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Thromboxane B2 (TXB2) and 12-hydroxy-5,8,10,14-eicosatetraenoic acid (12-HETE) formed from the endogenous and exogenous arachidonate during human platelet incubation, was evaluated by selected ion monitoring (SIM). TXB2 formed from endogenous substrate accounted for about one third of the total, whereas the great part of 12-HETE derived from exogenous arachidonate. These data indicate that under the tested conditions the pool of arachidonate that acts as substrate for cyclo-oxygenase is different from the pool that acts as substrate for lipoxygenase and that the arachidonate released from phospholipids is preferentially utilized by cyclo-oxygenase.

Simultaneous addition of diamide (azodicarboxylic acid-bis-dimethylamide, DIA), a SH-oxidizing agent, and collagen causes a deaggregation of otherwise irreversibly aggregating platelets. Thromboxane B2 (TXB2) and 12-HE-TE formation is inhibited depending on the concentration ratio between collagen and DIA. Thus, at 0.25 mM DIA and 20 micrograms/ml collagen neither TXB2 nor 12-HETE were measurable, but a full scale reversible aggregation is induced. Deaggregation is further attained by adding DIA to collagen-induced aggregates at a time, when maximum amplitude has been achieved. Investigation of arachidonic acid (AA) metabolites under these conditions revealed no influence of DIA on AA metabolism. Therefore, AA metabolization seems to play a minor role in collagen-induced aggregation and DIA-induced deaggregation. Polymerization of certain cytoskeletal proteins of the platelets, after addition of DIA, parallels DIA-induced deaggregation. DIA inhibits endogenous AA release, probably by interaction with platelet plasma membrane. DIA seems to inhibit the release of the alpha-granula protein thrombospondin.

The effects of a single oral administration of acetylsalicylic acid (500 mg), indomethacin (50 mg) and piroxicam (40 mg) to healthy volunteers on functional and biochemical parameters of platelets, polymorphonuclear (PMN) and mononuclear (MNL) leukocytes were evaluated. Blood was collected before and two hours after the drug intake and blood cells separated according to conventional techniques. The considered drugs almost completely suppressed the aggregation of platelets, whereas they did not affect either PMN and MNL aggregation. Superoxide anion generation by leukocytes was significantly increased after aspirin intake, whilst a significant reduction (PMN), or no effect (MNL) was observed after piroxicam and indomethacin respectively. The formation of arachidonate metabolites via the 5-lipoxygenase pathway by PMN and MNL challenged with 10pM A23187 was unchanged following aspirin and indomethacin. In this respect a selective increase of 5-HETE and LT& synthesis by MNL only was detected after piroxicam administration. The three drugs similarly reduced TXB2 synthesis by platelets and PMN (-80% for aspirin and indomethacin, and -40% for piroxicam). As far as MNL is concerned, aspirin inhibited this metabolite by 80%, while indomethacin reduced it by 40% only. In contrast piroxicam increased TXB2 synthesis by stimulated MNL. It can be concluded that the considered antiinflammatory drugs 1) differently affect the cyclooxygenase enzyme in platelets and leukocytes; 2) at variance with the situation in platelets, the inhibition of thromboxane formation by leukocytes is not related to modifications of cellular function; 3) the formation of arachidonate metabolites via the 5- lipoxygenase pathway is affected by piroxicam only.

The established mouse epidermis-derived cell line HEL/30 was incubated in the presence of 3H arachidonic acid (AA) for 1 h. After medium removal, cells were reincubated with fresh medium in the presence or absence of the calcium ionophore A23187 and tumor promoter 12-O-tetradecanoyl-phorbol-13-acetate (TPA). The AA metabolites formed were extracted from cell-free medium and analyzed using TLC and HPLC. The distribution of the recovered radioactivity showed PGE2, 15-hydroxy-eicosatetraenoic acid (15-HETE), and leukotriene B4 (LTB4), as major products of AA metabolism. The presence of calcium ionophore A23187 increased the release of radioactivity, without affecting the profile of metabolites present in the medium. TPA elicited a preferential increase of cycloxygenase metabolism, this effect being reversed by indomethacin. 5,8,11,14-eicosatetraynoic acid (ETYA) almost completely inhibited LT and HETE formation in A23187 and TPA-treated cells. The results show that HEL/30 cells are able to metabolize AA via both cyclo- and lipoxygenase pathways and that these activities can be modified by chemical means. This cell line might be a suitable tool for studying the involvement of arachidonic acid cascade in cell response to exogenous stimuli.

