Targeted profiling of arachidonic acid and eicosanoids in rat tissue by UFLC-MS/MS: Application to identify potential markers for rheumatoid arthritis.

We describe a method for the targeted analysis of bioactive arachidonic acid metabolites through cyclooxygenase (COX) and lipoxygenase (LOX) pathway in knee joint, liver, kidney, spleen and heart using an ultra-fast liquid chromatography-tandem mass (UFLC-MS/MS) method. Method validation was investigated, including linearity, precision, accuracy, matrix effect, extraction recovery and stability for the simultaneous analysis of prostaglandins (PGs), thromboxanes (TXs), leukotrienes (LTs) and hydroxyeicosatetraenoic acids (HETEs). The method enables us to chromatographically separate branched-chain species from their straight-chain isomers as well as separate biologically important eicosanoids. The concentrations of the following major eicosanoids were significantly increased in rheumatoid arthritis model rats than in normal ones: 5-HETE, 8-HETE, 12-HETE, 15-HETE, PGF2α, TXB2, 5-HpETE, LTE4, PGE2, PGD2, LTB4. Further multivariate data analysis (partial least square-discriminant analysis) showed COX products (PGs, TXs) were readily distributed towards liver and kidney, LOX products (LTs, HETEs) towards knee joint and spleen, and heart had no characteristic metabolites. The method described here offers a useful tool for the evaluation of complex regulatory eicosanoids responses in RA disease states and provides support for use of dual inhibitors of COX and LOX enzymes on RA treatment.

Metabolism of 5,6-epoxyeicosatrienoic acid by the human platelet. Formation of novel thromboxane analogs.

Radiolabeled cis-(+-)-5,6-epoxyeicosatrienoic acid (5(6)-EpETrE) was incubated with a suspension of isolated human platelets in order to study its metabolic fate. The epoxide slowly disappeared from the suspension and was completely metabolized within 30 min. After extraction and analysis by reverse-phase high performance liquid chromatography, seven metabolites were found. Addition of either indomethacin (0.01 mM, cyclooxygenase inhibitor) or BW755C (0.1 mM, cyclooxygenase/lipoxygenase inhibitor) to the incubations blocked the formation of four and six metabolites, respectively, 1,2-Epoxy-3,3,3-trichloropropane (inhibitor of microsomal epoxide hydrolase) failed to inhibit the formation of 5,6-dihydroxyeicosatrienoic acid (5,6-DiHETrE), a hydrolysis product of the precursor 5(6)-EpETrE. The metabolites were characterized by UV spectroscopy, negative ion chemical ionization liquid chromatography/mass spectrometry, gas chromatography/mass spectrometry and, in one instance, coelution with synthetic standard. Three primary platelet metabolites were structurally determined to be 5,6-epoxy-12-hydroxyeicosatrienoic acid, 5,6-epoxy-12-hydroxyheptadecadienoic acid, and a unique bicyclic metabolite, 5-hydroxy-6,9-epoxy-thromboxane B1, which originated from intramolecular hydrolysis of 5,6-epoxythromboxane-B1. This thromboxane analog was partially separated into stereoisomers and coeluted with the racemic synthetic standard in gas chromatography/mass spectrometry and liquid chromatography/mass spectrometry. Three other metabolites were characterized as 5,6,12-trihydroxyeicosatrienoic acid, 5,6,12-trihydroxyheptadecadienoic acid, and 5,6-dihydroxythromboxane-B1, and resulted from the hydrolysis of the corresponding epoxides rather than from the metabolism of 5,6-DiHETrE. The latter was not metabolized by platelet cyclooxygenase or lipoxygenase. The biosynthesis of two cyclooxygenase metabolites indicated the formation of unstable 5,6-epoxythromboxane-A1 as an intermediate precursor. Platelet aggregation was not induced by 5(6)-EpETrE, although responsiveness to arachidonic acid was reduced following preincubation with the epoxide. The platelet metabolites of 5(6)-EpETrE might be useful in assessing its in vivo production in humans.

11-Dehydrothromboxane B2: a quantitative index of thromboxane A2 formation in the human circulation.

