxo-ETE induces pulmonary eosinophilia in an integrin-dependent manner in Brown Norway rats.

We have shown previously that the 5-lipoxygenase product 5-oxo-6,8, 11,14-eicosatetraenoic acid (5-oxo-ETE) is a highly potent eosinophil chemoattractant in vitro. To determine whether this substance can induce pulmonary eosinophil infiltration in vivo, it was administered to Brown Norway rats by tracheal insufflation. Eosinophils were then counted in lung sections that had been immunostained with an antibody to eosinophil major basic protein. 5-Oxo-ETE induced a dramatic increase in the numbers of eosinophils (ED50, 2.5 microg) around the walls of the airways, which reached maximal levels (five times control levels) between 15 and 24 h after administration, and then declined. LTB4 also induced pulmonary eosinophil infiltration with a similar ED50 but appeared to be somewhat less effective. In contrast, LTD4 and LTE4 were inactive. 5-Oxo-ETE-induced eosinophilia was unaffected by the LTB4 and PAF antagonists LY255283 and WEB 2170, respectively. However, it was inhibited by approximately 75% by monoclonal antibodies to CD49d (VLA-4) or CD11a (LFA-1) but was not significantly affected by an antibody to CD11b (Mac-1). In conclusion, 5-oxo-ETE induces pulmonary eosinophilia in Brown Norway rats, raising the possibility that it may be a physiological mediator of inflammation in asthma.

Identification of two major F2 isoprostanes, 8,12-iso- and 5-epi-8, 12-iso-isoprostane F2alpha-VI, in human urine.

Isoprostanes (iPs) are nonenzymatic, free radical-derived compounds isomeric with enzymatically formed eicosanoids such as prostaglandins, leukotrienes, and thromboxanes. One group formed by the auto-oxidation of arachidonic acid, the F2-iPs, consists of four classes of isomers of prostaglandin F2alpha (PGF2alpha). They are relatively abundant in human urine. This fact, along with their chemical stability and excellent characteristics for quantitation by gas chromatography/mass spectrometry, has made them attractive indices of oxidative stress in humans. We developed a specific assay using gas chromatography/mass spectrometry for the first identified F2-iP, iPF2alpha-III (previously called 8-iso-PGF2alpha or 8-epi-PGF2alpha), which demonstrated the utility of monitoring a specific isomer. Recently, we described an assay for another isomer, iPF2alpha-VI, which is present in urine in greater concentration than iPF2alpha-III and which is particularly amenable to quantitation. We now describe the identification in human urine of two more isomers, 8,12-iso-iPF2alpha-VI and 5-epi-8, 12-iso-iPF2alpha-VI, using high performance liquid chromatography/tandem mass spectrometry and gas chromatography/mass spectrometry. These compounds are each present in approximately 5-fold greater concentrations than iPF2alpha-VI (approximately 20-fold greater than iPF2alpha-III). They share the unique chemical characteristics of class VI compounds, which make them attractive targets for quantitation by gas chromatography/mass spectrometry and immunoassay development.

Total synthesis of 17,17,18,18-d4-iPF2alpha-VI and quantification of iPF2alpha-VI in human urine by gas chromatography/mass spectrometry.

Isoprostanes are a new class of natural products formed in humans as a result of free-radical-catalyzed lipid peroxidation of polyunsaturated fatty acids. These endogenous compounds are isomeric with biologically active prostaglandins and have great promise as markers of oxidant stress in vivo. iPF2alpha-III (previously 8-iso-PGF2alpha), an isoprostane from Class III (previously known as Class IV), has been used as an index of free-radical-induced oxidative stress. This isoprostane is also produced by the cyclooxygenase enzymes COX1 and COX2. We are proposing a new reliable index of oxidative stress based on iPF2alpha-VI (previously IPF2alpha-I), a new Class VI isoprostane we recently discovered. The advantages of iPF2alpha-VI are that it is several fold more abundant in urine than iPF2alpha-III, hence allowing more accurate determinations. Equally, the proximity of the C-5 OH function to the carboxylic acid allows the formation of the lactone 35 which is easier to purify from other iPs which cannot form such lactones. We have performed the first total synthesis of d4-iPF2alpha-VI by using two synthons, (3,3,4,4-d4)-hexylphosphonium bromide 23 prepared from 5-hexynol and syn-anti-syn lactone 25 synthesized from d-glucose. We have developed two variants of a sensitive GC/MS assay using the synthetic d4-iPF2alpha-VI as an internal standard to determine the levels of endogenous iPF2alpha-VI in biological fluids. Quantification of iPF2alpha-VI formed in vivo may be a more reliable index to assess oxidant stress in humans.