Deuterated arachidonic acid (AA, [2H8]20:4 n-6) 1-25 microM, is converted to other fatty acids, as evaluated by gas chromatography-mass spectrometry, in THP-1 cells. The major products, in the 1 to 10 microM range, are 22:4 (elongated) and 20:3 (reduced in 5). At 25 microM, 24:4, 24:5 and 22:5 accumulate, with [2H8]/[2H0] ratios higher than in AA. At high AA concentration preferential conversion to elongated fatty acids with 5 unsaturations, through a 6 desaturase takes place and the 4-desaturated 22:5 appears to be formed through beta-oxidation of 24:5.

The idea that angiographic assessment of coronary atherosclerosis is a poor predictor of the occurrence of clinical complications is being appreciated more and more. Over a certain age, atherosclerotic lesions in the coronary arteries become common in humans [1]. Clinical manifestations of coronary artery disease are, on the contrary, relatively uncommon phenomena, sometimes occurring erratically and unpredictably in the patient’s life. This is especially true of the most catastrophic clinical complications of coronary artery disease, namely unstable angina, myocardial infarction, and sudden cardiac death. On the other hand, a long established and more understandable relationship exists between the angiographic degree of coronary artery lesions and chronic stable angina. Here, the progressive reduction of coronary reserve as a function of the degree of lumen obstruction can easily explain the occurrence of myocardial ischemia in conditions of an increased myocardial oxygen demand. However, stable angina is rarely an unmanageable situation, and we are mostly faced with the problem of unstable events only loosely correlated with quantitative assessment of lumen reductions. Given the strong evidence still relating the proven mechanisms of “coronary instability” — thrombosis and alterations in the vasomotor tone — to atherosclerotic plaques in the coronary arteries [2–6], one is left with the recognition of the limitations of quantitative assessment of lumen reduction as given by angiography, and with the need to explore qualitative features of the atherosclerotic plaque in search for hints that predict the “activation” of the disease.

Myocardial ischemia was induced in perfused paced isovolumic left heart preparation of the rabbit by reducing, for a period of 40 min, the flow rate from 20 ml/min to 0.2 ml/min (severe model) and to 1 ml/min (moderate model). The relationship between prostaglandin biosynthesis and cardiac ischemic damage was evaluated in the two experimental models. The results obtained indicate that the total amount of 6-keto-PGF1 alpha generated increases with the severity of the ischemia, particularly during the 20 min of reperfusion (moderate model 81.8 +/- 13.7 ng: severe model 375 +/- 102 ng). The inhibition of the prostaglandin synthesis, prostaglandin-E2, and 6-keto-prostaglandin-F1 alpha (PGE2 and 6-keto-PGF1 alpha levels below the detection limits) by Aspirin (20 micrograms/ml) and Indomethacin (1 microgram/ml) in moderate myocardial ischemia was correlated with greater increments in resting diastolic tension (nearly 100% and 40%, respectively). This phenomenon was also associated to a further decrease on cardiac contractility and increase on coronary perfusion pressure upon reperfusion. On the contrary drugs which stimulated prostaglandin generation in myocardial tissue, such as Defibrotide (400 micrograms/ml), completely protected the organ from ischemia. U-60257 (3 micrograms/ml) and FPL-55712 (2 micrograms/ml), compounds, which respectively inhibits biosynthesis and the effects of leukotrienes, displayed a beneficial activity on this moderate model of ischemia. The present data suggests that the deleterious effect of nonsteroidal antiinflammatory drugs in low flow myocardial ischemia and reperfusion damage may be associated with removal of PGI2 and PGE2 from ischemic myocardium.

