Epoxyeicosatrienoic acid metabolism in arterial smooth muscle cells

Epoxyeicosatrienoic acids (EETs) are eicosanoids synthesized from arachidonic acid by the cytochrome P450 eposygenase pathway. The present studies demonstrate that 8,9-, 11,12-, and 14,15-EET are rapidly taken up by porcine aortic smooth muscle cells. About half of the uptake is incorporated into phospholipids, and saponification indicates that most of this remains in the form of EET. The EETs also are converted to the corresponding dihydroxyeicosatrienoic acids (DHETs) and during prolonged incubations, additional metabolites that do not retain the EET carboxyl group are formed. Most of these products are released into the medium. However, some DHET and metabolites less polar than EET are incorporated into the phospholipids, and a small amount of unesterified EET is also present in the cells. The incorporation of 14,15-EET and its conversion to DHET did not approach saturation until the concentration exceeded 10-20 microM, indicating that vascular smooth muscle has a large capacity to utilize this EET. These findings suggest that certain vasoactive effects of EETs may be due to their incorporation by smooth muscle cells. Furthermore, through conversion to DHET and other oxidized metabolites, smooth muscle apparently has the capacity to inactivate EETs that are either formed in or penetrate into the vascular wall.     

Potentiation of endothelium-dependent relaxation by epoxyeicosatrienoic acids

Epoxyeicosatrienoic acids (EETs) are potent endothelium-derived vasodilators formed from cytochrome P-450 metabolism of arachidonic acid. EETs and their diol products (DHETs) are also avidly taken up by endothelial cells and incorporated into phospholipids that participate in signal transduction. To investigate the possible functional significance of EET and DHET incorporation into cell lipids, we examined the capacity of EETs and DHETs to relax porcine coronary arterial rings and determined responses to bradykinin (which potently activates endothelial phospholipases) before and after incubating the rings with these eicosanoids. 14,15-EET and 11,12-EET (5 mumol/L) produced 75 +/- 9% and 52 +/- 4% relaxation, respectively, of U46619-contracted rings, whereas 8,9-EET and 5,6-EET did not produce significant relaxation. The corresponding DHET regioisomers produced comparable relaxation responses. Preincubation with 14,15-EET, 11,12-EET, 14,15-DHET, and 11,12-DHET augmented the magnitude and duration of bradykinin-induced relaxation, whereas endothelium-independent relaxations to aprikalim and sodium nitroprusside were not potentiated. Pretreatment with 2 mumol/L triacsin C (an inhibitor of acyl coenzyme A synthases) inhibited [3H]14,15-EET incorporation into endothelial phospholipids and blocked 11,12-EET- and 14,15-DHET-induced potentiation of relaxation to bradykinin. Exposure of [3H]14,15-EET-labeled endothelial cells to the Ca2+ ionophore A23187 (2 mumol/L) resulted in a 4-fold increased release of EET and DHET into the medium. We conclude that incorporation of EETs and DHETs into cell lipids results in potentiation of bradykinin-induced relaxation in porcine coronary arteries, providing the first evidence that incorporated EETs and DHETs are capable of modulating vascular function.     

Susceptibility of plasmenyl glycerophosphoethanolamine lipids containing arachidonate to oxidative degradation

Plasmenyl phospholipids (1-alk-1'-enyl-2-acyl-3-glycerophospholipids, plasmalogens) are a structurally unique class of lipids that contain an alpha-unsaturated ether substituent at the sn-1 position of the glycerol backbone. Several studies have supported the hypothesis that plasmalogens may be antioxidant molecules that protect cells from oxidative stress. Because the molecular mechanisms responsible for the antioxidant properties of plasmenyl phospholipids are not fully understood, the oxidation of plasmalogens in natural mixtures of phospholipids was studied using electrospray tandem mass spectrometry. Glycerophosphoethanolamine (GPE) lipids from bovine brain were found to contain six major molecular species (16:0p/18:1-, 18:1p/18:1-, 18:0p/20:4-, 16:0p/20:4, 18:0a/20:4-, and 18:0a/22:6-GPE). Oxidation of GPE yielded lyso phospholipid products derived from plasmalogen species containing only monounsaturated sn-2 substituents and diacyl-GPE with oxidized polyunsaturated fatty acyl substituents at sn-2. The only plasmalogen species remaining intact following oxidation contained monounsaturated fatty acyl groups esterified at sn-2. The mechanism responsible for the rapid and specific destruction of plasmalogen GPE may likely involve unique reactivity imparted by a polyunsaturated fatty acyl group esterified at sn-2. This structural feature may play a central role determining the antioxidant properties ascribed to this class of phospholipids.     