Calcium/calmodulin-dependent conversion of 5-oxoeicosanoids to 6, 7-dihydro metabolites by a cytosolic olefin reductase in human neutrophils.

We previously showed that 6-trans isomers of leukotriene B4 but not leukotriene B4 itself are converted to dihydro metabolites by human neutrophils. The first step in the formation of these metabolites is oxidation of the 5-hydroxyl group by 5-hydroxyeicosanoid dehydrogenase. The objective of the present investigation was to characterize the second step in the formation of the dihydro metabolites, reduction of an olefinic double bond. We found that the olefin reductase reduces the 6,7-double bond of 5-oxoeicosanoids, is localized in the cytosolic fraction of neutrophils, and requires NADPH as a cofactor. Neutrophil cytosol converts a variety of both 5-oxo- and 15-oxoeicosanoids to dihydro products. However, conversion of 5-oxoeicosanoids to their 6,7-dihydro metabolites is inhibited by EGTA and a calmodulin antagonist and stimulated by the addition of calcium and calmodulin, whereas the reduction of 15-oxoeicosanoids to their 13,14-dihydro metabolites is slightly inhibited by calcium. Furthermore, eicosanoid Delta6- and Delta13-reductases could be separated by chromatography on DEAE-Sepharose. 5-Oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE) is converted by the Delta6-reductase to 6,7-dihydro-5-oxo-ETE, which is 1000 times less potent than 5-oxo-ETE in mobilizing calcium in neutrophils. We conclude that neutrophils contain both 5-oxoeicosanoid Delta6-reductase and prostaglandin Delta13-reductase. Metabolism of 5-oxo-ETE by the Delta6-reductase results in loss of its biological activity.

IPF2alpha-I: an index of lipid peroxidation in humans.

Isoprostanes are prostaglandin isomers produced from arachidonic acid by a free radical-catalyzed mechanism. Urinary excretion of 8-iso-prostaglandin F2alpha, an isomer of the PGG/H synthase (cyclooxygenase or COX) enzyme product, prostaglandin F2alpha (PGF2alpha), has exhibited promise as an index of oxidant stress in vivo. We have developed a quantitative method to measure isoprostane F2alpha-I, (IPF2alpha-I) a class I isomer (8-iso-PGF2alpha is class IV), using gas chromatography/mass spectrometry. IPF2alpha-I is severalfold as abundant in human urine as 8-iso-PGF2alpha, with mean values of 737 +/- 20.6 pg/mg creatinine. Both isoprostanes are formed in a free radical-dependent manner in low density lipoprotein oxidized by copper in vitro. However, IPF2alpha-I, unlike 8-iso-PGF2alpha, is not formed in a COX-dependent manner by platelets activated by thrombin or collagen in vitro. Similarly, COX inhibition in vivo has no effect on IPF2alpha-I. Neither serum IPF2alpha-I, an index of cellular capacity to generate the isoprostane, nor urinary excretion of IPF2alpha-I, an index of actual generation in vivo, is depressed by aspirin or indomethacin. In contrast, both serum thromboxane B2 and urinary excretion of its 11-dehydro metabolite are depressed by the COX inhibitors. Although serum 8-iso-PGF2alpha formation is substantially depressed by COX inhibitors, urinary excretion of the compound is unaffected. Urinary IPF2alpha-I is elevated in cigarette smokers compared with controls (1525 +/- 180 versus 740 +/- 40 pg/mg creatinine; P < 0.01) and is highly correlated with urinary 8-iso-PGF2alpha (r = 0.9; P < 0.001). Urinary IPF2alpha-I is a novel index of lipid peroxidation in vivo, which can be measured with precision and sensitivity. It is an abundant F2-isoprostane formed in a free radical- but not COX-dependent manner. Although 8-iso-PGF2alpha may be formed as a minor product of COX, this pathway contributes trivially, if at all, to levels in urine. Urinary excretion of both isoprostanes is elevated in cigarette smokers.

High-pressure liquid chromatography of oxo-eicosanoids derived from arachidonic acid.