In human plasma, 11-dehydrothromboxane (TX) B2 is a major long lived metabolite (t1/2 45 min) formed from infused TXB2, the hydration product of biologically active TXA2. Plasma concentrations of TXB2 itself are readily confounded by ex vivo platelet activation and, theoretically, an enzymatic derivative of this compound, not subject to formation in whole blood, would more accurately reflect TXA2 formation in vivo. To address this hypothesis, we developed a sensitive assay for both 11-dehydro-TXB2 and TXB2, using capillary gas chromatography/negative-ion chemical ionization mass spectrometry. We established that whole blood possesses a minor capacity to form 11-dehydro-TXB2, attributable to nonenzymatic formation in erythrocytes. However, the nonenzymatic formation of 11-dehydro-TXB2 was not a practical limitation to its use as an index of TX biosynthesis. Blood was drawn from healthy volunteers (i) via an indwelling catheter at the time of insertion and at 30, 60, 90, 180, and 240 min thereafter and (ii) via separate venipunctures at 0 time and at 90 and 240 min thereafter. Plasma TXB2 drawn via the catheter at baseline (66 +/- 63 pg/ml) was substantially greater than the maximal estimate of endogenous TXB2 (1-2 pg/ml) in plasma [Patrono, C., Ciabattoni, G., Pugliese, F., Perruci, A., Blair, I. A. & FitzGerald, G. A. (1986) J. Clin. Invest. 77, 590-594] and increased in magnitude and variance over time (339 +/- 247 pg/ml at 240 min). By contrast, 11-dehydro-TXB2 did not change significantly in the sequential catheter samples or in the samples drawn by separate venipuncture. Basal plasma concentrations in volunteers were depressed by pretreatment with 325 mg of aspirin. Furthermore, the range of concentrations in patients with severe atherosclerosis in whom urinary 2,3-dinor-TXB2 was increased was significantly higher (5-50 pg/ml, P less than 0.01) than in healthy subjects (0.9-1.8 pg/ml). Concentrations of 11-dehydro-TXB2 were increased in patients who had recently suffered a pulmonary embolism to a greater extent than either the 11-dehydro-13,14-dihydro-15-keto-TXB2 or the 2,3-dinor-TXB2 metabolites in plasma. These results indicate that plasma TXB2 is readily confounded by platelet activation ex vivo. Measurement of enzymatic metabolites of TXB2 minimizes this problem. The 11-dehydro metabolite is the most appropriate analytic target to detect phasic release of TXA2 in the human circulation, such as might occur in human syndromes of platelet activation.

Simultaneous extraction and preparation for high-performance liquid chromatography of prostaglandins and phospholipids.

A method for the maximum recovery of prostaglandins from brain tissue with simultaneous recovery of neutral lipids and phospholipids was developed. Hexane:2-propanol was used to extract lipids from bovine brain. This method, which does not require a washing step to remove nonlipid contaminants, was compared to extraction according to Folch et al. [(1957) J. Biol. Chem. 226, 497-509] for efficiency of lipid extraction. Recoveries of prostaglandins were 12-37% greater with hexane:2-propanol than with the Folch extraction procedure with washing. The ratios of cholesterol to lipid phosphorus and absolute phospholipid recoveries were comparable for the two methods. A new elution sequence was devised for separation of lipid classes on silicic acid columns. The elution sequence was chloroform (neutral lipids and free fatty acids), methyl formate (prostaglandins and cerebrosides), acetone (remaining glycolipids), and methanol (phospholipids). Reverse-phase HPLC of the methyl formate fraction was used to separate the prostaglandins. The method permits simultaneous quantitative recovery of prostaglandins and phospholipids (which contain the 20:4(n-6) precursor for prostaglandin synthesis), and therefore allows changes in phospholipid composition and prostaglandin synthesis to be studied in the same tissue sample.

Thromboxane A3 (TXA3) is formed in human platelets after dietary eicosapentaenoic acid (C20:5 omega 3).

Platelet-rich plasma of subjects, who had ingested cod liver oil containing 10% eicosapentaenoic acid (C20:5 omega 3), the precursor of trienoic prostanoids, was stimulated ex vivo with collagen. Formation of thromboxane B3, the hydrolysis product of non-aggregatory thromboxane A3, from endogenous eicosapentaenoic acid was demonstrated by combined capillary gas chromatography-mass spectrometry. Concomitantly platelet aggregation in platelet-rich plasma upon low doses of collagen and associated thromboxane B2 formation from endogenous arachidonic acid were reduced. We conclude that both the formation of inactive thromboxane A3 as well as the reduction of thromboxane A2 may contribute to the reduced platelet reactivity after dietary eicosapentaenoic acid.

An improved method for separation of thromboxane B2 by reversed phase liquid chromatography.

Column efficiency for thromboxane B2 (TXB2) is 10 times lower than for prostaglandins when chromatographed on octadecyl-silica columns. We described the use of a new non-silica reversed phase support which brings the column efficiency for TXB2 in the range of the prostaglandins.

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

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

Extraction and purification of prostaglandins and thromboxane from biological samples for gas chromatographic analysis.

An efficient extraction procedure for the isolation of prostaglandins (PGs) from biological samples for their subsequent quantification by gas chromatography-electron capture detection (GC-ECD) is described. PGs were extracted from lung, kidney, spleen and stomach fundus into ethyl acetate at different pHs. The highest recovery and least extraction of contaminating pigments was obtained at pH 4.5. Pigments and other contaminants are removed by thin layer chromatography using a solvent system chloroform-isopropyl alcohol-ethanol-formic acid (45:5:0.5:0.3). The isolated PGs were determined by GC-ECD after appropriate derivatization. The overall recovery of PGs using this procedure is 60%.

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

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

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

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

Identification of thromboxane B2 in guinea-pig uterine homogenates.

On the basis of gas chromatographic and mass spectrometric evidence, thromboxane B2 has been identified in incubates of homogenised guinea-pig uterus.