Development and validation of a high-performance liquid chromatography-electrospray mass spectrometry method for the simultaneous determination of 23 eicosanoids

Inflammation is implicated in the pathogenesis of a number of diseases, including cardiovascular disease. Current research is focused on developing assays to search for biomarkers for inflammation. Eicosanoids are the oxidative metabolites of arachidonic acid (eicosatetraenoic acid, AA), a long chain polyunsaturated fatty acid common in Western diets. AA can be oxidized by one of three pathways to form prostaglandins (PGs), leukotrienes (LTs), or a number of hydroxyl and epoxy compounds. These eicosanoids have a variety of physiological functions, including regulating inflammation. We have developed a method utilizing LC-MS to separate and quantitate 23 different eicosanoids from all the three oxidative pathways. The eicosanoids were separated using a gradient elution of acetonitrile with 0.1% formic acid (v/v) and water with 0.1% formic acid (v/v) at a flow rate of 1 mL/min with a Symmetry C18 column (250 mm x 4.6 mm). Deuterated eicosanoids were used as internal standards for quantitation. Mass spectrometric detection was carried out using an Agilent 1100-series LC-MSD with an electrospray ionization interface. Electrospray ionisation (ESI) mass spectra were acquired using negative ionization and selective ion monitoring. The method was validated and shown to be sensitive (LOQ at pg levels for most compounds), accurate (recovery values 75-120%) and precise (R.S.D.<20 for all compounds) with a linear range over several orders of magnitude. The method was applied to rat kidney tissue and shown to be indicative of the eicosanoid levels within a specific organ. The analysis of eicosanoids can provide insight into the inflammatory mechanisms associated with cardiovascular disease.     

外源性生长抑素对正常和急性坏死型胰腺炎胰血流量的影响

目的:研究外源性生长抑素(施他宁)对正常胰腺和急性坏死型胰腺炎(ANP)胰血流量的影响及其机制。方法:以LS-Ⅲ型计算机化组织血流仪测定施他宁影响大鼠胰腺组织血流的变化、牛磺胆酸钠诱发ANP模型、放免法测定血浆二十碳烯酸类代谢物和普通病理及电镜观察。结果:正常鼠施他宁给药后胰血流量显著低于给药前;诱发ANP后,胰血流量显著降低,但施他宁+ANP组血流量显著高于ANP组;ANP组发病6h血栓素B₂(TXB₂)高于假手术对照组的4.5倍,施他宁+ANP组各时点TXB₂均显著低于ANP组,6h6-酮-前列腺素-F_1α(6-Keto-PGF_1α)及各时点TXB₂／6-Keto-PGF_1α比值则显著高于ANP组;病理观察示施他宁+ANP组腺细胞坏死积分和微血管内微血栓显著少于ANP组。结论:外源性生长抑素使正常鼠胰血流量下降,但施他宁+ANP组胰血流量显著高于ANP组,其机制可能是二十碳烯酸类异常代谢的矫正,从而改善胰组织微循环、并起细胞保护作用所致。     

HMGB1-Like Dorsal Switch Protein 1 Triggers a Damage Signal in Mosquito Gut to Activate Dual Oxidase via Eicosanoids

Several mosquitoes transmit human pathogens by blood feeding, with the gut being the main entrance for the pathogens. Thus, the gut epithelium defends the pathogens by eliciting potent immune responses. However, it was unclear how the mosquito gut discriminates pathogens among various microflora in the lumen. This study proposed a hypothesis that a damage signal might be specifically induced by pathogens in the gut. The Asian tiger mosquito, Aedes albopictus, encodes dorsal switch protein 1 (Aa-DSP1) as a putative damage-associated molecular pattern (DAMP). Aa-DSP1 was localized in the nucleus of the midgut epithelium in naïve larvae. Upon infection by a pathogenic bacterium, Serratia marcescens, Aa-DSP1 was released to hemocoel and activated phospholipase A₂ (PLA₂). The activated PLA₂ increased the level of prostaglandin E₂ (PGE₂) in the gut and subsequently increased Ca²⁺ signal to produce reactive oxygen species (ROS) via dual oxidase (Duox). Inhibition of Aa-DSP1 via RNA interference or specific inhibitor treatment failed to increase PGE₂/Ca²⁺ signal upon the bacterial infection. Thus, the inhibitors specifically targeting eicosanoid biosynthesis significantly prevented the upregulation of ROS production in the gut and enhanced mosquito mortality after the bacterial infection. However, such inhibitory effects were rescued by adding PGE₂. These suggest that Aa-DSP1 plays an important role in immune response of the mosquito gut as a DAMP during pathogen infection by triggering a signaling pathway, DSP1/PLA₂/Ca²⁺/Duox.     

