A Comprehensive Literature Review on Cardioprotective Effects of Bioactive Compounds Present in Fruits of Aristotelia chilensis Stuntz (Maqui).

Some fruits and vegetables, rich in bioactive compounds such as polyphenols, flavonoids, and anthocyanins, may inhibit platelet activation pathways and therefore reduce the risk of suffering from CVD when consumed regularly. Aristotelia chilensis Stuntz (Maqui) is a shrub or tree native to Chile with outstanding antioxidant activity, associated with its high content in anthocyanins, polyphenols, and flavonoids. Previous studies reveal different pharmacological properties for this berry, but its cardioprotective potential has been little studied. Despite having an abundant composition, and being rich in bioactive products with an antiplatelet role, there are few studies linking this berry with antiplatelet activity. This review summarizes and discusses relevant information on the cardioprotective potential of Maqui, based on its composition of bioactive compounds, mainly as a nutraceutical antiplatelet agent. Articles published between 2000 and 2022 in the following bibliographic databases were selected: PubMed, ScienceDirect, and Google Scholar. Our search revealed that Maqui is a promising cardiovascular target since extracts from this berry have direct effects on the reduction in cardiovascular risk factors (glucose index, obesity, diabetes, among others). Although studies on antiplatelet activity in this fruit are recent, its rich chemical composition clearly shows that the presence of chemical compounds (anthocyanins, flavonoids, phenolic acids, among others) with high antiplatelet potential can provide this berry with antiplatelet properties. These bioactive compounds have antiplatelet effects with multiple targets in the platelet, particularly, they have been related to the inhibition of thromboxane, thrombin, ADP, and GPVI receptors, or through the pathways by which these receptors stimulate platelet aggregation. Detailed studies are needed to clarify this gap in the literature, as well as to specifically evaluate the mechanism of action of Maqui extracts, due to the presence of phenolic compounds.

Lipidomic methodologies for biomarkers of chronic inflammation in nutritional research: ω-3 and ω-6 lipid mediators.

The evolutionary history of hominins has been characterized by significant dietary changes, which include the introduction of meat eating, cooking, and the changes associated with plant and animal domestication. The Western pattern diet has been linked with the onset of chronic inflammation, and serious health problems including obesity, metabolic syndrome, and cardiovascular diseases. Diets enriched with ω-3 marine PUFAs have revealed additional improvements in health status associated to a reduction of proinflammatory ω-3 and ω-6 lipid mediators. Lipid mediators are produced from enzymatic and non-enzymatic oxidation of PUFAs. Interest in better understanding the occurrence of these metabolites has increased exponentially as a result of the growing evidence of their role on inflammatory processes, control of the immune system, cell signaling, onset of metabolic diseases, or even cancer. The scope of this review has been to highlight the recent findings on: a) the formation of lipid mediators and their role in different inflammatory and metabolic conditions, b) the direct use of lipid mediators as antiinflammatory drugs or the potential of new drugs as a new therapeutic option for the synthesis of antiinflammatory or resolving lipid mediators and c) the impact of nutritional interventions to modulate lipid mediators synthesis towards antiinflammatory conditions. In a second part, we have summarized methodological approaches (Lipidomics) for the accurate analysis of lipid mediators. Although several techniques have been used, most authors preferred the combination of SPE with LC-MS. Advantages and disadvantages of each method are herein addressed, as well as the main LC-MS difficulties and challenges for the establishment of new biomarkers and standardization of experimental designs, and finally to deepen the study of mechanisms involved on the inflammatory response.

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.

Enhanced performance for the analysis of prostaglandins and thromboxanes by liquid chromatography-tandem mass spectrometry using a new atmospheric pressure ionization source.

Eicosanoids, including prostaglandins and thromboxanes are lipid mediators synthetized from polyunsaturated fatty acids. They play an important role in cell signaling and are often reported as inflammatory markers. LC-MS/MS is the technique of choice for the analysis of these compounds, often in combination with advanced sample preparation techniques. Here we report a head to head comparison between an electrospray ionization source (ESI) and a new atmospheric pressure ionization source (UniSpray). The performance of both interfaces was evaluated in various matrices such as human plasma, pig colon and mouse colon. The UniSpray source shows an increase in method sensitivity up to a factor 5. Equivalent to better linearity and repeatability on various matrices as well as an increase in signal intensity were observed in comparison to ESI.