Formation of lithiated adducts of glycerophosphocholine lipids facilitates their identification by electrospray ionization tandem mass spectrometry

Electrospray ionization (ESI) tandem mass spectrometry (MS) has simplified analysis of phospholipid mixtures, and, in negative ion mode, permits structural identification of picomole amounts of phospholipid species. Collisionally activated dissociation (CAD) of phospholipid anions yields negative ion tandem mass spectra that contain fragment ions representing the fatty acid substituents as carboxylate anions. Glycerophosphocholine (GPC) lipids contain a quaternary nitrogen moiety and more readily form cationic adducts than anionic species, and positive ion tandem mass spectra of protonated GPC species contain no abundant ions that identify fatty acid substituents. We report here that lithiated adducts of GPC species are readily formed by adding lithium hydroxide to the solution in which phospholipid mixtures are infused into the ESI source. CAD of [MLi+] ions of GPC species yields tandem mass spectra that contain prominent ions representing losses of the fatty acid substituents. These ions and their relative abundances can be used to assign the identities and positions of the fatty acid substituents of GPC species. Tandem mass spectrometric scans monitoring neutral losses of the head-group or of fatty acid substituents from lithiated adducts can be used to identify GPC species in tissue phospholipid mixtures. Similar scans monitoring parents of specific product ions can also be used to identify the fatty acid substituents of GPC species, and this facilitates identification of distinct isobaric contributors to ions observed in the ESI/MS total ion current.     

The relationship of hydroxyeicosatetraenoic acids and F2-isoprostanes to plaque instability in human carotid atherosclerosis

Evidence for increased oxidant stress has been reported in human atherosclerosis. However, no information is available about the importance of in situ oxidant stress in relation to plaque stability. This information is relevant because the morbidity and mortality of atherosclerosis are essentially the consequences of acute ischemic syndromes due to unstable plaques. We studied 30 carotid atherosclerotic plaques retrieved by endarterectomy from 18 asymptomatic (stable plaques) and 12 symptomatic patients (unstable plaques). Four normal arteries served as controls. After lipid extraction and ester hydrolysis, quantitation of different indices of oxidant stress were analyzed, including hydroxyeicosatetraenoic acids (HETEs), epoxyeicosatetraenoic acids (EETs), ketoeicosatetraenoic acids (oxo-ETEs), and F2-isoprostanes using online reverse-phase high-performance liquid chromatography tandem mass spectrometry (LC/MS/MS). All measurements were carried out in a strictly double-blind procedure. We found elevated levels of the different compounds in atherosclerotic plaques. Levels of HETEs were 24 times higher than EETs, oxo-ETEs, or F2-isoprostanes. Levels of HETEs, but not those of EETs, oxo-ETEs or F2-isoprostanes, were significantly elevated in plaques retrieved from symptomatic patients compared with those retrieved from asymptomatic patients (1, 738 +/- 274 vs. 1,002 +/- 107 pmol/ micromol lipid phosphorous, respectively; P < 0.01). One monooxygenated arachidonate species, 9-HETE, which cannot be derived from known enzymatic reactions, was the most abundant and significant compound observed in plaques, suggesting that nonenzymatic lipid peroxidation predominates in advanced atherosclerosis and may promote plaque instability.     