Release of prostaglandin E2 and leukotriene C4/D4 from airway segments isolated from rats after exposure to ozone for 20 months

Metabolites of arachidonic acid have been implicated as mediators of some of the pulmonary effects observed after acute exposure to ozone. Accordingly, recent studies have focused on the effects of acute ozone exposure on the arachidonic acid cascade, however, whether eicosanoid metabolism is altered after chronic exposure to ozone is unknown. To begin to address this issue, we examined the effects of near-lifetime exposure to ozone on release of prostaglandin E₂ (PGE₂) and leukotriene C₄/D₄ from airway segments isolated from exposed Fischer-344 rats. Airway segments representing approximately eighth to tenth generation airways were isolated from rats of both genders that had been exposed for 6 h per day, 5 days per week for 20 months to filtered air or 0.12, 0.5 or 1.0 parts per million (ppm) ozone. Basal and stimulated release of eicosanoids were measured in the medium surrounding airway segments using enzymoimmunoassay. Basal release of PGE₂ was detected in the medium surrounding airway segments and this release was unaffected by ozone exposure. Incubation of the segments with the calcium ionophore, A23187, increased the release of the prostaglandin; the A23187-induced release of PGE₂ was significantly enhanced in airway segments isolated from rats in the 1.0 ppm exposure group. Basal release of leukotriene C₄/D₄ was not detected in the medium surrounding airway segments regardless of ozone exposure. Measurable amounts of the leukotriene were released during incubation with A23187, however, ozone was without affect on these levels. The results suggest that the cyclooxygenase pathway of the arachidonic acid cascade appears to be affected by ozone exposure. Which of the processes of prostaglandin production and release are affected by chronic ozone exposure remains to be determined.     

Release of calcitonin gene-related peptide in cardiac anaphylaxis

We have investigated the antigen-stimulated release of calcitonin gene-related peptide (CGRP) from ovalbumin-sensitized guinea-pig isolated hearts and the interaction with other mediators of anaphylaxis released concomitantly. It was found that antigen challenge caused a significant increase of CGRP release (from basal 31.2 ± 2.9 to 51.6 ± 4.9 fmol/5 min). Anaphylactic CGRP release was significantly attenuated in the presence of the cyclooxygenase inhibitor indomethacin while the 5-lipoxygenase inhibitor Bay-X1005 ((R)-2-[4-quinolin-2-yl-methoxy)phenyl]-2-cyclopentyl acetic acid) had no significant effect. Combined treatment with the histamine receptor (H₁,H₂) antagonists mepyramine and cimetidine also significantly attenuated anaphylactic release of CGRP. Under control conditions antigen injection increased release of cysteinyl-leukotrienes (LT), thromboxane (TXB₂) and 6-keto-prostaglandin (PG)F_1α from basal values of 0.96 ± 0.09, 2.7 ± 0.7 and 3.4 ± 0.28 ng/5 min respectively, to 5.9 ± 0.9, 48.4 ± 3.4 and 6.9 ± 1.4 ng/5 min. Indomethacin abolished the release of cyclooxygenase products of arachidonate metabolism and simultaneously increased cysteinyl-LT release significantly (8.8 ± 1.4 ng/5 min). Conversely Bay-X1005 completely abolished cysteinyl-LT release and had no significant effect on anaphylactic release of TXB₂ and 6-keto-PGF_1α. Simultaneous blockade of H₁ and H₂ receptors abolished release of 6-keto-PGF_1α, while release of TXB₂ and cysteinyl-LT was not significantly affected. The results indicate that CGRP is not a primary mediator of the immediate hypersensitivity reaction of the heart, but is in turn released by arachidonic acid metabolites of the cyclooxygenase pathway and histamine. In contrast, LT obviously do not contribute to anaphylactic CGRP release. CGRP is a potent coronary vasodilator and could act as endogenous functional antagonist of vasoconstrictor mediators also released during cardiac anaphylaxis such as cysteinyl-LT, platelet activating factor and TXA₂. Received: 8 May 1996 / Accepted: 11 October 1996     