Simple New Method of Detecting Lies By Identifying Invisible Unique Physiological Reflex Response Appearing Often Less Than 10-15 Seconds on the Specific Parts of Face of Lying Person; Quick Screening of Potential Murderers & Problematic Persons.

Frequently, we cannot find any significant visible changes when somebody lies, but we found there are significant invisible changes appearing in specific areas of the face when somebody lies and their location often depends on whether the lie is serious with or without physical violence involvement. These abnormalities were detected non-invasively at areas: 1) lobules and c) a small round area of each upper lateral side of forehead; 2) the skin between the base of the 2 orifices of the nose and the upper end of upper lip and 3) Alae of both sides of nose. These invisible significant changes usually last less than 15 seconds after telling a lie. In these areas, Bi-Digital O-Ring Test (BDORT), which received a U.S. Patent in 1993, became significantly weak with an abnormal value of (-)7 and TXB2, measured non-invasively, was increased from 0.125-0.5ng to 12.5-15ng (within the first 5 seconds) and then went back down to less than 1ng (after 15 seconds). These unique changes can be documented semi-permanently by taking photographs of the face of people who tell a lie, within as short as 10 seconds after saying a lying statement. These abnormal responses appear in one or more of the above-mentioned 3 areas 1), 2) & 3). At least one abnormal pupil with BDORT of (-)8-(-)12 & marked reduction in Acetylcholine and abnormal increase in any of 3 Alzheimer's disease associated factors Apolipoprotein (Apo) E4, ß-Amyloid (1-42), Tau protein, viral and bacterial infections were detected in both pupils and forehead of murderers and people who often have problems with others. Analysis of well-known typical examples of recent mass murderers was presented as examples. Using these findings, potential murderers and people who are very likely to develop problems with others can be screened within 5-10 minutes by examining their facial photographs and signatures before school admission or employment.

Soluble CD40L release as test for functional platelet loss.

BACKGROUND: During platelet storage, alterations of the platelet function, 'platelet storage lesion', can be observed resulting in a reduced platelet viability. The release of soluble CD40 ligand (sCD40L) by platelets reflects different aspects of platelet metabolism and activity. Therefore, we used the sCD40L release to test for functional platelet loss in platelet products during storage in comparison to the formation of thromboxane (TXB2). METHODS: On day 1, 3, and 5 in single donor apheresis platelet products (n = 8) under routine storage conditions, sCD40L (measured by ELISA) and TXB2 (measured by RIA) were determined after platelet stimulation (recalcification and clot formation). Results were related to a therapeutic unit (TU = 2 x 10*11 platelets). RESULTS: In platelet-rich plasma of the donors, sCD40L release was 42.5 +/- 7.1 ng/TU and TXB2 formation 2,183 +/- 576 ng/TU. On day 1, 3, and 5 sCD40L release was reduced to 95%, 64%, and 57% and TXB2 formation to 92%, 80%, and 65% of the respective control values. CONCLUSIONS: In single donor apheresis PCs, sCD40L release and TXB2 formation showed a comparable course over storage time and were reduced to about 60% of the respective control values after a storage period of 5 days. These findings are in line with literature data indicating that a functional platelet loss of about 30% will occur after 5 days of storage. Overall, sCD40L release could be easily induced by recalcification and clot formation and can be used as a marker for functional platelet loss.

Production of prostaglandins, isoprostanes and thromboxane by Aspergillus fumigatus: identification by gas chromatography-tandem mass spectrometry and quantification by enzyme immunoassay.

Aspergillus fumigatus has been reported to produce prostaglandin (PG)-like substances. The molecular structure of these fungal eicosanoids is however still unknown. By using the gas chromatography-tandem mass spectrometry (GC-MS/MS) methodology we identified a number of eicosanoids that were formed upon incubation of the precursor arachidonic acid ethyl ester (AAE, 10 µM) with three strains of A. fumigatus. The eicosanoids identified include the prostaglandins (PG) PGE(2), 6-keto-PGF(1α) (the stable hydrolysis product of prostacyclin PGI(2)) and PGF(2α), the isoprostanes 15(S)-8-iso-PGF(2α) and 15(S)-8-iso-PGE(2), and thromboxane B(2) (TxB(2), the stable hydrolysis product of TxA(2)). These eicosanoids are identical with those produced by cyclooxygenases (COX) in humans. The biosynthesis of all of these eicosanoids could not be inhibited by the human COX inhibitors indomethacin (100 µM), acetylsalicylic acid (100 µM) or the non-selective human lipoxygenase (LOX) inhibitor nordihydroguaiaretic acid (100 µM). By contrast, the selen-containing antioxidant ebselen (100 µM) was found to inhibit their synthesis. Our results suggest that mammals and fungi employ different eicosanoid biosynthesis pathways. As some of the detected eicosanoids are potent immunomodulators and bronchoconstrictors, they could play a possible role in pulmonary diseases associated with A. fumigatus infection.

