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.     

Sexual changes in men with urological disorders

Electrospray ionization (ESI) and tandem mass spectrometry (MS/MS) were used to analyze epoxyeicosatrienoic acids (EETs) and monohydroxyeicosatetraenoic acids (HETEs) isolated from human red blood cell membranes following base hydrolysis. ESI results in the formation of an abundant isobaric carboxylate anion at m/z 319 for both of these oxidized metabolites of arachidonic acid. The product ion spectra from the collision-induced dissociation of this carboxylate anion could be used to identify each of the isomeric eicosanoids from the unique fragment ions of each eicosanoid. The observed product ion spectra were identical with those previously obtained by fast atom bombardment ionization; however, ESI required less EET and HETE for analysis. Both EET and HETE phospholipids were present in human red blood cells (RBCs) and their abundance could be substantially increased by treatment under conditions that would induce free radical oxidation of membrane phospholipids. Following incubation of human RBCs with tert-butyl hydroperoxide (tBuOOH), phospholipids were extracted and purified by normal-phase high-performance liquid chromatography (HPLC) as to glycerophospholipid class containing ethanolamine (GPE), serine (GPS) and choline (GPC) as the polar head group. Each class of phospholipid was hydrolyzed to yield the free carboxylic acid prior to on-line HPLC/ESI-MS/MS analysis. The formation of oxidized arachidonic acid esterified to phospholipids in treated RBCs was found to increase significantly for both esterified EETs in GPE, GPS and GPC which increased 49-, 34- and 59-fold, respectively, and also for esterified HETEs in GPE, GPS and GPC which increased 3-, 4- and 11-fold, respectively, compared with untreated RBCs. These results provide the first characterization of EETs formed non-enzymatically as intact phospholipids in a lipid peroxidation model system.     

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.     

Cytochrome P450 metabolites of arachidonic acid: rapid incorporation and hydration of 14,15-epoxyeicosatrienoic acid in arterial smooth muscle cells

Arachidonic acid is converted to epoxyeicosatrienoic acids (EETs) by cytochrome P450 monooxygenases. EETs produce arterial vasodilatation, and recent evidence suggests that they are endothelium-derived hyperpolarizing factors. In porcine coronary arteries contracted with a thromboxane mimetic agent, we find that relaxation is rapidly initiated by exposure to 14,15-EET. The relaxation slowly increases in magnitude, resulting in a response which is sustained for more than 10 min. Cultured porcine aortic smooth muscle cells rapidly take up [3H]14,15-EET. After 3 min, radioactivity is present in neutral lipids, phosphatidylcholine, and phosphatidylinositol. The cells also convert 14,15-EET to 14,15-dihydroxyeicosatrienoic acid (14,15-DHET), and some DHET is detected in the medium after only 1 min of incubation. Like 14,15-EET, 14,15-DHET produces relaxation of the contracted coronary artery rings. These findings suggest that the incorporation into phospholipids and conversion to 14,15-DHET can occur at a rate that is fast enough to modulate the vasorelaxation produced by 14,15-EET.     

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.     

Role of P-450 arachidonic acid epoxygenase in the response of cerebral blood flow to glutamate in rats

BACKGROUND AND PURPOSE: Glutamate, a major excitatory neurotransmitter in the brain, has been implicated in the hyperemic response to increases in the activity of neurons, but the mechanism of glutamate-induced dilation of cerebral blood vessels is unknown. Glutamate has been shown to enhance the release of arachidonic acid (AA) in brain tissue and cultured astrocytes. We have previously shown that astrocytes metabolize AA to vasodilator products, epoxyeicostrienoic acids (EETs), and express a P-450 AA epoxygenase, P-450 2C11. We tested the hypothesis that glutamate-induced dilation of cerebral arterioles is mediated in part by changes in the formation and release of EETs by perivascular astrocytes. METHODS: Primary astrocyte cultures were prepared from 3-day-old rat pups. The cells were labeled with [14C]AA, and the effect of glutamate on the formation of EETs from [14C]AA by cultured astrocytes was studied. The expression of P-450 2C11 protein in the microsomal fractions of cultured astrocytes was assessed by Western blot. In vivo cerebral blood flow measurements were made in adult rats by laser-Doppler flowmetry after administration of glutamate into the subdural space of the rat before and after treatment with miconazole. RESULTS: Glutamate treatment (100 mumol/L for 30 minutes) induced a threefold increase in the formation of EETs from [14C]AA by cultured astrocytes, and the increase was inhibited by miconazole (20 mumol/L), an inhibitor of P-450 AA epoxygenase. Treatment with glutamate (100 mumol/L) for 12 hours increased the expression of P-450 2C11 protein in the microsomal fraction of cultured astrocytes. The response of laser-Doppler cerebral blood flow to administration of glutamate (500 mumol/L) into the subdural space of the rat was significantly attenuated after treatment with miconazole (20 mumol/L for 30 minutes). CONCLUSIONS: These findings suggest a role for a P-450 AA epoxygenase in astrocytes in the coupling between the metabolic activity of neurons and regional blood flow in the brain.     