Cytochrome P-450-dependent arachidonate metabolites and renal functions in the Lyon hypertensive rat

1. The present work aimed to assess the role of cytochrome P-450 (CP-450) metabolites of arachidonic acid such as epoxy-eicosatrienoic (EET) and hydroxyeicosatetraenoic acids (HETE) in the renal vasoconstriction and decreased natriuresis exhibited by genetically hypertensive (LH) rats of the Lyon strain. 2. The experiment was performed on single-pass isolated perfused kidney preparations from 8-week-old male LH rats and their low blood pressure (LL) controls. The effects of miconazole (an inhibitor of the formation of EET) and of 17-octadecynoic acid (17-ODYA, an inhibitor of both EET and HETE synthesis) were studied before and after stimulation of the kidneys with two noradrenaline (NA) infusions (65 and 110 nmol/L). 3. Unstimulated LH kidneys (n = 12) differed from LL (n = 12) by increased vascular resistance (RVR) and decreased glomerular filtration rate and urinary sodium excretion (UNaV). 4. Miconazole (1 mumol/L) did not change the functions of LH and LL unstimulated kidneys, but blunted the vasoconstrictor response to NA (110 nmol/L), the difference being significant in LH kidneys only (1.7 +/- 0.2 vs 3.6 +/- 1.2 mmHg/mL per min per g; P < 0.05). 5. Addition of 17-ODYA (3 mumol/L) to miconazole did not modify RVR in LH and LL kidneys or the response to NA infusion. On the contrary, it increased UNaV, the differences being significant in LH kidneys only (22.9 +/- 1.4 vs 17.5 +/- 1.4 mumol/min per g; P < 0.05 after NA 110 nmol/L). 6. It is suggested that EET may contribute to the elevated RVR and HETE to the reduced ability to excrete sodium, of LH kidneys.     

Differentiating keratinocytes express a novel cytochrome P450 enzyme, CYP2B19, having arachidonate monooxygenase activity

The novel cytochrome P450, CYP2B19, is a specific cellular marker of late differentiation in skin keratinocytes. CYP2B19 was discovered in fetal mouse skin where its onset of expression coincides spatially (upper cell layer) and temporally (day 15.5) with the appearance of loricrin-expressing keratinocytes during the stratification stage of fetal epidermis. CYP2B19 is also present postnatally in the differentiated keratinocytes of the epidermis, sebaceous glands, and hair follicles. CYP2B19 mRNA is tightly coupled to the differentiated (granular cell) keratinocyte phenotype in vivo and in vitro. In primary mouse epidermal keratinocytes, it is specifically up-regulated and correlated temporally with calcium-induced differentiation and expression of the late differentiation genes loricrin and profilaggrin. Recombinant CYP2B19 metabolizes arachidonic acid and generates 14,15- and 11, 12-epoxyeicosatrienoic (EET) acids, and 11-, 12-, and 15-hydroxyeicosatetraenoic (HETE) acids (20, 35, 18, 7, and 7% of total metabolites, respectively). Arachidonic acid metabolism was stereoselective for 11S,12R- and 14S,15R-EET, and 11S-, 12R-, and 15R-HETE. The CYP2B19 metabolites 11,12- and 14,15-EET are endogenous constituents of murine epidermis and are present in similar proportions to that generated by the enzyme in vitro, suggesting that CYP2B19 might be the primary enzymatic source of these EETs in murine epidermis.     

Pharmacological evaluation of an epoxide as the putative hyperpolarizing factor mediating the nitric oxide-independent vasodilator effect of bradykinin in the rat heart

A cytochrome P450-derived metabolite of arachidonic acid, namely an epoxyeicosatrienoic acid (EET), has many of the properties of a hyperpolarizing factor that mediates endothelium-dependent, nitric oxide-independent vasodilation. As there are four EET regioisomers, we used pharmacological criteria, based on previous observations with bradykinin (BK), to evaluate which, if any, of the EETs could be considered a potential mediator of vasodilator responses to BK in the rat isolated heart treated with indomethacin and nitroarginine to eliminate prostaglandin and nitric oxide components of the response. Nifedipine, used as a probe for dilator mechanisms dependent on closure of voltage-dependent Ca++ channels, almost abolished the vasodilator effect of cromakalim and attenuated those of BK and 5,6 EET. The vasodilator effects of the other EETs were not reduced and were excluded from consideration as mediators of BK-induced vasodilation. The vasodilator effect of 5,6 EET, as with that of BK, was markedly reduced by charybdotoxin but not iberiotoxin, suggesting the contribution of a similar type K+ channel to the vascular response to both agents. As expected for a putative endothelium- and cytochrome P450-derived mediator, the coronary vasodilator effect of 5,6 EET was not affected by either removal of the endothelium or inhibition of cytochrome P450 with clotrimazole, interventions that virtually abolished the vasodilator activity of BK. Thus, of the four EET regioisomers, 5,6 EET is the most likely mediator of the vasodilator effect of BK in the isolated heart under these experimental conditions.     

