Mass spectrometric quantitation and analysis of leukotrienes and other 5-lipoxygenase metabolites

Detailed studies fo the 5-lipoxygenase pathway of arachidonic acid metabolism is a difficult challenge, but nonetheless, an important pursuit. The leukotriense are perplexing compounds to quantitate due, in part, to their production in very small quantities by only certain cells, as well as to their chemical/biochemical instability. Several mass spectrometric techniques have been developed to quantitate 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene b₄ (LTB₄). The mass spectral properties of terbutyldimethylsilyl derivatives of LTB₄ are reported here which are quite favorable for electron impact ionization. Catalytic reduction of LTB₄ prior to derivatization greatly improved capillary gas chromatographic behavior as well as electron impact mass spectral properties. Subpicomole quantities could be readily detected by selected ion recording of the M-57 ion, which is the most abundant ion in the mass spectrum. Lipoxygenase products labeled with oxygen-18 at the carboxyl moiety are uniquely stable to catalytic reduction and, thus, may serve as useful internal standards.     

Selective feed-back inhibition of the 5-lipoxygenation of arachidonic acid in human T-lymphocytes

Purified human T-lymphocytes exhibit 5-lipoxygenase activity as demonstrated by the conversion of arachidonic acid to 5-hydroxy-eicosatetraenoic acid (5-HETE), 5(S),12(R)-di-hydroxy-eicosa-6,14 cis-8,10 trans-tetraenoic acid (leukotriene B₄), and 5,12-di-HETE isomers of leukotriene B₄ that lack a 6-cis double bond. The concentrations of leukotriene B₄, 5-HETE, 11-HETE and 15-HETE in suspensions of T-lymphocytes were increased significantly by concanavalin A and by the calcium ionophore A23187. Preincubation of T-lymphocytes with 15-HETE at μM concentrations, characteristic of suspensions of stimulated lymphocytes, inhibited selectively the increases in the levels of 5-HETE and leukotriene B₄, but not of 11-HETE and prostaglandin E₂.     

Stable isotope dilution gas chromatography/mass spectrometry of prostaglandins and leukotrienes

The group of arachidonic acid metabolites comprising the prostaglandins, thromboxanes, and leukotrienes (eicosanoids) are extremely potent, biologically active compounds. Their properties include proaggregatory anti-aggregatory activity for platelets, chemotactic activity for neutrophils, vasoactive activity, and contractile activity to smooth muscle. In order to determine the role of these substances in pathophysiological conditions, it is essential to have highly sensitive methods available for their analysis. It is generally accepted that combined gas chromatography/mass spectrometry is the most specific technique available for the quantitative analysis of eicosanoids. However, methods based on electron impact ionization and positive ion chemical ionization are relatively insensitive, and many investigators have preferred the use of less specific but more sensitive methods based on radioimmunoassay. We have explored the use of negative ion chemical ionization mass spectrometry to improve sensitivity coupled with capillary column chromatography to maximize specificity. Conversion of the terminal carboxyl group (present in all eicosanoids) to the pentafluorobenzyl ester derivative confers excellent electron capturing properties to the molecule. The derivative undergoes highly efficient thermal electron capture in the gas phase, and any fragmentation that occurs subsequently is directed almost entirely away from the analyte molecule. The stabilized carboxylate anion that results carries at least 30% of the total ion current. Using selected ion monitoring techniques it is possible to detect eicosanoids in the range 1–8 pg on column. This methodology has been applied to the development of stable isotope dilution assays for plasma 6-oxo-prostaglandin (PG) F_1α (1) and for the simultaneous analysis of six biologically important PGs in biological fluids (2). In addition, stable isotope dilution techniques have been developed for the analysis of serum thromboxane B₂ and serum leukotriene B₄ (3). The application of this technology to understanding the role of arachidonic acid metabolism in humans will be discussed.     