Mass spectrometry-based quantitative analysis and biomarker discovery.

Mass spectrometry-based quantitative analysis and biomarker discovery using metabolomics approach represent one of the major platforms in clinical fields including for the prognosis or diagnosis, assessment of severity and response to therapy in a number of clinical disease states as well as therapeutic drug monitoring (TDM). This review first summarizes our mass spectrometry-based research strategy and some results on relationship between cysteinyl leukotriene (cysLT), thromboxane (TX), 12-hydroxyeicosatetraenoic acid (12-HETE) and other metabolites of arachidonic acid and diseases such as atopic dermatitis, rheumatoid arthritis and diabetes mellitus. For the purpose of evaluating the role of these metabolites of arachidonic acid in disease status, we have developed sensitive determination methods with simple solid-phase extraction and applied in clinical settings. In addition to these endogenous compounds, using mass spectrometry, we have developed actually applicable quantitative methods for TDM. Representative example was a method of TDM for sirolimus, one of the immunosuppressant agents for a recipient of organ transplant, which requires rigorous monitoring of blood level. As we recognized great potential in mass spectrometry during these researches, we have become interested in metabolomics as the non-targeted analysis of metabolites. Now, established strategy for the metabolomics investigation applies to samples from cells, animals and humans to separate groups based on altered patterns of metabolites in biological fluids and to identify metabolites as potential biomarkers discriminating groups. We would be honored if our research using mass spectrometry would contribute to provide useful information in the field of medical pharmacy.

The effect of consuming meat enriched in walnut paste on platelet aggregation and thrombogenesis varies in volunteers with different apolipoprotein A4 genotype / El efecto del consumo de carne enriquecida en pasta de nuez sobre la agregacion plaquetaria y la trombogenesis varia en voluntarios con diferente genotipo para la apolipoproteina A4

Background and aim: Low-fat meat (LM) has been considered adequate under a cardiovascular disease point of view. Meat enriched in walnut paste (WM) consumption produces beneficial antithrombogenic effects but with striking inter-individual variability that may be related to gene polymorphism. Variants in the APOA4 gene (APOA4) polymorphism are known to affect the cardiovascular risk. This study aimed to compare the effects of consumption of WM and LM on platelet aggregation, production of thromboxane A2 (TXA2) and prostacyclin I2 (PGI2), and the TXA2/PGI2 ratio in 22 volunteers with different APOA4 polymorphism. Subjects and Methods: Six volunteers carried the Gln allele (APOA4-2) while 16 were homozygous for the His allele (APOA4-1). Platelet aggregation, TXA2 (measured as TXB2), PGI2 (measured as 6-keto-PGF1α), and the thrombogenic ratio (TXB2/6-keto-PGF1α) were determined at baseline and at weeks 3 and 5 for the WM and LM dietary periods. Results: Platelet aggregation decreased significantly (P<0.05) more in APOA4-1 than in APOA4-2 volunteers at 3-wk WM period, while TXB2 levels dropped more in APOA4-2 than in APOA4-1 volunteers at 5-wk WM period. TXB2 levels and the TXB2/6-keto-PGF1α ratio decreased significantly more (P<0.05) after 5 wk treatment in APOA4-2 than in APOA4-1 carriers on the WM diet than on the LM counterpart. However, 6-keto-PGF1α levels increased more (P<0.05) in APOA4-1 than in APOA4-2 volunteers after the 5-wk WM period than after the 5-wk LM diet. Conclusions: Present results suggest that consumption of WM with respect to LM decrease the thrombogenic risk more in Gln carriers than in His/His (AU)