The effects of a single oral administration of acetylsalicylic acid (ASA, 500 mg), indomethacin (Indo, 50 mg) and sodium salicylate (SA, 400 mg) to healthy volunteers on platelet formation of 12-hydroxyeicosatetraenoic acid (12-HETE) were evaluated. Blood was collected before, 2, 6 and 24 hours after drug administrations. Platelet rich plasma (PRP) samples were stimulated with 20 μg/ml collagen and 12-HETE levels were determined by selective ion monitoring. The effects of ASA on the same parameter were also evaluated in vitro in PRP and in washed platelet (WP) samples in the absence and in the presence of platelet poor plasma (PPP, 25-100%) or bovine serum albumin (BSA, 10-40 mg/ml). In subjects who ingested ASA, the formation of 12-HETE in PRP stimulated with collagen was significantly inhibited 2 and 6 hours after the drug administration. At 24 h 12-HETE synthesis tended to return toward basal values. In contrast the administration of a single dose of Indo or of SA did not significantly affect 12-HETE synthesis by stimulated PRP. ASA (3 mM) added in vitro inhibited 12-HETE formation in PRP but not in WP. In addition ASA inhibited 12-HETE synthesis in washed platelets resuspended either in PPP (25-100 %) or in BSA (10-40 mg/ml). It is concluded ASA, but not Indo or SA, orally administered to normal subjects inhibits 12-HETE synthesis in collagen stimulated PRP. The results obtained in vitro suggest that albumin and/or some albumin component may be responsible for the inhibitory effect of ASA on platelet 12-HETE syntesis.

Die Vorstellung, das die angiographische Erfassung der Koronarsklerose ein schlechter Pradiktor fur das Auftreten von klinischen Komplikationen ist, wird mehr und mehr geteilt. Ab eines bestimmten Alters sind arteriosklerotische Lasionen der Koronararterien beim Menschen haufig (3). Die klinischen Manifestationen der koronaren Herzkrankheit (KHK) sind ganz im Gegensatz dazu relativ seltene Ereignisse, die manchmal unregelmasig und unvorhersehbar im Leben des Patienten auftreten. Dies trifft ganz besonders auf die katastrophalen klinischen Komplikationen der KHK zu, namlich die instabile Angina, den Myokardinfarkt und den plotzlichen Herztod. Andererseits existiert ein lang bekannter und verstandlicher Zusammenhang zwischen dem angiographischen Ausmas der koronar-sklerotischen Lasionen und der chronisch-stabilen Angina. Aufgrund der progressiven Verminderung der Koronarreserve als einer Funktion des Ausmases der Lumenobstruktion, ist das Vorkommen der Myokardischamie unter den Bedingungen eines verstarkten Sauerstoffbedarfs des Herzmuskels leicht zu erklaren.

The effects of single oral administrations of acetylsalicylic acid (ASA, 500 mg), indomethacin (Indo, 50 mg) and sodium salicylate (NaSal, 400 mg) on platelet aggregation and on the thromboxane B2 (TXB2) and 12-hydroxyeicosatetraenoic acid (12-HETE) synthesis by platelet rich plasma (PRP) stimulated with collagen were evaluated. While both ASA and Indo significantly inhibited TXB2 synthesis and platelet aggregation, significant reduction of 12-HETE formation at 2 and 6 h after the administration of the drug, was detected only in subjects who ingested ASA. NaSal did not affect any of the tested parameters. The comparison of the effect of ASA (200 mg) on 12-HETE synthesis in washed platelets and PRP shows that the drug is able to affect this parameter only in PRP. To obtain a constant inhibition of 12-HETE synthesis in PRP over a 24 h period, a repeated ASA treatment schedule was assessed (ASA 200 mg every 6 h for 5 times). TXB2 synthesis in PRP was almost completely suppressed at 2 h after the first ASA administration and inhibition remained constant up to 48 h after the last ASA intake. As far as 12-HETE synthesis by stimulated PRP is concerned, a significant reduction of this parameter was detected at 4 h after the first drug administration and the levels remained almost constant following the repeated administrations during a 24 h period. These data indicate that ASA, but not Indo and NaSal, significantly affect not only TXB2 synthesis but also 12-HETE formation in PRP. The lack of the effect of ASA administration on 12-HETE, found when studies were carried out in washed platelets, indicates that the drug requires the presence of plasma factors for its activity on the formation of 12-lipoxygenase products by platelets.