The identification of hydroxy fatty acids in psoriatic skin

Psoriasis is a common chronic inflammatory and proliferative skin disease characterised by epidermal neutrophil infiltration which may be induced by chemotactic substances in the involved epidermis. Superficial psoriatic scale was shown to contain biologically active amounts of leukotriene B₄ and monohydroxy-eicosatetraenoic acid (HETE)- like material as determined by assay for chemokinetic activity in high performance liquid chromatography (HPLC) fractions of scale extracts. Extracts of scale and chamber fluid from abraded lesional and uninvolved psoriatic skin were purified by HPLC and appropriate fractions were analysed by gas chromatography - mass spectrometry (GC-MS). The following monohydroxy metabolites of arachidonic, linoleic and 11,14-eicosadienoic acids were identified : 15-HETE, 12-HETE, 11-HETE, 9-HETE, 8-HETE, 5-HETE, 13-hydroxy-octadecadienoic acid (13-HODD), 9-HODD and 15-hydroxy-eicosadienoic acid (15-HEDE). The results suggested that 12-HETE, 13-HODD and 9-HODD are the most abundant monohydroxy fatty acids in the psoriatic skin extracts described above. Assays of 13-HODD, 9-HODD and 15-HEDE for chemokinetic activity were negative with concentrations up to 10^?4M. The biological significance of these three compounds in not known, but some of the hydroxylated metabolites of arachidonic acid may, by virtue of their chemotactic properties, be relevant to the pathogenesis of the psoriatic neutrophil infiltrate.     

Activation of the abl oncogene in murine and human leukemias

Deuterium-labelled standards of four regionally isomeric epoxyeicosatrienoic acids (EETs) and their hydrolysis products, the dihydroxyeicosatrienoic acids (DHETs), have been prepared and analyzed by capillary column gas chromatography (GC)-negative ion (NI)-methane chemical ionization (MCI)-mass spectrometry (MS) as the pentafluorobenzyl esters. As little as 40 pg of these compounds were readily visualized by these methods, and the deuterium-labelled standards were used in a stable isotope dilution mass spectrometric assay which was linear from near the detection limit over several orders of magnitude. NADPH-dependent synthesis of both EETs and DHETs from arachidonate by hepatic microsomal cytochrome P-450-mono-oxygenase activity was demonstrable with these methods and was significantly suppressed by the compound BW755C (500 microM), but not by eicosa-5,8,11,14-tetraynoic acid (ETYA, 20 microM) or by nordihydroguaiaretic acid (NDGA, 50 microM). All three compounds suppress glucose-induced insulin secretion and 12-hydroxyeicosatetraenoic acid (12-HETE) synthesis by isolated pancreatic islets with similar concentration dependence. Microsomes derived from isolated pancreatic islets synthesized less than 3% of the EET and DHET compounds as a comparable amount of hepatic microsomes. Intact islets synthesized less than 3% by mass of the EET and DHET compounds compared to the mass of 12-HETE produced by the islets. Islets also failed to convert 3H-labelled arachidonate to 3H-labelled EETs or DHETs under conditions where conversion to [3H]12-HETE and to [3H]prostaglandin E2 (but not to [3H]leukotriene C4, D4, or E4) was clearly demonstrable. Neither exogenous EETs nor leukotriene C4 stimulated insulin secretion from the isolated islets or reversed the suppression of glucose-induced secretion by the lipoxygenase inhibitor BW755C. The cytochrome P-450-monooxygenase inhibitor, metyrapone (50 microM), did not influence insulin secretion from the isolated islets under conditions where the lipoxygenase inhibitor, NDGA, suppressed glucose-induced secretion. These observations argue against the recently suggested hypothesis that EETs derived from arachidonate by monooxygenase action participate in glucose-induced insulin secretion by isolated pancreatic islets.     

The effects of lipoxygenase metabolites of arachidonic acid on human myometrial contractility

The effects of the lipoxygenase products of arachidonic acid, 5- and 12-hydroxyeicosatetraenoic acid (5- and 12-HETE) and leukotriene B₄ (LTB₄), on the spontaneous contractility of lower uterine segment human myometrial strips obtained prior to labour have been studied . 5-HETE gave a dose- dependent (10–500ng) increase in both the rate of contractions and overall contractility of myometrial strips while 12-HETE and LTB₄ had no effect at the same concentrations. Prostaglandin F₂ (50ng) contracted all myometrial strips in a similar pattern to 5-HETE but was approximately 10 times more potent. The effect of 5-HETE may be direct or perhaps indirect via interaction with the cyclo-oxygenase pathway. The findings do not disprove the contention that the onset of parturition may be characterized by a switch in arachidonic acid metabolism in intra-uterine tissues from lipoxygenase to cyclo-oxygenase products.     