Antecedentes y objetivos: La carne con bajo contenido graso (LM) se considera adecuada bajo el punto de vista cardiovascular. La ingesta de carne enriquecida en pasta de nuez (WM), mejora los efectos antitrombogénicos con una variabilidad inter-individual que puede estar relacionada con el polimorfismo genético. Variaciones en los genes APOA4 (APOA4) del polimorfismo afectan el riesgo cardiovascular. Este estudio compara los efectos de la ingesta de WM y LM sobre la agregación plaquetaria, la producción de tromboxano A2 (TXA2) y prostaciclina I2 (PGI2), y el cociente TXA2/PGI2 ratio en 22 voluntarios con diferentes polimorfismos APOA4. Material y métodos: Seis voluntarios portaban el alelo Gln (APOA4-2) frente a los 16 homozigotos para el alelo His (APOA4-1). La agregación plaquetaria, el TXA2 (medido como TXB2), la PGI2 (medida como 6-keto-PGF1α), y el cociente trombogenético (TXB2/6-keto-PGF1α) se determinaron al comienzo y en las semanas 3 y 5 de los periodos de WM y LM. Resultados: La agregación plaquetaria disminuyó significativamente más (P <0.05) en los voluntarios APOA4-1 que en los APOA4-2 en la semana 3 del periodo WM. El descenso de los niveles de TXB2 fue mayor para los voluntarios APOA4-2 que para los APOA4-1 en la semana 5 del periodo WM. Después de 5 semanas con dieta WM, la concentración de TXB2 y el cociente TXB2/6-keto- PGF1α disminuyeron significativamente más (P<0.05) en los individuos APOA4-2 que en los APOA4-1 que con la dieta LM. Sin embargo, después de 5 semanas, la dieta WM con respecto a la LM incrementó más (P <0.05) los niveles de 6-keto-PGF1α en los voluntarios APOA4-1 que en los APOA4-2. Conclusiones: Estos resultados sugieren que la ingesta de WM en comparación d LM, disminuye más el riesgo trombogénico en los voluntarios portadores de Gln que en los que His/His (AU)

Determination of thromboxanes, leukotrienes and lipoxins using high-temperature capillary liquid chromatography-tandem mass spectrometry and on-line sample preparation.

An on-line strong cation-exchange (SCX)-reversed-phase (RP) capillary liquid chromatographic (cLC) method with ion-trap tandem mass spectrometric (IT-MS/MS) detection for the simultaneous determination of thromboxane (TX) B(2), TXB(3), leukotriene (LT) B(4), LTD(4) and lipoxin (LX) A(4) in cell culture supernatants was developed and validated. In the present method, a high temperature (70 degrees C) was used for the separation on the analytical column to obtain efficient chromatography of the thromboxanes. An on-line sample preparation was performed, where peptides/proteins contained in the matrix were removed by the SCX column. Sample pre-treatment included dilution and filtration, and the analysis time including all sample preparation steps was 60min per sample. Limits of detection in the range of 1-4ng/mL cell culture supernatant, recoveries between 30% and 100%, within day precisions of less than 20% RSD and between day precisions of less than 30% RSD were obtained. Human mesenchymal stem cells (hMSCs) were stimulated with cytokine-containing supernatants derived from activated human T lymphocytes, and thromboxane, leukotriene and lipoxin production was analysed using the developed method. TXB(2) was found in cultures from both non-differentiated and differentiated hMSCs that were stimulated with a cytokine-containing supernatant obtained from activated T-cells.

Estrogen receptor-beta mediates cyclooxygenase-2 expression and vascular prostanoid levels in human placental villous endothelial cells.

OBJECTIVE: Regulation of fetoplacental blood flow is likely mediated by factors such as prostanoids. Estrogen and its receptors affect prostanoid biosynthesis. Previously, we demonstrated that villous endothelial cells express estrogen receptor-beta (ESR2), and we sought to determine its role in the mediation of fetoplacental vascular function. STUDY DESIGN: Villous endothelial cells from uncomplicated pregnancies were isolated, cultured, and treated with estrogen. RNA interference, real-time polymerase chain reaction, Western blotting, and enzyme immunoassays were performed. RESULTS: Cyclooxygenase-2 (COX-2) expression levels were not altered consistently by estrogen. RNA interference of ESR2 led to a concomitant decrease in COX-2 messenger RNA (P < .0001) and protein (P < .05) in the presence and absence of estradiol. ESR2 knock-down also led to diminished prostacyclin and thromboxane concentrations in the absence of estradiol (P < .005). CONCLUSION: ESR2 mediates COX-2 expression levels and both prostacyclin and thromboxane concentrations in the basal state, which suggests the possibility of ligand-independent regulation of COX-2 activity and prostaglandin H2 substrate availability. Further investigation regarding ESR2 regulation of prostanoid biosynthesis and its effects on the fetoplacental vasculature is warranted.

