196,200 research outputs found
Scavenging of free radicals by tenoxicam: A participating mechanism in the antirheumatic/antiinflammatory efficacy of the drug
No full textThe radical scavenging activity of tenoxicam against hydroxyl (HO.), superoxide (O2(.-)), and peroxyl (LOO(.)) radicals, all of the involved in the inflammatory reactions, has been tested in different cell-free systems and by different techniques. Tenoxicam is a good scavenger of both HO. radicals (IC50 = 56.7 mu M), as determined by Electron Spin Resonance (ESR) spectroscopy with the spin trapping (5,5-dimethyl-1-pyrroline N-oxide, DMPO) technique, and O2(.-)radicals generated by the phenazine methosulfate/reduced beta-nicotinamide adenine dinucleotide (PMS/NADH) system. The high reactivity of the drug towards HO. was confirmed by the rate constant of reaction with HO. (k congruent to 10(10) M(-1) s(-1)), determined by competition kinetic studies with N,N-dimethyl-4-nitrosoaniline. In addition at a mu M level (1-5 mu M) it dose-dependently prevents the phycoerythrin peroxidation induced by the water-soluble azo-initiator 2,2-azobis(2-amidinopropane) dihydrochloride (ABAP), indicating a quenching effect on aqueous peroxyl radicals. The HO.-entrapping capacity was confirmed in models more close to the in vivo situation: tenoxicam inhibits the HO.-induced depolymerization of hyaluronic acid already at 15 mu M and the HO.-driven lipid peroxidation in phosphatidylcholine liposomes (PCL) with an IC50 of 10 mu M. In this membrane model it delays at 1-10 mu M level the decomposition of phosphatidylcholine hydroperoxides to short-chin alkenals (markers: total carbonyl functions as 2,4-dinitrophenylhydrazones and conjugated dienes). The high susceptibility of the drug to HO. attack is also demonstrated by its extensive degradation (HPLC studies) when irradiated with HO. radicals. The antioxidant component of tenoxicam evidenced in this study sheds some light on the hitherto undefined mechanism of the antiinflammatory action of the drug
In vitro metabolism of a nitroderivative of acetylsalicylic acid (NCX4016) by rat liver: LC and LC-MS studies
No full textThe metabolism of a nitroderivative of acetylsalicylic acid, benzoic acid, 2-(acetyloxy)-3-[(nitrooxy)methyl]phenyl ester (NCX4016), the lead compound of a new class of NO-releasing non steroidal-antiinflammatory drugs has been studied in vitro in rat liver subcellular fractions (S 9000xg, microsomes, cytosol). Samples were extracted with CH3CN (2 vol.) containing 1% H3PO4 (2 M), vortexed for 3 min and then centrifuged for 5 min at 5000 rpm. Supernatants were diluted with 0.02 M phosphoric acid and analysed by reverse-phase LC. Linearity of calibration for NCX4016 and metabolites was observed over the range 0.25-50 microg/ml with coefficients of determination greater than 0.9996. Extraction efficiency from spiked liver samples ranged from 85 to 95% for all the analytes. In the S 9000xg fraction, NCX4016 undergoes rapid metabolization, with the formation of salicylic acid (SA) and [3-(nitrooxymethyl)phenol] (HBN). HBN is then rapidly metabolised to 3-hydroxybenzylalcohol (HBA), and mainly to a new metabolic species, whose formation takes place specifically in the liver cell cytosol. LC-MS analysis (electrospray ionisation) of the cytosol extract in negative and positive-ion modes furnished deprotonated [M-H]- and protonated [M+H]+ molecular ions at m/z 412 and 414, respectively, accompanied by the typical clusters with sodium. MS/MS analysis in negative-ion mode, by selection and collision of the ion at m/z 412, gave a fragmentation pattern characterized by the ions at m/z 272 and 254, which allowed to assign the structure of 1-(glutathion-S-yl)methylene-3-hydroxy-benzene, a conjugated product between GSH and the benzyl carbon atom of HBN. In rat liver cytosol HBN is completely metabolised to this thioether adduct within 30 min incubation; the process is enzymatically mediated by GSH transferase and strictly dependent on GSH availability. The relevance of this new metabolic pathway in NCX4016 detoxification by rat liver is discussed
Hydroxynimesulide, the main metabolite of nimesulide, prevents hydroperoxide/hemoglobin-induced hemolysis of rat erythrocytes