Characterization of N-linked glycans by supercritical fluid chromatography-mass spectrometry

N-Linked glycans have been characterized by supercritical fluid chromatography (SFC) and SFC-MS using positive- and negative-ion chemical ionization. Four common oligosaccharide derivatives have been prepared and their chromatographic properties assessed on three SFC columns of varying polarity. Carbon dioxide has been used as the SFC mobile phase, with ammonia or CO2 added to the ion source for positive- and negative-ion chemical ionization, respectively. Direct SFC-MS interfacing allows the analytical manipulations of single-ion monitoring, total-ion plots, background subtraction, library searches, and spectral reconstruction algorithms. Positive ammonia chemical ionization yields abundant molecular-weight information, (MH)+, and (MNH4)+ with little or no fragmentation. To capitalize on sensitivity, samples were prepared with the pentafluorobenzyl aminobenzoate reagent, acetylated, and analyzed by SFC-NICI-MS. This modification improves column efficiency and resolution and greatly enhances detecting sensitivity. These "soft" ionization conditions provide abundant molecular-weight-related anions for collision-induced dissociation and subpicogram detection.     

A human epithelial cell line, intestine 407, can produce 5-hydroxyeicosatetraenoic acid and leukotriene B4

The present study was carried out to further characterize the role of non-inflammatory cells in the inflammatory process. More specifically, we have investigated whether human epithelial cells can generate inflammatory lipid mediators via activation of the 5-lipoxygenase pathway. The cells were stimulated with the calcium ionophore A23187 (5 μM) for different periods of time, after which the production of eicosanoids was determined by gradient reverse-phase high performance liquid chromatography (RP-HPLC) and rapid spectral detection, permitting continuous ultraviolet spectroscopy. In both non-prelabeled cells and cells prelabeled with [1-^14Carachidonic acid, cell stimulation for 30 min or more resulted in the production of two important 5-lipoxygenase products: 5-hydroxyeicosatetraenoic acid (5-HETE) and leukotriene B₄ (LTB₄). Stimulation for 15 min or less, however, led solely to the formation of 5-HETE. The identities of 5-HETE and LTB₄ were confirmed by HPLC retention times and UV spectra, as well as by gas chromatography-mass spectrometry for 5-HETE and radioimmunoassay for LTB₄. It can therefore be concluded that human epithelial cells in general can produce important inflammatory mediators, which suggests that epithelial cells may play a more active role in the inflammatory process than is normally assumed.     

Stimulus-specificity of the chemotactic deactivation of human neutrophilis by lipoxygenase products of arachidonic acid

Preincubation of human neutrophils with chemotactic concentrations of 5(S)-hydroxy-eicosatetraenoic acid (5-HETE) or 5(S), 12(R)-dihydroxy-6, 14 cis-8, 10 trans-eicosatetraenoic acid (leukotriene B₄) induces a state of preferential chemotactic unresponsiveness to the homologous factor, termed deactivation, and less suppression of the responses to other chemotactic stimuli. The ratio of the concentration required for maximal chemotactic deactivation of neutrophils to that which stimulates chemotaxis optimally is greater for 5-HETE and leukotriene B₄ than for peptide and protein factors. In contrast to other chemotactic factors, 5-hydroperoxy-eicosatetraenoic acid (5-OOHETE) induces neutrophil chemotactic deactivation that is independent of the nature of the subsequent stimulus and is more slowly reversible after elimination of the fluid-phase deactivating factor. The unique characteristics of the chemotactic deactivation of human neutrophils by 5-OOHETE may be attributable in part to its endogenous metabolism to potent deactivating factors or to covalent derivatization of subcellular structures of the neutrophils by the highly reactive 5-OOHETE.     