DNA adducts of 2,3-epoxy-4-hydroxynonanal: detection of 7-(1', 2'-dihydroxyheptyl)-3H-imidazo[2,1-i]purine and 1,N6-ethenoadenine by gas chromatography/negative ion chemical ionization/mass spectrometry

2,3-Epoxy-4-hydroxynonanal (EH) is a bifunctional aldehyde formed by epoxidation of trans-4-hydroxy-2-nonenal, a peroxidation product of omega-6 polyunsaturated fatty acids. EH is mutagenic and tumorigenic and capable of modifying DNA bases forming etheno adducts in vitro. Recent studies showed that etheno adducts are present in tissue DNA of humans and untreated rodents, suggesting a potential endogenous role of EH in their formation. A sensitive assay is needed so we can determine whether EH is involved in etheno adduct formation in vivo and study the biological significance of the etheno adducts in DNA. In this study, we developed a gas chromatography/negative ion chemical ionization/mass spectrometry assay for the analysis of 1, N6-ethenoadenine (epsilonAde) and 7-(1', 2'-dihydroxyheptyl)-3H-imidazo[2,1-i]purine (DHH-epsilonAde) in DNA; both are products from the reaction of adenine with EH. The assay entails the following sequence of steps: (1) addition of [15N5]epsilonAde and [15N5]DHH-epsilonAde to DNA as internal standards, (2) acid hydrolysis of DNA, (3) adduct enrichment by C18 solid phase extraction (SPE), (4) derivatization by pentafluorobenzylation (PFB), (5) separation of PFB-epsilonAde and PFB-DHH-epsilonAde on a Si SPE column, (6) acetonide (ACT) formation of PFB-DHH-epsilonAde, and (7) GC/MS analysis with selective ion monitoring (SIM). The limit of detection by on-column injection for PFB-epsilonAde monitoring of the (M - PFB)- ion at m/z 158 was 30 amol and for ACT-PFB-DHH-epsilonAde monitoring of the (M - PFB)- ion at m/z 328 was 0.4 fmol; the detection limits for the entire assay were 6.3 fmol for epsilonAde and 36 fmol for DHH-epsilonAde. In calf thymus DNA modified with EH at 37 degreesC for 50 h, both epsilonAde and DHH-epsilonAde were detected at high levels by this method, 4.5 +/- 0.7 and 90.8 +/- 8.7 adducts/10(3) adenine, respectively. These levels were also verified by HPLC fluorescence analysis, indicating that EH extensively reacts with adenine in DNA, forming etheno adducts. The high sensitivity of the assay suggests that it may be used in the analysis of ethenoadenine adducts in vivo.     

Acetylcholine in human CSF: methodological considerations and levels in dementia of Alzheimer type