The formation of the major metabolic products of endogenous arachidonic acid (AA) via cyclooxygenase and lipoxygenase pathways in platelets from normal and type IIA hypercholesterolemic subjects was evaluated. 12-Hydroxyeicosatetraenoic acid (12-HETE) and thromboxane B2(TXB2) were determined by selected ion monitoring (SIM) after extraction and purification of collagen stimulated platelet-rich plasma (PRP). The levels of both arachidonic acid metabolites in the non-stimulated PRP of control and type IIA subjects were below the detection limit of the method, rising significantly after collagen stimulation. Both 12-HETE and TXB2 levels in collagen-stimulated PRP samples from the patients were significantly higher than levels in controls (P less than 0.001). In view of the key role of 12-HETE in mediating smooth muscle cell migration and proliferation and in stimulating macrophage activity, these data may provide information for the understanding of the elevated incidence of thrombosis and atheromatous lesion in patients with type IIA hypercholesterolemia.

Interest in the quantitative analysis of flavouring agents in alcoholic beverages arises from the possible toxicological consequences deriving from their use. The method reported here, based on gas chromatography with selected-ion monitoring mass spectrometry (SIM), allows the simultaneous measurement of alpha, beta-thujone, pulegone, safrole, coumarin and beta-asarone at the ppb level in alcoholic beverages.

Background Over the past fifteen years, we have demonstrated that cortisol and dehydroepiandrosterone (DHEA) have opposite effects on the regulation of protein kinase C (PKC) activity in the context of the immune system. The anti-glucocorticoid effect of DHEA is also related to the regulation of splicing of the glucocorticoid receptor (GR), promoting the expression of GRβ isoform, which acts as a negative dominant form on GRα activity. Moreover, it is very well known that DHEA can be metabolized to androgens like testosterone, dihydrotestosterone (DHT), and its metabolites 3α-diol and 3β-diol, which exert their function through the binding of the androgen receptor (AR). Based on this knowledge, and on early observation that castrated animals show results similar to those observed in old animals, the purpose of this study is to investigate the role of androgens and the androgen receptor (AR) in DHEA-induced expression of the PKC signaling molecule RACK1 (Receptor for Activated C Kinase 1) and cytokine production in monocytes. Results Here we demonstrated the ability of the anti-androgen molecule, flutamide, to counteract the stimulatory effects of DHEA on RACK1 and GRβ expression, and cytokine production. In both THP-1 cells and human peripheral blood mononuclear cells (PBMC), flutamide blocked the effects of DHEA, suggesting a role of the AR in these effects. As DHEA is not considered a direct AR agonist, we investigated the metabolism of DHEA in THP-1 cells. We evaluated the ability of testosterone, DHT, and androstenedione to induce RACK1 expression and cytokine production. In analogy to DHEA, an increase in RACK1 expression and in LPS-induced IL–8 and TNF–α production was observed after treatment with these selected androgens. Finally, the silencing of AR with siRNA completely prevented DHEA-induced RACK1 mRNA expression, supporting the idea that AR is involved in DHEA effects. Conclusions We demonstrated that the conversion of DHEA to active androgens, which act via AR, is a key mechanism in the effect of DHEA on RACK1 expression and monocyte activation. This data supports the existence of a complex hormonal balance in the control of immune modulation, which can be further studied in the context of immunosenescence and endocrinosenescence.

Transcription regulation by bile acids is far more complicated than it appeared at first when several groups began their investigations in the early '90. It has become clear now that bile acids regulate the transcription of genes involved in bile acid synthesis, transport and other metabolic pathways via multiple mechanisms that involve transcription factors, nuclear receptors, coregulators, chromatin and the related modifying enzyme complexes. At a first look this might seem surprising but if one considers the physical-chemical properties of these molecules it should be evident that, due to their detergent properties, bile acids may be harmful if they reach high concentrations in the liver and intestine. Therefore, living organisms have developed biochemical mechanisms that finely tune the concentration of bile acids according to the body needs and in response to environmental challenges. In this review, we will discuss the most recent evidences on the mechanisms through which bile acids regulate gene transcription, including the function of nuclear receptors and emphasizing the emerging role of chromatin and the associated modifying enzymes.

Olive oil phenolic constituents have been shown, in vitro, to be endowed with potent biological activities including, but not limited to, an antioxidant action. To date, there is no information on the absorption and disposition of such compounds in humans. We report that olive oil phenolics, namely tyrosol and hydroxytyrosol, are dose-dependently absorbed in humans after ingestion and that they are excreted in the urine as glucuronide conjugates. Furthermore, an increase in the dose of phenolics administered increased the proportion of conjugation with glucuronide.