Metabolism of leukotriene B4 by guinea pig eosinophils

The metabolism of leukotriene B₄ (5(S),12(R)-dihydroxy-6-cis-8,10-trans-14-cis-eicosatetraenoic acid) by isolated guinea pig eosinophils was investigated. Incubation of guinea pig eosinophils with [³H]-leukotriene B₄ resulted in the rapid conversion of leukotriene B₄ to several more polar metabolites. Two of these metabolites were identified by ultraviolet spectroscopy and gas chromatography-mass spectrometry as the omega oxidation products 5(S),12(R),20-trihydroxy-6,8,10,14-eicosatetraenoic acid (20-hydroxy-leukotriene B₄) and 5(S),12(R),19-trihydroxy-6,8,10,14-eicosatetraenoic acid (19-hydroxy-leukotriene B₄). Two novel metabolites, 5(S),12(R),18,19-tetrahydroxy-6,8,10,14 eicosatetraenoic acid (18,19-dihydroxy-leukotriene B₄) and 5(S),12(R)-dihydroxy-1,18-dicarboxylic-6,8,10,14,16-octadecapentaenoic acid (Δ16,17–18-carboxy-19,20-dinor-leukotriene B₄) were tentatively identified. The identification of these compounds indicates that guinea pig eosinophils are capable of metabolizing leukotriene B₄ by both omega and beta oxidation. This catabolic activity may play a role in modulating inflammatory reactions by removing the chemoattractant leukotriene B₄ from inflammatory sites.     

The effect of n-3 polyunsaturated fatty acids on leukotriene B₄ and leukotriene B₅ production from stimulated neutrophil granulocytes in patients with chronic kidney disease

The proinflammatory leukotriene B₄ (LTB₄) may be of importance in the progression of chronic kidney disease (CKD). We investigated whether n-3 polyunsaturated fatty acids (PUFA) decrease LTB₄ and increase the formation of the less inflammatory leukotriene B₅ (LTB₅) in patients with CKD.Fifty-six patients with CKD stage 2-5 were randomised to 2.4 g n-3 PUFA or olive oil for 8 weeks. Compared to controls, n-3 PUFA significantly decreased release of LTB₄ (p<0.001) and 5-hydroxyeicosatetraenoic acid (5-HETE) (p<0.01) and significantly increased release of LTB₅ (p<0.001) and 5-hydroxyeicosapentaenoic acid (5-HEPE) (p<0.001) from stimulated neutrophil granulocytes. Kidney function evaluated by creatinine clearance and proteinuria did not improve. In conclusion, n-3 PUFA supplementation for 8 weeks in patients with CKD stage 2-5 significantly decreased LTB₄ and 5-HETE and significantly increased LTB₅ and 5-HEPE. No effect was seen on kidney function.     

Formation of leukotrienes and other hydroxy acids during platelet-neutrophil interactions in vitro

Interactions of human platelets with neutrophils were studied in suspensions of [³H]arachidonate-labeled platelets and unlabeled neutrophils stimulated with ionophore A23187. Several radioactive arachidonate metabolites, not produced by platelets alone, were detected, including [³H]-labeled leukotriene B₄ (LTB₄), dihydroxyeicosatetraenoic acid (DHETE) and 5-hydroxyeicosatetraenoic acid (5-HETE). When [³H]12-HETE, a platelet product, was added to stimulated neutrophils, DHETE was formed. Similarly, when [³H]5-HETE, a neutrophil product, was added to stimulated platelets, DHETE was the major product. These results suggest that upon stimulation: 1) platelet-derived arachidonate may serve as precursor for the neutrophil-derived eicosanoids LTB₄ and 5-HETE, and 2) that platelet-derived 12-HETE can be converted to DHETE by human neutrophils. The present investigation documents cell-cell interactions via the lipoxygenase pathway, which may be important in hemostasis, thrombosis and inflammation.     

15-Hydroxyeicosatetraenoic Acid Inhibits Superoxide Anion Generation by Human Neutrophils: Relationship to Lipoxin Production

Human neutrophils can aggregate, degranulate, and release mediators of inflammation including oxygen radicals and lipoxygenase (LO)-derived products of arachidonic acid. The regulation of 5– and 15-lipoxy-genases appears to be important since their products (e.g. leukotrienes and lipoxins) display unique spectra of bioactions. Addition of 15-HETE. a product of the 15-LO, to neutrophils in suspension dramatically shifted the LO products generated and led to a dose-dependent increase in lipoxins, while the production of leukotriene B₄ and its μ-oxidation products (i.e. 20-COOH-LTB₄ and 20-OH-LTB₄) was inhibited. Exogenous 15-HETE also dose-dependently inhibited the generation of superoxide anions induced by either the chemotactic peptide f-met-leu-phe or the divalent cation ionophore A23187. Neither lipoxin A, nor lipoxin B₄ (10^?8?10^?6M) inhibited O₂^?_? generation induced by either f-met-leu-phe or A23187. These results indicate that in addition to serving as a substrate for lipoxin generation, 15-HETE also inhibits superoxide anion generation by human neutrophils. Together they provide further evidence to suggest that products of the 15-lipoxygenase may serve a regulatory role at inflammatory loci.     

