Simultaneous analysis of tramadol, O-desmethyltramadol, and N-desmethyltramadol enantiomers in rat plasma by high-performance liquid chromatography-tandem mass spectrometry: application to pharmacokinetics

Tramadol (T) is available as a racemic mixture of (+)‐trans‐T and (−)‐trans‐T. The main metabolic pathways are O‐demethylation and N‐demethylation, producing trans‐O‐desmethyltramadol ( M1 ) and trans‐N‐desmethyltramadol ( M2 ) enantiomers, respectively. The analgesic effect of T is related to the opioid activity of (+)‐trans‐T and (+)‐ M1 and to the monoaminergic action of (+/−)‐trans‐T. This is the first study using tandem mass spectrometry as a detection system for the simultaneous analysis of trans‐T, M1 , and M2 enantiomers. The analytes were resolved on a Chiralpak® AD column using hexane:ethanol (95.5:4.5, v/v) plus 0.1% diethylamine as the mobile phase. The quantitation limits were 0.5 ng/ml for trans‐T and M1 and 0.1 ng/ml for M2 . The method developed and validated here was applied to a pharmacokinetic study in rats. Male Wistar rats (n = 6 at each time point) received a single oral dose of 20 mg/kg racemic trans‐T. Blood samples were collected up to 12 h after drug administration. The kinetic disposition of trans‐T and M2 was enantioselective (AUC_(+)/(−) ratio = 4.16 and 6.36, respectively). The direction and extent of enantioselectivity in the pharmacokinetics of trans‐T and M2 in rats were comparable to data previously reported for healthy volunteers, suggesting that rats are a suitable model for enantioselective studies of trans‐T pharmacokinetics. Chirality, 2011. © 2010 Wiley‐Liss, Inc.     

Protein expression changes in skeletal muscle in response to growth promoter abuse in beef cattle

The fraudulent treatment of cattle with growth promoting agents (GPAs) is a matter of great concern for the European Union (EU) authorities and consumers. It has been estimated that 10% of animals are being illegally treated in the EU. In contrast, only a much lower percentage of animals (<0.5%) are actually found as being noncompliant by conventional analytical methods. Thus, it has been proposed that methods should be developed that can detect the use of the substances via the biological effects of these substances on target organs, such as the alteration of protein expression profiles. Here we present a study aimed at evaluating if a correlation exists between the treatment with GPAs and alterations in the two-dimensional electrophoresis (2DE) protein pattern obtained from the biceps brachii skeletal muscle from mixed-bred cattle. After image analysis and statistical evaluation, protein spots that differentiate between treated and control groups were selected for analysis by mass spectrometry. A set of proteins could be defined that accurately detect the use of glucocorticoids and β(2)-agonists as growth promoters through the changes caused in muscle differentiation. As a further validation, we repeated the analysis using an independent set of samples from a strain of pure-bred cattle and verified these proteins by Western blot analysis.     

Seasonal variation of Ralstonia solanacearum biovar 2 populations in a Spanish river: recovery of stressed cells at low temperatures

The presence of Ralstonia solanacearum biovar 2 in the watercourses of European countries is increasing, but little is known about its ecology in aquatic habitats. The detection of this pathogen in 2000 in one Spanish river led us to study its population density at different locations on the river over a period of 3 years. During 2000 and 2001, the pathogen was recovered at low densities (10 to 80 CFU/ml) by direct plating on modified SMSA agar from water samples at 14 degrees C or higher, but its isolation was usually unsuccessful at temperatures below 9 degrees C. To monitor the pathogen's abundance in winter, we used two liquid selective media for enrichment (at 29 and 35 degrees C) and compared them by using spiked river water samples: modified Wilbrink broth (MWB) was more efficient than modified SMSA broth for double-antibody-sandwich indirect enzyme-linked immunosorbent assay (DASI-ELISA) detection of R. solanacearum. Enrichment in MWB at both temperatures allowed us to recover R. solanacearum cells that were nonculturable on solid media up to 25 days after their entry into the viable but nonculturable state. When we applied this technique to water samples during the cold months of 2001 and 2002, we obtained the best detection results by the most-probable-number method after enrichment at 35 degrees C with MWB. The enrichment protocol was combined with DASI-ELISA and validated by Co-PCR to detect both naturally and artificially starved and cold-stressed cells in water, which were still infective. Overall, the data from this study demonstrate the effects of temperature variation on the population and culturability of R. solanacearum cells on solid media and their survival at low temperatures.     

Attenuation of lipid peroxidation and hyperlipidemia by quercetin glucoside in the aorta of high cholesterol-fed rabbit

Antioxidative activity of dietary flavonoids is suggested to be, at least partly, responsible for a wide variety of their biological effects relating to anti-atherosclerosis. However, it is not known whether dietary flavonoids reach to the target site and act as antioxidants. In this study, we tried to evaluate the antioxidative effect of quercetin 3-O-beta-D-glucoside (Q3G), a typical flavonoid present in vegetables, in rabbit aorta. New Zealand White rabbits were fed a control diet (control group), 2.0% cholesterol diet (HC group) and 2.0% cholesterol plus 0.1% Q3G (HC + Q3G group) for one month. The amounts of total cholesterol, triacylglycerol and total fatty acids in both the plasma and aorta were significantly lower in the HC + Q3G group as compared with the HC group. Quercetin was detected in the aorta of the HC + Q3G group after enzymatic deconjugation, indicating that quercetin accumulated as conjugated metabolites in the aorta. The contents of TBA-reacting substances (TBARS) and cholesteryl ester hydroperoxides (CEOOH) in the aorta of the HC + Q3G group were significantly lower than those in the HC group. The aorta of HC + Q3G group was more resistant than that of HC group in copper ion-induced lipid peroxidation ex vivo. HC + Q3G group accumulated a higher amount of vitamin E per total cholesterol than HC group in the aorta. These results strongly suggest that quercetin glucosides accumulate in the aorta as their metabolites and attenuate lipid peroxidation occurring in the aorta, along with the attenuation of hyperlipidemia.     

Exploring the genome of Trypanosoma vivax through GSS and in silico comparative analysis

A survey of the Trypanosoma vivax genome was carried out by the genome sequence survey (GSS) approach resulting in 1,086 genomic sequences. A total of 455 high-quality GSS sequences were generated, consisting of 331 non-redundant sequences distributed in 264 singlets and 67 clusters in a total of 135.5 Kb of the T. vivax genome. The estimation of the overall G+C content, and the prediction of the presence of ORFs and putative genes were carried out using the Glimmer and Jemboss packages. Analysis of the obtained sequences was carried out by BLAST programs against 12 different databases and also using the Conserved Domain Database, InterProScan, and tRNAscan-SE. Along with the existing 23 T. vivax entries in the GenBank, the 32 putative genes predicted and the 331 non-redundant GSS sequences reported herein represent new potential markers for the development of PCRbased assays for specific diagnosis and typing of Trypanosoma vivax.