Rapid sample preparation and simultaneous quantitation of prostaglandins and lipoxygenase derived fatty acid metabolites by liquid chromatography-mass spectrometry from small sample volumes.

BACKGROUND: Fatty acid metabolites play a key role in numerous physiological and pathological processes. A rapid liquid chromatography-mass spectrometry assay for the simultaneous determination of prostanoids, isoprostane and lipoxygenase (LOX) derived fatty acid metabolites in a small biological sample of only 20 microL was developed. METHODS: Human plasma samples were applied to a filter spot, extracted without prior derivatization and analyzed within 13 min. Detection of metabolites was performed on a triple quadrupole mass spectrometer in negative multiple-reaction monitoring detection mode. Application of this assay to various biological matrices was performed. RESULTS: The validated assay was linear over the concentration range of 5-500 nmol/L for prostanoids and isoprostane, 50-5000 nmol/L for LOX-derived metabolites and 400-40,000 nmol/L for fatty acids. Limits of quantitation were 0.4-233 nmol/L, depending on the metabolite. Plasma samples from diabetic patients and controls showed significant increases in (+/-)9-HODE and 15(S)-HETE with p-values of 0.019 and 0.024, respectively. CONCLUSIONS: The small amount of 20 microL sample volume used in this assay and the demonstrated application to various sample types makes it an ideal routine analysis method for fatty acid metabolites. The resulting values for LOX-derived metabolites in diabetes mellitus type 2 samples support earlier findings about the role of lipid oxidation products in diabetes.

Predominant basal directional release of thromboxane, but not prostacyclin, by placental trophoblasts from normal and preeclamptic pregnancies.

OBJECTIVE: To investigate apical and basal releases of thromboxane (TX) and prostacyclin (PGI2) by trophoblasts (TCs) from normal and preeclamptic (PE) placentas. METHODS: TCs isolated from normal and PE placentas were incubated in cell culture inserts for 48h. Medium from the upper (apical) and the lower (basal) chambers were then collected separately and measured for TX and PGI2 by their stable metabolites of TXB2 and 6-keto PGF1alpha by ELISA. Apical and basal releases of TX and PGI were also examined with apical exposure of TCs to arachidonic acid (AA)+/-aspirin at different concentrations. Villous tissue expressions for PGI synthase, TX synthase and TX (TP) receptor were examined by immunohistochemistry. RESULTS: (1) TXB2, but not 6-keto PGF1alpha, concentrations were significantly higher in the lower than in the upper chambers with both normal and PE TCs (p<0.01); (2) apical exposure of TCs to AA resulted in a significant increase in TX release towards both the upper and the lower chambers in normal TCs (p<0.01), but only a significant increase in the upper chamber in PE TCs (p<0.01); (3) aspirin could attenuate AA-induced TX release both in the upper and the lower chambers in normal, but not in PE, TCs (p<0.01), respectively; (4) there were no differences in 6-keto PGF1alpha productions both in normal and PE TCs treated with AA+/-aspirin; (5) intense staining of TX synthase and TP receptor was seen in syncytiotrophoblast layer, villous core vessels and stromal cells in preeclamptic placental tissue sections. CONCLUSION: Predominant basal release of TX together with intense staining of TX synthase and TP receptor in trophoblasts, stromal cells and villous core vessels are found in placentas from PE. We speculate if predominant basal release of TX by TCs occurs in vivo as we found in our in vitro culture condition, basal released TX may play a significant role in increased placental vasoconstriction such as in PE.

Role of thromboxane in producing hepatic injury during hepatic stress.

