A quantitiative LC-MS/MS method for the measurement of arachidonic acid, prostanoids, endocannabinoids, N-acylethanolamines and steroids in human plasma.

Free arachidonic acid is functionally interlinked with different lipid signaling networks including those involving prostanoid pathways, the endocannabinoid system, N-acylethanolamines, as well as steroids. A sensitive and specific LC-MS/MS method for the quantification of arachidonic acid, prostaglandin E2, thromboxane B2, anandamide, 2-arachidonoylglycerol, noladin ether, lineoyl ethanolamide, oleoyl ethanolamide, palmitoyl ethanolamide, steroyl ethanolamide, aldosterone, cortisol, dehydroepiandrosterone, progesterone, and testosterone in human plasma was developed and validated. Analytes were extracted using acetonitrile precipitation followed by solid phase extraction. Separations were performed by UFLC using a C18 column and analyzed on a triple quadrupole MS with electron spray ionization. Analytes were run first in negative mode and, subsequently, in positive mode in two independent LC-MS/MS runs. For each analyte, two MRM transitions were collected in order to confirm identity. All analytes showed good linearity over the investigated concentration range (r>0.98). Validated LLOQs ranged from 0.1 to 190ng/mL and LODs ranged from 0.04 to 12.3ng/mL. Our data show that this LC-MS/MS method is suitable for the quantification of a diverse set of bioactive lipids in plasma from human donors (n=32). The determined plasma levels are in agreement with the literature, thus providing a versatile method to explore pathophysiological processes in which changes of these lipids are implicated.

Chapter III: Management of cardiovascular risk factors and medical therapy

Critical limb ischaemia (CLI) is a particularly severe manifestation of lower limb atherosclerosis posing a major threat to both limb and life of affected patients. Besides arterial revascularisation, risk-factor modification and administration of antiplatelet therapy is a major goal in the treatment of CLI patients. Key elements of cardiovascular risk management are smoking cessation and treatment of hyperlipidaemia with dietary modification or statins. Moreover, arterial hypertension and diabetes mellitus should be adequately treated. In CLI patients not suitable for arterial revascularisation or subsequent to unsuccessful revascularisation, parenteral prostanoids may be considered. CLI patients undergoing surgical revascularisation should be treated with beta blockers. At present, neither gene nor stem-cell therapy can be recommended outside clinical trials. Of note, walking exercise is contraindicated in CLI patients due to the risk of worsening pre-existing or causing new ischaemic wounds. CLI patients are oftentimes medically frail and exhibit significant comorbidities. Co-existing coronary heart and carotid as well as renal artery disease should be managed according to current guidelines. Considering the above-mentioned treatment goals, interdisciplinary treatment approaches for CLI patients are warranted. Aim of the present manuscript is to discuss currently existing evidence for both the management of cardiovascular risk factors and treatment of co-existing disease and to deduct specific treatment recommendations.

Preischemic iloprost application for improvement of graft preservation: which route is superior in experimental pig lung transplantation: inhaled or intravenous?

BACKGROUND: Optimal allograft protection is essential in lung transplantation to reduce postoperative organ dysfunction. Although intravenous prostanoids are routinely used to ameliorate reperfusion injury, the latest evidence suggests a similar efficacy of inhaled prostacyclin. Therefore, we compared donor lung-pretreatment using inhaled lioprost (Ventavis) with the commonly used intravenous technique. METHODS: Five pig lungs were each preserved with Perfadex and stored for 27 hours without (group 1) or with (group-2, 100 prior aerosolized of iloprost were (group 3) or iloprost (IV). Following left lung transplantation, hemodynamics, Po(2)/F(i)o(2), compliance, and wet-to-dry ratio were monitored for 6 hours and compared to sham controls using ANOVA analysis with repeated measures. RESULTS: The mortality was 100% in group 3. All other animals survived (P < .001). Dynamic compliance and PVR were superior in the endobronchially pretreated iloprost group as compared with untreated organs (P < .05), whereas oxygenation was comparable overall W/D-ratio revealed significantly lower lung water in group 2 (P = .027) compared with group 3. CONCLUSION: Preischemic alveolar deposition of iloprost is superior to IV pretreatment as reflected by significantly improved allograft function. This strategy offers technique to optimize pulmonary preservation.