Identification and relative quantitation of F2-isoprostane regioisomers formed in vivo in the rat

F2-isoprostanes are a complex mixture of isomers formed in four regioisomeric family types by free radical-initiated oxidation of arachidonic acid present in membrane phospholipids. F2-isoprostanes isolated from the livers of rats treated with carbon tetrachloride were separated by initial reverse phase HPLC and detected using electrospray ionization mass spectrometry with the characteristic loss of 44 u (C2H4O) from the common 1,3-diol cyclopropane ring found in these eicosanoids. Collision induced decomposition of the carboxylate anions from the separated F2-isoprostanes formed abundant ions characteristic for regioisomers of Type I (m/z 115), Type III (m/z 127), and Type IV (m/z 193), which made possible characterization of these three family subtypes by LC/MS/MS. Capillary GC/MS was employed to further identify the F2-isoprostane regioisomers using electron ionization mass spectrometry and to obtain characteristic mass spectra of the pentafluorobenzyl ester trimethylsilyl ether derivatives. Quantitation of the F2-isoprostanes separated by both reverse-phase HPLC and capillary GC/MS was carried out using negative ion chemical ionization mass spectrometry. The most abundant isomers identified were Type I and IV regioisomers constituting 33 and 25% of the total products, respectively. As expected, the Type II and III regioisomer products were of less abundance. Over 45 F2-isoprostanes could be separated in this complex mixture, suggesting random production of each regioisomeric subtype in this in vivo model.     

Giant perimedullary arteriovenous fistulas of the spine: clinical and radiologic features and endovascular treatment

Little is known about the epoxygenase pathway of the arachidonic acid cascade in uterine tissues. In this paper, we describe the formation of epoxyeicosatrienoic acids (EETs) and dihydroxyeicosatrienoic acids (DHETs) in human term placenta after uncomplicated pregnancies. Metabolism of [3H]-arachidonic acid was analyzed in short term tissue cultures of placenta by reverse phase HPLC. Major metabolites coeluted with authentic EETs and DHETs. The formation of EETs was inhibited by carbon monoxide. In non-radioactive incubations with biopsies from seven different placentas, sufficient material for GC/MS analysis was obtained. The combined media were purified by solid phase extraction and reverse phase HPLC. The fraction coeluting with DHETs was derivatized with pentafluorobenzylbromide (PFB) and bis-(trimethylsilyl)-trifluoroacetylacetamide (BSTFA) and analyzed by GC/NICI/MS/MS. 11, 12-DHET and 14, 15-DHET were identified by their mass spectra displaying specific fragments at m/z 149 and m/z 189, respectively. Our results suggest that the epoxygenase pathway is active in human term placenta.     

Phosphorus metabolism during growth of lymphoma in mouse liver: a comparison of 31P magnetic resonance spectroscopy in vivo and in vitro