Hepatic injury after hepatic stress is caused by several mechanisms, including inflammatory reaction and microcirculatory disturbance. Levels of thromboxane, a vasoconstrictive eicosanoid, have been shown to increase in systemic circulation after different types of hepatic stress such as endotoxemia, hepatic ischemia-reperfusion, hepatectomy, liver transplantation, hemorrhagic shock and resuscitation, hepatic cirrhosis, and alcoholic liver injury. The production of thromboxane from the liver is also enhanced under these stresses, which may act on the liver in an autocrine or a paracrine fashion. Kupffer cells, resident hepatic macrophages, may be a major source of stress-induced thromboxane, although other cell types in the liver such as sinusoidal endothelial cells and hepatocytes may also produce this eicosanoid. Thromboxane induces hepatic damage through vasoconstriction, platelet aggregation, induction of leukocyte adhesion, up-regulation of proinflammatory cytokines, and induction of other vasoconstrictor release. In this regard, administration of cyclooxygenase inhibitor, specific thromboxane synthase inhibitor, and specific thromboxane receptor antagonists has been shown to protect from severe hepatic injury elicited by these hepatic stresses. Furthermore, blockade of Kupffer cell function by administration of gadolinium chloride showed salutary effects in preventing hepatic damage in bile duct ligation models. This review article summarizes the recent knowledge of the role of thromboxane in various types of hepatic stress and the effects of thromboxane inhibitors in these models.

Ácidos grasos omega-3 en las lesiones deportivas ¿una posible ayuda terapéutica? (i) / Omega-3 fatty acid: a possible therapeutic aid? (i)

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Exudation of plasma and production of thromboxane in human bronchi after local bradykinin challenge.

Plasma exudation has been suggested to be an important component of the inflammatory response in asthma. Bradykinin elicits many of the features of asthma, including bronchoconstriction, cough, plasma exudation and mucus secretion. In an attempt to quantify local plasma exudation, we have employed a novel low-trauma technique with the aim of challenging and lavaging a central part of the bronchial tree, by selecting a medium sized bronchus. A fibreoptic bronchoscopy was performed in non-smoking healthy volunteers. The instrument was placed proximally in the right upper lobe bronchus. A plastic catheter, equipped with an inflatable latex balloon, was inflated with air (2-4 cmH2O). A solution (100 microl of either two different concentrations of bradykinin: 0.09 and 0.9 mg ml(-1) or normal saline) was instilled through the catheter and distal to the balloon. Eight minutes later a lavage procedure with 10 ml of saline was performed through the catheter. The procedure was then repeated twice, with the other solutions, but from the lingular and middle lobe bronchi. All solutions were given in a blinded fashion, and two different studies were performed. Lavage concentrations of albumin and IgG were quantified as measurements of plasma exudation. In our first study we found that bradykinin challenge significantly increased concentrations of albumin and IgG. In study two, there was no numeric increase in plasma proteins after local bradykinin challenge, but the concentration of thromboxane was significantly increased in lavages from bradykinin-challenged bronchi. Thus, local bronchial administration of bradykinin has the capacity to induce exudation of large plasma macromolecules into the bronchial lumen, as well as local thromboxane production.

Cytokine-induced bronchoconstriction in precision-cut lung slices is dependent upon cyclooxygenase-2 and thromboxane receptor activation.

Cytokines play an essential role in the regulation of inflammatory responses. The effects of cytokines on lung functions are less well known and their study in vivo is complicated by the attraction of leukocytes to the inflamed sites. Recently the model of precision-cut lung slices was developed, where viable lung slices with an intact microanatomy are taken into culture and where bronchoconstriction can be followed by observing single airways under the microscope. We used this model to study the direct effects of cytokines on airway tonus in the absence of blood-derived leukocytes. Incubation of precision-cut lung slices with a mixture of tumor necrosis factor (TNF)-alpha, interleukin (IL)-1beta, and interferon (IFN)-gamma resulted in contraction of airways, which was accompanied by expression of cyclooxygenase (Cox)-2 and thromboxane release into the supernatant. The thromboxane receptor antagonist SQ29548 completely prevented the cytokine-induced bronchoconstriction, whereas the 5-lipoxygenase inhibitor AA681 had no effect on cytokine-induced bronchoconstriction. Preventing the expression of Cox-2 by dexamethasone or blocking Cox-2 activity with the selective Cox-2 inhibitor NS398 attenuated both thromboxane formation and bronchoconstriction. Incubation of lung slices with each of the cytokines alone caused no bronchoconstriction; in fact, IL-1 alone rather dilated the airways. However, simultaneous incubation with TNF and IL-1beta caused a bronchoconstriction that was not further enhanced by IFN-gamma. We conclude that TNF-alpha and IL-1beta synergistically cause bronchoconstriction by induction of Cox-2 and subsequent activation of the thromboxane receptor. Our study raises the possibility that TNF and IL-1 may contribute to bronchospasm during inflammatory lung diseases.

