LC-MS/MS assay for the simultaneous determination of tocopherols, polyunsaturated fatty acids and their metabolites in human plasma and serum.

The role of vitamin E in both enzymatic and free radical-dependent metabolism of polyunsaturated fatty acids (PUFAs) has been well demonstrated. This study proposed a new LC-MS/MS method to quantify the main vitamin E forms, their metabolites and main PUFA species in human blood, since, at present, there are not procedures able to simultaneously determine these two classes of compounds. After the optimization of sample treatment and reverse-phase separation conditions, tandem mass spectrometry detection was evaluated experimenting both positive and negative electrospray ionisation modes. The procedure was also preliminarily adapted to assess five arachidonic acid-derived eicosanoids that could be under the influence of vitamin E function, such as LTB4 (leukotriene B4), 20-HETE (20-hydroxyeicosatetraenoic acid) and their ω-oxidation metabolites. After the validation study, the performance characteristics were confirmed analysing a certified reference material (SRM® 1950 - frozen human plasma by NIST). Finally, an application of the method in the analysis of lipid abnormalities of chronic kidney disease patients was shown.

Changes in erythrocyte polyunsaturated fatty acids and plasma eicosanoids level in patients with asthma.

BACKGROUND: To investigate the changes of polyunsaturated fatty acids (PUFAs) and their downstream eicosanoids in patients with asthma, the levels of erythrocyte membrane lipids and plasma lipid metabolites were examined. METHODS: Erythrocyte membrane lipids were extracted and esterificated, and then fatty acid compositions were determined by gas chromatography. The concentrations of six eicosanoids of PGE2, TXA2, LTB4, PGE1, 6-k-PGF1α and PGF2α in plasma were measured by ELISA. RESULTS: The results showed that the contents of erythrocyte membrane fatty acids in patients with asthma were mainly composed of C16:0, C18:0, C18:1, C18:2(n-6), and C20:4(n-6). The ratio n-6/n-3 PUFAs in patients and health persons were (4.42 ± 1.33):1 and (3.21 ± 0.79):1 (p < 0.01), showing statistically significant differences. ELISA results showed that the levels of plasma PGE2, TXB2, and PGE1 in patients were higher than health persons; and the levels of eicosanoids of PGF2α and 6-k-PGF1α were significantly lower in patient group than healthy group (p < 0.05), but LTB4 was no obvious difference (p = 0.09). Increased ratio of n-6/n-3 PUFAs is consistent to the increased levels of pro-inflammatory PGE2 and TXB2 and anti-inflammatory PGE1 originated from C20:4(n-6) and C18:2(n-6), indicating that increased ratio of n-6/n-3 PUFAs and eicosanoids from n-6 PUFAs might promote the progress of airway inflammation of asthma. CONCLUSION: Changes of erythrocyte fatty acids, n-6/n-3 PUFAs ratio and the levels of plasma PGE2, TXB2, and PGE1 in patients with asthma were relevant to airway inflammation in some extent. Therefore, it could be proposed that increase of n-3/n-6 PUFAs ratio by diet supplementation of n-3 PUFAs might effectively improve airway inflammation in asthma.

Study of the role of cytosolic phospholipase A2 alpha in eicosanoid generation and thymocyte maturation in the thymus.

The thymus is a primary lymphoid organ, home of maturation and selection of thymocytes for generation of functional T-cells. Multiple factors are involved throughout the different stages of the maturation process to tightly regulate T-cell production. The metabolism of arachidonic acid by cyclooxygenases, lipoxygenases and specific isomerases generates eicosanoids, lipid mediators capable of triggering cellular responses. In this study, we determined the profile of expression of the eicosanoids present in the mouse thymus at different stages of thymocyte development. As the group IVA cytosolic phospholipase A2 (cPLA2α) catalyzes the hydrolysis of phospholipids, thereby generating arachidonic acid, we further verified its contribution by including cPLA2α deficient mice to our investigations. We found that a vast array of eicosanoids is expressed in the thymus, which expression is substantially modulated through thymocyte development. The cPLA2α was dispensable in the generation of most eicosanoids in the thymus and consistently, the ablation of the cPLA2α gene in mouse thymus and the culture of thymuses from human newborns in presence of the cPLA2α inhibitor pyrrophenone did not impact thymocyte maturation. This study provides information on the eicosanoid repertoire present during thymocyte development and suggests that thymocyte maturation can occur independently of cPLA2α.