Large phosphomonoester (PME) signals are detected in the phosphorus magnetic resonance spectra (31P MRS) of many neoplastic and rapidly dividing tissues. In addition, alterations in phosphodiester (PDE) signals are sometimes seen. The present study of a murine lymphoma growing in liver showed a positive correlation between the hepatic PME/PDE ratio measured in vivo by 31P MRS at 4.7 T and the degree of lymphomatous infiltration in the liver, quantified by histology. High-resolution 31P MRS of liver extracts at 9.7 T showed that the PME peak consists largely of phosphoethanolamine (PE) and to a lesser extent of phosphocholine (PC). The concentration of both PE and PC increased positively with lymphomatous infiltration of the liver. In vivo, the PDE peak contains signals from phospholipids (mostly phosphatidylethanolamine and phosphatidylcholine) and the phospholipid breakdown products glycerophosphoethanolamine (GPE) and glycerophosphocholine (GPC). Low levels of GPE and GPC were detected in the aqueous extracts of the control and infiltrated livers; their concentrations remained unchanged as the infiltration increased. The total concentration of phospholipids measured by 31P MRS of organic extracts decreased about 3-fold as the infiltration increased to 70%. Thus, our data showed that the increased PME/PDE ratio in vivo is due to both an increase in the PME metabolites and a decrease in the PDE metabolites. We propose that this ratio can be used as a non-invasive measure of the degree of lymphomatous infiltration in vivo.     

Study of mechanisms involved in the collision-induced dissociation of carboxylate anions from glycerophospholipids using negative ion electrospray tandem quadrupole mass spectrometry

The collision-induced dissociation of the carboxylate anions from human blood phosphatdilycholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylinositol (PI) and phosphatidic acid (PA) containing the C18:0 (sn-1) and C20:4 (sn-2) fatty acyl residues was studied using normal phase liquid chromatography coupled with negative ion electrospray tandem mass spectrometry. The product ion peak area ratio of C18:0 to C20:4 was calculated for each phospholipid species and was found to increase with increasing collision energy for all classes. For the phospholipids with a net neutral charge (PE, PC) there was a preferential loss of the sn-2 carboxylate anion (C20:4) at low collision energy, while at higher energy there was a preferential loss of the sn-1 carboxylate anion (C18:0). For the phospholipids with a net negative charge (PI, PA, PS) the intensity of the sn-1 carboxylate anion peak was equal to or higher than the sn-2 carboxylate anion peak at the energies measured. At a given collision energy the product ion peak area ratio decreased in the order PA > or = PS > PI. Studying PS and PE species at different collision energies, it was found that for both classes the increase in the abundance ratio with increasing collision energy was largely dependent on the chain length and degree of unsaturation of the sn-2 acyl chain.     

(Carboxyalkyl)pyrroles in human plasma and oxidized low-density lipoproteins

Free-radical oxidation of human plasma low-density lipoprotein (LDL) produces (carboxyalkyl)pyrrole (CAP) epitopes that were detected with enzyme-linked immunosorbent assays using antibodies raised against keyhole limpet hemocyanin (KLH)-bound 2-(omega-carboxyheptyl)-pyrrole (CHP) and 2-(omega-carboxypropyl)pyrrole (CPP). These antibodies exhibit high structural selectivity (< 0.5% cross-reactivity) in competitive binding inhibition assays with the corresponding human serum albumin (HSA)-bound pyrroles. No cross-reactivity was detected for HSA-bound 2-pentylpyrrole, an epitope that is generated by a reaction of 4-hydroxy-2-nonenal (HNE) with protein lysyl residues. Oxidation of either arachidonic or linoleic acid in the presence of HSA produced an HNE-derived 2-pentylpyrrole epitope. However, only oxidation of linoleic acid formed HSA-bound CHP, while only oxidation of arachidonic acid generated HSA-bound CPP. Since ester hydrolysis with KOH markedly elevated levels of immunoreactive epitopes detected in oxidized LDL, the CAPs are presumably generated by reactions of oxidized cholesteryl esters, triglycerides, and phospholipids with LDL protein, and only some of these oxidized esters are hydrolyzed, e.g., by phospholipase activity associated with LDL. Protein-bound CHP immunoreactivity was detected in human plasma, and levels are significantly elevated in renal failure and atherosclerosis patients compared with healthy volunteers. This provides the first evidence for the biological occurrence of protein-bound CAPs in vivo and further suggests that free-radical oxidation of polyunsaturated lipids produces hydroxyalkenal carboxylate esters whose gamma-hydroxy-alpha,beta-unsaturated aldehyde functionality and reactivity resemble that of HNE.     