No full textThe protective effect of hydroxynimesulide, the main metabolite of the nonsteroidal antiinflammatory drug nimesulide, on red blood cell (RBCs, 0.2%; 3.5x10(7) cells/ml) hemolysis induced by cumene hydroperoxide (CuOOH; 50 mu M) was evaluated by turbidimetric and morphological analyses. Hydroxynimesulide inhibits the CuOOH-induced hemolysis in a dose dependent fashion: the protective effect, calculated after 150 min incubation (100% hemolysis in the controls), starts at 1 mu M (% hemolysis 85.2 +/- 3.4%) and increases at the higher concentrations (63.5 +/- 3.9% at 5 mu M; 43.5 +/- 6.3% at 10 mu M; and, 14.5 +/- 4.3% at 20 mu M). In addition, in the samples protected with 10 mu M and 20 mu M, there is a significant delay (30 and 60 min) in the onset of the hemolytic response. Inhibition of hemolysis is the result of a protection of RBC membrane integrity both on lipid (cis-Parinaric acid fluorescence quenching was delayed by 53 +/- 10 sec vs. the controls at 1 mu M, by 115 +/- 15 sec at 5 mu M, with a lag phase of 240 +/- 18 sec at 10 mu M) and protein constituents, as determined by SDS-PAGE electrophoresis. In hemolysis experiments, the efficacy of hydroxynimesulide is comparable to that of alpha-tocopherol and a cooperative interaction between hydroxynimesulide and alpha-tocopherol (both at 10 mu M) has been observed These results indicate that hydroxynimesulide protects RBC membranes by directly quenching reactive oxygen species generated by hemoglobin/peroxide interaction. Evidence for a direct radical scavenging intervention of the metabolite comes from HPLC studies, which demonstrate a time-dependent consumption of hydroxynimesulide, with the concomitant formation of two main reaction (addition/oxidation) products
Profiling histidine-containing dipeptides in rat tissues by liquid chromatography/electrospray ionization tandem mass spectrometry
No full textThe histidine-containing dipeptides carnosine (CAR) and structurally related anserine (ANS) and homocarnosine (HCAR), widely distributed in vertebrate organisms, have recently been proposed as endogenous quenchers for highly cytotoxic alpha,beta-unsaturated aldehydes generated by peroxidation. A sensitive, selective, specific and rapid liquid chromatographic/electrospray ionization tandem mass spectrometric assay was developed and validated for the simultaneous determination of these peptides in biological matrices in order to establish their plasma/tissue distribution. Samples (plasma or tissue homogenates from male rats) were prepared by protein precipitation with HClO(4) (1 : 1, v/v) containing H-Tyr-His-OH as internal standard. The supernatant was separated on a Phenomenex Sinergy polar-RP column with a mobile phase of water-acetonitrile-heptafluorobutyric acid (9 : 1 : 0.01, v/v/v) at a flow-rate of 0.2 ml min(-1), with a run time of 10 min. Detection was effected on an ion trap mass spectrometer equipped with an electrospray ionization interface operating in positive ionization mode. The acquisitions were in the multiple reaction monitoring mode using the following precursor --> product ion combinations: H-Tyr-His-OH (internal standard) m/z 319 --> 301; CAR m/z 227 --> 210 + 209; ANS m/z 241 --> 224 + 197 + 170; HCAR m/z 241 --> 156. The method was validated over the concentration range 15-1000 nmol g(-1) and the limit of quantification (LOQ) and limit of detection (LOD) were 12.5 and 4.2 pmol injected, respectively. The intra- and inter-day precisions were <10% (< or =17.47% at the LOQ) and the intra- and inter-assay accuracies were within +/-10% for all concentrations. The mapping profile in rat tissue gave the following results: the highest concentrations of CAR and ANS were found in skeletal muscles (soleus, gastrocnemius, tibialis), followed by the heart, cerebellum and brain (ANS below the LOQ). HCAR was found only in the brain and cerebellum. No histidine-containing dipeptides were detectable in plasma, liver, kidney and lung
The inhibitory effect of pyrazinamide on microsomal monooxygenase activities is related to the binding to reduced cytochrome P-450
No full textThe antitubercular agent Pyrazinamide (PZA) binds to oxidized and reduced rat liver microsomal cytochrome P-450 with the binding characteristics typical of basic heterocyclic compounds. The PZA-cytochrome P-450(Fe2+) interaction, although characterized by rather weak affinity (Ks = 4 × 10-3M), has a PZA-cytochrome P-450(Fe2+) bond of considerable strenght and stability. PZA has an inhibitory effect on the aniline hydroxylase, p-nitroanisole O-demethylase and aminopyrine demethylase activities of rat liver microsomes. The PZA-cytochrome P-450(Fe2+) binding characteristics explain the observed inhibitory effect of the drug on the monooxygenase activities
[A rapid, specific and sensitive method for the identification of anthelmintic drug residues in milk and meat using mass spectrometry MS/MS]
No full textLoss of substrate binding capacity of the hepatic microsomal cytochrome P-450 in Fasciola hepatica infected rats: toxicological implications