Synthesis of leukotriene B4, and prostanoids by human alveolar macrophages: analysis by gas chromatography/ mass spectrometry

Human alveolar macrophages, obtained during diagnostic bronchoscoy, were maintained in monolayer culture. Challenge of these cells (>95% purity) with 1.2 mg/ml zymosan A particles (opsonized with human serum) was followed by a rapid release of leukotriene B₄ into the medium, 7.28 ± 5.99 ng/mg cell protein at 2 h mean ± S.D⁴, n = 4). Leukotriene B₄ was identified and measured by a novel technique employing capillary column gas chromatography coupled to negative ion chemical ionization mass spectrometry. The release of thromboxane B₂, prostaglandins D₂, E₂, F_2α and the lysosomal enzyme N-acetyl-β-D- glucosaminidase was also measured. Thromboxane B₂ was the most abundant metabolite of arachidonic acid released into the culture medium (65.2 ± 14.8 ng/mg cell protein 2 h after the addition of zymosanA, n = 4), and the synthesis of thromboxane B₂ was inhibited by >90% in 1 μM Na flurbiprofen. Inhibition of cyclooxygenase activity was accompanied by a 2-fold increase in leukotriene B₄ synthesis.     

Melanocyte-Stimulating Properties of Arachidonic Acid Metabolites: Possible Role in Postinflammatory Pigmentation

Normal human epidermal melanocytes became swollen and more dendritic with an increase in the amount of tyrosinase and immunoreactive b-locus protein when they were cultured for 2 days with the following arachidonic acid metabolites: prostaglandin (PG) D₂, leukotriene (LT) B₄, LTC₄, LTD₄, LTE₄, thromboxane (TX) B₂ and 12-hydroxy eicosatetraenoic acid (12-HETE). The effect of LTC₄ was particularly strong compared to that of PGE₂, about which we have previously reported. On the other hand, PGE₁, PGF_2α and 6-ketoPGF_1α did not show any significant stimulatory effect. These data suggest that arachidonate-derived chemical mediators, especially LTC₄, may be responsible for the induction of post-inflammatory hyperpigmentation of the skin.     

Micro-quantitative determination of ciprostene in plasma by gas chromatography—mass spectrometry coupled with an antibody extraction

A simple and highly sensitive method has been developed for the determination in plasma of ciprostene, 9β-methyl-6α-carbaprostaglandin I₂, using gas chromatography—mass spectrometry following solid-phase extraction on an immobilized antibody column. The anti-ciprostene antibody obtained from rabbit serum was coupled to an agarose support matrix, and the immobilized antibody thus prepared was used as an extraction phase for sample clean-up. The extracted drug was treated with pentafluorobenzyl bromide followed by bis(trimethylsilyl)trifluoroacetamide. The derivative was quantitatively analysed by negative-ion chemical ionization gas chromatography—mass spectrometry. The lower limit of quantitation was 50 pg/ml when 1 ml of human plasma was used. The plasma concentration of ciprostene in a dog treated with ciprostene at 2.5 μg/kg was determined successfully by this method.     

Analysis of F2-isoprostanes as indicators of non-enzymatic lipid peroxidation in vivo by gas chromatography-mass spectrometry: development of a solid-phase extraction procedure

Recently, it has been reported that a series of prostaglanding F₂-like compounds (F₂-isoprostanes) are produced in vivo during peroxidation of arachidonic acid by a mechanism independent of the cyclooxygenase pathway. Of these, 8-epi-PGF_α² is shown to be a potent vasoconstrictor. We describe an improved method for analysing F₂-isoprostanes in biological fluids. The method involves solid-phase extraction on an octadecylsilane (C₁₈) and an aminopropyl (NH₂) cartridge. After conversion to pentafluorobenzyl ester and trimethylsilyl ether derivatives, F₂-isoprostanes are analysed by negative-ion chemical ionization mass spectrometry using tetradeuterated PGF_α² as the internal standard. The limit of detection of the assay was 10 pg/ml, with a coefficient of variation ranging from 9.4 to 15.1%. Analysis of plasma samples from healthy volunteers (n = 7) revealed no quantifiable levels of free (unesterified) 8-epi-PGF_α². However, the plasma samples contained 58 to 166 pg/ml of 8-epi-PGF_α² when analyzed for the total (sum of free and esterifield)_ F₂-isoprostances. The main advantages of the method lie in the improved recovery, gas chromatographic separation and speed compared to existing techniques.     