Transfection of an active cytochrome P450 arachidonic acid epoxygenase indicates that 14,15-epoxyeicosatrienoic acid functions as an intracellular second messenger in response to epidermal growth factor

A common feature of most isolated cell systems is low or undetectable levels of bioactive cytochrome P450. We therefore developed stable transfectants of the renal epithelial cell line, LLCPKcl4, that expressed an active regio- and enantioselective arachidonic acid (AA) epoxygenase. Site-specific mutagenesis was used to convert bacterial P450 BM-3 into an active regio- and stereoselective 14S,15R-epoxygenase (F87V BM-3). In clones expressing F87V BM-3 (F87V BM-3 cells), exogenous AA induced significant 14S,15R-epoxyeicosatrienoic acid (EET) production (241. 82 ng/10(8) cells, >97% of total EETs), whereas no detectable EETs were seen in cells transfected with vector alone. In F87V BM-3 cells, AA stimulated [3H]thymidine incorporation and increased cell proliferation, which was blocked by the tyrosine kinase inhibitor, genistein, by the phosphatidylinositol 3 (PI-3) kinase inhibitors, wortmannin and LY294002, and by the mitogen-activated protein kinase kinase inhibitor, PD98059. AA also induced tyrosine phosphorylation of extracellular signal-regulated kinase (ERK) and PI-3 kinase that was inhibited by the cytochrome P450 BM-3 inhibitor, 17-ODYA. Epidermal growth factor (EGF) increased EET production in F87V BM-3 cells, which was completely abolished by pretreatment with either 17-ODYA or the phospholipase A2 (PLA2) inhibitor, quinacrine. Compared with vector-transfected cells, F87 BM-3 transfected cells demonstrated marked increases in both the extent and sensitivity of DNA synthesis in response to EGF. These changes occurred in the absence of significant differences in EGF receptor expression. As seen with exogenous AA, EGF increased ERK tyrosine phosphorylation to a significantly greater extent in F87V BM-3 cells than in vector-transfected cells. Furthermore, in these control cells, neither 17-ODYA nor quinacrine inhibited EGF-induced ERK tyrosine phosphorylation. On the other hand, in F87V BM-3 cells, both inhibitors reduced ERK tyrosine phosphorylation to levels indistinguishable from that seen in cells transfected with vector alone. These studies provide the first unequivocal evidence for a role for the AA epoxygenase pathway and endogenous EET synthesis in EGF-mediated signaling and mitogenesis and provide compelling evidence for the PLA2-AA-EET pathway as an important intracellular-signaling pathway in cells expressing high levels of cytochrome P450 epoxygenase.     

Electrospray ionization tandem mass spectrometric analysis of sulfatide. Determination of fragmentation patterns and characterization of molecular species expressed in brain and in pancreatic islets

The sphingolipid sulfatide is a component of myelin and some non-neuronal cells. Antibodies to sulfatide occur in some patients with autoimmune neuropathies and in patients with insulin-dependent diabetes mellitus (IDDM) caused by immunologic destruction of insulin-secreting pancreatic islet beta-cells. Distinct sulfatide molecular species may differ in immunogenicity, and facile means to identify sulfatide species in islets and other tissues obtainable in only small amounts could be useful. Electrospray ionization mass spectrometry (ESI/MS) permits structural determination of small quantities of phospholipids and is applied here to sulfatide analysis. We find that sulfatide standards are readily analyzed by negative ion ESI/MS, and tandem mass spectra of individual species exhibit some ions common to all species and other ions that reflect distinct fatty acid substituents in different sulfatide molecules. A signature ion cluster resulting from cleavage directed by the alpha-hydroxy group of sulfatide species with a hydroxylated fatty acid substituent identifies such species. Sulfatide profiles in tissue lipid extracts can be obtained by ESI/MS/MS scanning for common sulfatide ions and for ions reflecting fatty acid substituents. Islets are demonstrated to contain sulfatide and to exhibit a profile of species different from that of brain.     