Application of gas chromatography-mass spectrometry and gas chromatography-tandem mass spectrometry to assess in vivo synthesis of prostaglandins, thromboxane, leukotrienes, isoprostanes and related compounds in humans.

Prostaglandins, thromboxane, leukotrienes, isoprostanes and other arachidonic acid metabolites are structurally closely related, potent, biologically active compounds. One of the most challenging tasks in eicosanoids research has been to define the role of the various eicosanoids in human health and disease, and to monitor the effects of drugs on the in vivo synthesis of these lipid mediators in man. Great advances in instrumentation and ionization techniques, in particular the development of tandem mass spectrometry and negative-ion chemical ionization (NICI), in gas chromatography and also advances in methodologies for solid-phase extraction and sample purification by thin-layer chromatography and high-performance liquid chromatography have been made. Now gas chromatography-mass spectrometry (GC-MS) and GC-tandem MS in the NICI mode are currently indispensable analytical tools for reliable routine quantitation of eicosanoid formation in vivo in humans. In this article analytical methods for eicosanoids based on GC-MS and GC-tandem MS are reviewed emphasizing the quantitative measurement of specific index metabolites in human urine and its importance in clinical studies in man. Aspects of method validation and quality control are also discussed.

Inhibition of antigen-induced muscle contractions by inhibitors of thromboxane pathway in rat small intestine.

Rats were sensitized against egg albumin and the response of the longitudinal muscle from the proximal small intestine to the antigen was tested. Egg albumin (1-100 micrograms/ml) concentration-dependently induced a contraction of the longitudinal muscle in tissues from sensitized animals but not from nonsensitized animals. The response to the antigen was resistant to neuronal blockers like tetrodotoxin, atropine and hexamethonium. Inhibitors of thromboxane synthesis such as the cyclooxygenase inhibitor, indomethacin, the thromboxane synthase blocker, 1-benzylimidazole, or the combined cyclooxygenase/lipoxygenase/thromboxane synthase inhibitor, sulfasalazine, inhibited the contraction evoked by egg albumin. A similar concentration-dependent inhibition of the antigen response was observed with two thromboxane A2 receptor blockers, SK&F 88046 and KW-3635. None of these blockers affected the response to the muscarinic agonist, carbachol, excluding unspecific effects of the drugs on smooth muscle contractility. The effect of antigen was reduced by the mast cell stabilizing agent, quercetin, and by the histamine H1 receptor blocker, mepyramin. These drugs, however, also inhibited the response to carbachol. When contractions were stimulated directly by the stable thromboxane derivative, carbocyclic thromboxane A2, the smooth muscle proved to be more than three orders of magnitude more sensitive to this agonist of the thromboxane pathway compared to histamine. Consequently, thromboxane A2 seems to be one of the main mediators of anaphylactically induced longitudinal muscle contractions in the rat small intestine.

Measurement of thromboxane and prostacyclin in valvulotomized human saphenous veins.

The present study was done to determine the effect of the modified Hall valvulotome technique on endothelial injury by measuring TxB2 and 6-keto PGF1alpha, the stable metabolites of thromboxane and prostacyclin, respectively. It was hypothesized that increased levels of these cyclooxygenase products would be an excellent indicator of vascular endothelial injury in the presence of the modified Hall valvulotome. Eight segments of human distal saphenous veins were obtained, each measuring approximately 4 cm in length, with diameters of approximately 2 to 3 mm. From these original vein segments, two groups of smaller vein segments were examined, with each group consisting of eight segments, each segment measuring 2 cm in length. The first group of vein segments was designated as the control group, and the second group of vessels had a modified Hall valvulotome (2.5 mm size) inserted into each segment to simulate valvulotomy. After this procedure, all vein segments were analyzed for levels of thromboxane and prostacyclin by a standard radioimmunoassay procedure. Results from the present study indicate that the modified Hall valvulotome technique in human saphenous veins does not significantly increase the levels of the cyclooxygenase metabolites thromboxane and prostacyclin relative to control conditions. However, the ratio of TxB2 formation 6-keto PGF1alpha production was increased in the valvulotomized vessel segments, indicating possible platelet release of thromboxane. Therefore, even though there was increased thromboxane production relative to prostacyclin levels in the modified Hall valvulotome technique, it still appears that this type of valvulotomy is relatively noninsulting to the endothelial cell lining.