Prostaglandin D2-supplemented "functional eicosanoid testing and typing" assay with peripheral blood leukocytes as a new tool in the diagnosis of systemic mast cell activation disease: an explorative diagnostic study.

BACKGROUND: Systemic mast cell activation disease (MCAD) is characterized by an enhanced release of mast cell-derived mediators, including eicosanoids, which induce a broad spectrum of clinical symptoms. Accordingly, the diagnostic algorithm of MCAD presupposes the proof of increased mast cell mediator release, but only a few mediators are currently established as routine laboratory parameters. We thus initiated an explorative study to evaluate in vitro typing of individual eicosanoid pattern of peripheral blood leukocytes (PBLs) as a new diagnostic tool in MCAD. METHODS: Using the "functional eicosanoid testing and typing" (FET) assay, we investigated the balance (i.e. the complex pattern of formation, release and mutual interaction) of prostaglandin E2 (PGE2) and peptido-leukotrienes (pLT) release from PBLs of 22 MCAD patients and 20 healthy individuals. FET algorithms thereby consider both basal and arachidonic acid (AA)-, acetylsalicylic acid (ASA)-, and substance P (SP)-triggered release of PGE2 and pLT. The FET assay was further supplemented by analyzing prostaglandin D2 (PGD2), as mast cell-specific eicosanoid. RESULTS: We observed marked PGE2-pLT imbalances for PBLs of MCAD patients, as indicated by a markedly enhanced mean FET value of 1.75 ± 0.356 (range: 1.14-2.36), compared to 0.53 ± 0.119 (range: 0.36-0.75) for healthy individuals. In addition, mean PGD2 release from PBLs of MCAD patients was significantly, 6.6-fold higher than from PBLs of healthy individuals (946 ± 302.2 pg/ml versus 142 ± 47.8 pg/ml; P < 0.001). In contrast to healthy individuals, PGD2 release from PBLs of MCAD patients was markedly triggered by SP (mean: 1896 ± 389.7 pg/ml; P < 0.001), whereas AA and ASA caused individually varying effects on both PGD2 and pLT release. CONCLUSIONS: The new in-vitro FET assay, supplemented with analysis of PGD2, demonstrated that the individual patterns of eicosanoid release from PBLs can unambiguously distinguish MCAD patients from healthy individuals. Notably, in our analyses, the FET value and both basal and triggered PGD2 levels were not significantly affected by MCAD-specific medication. Thus, this approach may serve as an in-vitro diagnostic tool to estimate mast cell activity and to support individualized therapeutic decision processes for patients suffering from MCAD.

Inflammatory mediators in exhaled breath condensate of children with obstructive sleep apnea syndrome.

BACKGROUND: Upper airway inflammation is now recognized in adults with obstructive sleep apnea (OSA) syndrome. However, the role played by eicosanoids such as leukotrienes and prostaglandins is unclear. OBJECTIVE: To investigate whether eicosanoids are measurable in exhaled breath condensate (EBC), and to determine whether differences in these inflammatory mediators emerge among children with and without sleep-disordered breathing (SDB). METHODS: EBC was collected from 50 consecutive snoring children undergoing overnight polysomnography for suspected SDB, and from 12 nonsnoring control subjects. Prostaglandin E2 (PGE2), leukotriene B4 (LTB4), and cysteinyl leukotrienes (cys-LTs: leukotriene C4 [LTC4]/leukotriene D4 [LTD4]/leukotriene E4 [LTE4]) EBC levels were analyzed using enzyme-linked immunosorbent assay. RESULTS: LTB4 levels were elevated in children with an apnea-hypopnea index (AHI) > 5/h (SDB; 97.6 +/- 6.3 pg/mL) compared to children with an AHI < 5/h (mild SDB; 66.4 +/- 19.1 pg/mL; p < 0.01) and control subjects (27.8 +/- 3.7 pg/mL; p < 0.01). Similarly, cys-LT (LTC4/LTD4/LTE4) concentrations were also increased in SDB (45.1 +/- 10.6 pg/mL in SDB vs 27.6 +/- 8.3 pg/mL in mild SDB, and 15.7 +/- 7.6 pg/mL in control subjects; p < 0.01). In contrast, PGE2 concentrations were similar among the three groups. CONCLUSIONS: Inflammatory mediators such as leukotrienes and prostaglandins can be readily quantified in EBC collected from the upper airway of children. Disease severity-dependent increases in leukotriene concentrations (LTB4 and LTC4/LTD4/LTE4) emerge among children and may serve as a noninvasive tool in the clinical assessment of these children.