Eicosanoids are a large group of biologically active metabolites of arachidonic acid and related C20 fatty acids. Many of these compounds contain hydroxyl groups which can be converted to oxo groups by a variety of substrate-specific dehydrogenases. In many cases, this results in a reduction in potency, but in others, such as the oxidation of 5-hydroxyeicosatetraenoic acid to its oxo metabolite 5-oxo-eicosatetraenoic acid, there is a dramatic increase in biological activity. Thus, it is often very important to analyze the relative amounts of oxo- and hydroxy-eicosanoids formed by various cells and tissues. The present study was designed to compare the chromatographic behavior of oxo-eicosanoids and their hydroxy counterparts in commonly used mobile phases for reversed-phase and normal-phase HPLC. We examined three groups of eicosanoids: prostaglandins, leukotriene B4 and some of its metabolites, and monohydroxy-eicosanoids and their oxo metabolites. We found that in reversed-phase HPLC, the retention times of oxo-eicosanoids were longer than those of the corresponding hydroxy-eicosanoids in mobile phases containing acetonitrile as the major organic component, whereas the reverse was true for mobile phases containing methanol. Normal-phase HPLC using mobile phases containing hexane, isopropanol, and acetic acid gave excellent separation of oxo- and hydroxy-eicosanoids. Increasing the concentration of acetic acid in the mobile phase selectively reduced the retention times of oxo-eicosatetraenoic acids compared to monohydroxy-eicosatetraenoic acids, whereas the reverse was true for isopropanol. Differences in the chromatographic behavior of oxo- and hydroxy-eicosanoids can be useful clues in the structural characterization of these compounds, as illustrated by the chromatographic properties of a complex series of LTB4 metabolites formed by rat neutrophils.

Nomenclature of isoprostanes: a proposal.

We have proposed a nomenclature system for the isoprostanes, a new class of natural products formed in vivo by the free-radical peroxidation of polyunsaturated fatty acids. Our proposed nomenclature is based on the assignment of four isoprostanes, 1, 9, 17, and 25, as representatives of the four classes of isoprostanes derived from arachidonic acid (AA). We have attempted as much as possible to retain elements from the familiar prostaglandin nomenclature. In this proposal, we have used the abbreviation i.p. for isoprostane. We have classified isoprostane classes or types based on omega-carbon as being the starting reference. Roman numerals I-VI refer the six types of isoprostanes derived from eicosapentaenoic acid (EPA) and III-VI refer to the four types derived from AA. This nomenclature can be applied to isoprostanes derived from other PUFAs also.

The isoprostanes: a perspective.

The isoprostanes are a new class of natural products produced in vivo by a non-enzymatic free-radical-induced peroxidation of polyunsaturated fatty acid. In the case of arachidonic acid, for example, four classes of isoprostanes can be produced. Because of the specific structural features distinguishing them from other free-radical-generated products, e.g., HETEs, etc., the isoprostanes can provide an exclusive and selective index for the oxidant component of several inflammatory and degenerative diseases. The possible mechanisms of formation of the individual isoprostanes is discussed in detail. Class III products, such as 8-iso-PGF2 alpha and 8-iso-PGE2 have been shown to be vasoconstrictors and modulate platelet function. Several synthetic representatives from the four classes of arachidonic-acid-derived isoprostanes have already been prepared by total synthesis. These synthetic standards have been used for the identification and quantitation of these isoprostanes in biological fluids using gas chromatography/mass spectrometry methodology.

Effects of metabolites of leukotriene B4 on human neutrophil migration and cytosolic calcium levels.

Leukotriene B4 (LTB4) is metabolized by beta-oxidation, omega-oxidation and the 12-hydroxyeicosanoid dehydrogenase/delta 10-reductase pathway. We have investigated the effects of metabolites formed by the latter pathway on calcium mobilization and migration in human neutrophils and have compared their potencies with those of other LTB4 derivatives. 12-Oxo-LTB4 and 10,11-dihydro-LTB4 were 60 to 100 times less potent than LTB4 in stimulating neutrophils, whereas 10,11-dihydro-12-oxo-LTB4 and 10,11-dihydro-12-epi-LTB4 exhibited still lower potencies. The 6-trans isomers of 12-oxo-LTB4 and 10,11-dihydro-12-oxo-LTB4 were much less potent than the 6-cis compounds. The EC50 values for biologically and chemically (6-cis) synthesized 12-oxo-LTB4 were similar, indicating that the 6,7-double bond is retained in the cis configuration in the biologically formed compound. Methylation of LTB4 markedly reduced its effect on cytosolic calcium levels, whereas addition of a 3-hydroxyl group had a much more modest effect. Modifications of the omega end of the molecule also resulted in lower potencies for calcium mobilization. Nearly all of the compounds tested desensitized neutrophils to LTB4-induced calcium mobilization, which suggests that their effects were mediated by receptors for the latter compound. However, modifications in the carboxyl end of the molecule had smaller effects on desensitization than on calcium mobilization, whereas the reverse was true for modifications in the omega end of the molecule. This suggests that the structural requirements for agonist-induced desensitization to LTB4 may differ to some extent from the requirements for calcium mobilization.