Activation of human polymorphonuclear leukocytes by products derived from the peroxidation of human red blood cell membranes

Oxidation of red blood cell (RBC) ghost preparations initiated by tert-butyl hydroperoxide (tBuOOH) was employed to explore the formation of lipid products derived from endogenous phospholipids that specifically expressed biological activity toward the human polymorphonuclear leukocyte (PMN). Common measure of lipid peroxidation, thiobarbituric acid-reactive substances (TBARS) and the increased absorbance at 235 nm consistent with the formation of conjugated dienes, was observed following a 90-min incubation of RBC ghosts with tBuOOH. Saponification of phospholipids and separation of the resultant fatty acids by RP-HPLC permitted direct mass spectrometric analysis of oxidized fatty acids. Individual HPLC fractions were assayed for their ability to increase intracellular free calcium ion concentrations in human PMN to guide structural investigations. Two fractions were found to contain biologically active components, and tandem mass spectrometric analysis of the abundant ions observed in these fractions resulted in the characterization of several oxidized polyunsaturated fatty acids derived from arachidonic and linoleic acids. The major components in these fractions included 5-hydroxyeicosatetraenoic acid (5-HETE) and 5-hydroperoxyeicosatetraenoic acid (5-HpETE). The dose-dependent increases in intracellular calcium in the neutrophil using synthetic 5(rac)-HETE, 5(rac)-HpETE, and 5-oxo-ETE were found to have EC50's of 250, 6, and 3 nM, respectively. The quantity of 5-oxygenated arachidonate components present in oxidized RBC was consistent with the observed biological response elicited by fractions A and B. This study suggests that 5-HETE and 5-HpETE are abundant products of lipid peroxidation of cellular membranes and that these racemic products possess significant biological activity. Such compounds could play important roles as mediators of the cellular response to toxicologic stimuli that generate free radical species.     

Structures of bacitracin A and isolated congeners: sequencing of cyclic peptides with blocked linear side chains by electrospray ionization mass spectrometry

The bacitracin antibiotic complex consists principally of bacitracin A, a peptide antibiotic containing seven amino acid residues in a ring and five amino acid residues in a blocked side chain, together with a mixture of minor components presumably related but of unknown structures. A preparative high-performance liquid chromatographic method was developed for isolating the minor components A2, B1 and B2 which were then characterized by amino acid analysis, exact mass fast atom bombardment (FAB) mass spectrometry, FAB tandem mass spectrometry (MS/MS) and electrospray ionization (ESI) mass spectrometry. For bacitracins A (MW 1421), A2 (MW 1421), B1a (MW 1407), B1b (MW 1407), B2 (MW 1407) and F (MW 1419), the side chain sequences were determined by ESI MS/MS and ESI nozzle-skimmer collision-induced dissociation (CID) mass spectrometry and the ring sequences elucidated by ESI nozzle-skimmer CID MS/MS. Relative to bacitracin A, bacitracin A2a has the modified isoleucine residue at position 1 replaced by a modified allo-isoleucine residue, bacitracin B1a has the isoleucine residue at position 8 replaced by a valine residue, bacitracin B1b has the isoleucine residue at position 5 replaced by a valine residue and bacitracin B2 has the modified isoleucine residue at position 1 replaced by a modified valine residue. FAB tandem mass spectra were shown to be consistent with the above structural assignments for the isolated bacitracin components. Structures were also proposed for the trace bacitracin components C1 (MW 1393) and D1 (MW 1379) using ESI MS/MS data obtained from the analysis of the bacitracin complex without isolation.     