Four different methods to measure acetylcholine (ACh) and choline (Ch) concentration, i.e. thermospray/mass spectroscopy (TS/MS), high pressure liquid chromatography/mass spectroscopy (HPLC/MS), high pressure liquid chromatography/electrochemical detection (HPLC/ECD) and gas chromatography/mass spectroscopy (GC/MS), both latter methods coupled to a solid phase extraction system were compared for their applicability to human lumbar cerebrospinal fluid (CSF). Furthermore, samples from 15 control persons and 11 patients with dementia of Alzheimer-type (DAT) were compared to search for an ACh deficit in the CSF in DAT. GC/MS was the most sensitive, but most laborious method, and HPLC/ECD was acceptably sensitive, reliable and more specific. TS/MS was not specific enough for CSF extracts and HPLC/MS was more specific, but far less sensitive. Thus, only GC/MS and HPLC/ECD were qualified to detect ACh in human CSF extracts. Comparison of GC/MS and HPLC/ECD revealed highly correlated levels of ACh (r = 0.999). Using HPLC/ECD, ACh concentrations were greatly reduced in the DAT group (3.75 +/- 1.40 pmol/ml CSF) as compared to the controls (6.14 +/- 1.39 pmol/ml CSF), but the difference between controls and DAT patients was not statistically significant due to the number of samples below detection limit (8 out of 11 samples in DAT, 7 out of 15 in controls). Ch concentrations were not statistically significant between the two groups. The data show that methodological limitations preclude a widespread clinical application of determining ACh in the human CSF. Despite of reductions of ACh in the CSF in DAT, the determination of Ach in the CSF is not suitable for diagnostic purposes.     

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.     

The interaction of myristylated peptides with the catalytic domain of protein kinase C revealed by their sequence palindromy and the identification of a myristyl binding site

The nature of products of contamination intake were investigated in cattle dosed with [14C]di-n-butylphthalate (DBP). Radio-labelled metabolites were extracted from bile, faeces, plasma and urine onto solid-phase media, fractionated by ion-exchange chromatography, separated by reverse phase HPLC and analysed by negative ion atmospheric pressure chemical ionization mass spectrometry(n) (LCQ, Finnigan). All matrices contained a common major metabolite [deprotonated molecular ion (M-H)- m/z 221] which coeluted with and had an identical daughter ion spectrum to reference monobutylphthalate (MBP). MBP was metabolised to a beta-glucuronidase sensitive compound (M-H)- m/z 397 whose spectrum contained daughter ions (m/z 175 and 221) consistent with the parent glucuronide. A further three beta-glucuronidase resistant radio-labelled metabolites were also produced (M-H- m/z 165, 193 and 237); comparison of daughter ion spectra with those of reference MBP and phthalic acid indicated identity with phthalic acid, monoethylphthalate (MEP) and monohydroxybutylphthalate (MHBP) respectively. The presence of a benzoate daughter ion (m/z 121) in all spectra was indicative of side chain biotransformation. Both MBP and MEP contained a phthalate daughter ion (m/z 165) indicating loss of a butyl and ethyl side chain respectively. A daughter ion of m/z 89 derived from the side chain provided evidence that the third metabolite was MHBP. Incubation of DBP with isolated bovine hepatocytes produced the same metabolites and provided relatively clean samples for LC/MSn analysis. Detection of these DBP metabolites in meat or dairy food products will provide evidence for environmental exposure and biotransformation in vivo, whereas the presence of the parent compound would suggest contamination during food processing and packaging.     

Identification and determination of phase I metabolites of propafenone in rat liver perfusate

Propafenone (PF) is a class 1C antiarrhythmic agent. To study the mechanisms of PF interactions with dietary nutrients in isolated, perfused rat livers, metabolites of PF in liver perfusate were identified and an analytical method was developed for these metabolites plus parent drug. Identification of phase I metabolites was performed using HPLC/MS equipped with a Lichrospher RP-18 column and tandem mass spectrometry (MS/MS) with electrospray and atmospheric pressure chemical ionizations. Three major metabolite peaks, whose protonated molecular ions were m/z 358, 358 and 300, were identified as a propafenone derivative hydroxylated in the omega-phenyl ring (omega-OH-PF), 5-hydroxypropafenone (5-OH-PF), and N-despropylpropafenone (N-des-PF). The levels of omega-OH-PF, 5-OH-PF, N-des-PF and PF were determined simultaneously by HPLC with UV detection at 210 nm and a mobile phase of 0.03% triethylamine and 0.05% phosphoric acid in water-acetonitrile-methanol (45:20:35, v/v/v) after extraction with 5 ml diethyl ether at pH 10.0 and evaporation of solvent under nitrogen. The results revealed that omega-OH-PF, which was not found in humans, was the major metabolite of PF in rat liver perfusate, not 5-OH-PF which is the major metabolite in human plasma.     