No full textExperimental fascioliasis in the rat is responsible for a dramatic decrease in the drug-metabolizing ability of the hepatic monooxygenase system. The present investigation, through a spectroscopic study of hexobarbital interaction with microsomal cytochrome P-450 and in vitro and in vivo studies of hexobarbital metabolism in the rat, demonstrates that this decrease is due to an alteration in the structure of the hemoprotein (loss of substrate binding capacity of cytochrome P-450 followed by denaturation). These results might be responsible for a decreased safety margins for those flukicidal agents that are detoxified by the monooxygenase pathway, and might explain the accumulation problems frequently associated with chemotherapy of Fasciola hepatica
Un metodo rapido, specifico e sensibile di identificazione dei residui di farmaci antielmintici nel latte e carne mediante spettrometria di massa MS/MS
No full textAPPLICATION OF COLLISIONALLY ACTIVATED DECOMPOSITION MASS-ANALYZED ION KINETIC-ENERGY SPECTROMETRY TO A SURVEY OF SHEEP MILK FOR BENZIMIDAZOLE RESIDUES IN RELATION TO WITHDRAWAL PERIODS
No full textA collisional spectroscopy procedure for simultaneous detection of five widely employed benzimidazole anthelmintics (laevamisole, thiabendazole, mebendazole, fenbendazole, febantel) in sheep milk was developed. The method which involves injection of crude milk extracts and selection and collision of the most abundant ionic species (M+. or fragments) obtained under electron impact ionization, is highly suitable for multi-residue analysis because of its sensitivity (limits of detection in the range of 0.6-2.8 p.p.b) and its rapidity (more than five samples per hour can be processed). The collisional approach was applied successfully for monitoring of anthelmintic residues in sheep milk
Determination of histidine-dipeptides and the corresponding 4-hydroxy-2-nonenal Michael adducts by liquid chromatography/electrospray ionization tandem mass spectrometry in rat tissues
No full textCarnosine (CAR) and structurally related anserine (ANS) and homocarnosine (HCAR) are histidine-containing dipeptides widely distributed in vertebrate organisms. Although their biological role still remains unknown, it has been proposed that they act as highly effective quenchers of alfa,beta-unsaturated aldehydes such as 4-hydroxy-2-nonenal, nonenal, and acrolein. We recently proposed the reaction mechanism and found that the Michael adduct is the main reaction product (1-3). The aim of this work was to develop and validate a simple, sensitive and selective LC-MS/MS method to quantitate both histidine-dipeptides (HD) and the corresponding HNE-Michael adducts (HD-HNE) in biological matrices (rat tissues). Tissue homogenates (1:3 in PBS) from male Wistar rats were spiked with Tyr-His as internal standard (100 microM, final concentration) and deproteinized with 0.7 mM HClO4 (1:1 v/v). The supernatant was then separated on a Phenomenex Sinergy polar-RP column with a mobile phase consisting of water-acetonitrile-heptafluorobutyric acid (9:1:0.01 v/v/v) (phase A) and acetonitrile (phase B) at a flow rate of 0.2 ml min−1 and with a gradient elution. Detection was performed on an ion trap mass spectrometer equipped with an ESI interface operating in positive ionization mode. The acquisitions were performed in MRM mode using the following precursor > product ion combinations: Tyr-His (IS): m/z 319->301; CAR: m/z 227->210; ANS m/z 241->170,197,224; HCAR: m/z 241->156; carnosine-HNE Michael adduct (CHMA) m/z 383->366; anserine-HNE Michael adduct (AHMA) m/z 397->197, 241, 326, 353, 380. For HD analytes, the method was validated over the concentration range 15-1,000 nmoles g-1 (wet tissue) and the LOQ and LOD were 12.5 and 4.2 pmoles injected, respectively. The intra- and inter-day precisions (CV%) were < 10% (< 17.47% at the LOQ); intra- and inter-assay accuracy (RE%) were within ± 10% for all the concentrations. For HD-HNE analytes, the method was validated over the concentration range 10-100 nmoles g-1 and the LOQ and LOD were 8.1 and 3.0 pmoles injected, respectively. The mapping profile in rat tissue gave the following results: the highest concentrations of CAR and ANS were found in skeletal muscles (soleus, gastrocnemius, tibialis), ranging from 2.606 to 5.280 and 5.415-7.861 μmoles g-1 wet tissue respectively, followed by heart (67.03 and 64.50 nmoles g-1), cerebellum (48.41 and 57.48 nmoles g-1) and brain (27.35 nmoles g-1 CAR; ANS under LOQ). HCAR was found only in brain (51.14 nmoles g-1) and cerebellum (77.64 nmoles g-1). HD-HNE were under the LOD in all the tissue samples analysed.
The present method will be used to profile HD and HD-HNE levels in several physiological (aging, endurance exercise) and pathological (inflammation, ischemia-reperfusion damage) conditions in order to reach the following goals: 1) to gain a deeper insight into the biological role of HD as detoxyfing agents of cytotoxic unsaturated aldehydes and to correlate the HD content to the carbonylation damage; 2) to find out whether HD-HNE can be used as specific, reliable and unequivocal markers of oxidative stress
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