Epoxygenase metabolites of arachidonic acid affect electrophysiologic properties of rat tracheal epithelial cells1

Epoxyeicosatrienoic acids (EETs) and dihydroxyeicosatrienoic acids, products of the cytochrome P450 arachidonic acid epoxygenase pathway, have been shown to affect electrolyte transport in the kidney; however, the effects of these compounds on airway epithelial ion transport have not been investigated. Intact rat tracheas and primary cultures of rat tracheal epithelial cells were mounted in Ussing chambers to monitor changes in transepithelial voltage (Vt), short circuit current (Isc) and electrical resistance (Rt), with or without the addition of increasing concentrations (10(-9)-10(-6) M) of arachidonic acid, each of the four regioisomeric EETs and each of the corresponding dihydroxyeicosatrienoic acids. In intact tracheas, 11,12-EET caused dose-dependent decreases in Vt and Isc (DeltaVt = 0. 4 +/- 0.1 mV, DeltaIsc = -16.9 +/- 5.4 microA/cm2 at 10(-6) M, P < . 05 vs. vehicle), whereas changes in Rt were not significantly different than vehicle alone. 11,12-dihydroxyeicosatrienoic acid caused less impressive decreases in Vt and Isc, although arachidonic acid and the other compounds tested were without significant effects. 11,12-EET induced similar changes in cultured tracheal epithelial cell electrical parameters at concentrations as low as 10(-9) M. The effects of 11,12-EET were highly stereoselective, with activity limited to 11(R),12(S)-EET, the least abundant rat lung enantiomer. Pretreatment with amiloride or mucosal exposure to sodium free media did not significantly alter the 11,12-EET-induced changes in Vt. In contrast, pretreatment with bumetanide abolished the 11,12-EET electrophysiologic effects, suggesting that these effects may be mediated through inhibition of a chloride conductive pathway. We conclude that arachidonic acid epoxygenase metabolites cause significant changes in rat airway electrical parameters and may be involved in the control of lung fluid and electrolyte transport.     

Genital chlamydial disease in an urban, primarily Hispanic, family planning clinic

A method for the separatory determination of diastereomeric isomers of glucuronic acid conjugates of ibuprofen having a carboxyl group at the chiral center by liquid chromatography (LC)/electrospray ionization (ESI)-mass spectrometry (MS) has been developed. The authentic specimens of acyl glucuronides of R(-)- and S(+)-ibuprofen were chemically synthesized by the Mitsunobu reaction. In the ESI mode, the glucuronides were characterized by an abundant quasi-molecular ion [M-H]-, and the formation of the negative ion was markedly influenced by a drift voltage. The resolution of diastereomeric isomers was achieved on a Develosil ODS-HG-5 column with 20 mM ammonium acetate (pH 5.0):acetonitrile (5:2, v/v) as a mobile phase where diastereomers were monitored with a corresponding quasi-molecular ion. After oral administration of racemic ibuprofen, a preferential excretion of (S)-ibuprofen glucuronide into the urine was observed.     

Molecular species of sphingomyelin: determination by high-performance liquid chromatography/mass spectrometry with electrospray and high-performance liquid chromatography/tandem mass spectrometry with atmospheric pressure chemical ionization

In a sphingomyelin-enriched sample of polar lipids from bovine milk, molecular species of intact sphingomyelin were separated by normal-phase high-performance liquid chromatography and detected by mass spectrometry (MS) for structural information. First, by using electrospray with positive ionization (ESI), protonated molecules ([M + H]+) were detected. Second, in atmospheric pressure chemical ionisation (APCI+), in-source fragmentation of sphingomyelin ions led to the formation of ceramide ions. With the ceramide ions as precursors, ions representative of both the long-chain base (LCB) parts and the fatty acid (FA) parts were detected in APCI-MS/MS via collision-induced decomposition (CID). Using this procedure, it was possible to determine the sphingomyelin molecular masses using ESI+ and then their respective LCB-FA combinations(s) using APCI+(-)MS/MS. At least 36 protonated molecules of intact sphingomyelin were detected in the bovine milk sample. The combinations found covered a range of molecular masses from 673 to 815 Da. The 12 most common protonated molecules (constituting approximately 90% of the total ion current in ESI) were composed of at least 25 different LCB-FA combinations. Saturated and unsaturated LCBs and FAs were detected in addition to hydroxy fatty acids. The most common LCBs were 16:1, 17:1, 18:1 and 19:1, whereas the most common FAs were 16:0, 22:0, 23:0 and 24:0. LCB-FA combinations of sphingomyelin from bovine brian, bovine erythrocytes and chicken egg yolk are also presented.