Rapid simultaneous analysis of prostaglandin E2, 12-hydroxyeicosatetraenoic acid and arachidonic acid using high performance liquid chromatography/electrospray ionization mass spectrometry

Arachidonic acid (AA) can be metabolized to a variety of lipid mediators including prostaglandins (PGE), and hydroxyeicosatetraenoic acids (HETE) by cyclooxygenase, lipoxygenase and cytochrome P450-dependent monooxygenase enzymatic pathways. Traditional experimental procedures to quantify these lipid mediators require purification, often by high performance liquid chromatography (HPLC), prior to derivatization for gas chromatography/mass spectrometry (GC/MS) analysis. This paper describes a rapid and simple technique for the simultaneous quantitative analysis of PGE2, 12-HETE, and AA by HPLC/electrospray ionization mass spectrometry on cultured human dermal fibroblast supernatants. Extension of the method to analyse 5-HETE and 15-HETE was investigated. The advantages of this method include minimal sample preparation and elimination of the problem associated with thermal stability for GC/MS analysis. A detection limit of 20pg on column for PGE2 and 5pg on column for 12-HETE and AA was determined.     

煤中天然有机物的高效液相色谱-质谱/质谱分析

运用红外光谱法测定褐煤氧化前后的结构变化,在1580cm-1及1400cm-1处的两个特征峰表明氧化煤中含有大量的羧酸类物质。以此为基础,采用温和的碱氧化方法对煤进行氧化处理,选择了合适的分离条件,建立了氧化煤中天然有机物质预分离样品的高效液相色谱方法,并通过电喷雾串联质谱(ESI(-)-MS/MS)法对其中一组质量数相差58的物质进行了细致的分析。选择m/z268为特征吸收峰,并依据煤的大分子结构,对分析得到的物质进行细致的结构推测,认为该物质应为含有两个乙酸基侧链的炔基喹啉,其化学式为C15H11NO     

Determination of dehydroepiandrosterone sulfate in human plasma by gas chromatography/mass spectrometry using a deuterated internal standard: a method suitable for routine clinical use

A capillary gas chromatographic-mass spectrometric method for the determination of dehydroepiandrosterone sulfate (DHEA-S) in human plasma is described. [7,7-2H2]DHEA-S is used as internal standard. After desulfation by methanolysis, heptafluorobutyryl derivatives are prepared, and selected ion monitoring of characteristic fragment ions--m/z 270 for DHEA and m/z 272 for [7,7-2H2]DHEA--carried out. Requiring small amounts of plasma, the rapid, convenient work-up and the application of bench-top GC/MS instrumentation proved our method to be suited for routine clinical use in adults and children. The method needs no complex corrections for isotope contributions and provides good accuracy and precision. Comparative values of samples assayed by our GC/MS procedure and by a direct RIA indicated that the RIA overestimates the true concentration.     

Recognition of chitin and proteins in invertebrate cuticles using analytical pyrolysis/gas chromatography and pyrolysis/gas chromatography/mass spectrometry

Flash pyrolysis/gas chromatography (py/GC) and py/GC/mass spectrometry (MS) have been utilized to characterize the cuticles of invertebrates chemically. Pyrolysis products have been identified and assigned to specific cuticular components. Acetylpyridones, acetamidofuran, 3-acetamido-5-methylfuran and 3-acetamido-(2 and 4)-pyrones are proposed as characteristic pyrolysis markers for chitin. Pyrolysis products displaying ions of m/z 70, 154, 168, 194 are thought to derive from diketopiperazine structures and provide potential markers for proteins and peptides in which proline, alanine, valine, arginine and glycine are the dominant amino acids. These products, constituting specific pyrolysis markers for invertebrate cuticles, may reflect the amino acid composition of their constituent structural proteins. The source of the various pyrolysis products of proteins has been verified by pyrolysis of reference proteins, peptides and amino acid mixtures. The presence of additional pyrolysis products related directly to histidine and catechol moieties is consistent with the chemical structure and composition proposed for arthropod cuticles based on recent work utilizing solid state 13C and 15N nuclear magnetic resonance. This study constitutes the first comprehensive chemical characterization of the pyrolysis products of invertebrate cuticles and provides the basis for future investigations requiring qualitative screening for cross-linked chitin and proteins in modern and fossil cuticles and in materials, e.g. geopolymers, that may be derived from them.     

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.     