Eicosanoids in exhaled breath condensates in the assessment of childhood asthma

The value of measurements of eicosanoids in exhaled breath condensate (EBC) for the evaluation of childhood asthma is still inconclusive most likely because of the limited value of the methods used. In this case-control study in 48 asthmatic and 20 healthy children, we aimed to characterize the baseline profile of the inflammatory mediators cysteinyl leukotrienes (cysLTs), 9(alpha)11(beta)PGF(2), PGE(2), PGF(2alpha), 8-isoprostane (8-iso-PGF(2alpha)) within EBC in asthmatic compared with healthy children using new methods. In addition, we investigated their relation to other inflammatory markers. The assessment included collection of EBC, measurement of fractional exhaled nitric oxide (FE(NO)) and evaluation of urinary excretion of leukotriene E(4.) cysLTs were measured directly in EBC by radioimmunoassay and prostanoids were measured using gas chromatography negative-ion chemical ionization mass spectrometry. Only cysLT levels were significantly higher in asthmatic compared with healthy children (p = 0.002). No significant differences in cysLTs were found between steroid naïve and patients receiving inhaled corticosteroids. In contrast, FE(NO) was significantly higher in steroid naïve compared with steroid-treated asthmatic and healthy children (p = 0.04 and 0.024, respectively). The diagnostic accuracy of cysLTs in EBC for asthma was 73.6% for the whole group and 78.2% for steroid-naïve asthmatic children. The accuracy to classify asthmatic for FE(NO) was poor (62.9%) for the whole group, but improved to 79.9% when only steroid-naïve asthmatic children were taken into consideration. cysLTs in EBC is an inflammatory marker which distinguishes asthmatics, as a whole group, from healthy children.

Phospholipid oxidation generates potent anti-inflammatory lipid mediators that mimic structurally related pro-resolving eicosanoids by activating Nrf2.

Exposure of biological membranes to reactive oxygen species creates a complex mixture of distinct oxidized phospholipid (OxPL) species, which contribute to the development of chronic inflammatory diseases and metabolic disorders. While the ability of OxPL to modulate biological processes is increasingly recognized, the nature of the biologically active OxPL species and the molecular mechanisms underlying their signaling remain largely unknown. We have employed a combination of mass spectrometry, synthetic chemistry, and immunobiology approaches to characterize the OxPL generated from the abundant phospholipid 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine (PAPC) and investigated their bioactivities and signaling pathways in vitro and in vivo. Our study defines epoxycyclopentenones as potent anti-inflammatory lipid mediators that mimic the signaling of endogenous, pro-resolving prostanoids by activating the transcription factor nuclear factor E2-related factor 2 (Nrf2). Using a library of OxPL variants, we identified a synthetic OxPL derivative, which alleviated endotoxin-induced lung injury and inhibited development of pro-inflammatory T helper (Th) 1 cells. These findings provide a molecular basis for the negative regulation of inflammation by lipid peroxidation products and propose a novel class of highly bioactive compounds for the treatment of inflammatory diseases.

Platelet receptors and patient responses: The contributions of Professor Stan Heptinstall to platelet research.

Stan Heptinstall's contributions to platelet research covered organising meetings at the national and European level as well as starting and maintaining the journal "Platelets". The major part of his research addressed problems of inhibition of platelet receptors and the effects of this on patient health. In particular, the effects of P2Y12 inhibitors on patients with acute cardiovascular problems were a major focus. Other studies included the effects of feverfew (Tanacetum parthenium) extracts on platelets, of direct anti-IIb/IIIa receptor (αIIbβ3) inhibitors and of prostanoids on platelet function. Recently, methods for assessing the effectiveness of platelet inhibition were investigated.