Gas exchange and pulmonary haemodynamic responses to fat emulsions in acute respiratory distress syndrome

Objective: To investigate the gas exchange and pulmonary haemodynamic responses to two different intravenous fat emulsions in patients with acute respiratory distress syndrome (ARDS). Design: Prospective, randomized, double-blind, placebo-controlled study. Setting: Intensive care unit in a university-affiliated hospital. Patients: 21 patients with ARDS [mean age, 57 ± 3 (SEM) years; Acute Physiology and Chronic Health Evaluation II, 20 ± 3; Murray's score, 2.85 ± 0.12] consecutively admitted. Interventions: Patients were assigned to three groups (n = 7 each): group A (LCT) received long-chain triglycerides (20 % LCT), group B (MCT/LCT), medium-chain triglycerides/long-chain triglycerides (20 % MCT/LCT: 50/50) and group C placebo (0.9 % sodium chloride, NaCl). The infusion was always given at the rate of 2 mg/kg min over a total period of 12 h, with a volume infusion of 500 ml in each group. Measurements: Data were collected before, immediately after and 12 h after infusion ceased. Pulmonary and systemic haemodynamic and gas exchange variables were measured at each time point. Serum triglyceride cholesterol, and non-esterified fatty acids levels were measured. Results: During LCT infusion, cardiac output, oxygen consumption and oxygen delivery increased (all p < 0.05), whereas pulmonary haemodynamics, arterial oxygen tension, mixed venous partial pressure of oxygen and venous admixture ratio remained essentially unaltered. No changes were observed following MCT/LCT infusion. Conclusions: The administration of LCT emulsion given at a slow rate did not alter arterial oxygenation because of the beneficial effect of a high cardiac output, hence offsetting the detrimental effect of increased O₂ consumption. Received: 1 December 1997 Accepted: 3 June 1998     

Role of thromboxane,prostaglandins and leukotrienes in endotoxic and septic shock

Intravenous bolus endotoxin elicits a marked but transient increase in plasma TxB₂ and 6-keto-PGF₁ in a large number of species. A smaller, delayed and more prolonged increase in TxB₂ and 6-keto-PGF₁ are reported in animals with septic shock, i.e., those with fecal peritonitis or cecal ligation. Thromboxane synthetase inhibitors or antagonists attenuate endotoxin-induced acute cardiopulmonary changes, the delayed increase in serum lysosomal enzymes, fibrin/fibrinogen degradation products and the thrombocytopenia in a number of species. While these drugs increase survival of rats or mice following endotoxin they do not alter survival of rats in septic shock. These results support the hypothesis that TxA₂ exerts a pathophysiologic effect in shock following bolus endotoxin. In contrast, nonsteroidal antiinflammatory drugs (NSAID) and dietary essential fatty acid deficiency increase survival of rats subjected to endotoxin shock, and survival time in models of septic shock. These results also suggest that some other cyclooxygenase product(s) is involved in septic shock due to fecal peritonitis or cecal ligation. Preliminary experimental studies indicate salutary effects of leukotriene inhibitors and antagonists in endotoxin shock and in models of acute pulmonary injury. Clinical studies have demonstrated elevated plasma TxB₂ and 6-keo-PGF₁ concentrations in patients with septic shock, and elevated LTD₄ in pulmonary edema fluid of patients with the adult respiratory distress syndrome. In view of these clinical and experimental results, clinical trials of NSAID and/or leukotriene inhibitors/antagonists should be considered.