Volunteer bias, sexuality, and personality

A gas chromatography-mass spectrometry (GC-MS) procedure was developed for the detection of 4-hydroxycoumarin anticoagulants and their metabolites in urine as part of a systematic toxicological analysis procedure for acidic drugs and poisons after extractive methylation. The part of the phase-transfer catalyst remaining in the organic phase was removed by solid-phase extraction on a diol phase. The compounds were separated by capillary GC and identified by computerized MS in the full scan mode. Using mass chromatography with the ions m/z 291, 294, 295, 309, 313, 322, 324, 336, 343 and 354, the possible presence of 4-hydroxycoumarin anticoagulants and/or their metabolites could be indicated. The identity of positive signals in such mass chromatograms was confirmed by comparison of the peaks underlying full mass spectra with the reference spectra recorded during this study. This method allowed the detection of therapeutic concentrations of phenprocoumon and warfarin in human urine samples. In absence of human urine, acenocoumarol, coumachlor, coumatetrayl, pyranocoumarin (cyclocumarol) could be detected only in rat urine.     

Identification of the 11,14,15- and 11,12, 15-trihydroxyeicosatrienoic acids as endothelium-derived relaxing factors of rabbit aorta

A number of endothelium-derived relaxing factors have been identified including nitric oxide, prostacyclin, and the epoxyeicosatrienoic acids. Previous work showed that in rabbit aortic endothelial cells, arachidonic acid was metabolized by a lipoxygenase to vasodilatory eicosanoids. The identity was determined by the present study. Aortic homogenates were incubated in the presence of [U-14C]arachidonic acid, [U-14C]arachidonic acid plus 15-lipoxygenase (soybean lipoxidase), or [U-14C]15-hydroxyeicosatetraenoic acid (15-HPETE) and analyzed by reverse phase high pressure liquid chromatography (RP-HPLC). Under both experimental conditions, there was a radioactive metabolite that migrated at 17.5-18.5 min on RP-HPLC. When the metabolite was isolated from aortic homogenates, it relaxed precontracted aortas in a concentration-dependent manner. Gas chromatography/mass spectrometry (GC/MS) of the derivatized metabolite indicated the presence of two products; 11,12,15-trihydroxyeicosatrienoic acid (THETA) and 11,14,15-THETA. A variety of chemical modifications of the metabolite supported these structures and confirmed the presence of a carboxyl group, double bonds, and hydroxyl groups. With the combination of 15-lipoxygenase, arachidonic acid, and aortic homogenate, an additional major radioactive peak was observed. This fraction was analyzed by GC/MS. The mass spectrum was consistent with this peak, containing both the 11-hydroxy-14, 15-epoxyeicosatrienoic acid (11-H-14,15-EETA) and 15-H-11,12-EETA. The hydroxyepoxyeicosatrienoic acid (HEETA) fraction also relaxed precontracted rabbit aorta. Microsomes derived from rabbit aortas also synthesized 11,12,15- and 11,14,15-THETAs from 15-HPETE, and pretreatment with the cyctochrome P450 inhibitor, miconazole, blocked the formation of these products. The present studies suggest that arachidonic acid is metabolized by 15-lipoxygenase to 15-HPETE, which undergoes an enzymatic rearrangement to 11-H-14,15-EETA and 15-H-11,12-EETA. Hydrolysis of the epoxy group results in the formation of 11,14,15- and 11,12,15-THETA, which relaxed rabbit aorta. Thus, the 15-series THETAs join prostacyclin, nitric oxide, and epoxyeicosatrienoic acids as new members of the family of endothelium-derived relaxing factors.     

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.     

Social senses: G-protein-coupled receptor signaling pathways in Dictyostelium discoideum

Incubation of phenol-induced cells of the yeast Candida maltosa SBUG 700 with mono- and dichlorophenols resulted in the formation of metabolites of the substrates and of further metabolites not related to the degradation pathway of the substrates. These additional compounds, identified as 4-hydroxyphenylacetic acid (4-HPA), phenylacetic acid (PA), indolylacetic acid (IA) and indolylethanol (i.e.) by means of HPLC and GC/MS, were not excreted in incubation experiments with glucose. The excretion of these metabolites of aromatic amino acid metabolism is not caused by toxic effects of the phenol derivatives, but seems to be a result of carbon and nitrogen starvation of yeast cells.