Inhibition of Icosanoid Production in MC/9 Mouse Mast Cells by n-3 Polyunsaturated Fatty Acids Isolated from Edible Marine Algae

The effects of two polyunsaturated fatty acids, 18:4n-3 and 16:4n-3 purified from the marine algae, Undaria pinnatifida and Ulva pertusa, on icosanoid production in MC/9 mouse mast cells were assessed. Both fatty acids suppressed the production of leukotriene B₄ (LTB₄), leukotriene C₄ (LTC₄), and 5-hydroxyeicosatetraenoic acid (5-HETE). The order of the suppressive activity for the two marine algae-derived fatty acids and three other common polyunsaturated fatty acids was as follows; 22:6n-3=18:4n-3=18:3n-3>20:5n-3=16:4n-3 for LTB₄; 22:6n-3=18:4n-3=18:3n-3>16:4n-3>20:5n-3 (no suppression) for LTC₄; 22:6n-3=18:4n-3>18:3n-3>20:5n-3=16:4n-3 for 5-HETE.     

Leukotriene B3, leukotriene B4 and leukotriene B5; Binding to leukotriene B4 receptors on rat and human leukocyte membranes

Specific high-affinity binding sites for [³H]-leukotriene B₄ have been identified on membrane preparations from rat and human leukocytes. The rat and human leukocyte membrane preparations show linearity of binding with increasing protein concentration, saturable binding and rapid dissociation of binding by excess unlabelled leukotriene B₄. Dissociation constants of 0.5 to 2.5 nM and maximum binding of 5000 fmoles/mg protein were obtained for [³H] leukotriene B₄ binding to these preparations. Displacement of [³H]-leukotriene B₄ by leukotriene B₄ was compared with displacement by leukotriene B₃ and leukotriene B₅ which differ from leukotriene B₄ only by the absence of a double bond at carbon 14 or the presence of an additional double bond at carbon 17, respectively. Leukotriene B₃ was shown to be equipotent to leukotriene B₄ in ability to displace [³H]-leukotriene B₄ from both rat and human leukocyte membranes while leukotriene B₅ was 20–50 fold less potent. The relative potencies for the displacement of [³]-leukotriene B₄ by leukotrienes B₃, B₄ and B₅ on rat and human leukocyte membranes were shown to correlate well with their potencies for the induction of the aggregation of rat leukocytes and the chemokinesis of human leukocytes.     

Formation of leukotrienes and hydroxy acids by human neutrophils and platelets exposed to monosodium urate

Monosodium urate (MSU) crystals stimulate the production of arachidonic acid metabolites by human neutrophils and platelets. Neutrophils exposed to MSU generated leukotriene B₄(LTB₄). 6- -LTB₄, 12- -6- -LTB₄, and 5S, 12S DHETE from endogenous sources of arachidonate. In addition to these metabolites both monohyroxyeicosatetraenoic acids (i.e., 5-HETE) and w-oxidation products (i.e., 20-COOH LTB₄) were formed by neutrophils exposed to MSU. Addition of exogenous arachidonic acid led to increased formation of each of these metabolites. When neutrophils were treated with colchicine (10 uM), LTB₄ but 5-HETE formation was impaired. (1-¹⁴C) Arachidonate-labeled platelets exposed to MSU released (1-¹⁴C)-arachidonate. (¹⁴C)-12 HETE, (¹⁴C)-HHT and (¹⁴C)-thromboxane B₂. Results indicate that MSU stimulates arachidonic acid metabolism in both human neutrophils and platelets. Moreover, they suggest not only that metabolites of arachidonate may be considered as possible candidates for mediators of inflammation in crystal-associated diseases, but that colchicine blocks the formation of LTB₄.