Functional eicosanoid test and typing (FET) in acetylsalicylic acid intolerant patients with urticaria.

One of the common side effects of acetylsalicylic acid (ASA) is the induction of pseudoallergic reactions that range from urticarial wheals to anaphylactic shock. At present there is no reliable detection method available for the diagnosis of ASA-hypersensitivity and its relation to clinical symptoms. The purpose of the present study was to evaluate the functional eicosanoid typing (FET) score taking into account several parameters of the equilibrium between prostaglandins (PG) and peptido-leukotriens (pLT). A total eicosanoid pattern score (TEP) ranging from 0.0 to 3.0, was defined that exhibited significant differences (p

Testing and typing of eicosanoid-patterns.

Eicosanoids are pleiotrope mediators with essential function in most biological processes. The network of inter- and intracellular signalling requires coordinated cellular information processing. The cross-talk is characterised by complex non-linear responses to combinations of different stimuli and cells, but little is known about the density of these interactions. Here I have analysed eicosanoid interactions carried out by functional eicosanoid testing and typing (FET) in leucocytes from healthy subjects and patients suffering from inflammatory diseases. The known eicosanoid pattern scoring was extended to metabolically linked prostaglandin E(2) and peptido-leukotrienes pathways, both alone and in all pair wise combinations, for basal, maximal synthesis capacity, acetylsalicylic acid, and neuropeptide modification. Eicosanoids fluctuated over twenty minutes context-dependent dynamically, demanding further data integration. The integration suggested that many stimuli converge for quantitative discrimination applying a total eicosanoid pattern score (TEP). Varying cellular activities affect FET and thereby TEP. The non-additive metabolic interactions were consistent with known mechanisms of metabolic pathway cross-talk. FET-based modelling of eicosanoid circuits most suitably reflects the fundamental impact of eicosanoids in maintaining cellular integrity of organ and body function. This might improve our present understanding of complex cellular eicosanoid interactions of inflammatory diseases and might be applied for diagnostic considerations.

International Union of Pharmacology XLIV. Nomenclature for the oxoeicosanoid receptor.

Oxoeicosanoids are a family of biologically active arachidonic acid derivatives that have been intimately linked with cellular migration. These metabolites are not only potent chemotaxins but also elicit oxygen radical production as well as induce secretory events in different cells. The most potent native ligand reported is 5-oxo-6,8,11,14-eicosatetraenoic acid (5-oxo-ETE), and the cell membrane receptor activated has now been cloned. This receptor is distinct from those receptors activated by either the prostaglandins or the leukotrienes. The purpose of this review is to briefly summarize the molecular evidence and highlight the significance of this receptor. In addition, an official nomenclature for this oxoeicosanoid receptor is proposed.

COX and LOX eicosanoids modulate platelet activation and procoagulation induced by two murine cancer cells.

Involvement of arachidonic acid cyclooxygenase (COX) and lipoxygenase (LOX) metabolites in platelet aggregation and coagulation induced by two varieties of cancer cells of murine transplantable tumors was studied. A lung alveolar carcinoma (LAC) and a fibrosarcoma (FS), induced platelet aggregation and plasma coagulation (P<0.05). Pretreatment of both tumor lines with a COX inhibitor did not block the tumor cell induced platelet aggregation (TCIPA). COX [12(S)-HTT] and LOX [12(S)-HETE], metabolites of washed platelets (WP), alone or co-incubated with LAC or FS cells, were analyzed. We observed higher 12(S)-HETE release with respect to 12(S)HHT when WP were co-incubated with LAC cells. With both neoplastic cell (NC) lines prothrombin time (PT) was shortened. Pretreatment of NC with iodoacetic acid, soybean trypsin inhibitor or Factor X-deficient plasma increased the PT. These results indicate that AA metabolites play a role on the procoagulation and platelet aggregation induced by mesenchymal and epithelial murine cancers.