26 research outputs found

    Urinary Analysis of Four Testosterone Metabolites and Pregnanediol by Gas Chromatography-Combustion-Isotope Ratio Mass Spectrometry after Oral Administrations of Testosterone

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    The most frequently used method to demonstrate testosterone abuse is the determination of the testosterone and epitestosterone concentration ratio (T/E ratio) in urine. Nevertheless, it is known that factors other than testosterone administration may increase the T/E ratio. In the last years, the determination of the carbon isotope ratio has proven to be the most promising method to help discriminate between naturally elevated T/E ratios and those reflecting T use. In this paper, an excretion study following oral administration of 40 mg testosterone undecanoate initially and 13 h later is presented. Four testosterone metabolites (androsterone, etiocholanolone, 5α-androstanediol, and 5β-androstanediol) together with an endogenous reference (5β-pregnanediol) were extracted from the urines and the δ13C/12C ratio of each compound was analyzed by gas chromatography-combustion-isotope ratio mass spectrometry. The results show similar maximum δ13C-value variations (parts per thousand difference of δ13C/12C ratio from the isotope ratio standard) for the T metabolites and concomitant changes of the T/E ratios after administration of the first and the second dose of T. Whereas the T/E ratios as well as the androsterone, etiocholanolone and 5α-androstanediol δ13C-values returned to the baseline 15 h after the second T administration, a decrease of the 5β-androstanediol δ-values could be detected for over 40 h. This suggests that measurements of 5β-androstanediol δ-values allow the detection of a testosterone ingestion over a longer post-administration period than other T metabolites δ13C-values or than the usual T/E ratio approac

    Detection of Exogenous GHB in Blood by Gas Chromatography-Combustion-Isotope Ratio Mass Spectrometry: Implications in Postmortem Toxicology

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    Because GHB (γ-hydroxybutyrate) is present in both blood and urine of the general population, toxicologists must be able to discriminate between endogenous levels and a concentration resulting from exposure. In this paper, we propose a procedure for the detection of exogenous GHB in blood by gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Following liquid-liquid and solid-phase extractions, GHB is derivatized to GHB di-TMS before analysis by GC-C-IRMS. Significant differences in the carbon isotopic ratio (Δδ13C-values > 13.5‰) were found between endogenous and synthetic GHB. Indeed, for postmortem blood samples with different GHB concentrations (range: 13.8-86.3 mg/L), we have obtained GHB δ13C-values ranging from −20.6 to −24.7‰, whereas δ13C-values for the GHB from police seizure were in the range −38.2 to −50.2‰. In contrast to the use of cut-off concentrations for positive postmortem blood GHB concentrations, this method should provide an unambiguous indication of the drug origi

    Source inference of exogenous gamma-hydroxybutyric acid (GHB) administered to humans by means of carbon isotopic ratio analysis: novel perspectives regarding forensic investigation and intelligence issues

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    γ-Hydroxybutyric acid (GHB) is an endogenous short-chain fatty acid popular as a recreational drug due to sedative and euphoric effects, but also often implicated in drug-facilitated sexual assaults owing to disinhibition and amnesic properties. Whilst discrimination between endogenous and exogenous GHB as required in intoxication cases may be achieved by the determination of the carbon isotope content, such information has not yet been exploited to answer source inference questions of forensic investigation and intelligence interests. However, potential isotopic fractionation effects occurring through the whole metabolism of GHB may be a major concern in this regard. Thus, urine specimens from six healthy male volunteers who ingested prescription GHB sodium salt, marketed as Xyrem®, were analysed by means of gas chromatography/combustion/isotope ratio mass spectrometry to assess this particular topic. A very narrow range of δ13C values, spreading from −24.81‰ to −25.06‰, was observed, whilst mean δ13C value of Xyrem® corresponded to −24.99‰. Since urine samples and prescription drug could not be distinguished by means of statistical analysis, carbon isotopic effects and subsequent influence on δ13C values through GHB metabolism as a whole could be ruled out. Thus, a link between GHB as a raw matrix and found in a biological fluid may be established, bringing relevant information regarding source inference evaluation. Therefore, this study supports a diversified scope of exploitation for stable isotopes characterized in biological matrices from investigations on intoxication cases to drug intelligence programme

    Directed molecular evolution of cytochrome c peroxidase

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    Cytochrome c peroxidase (CCP) from Saccharomyces cerevisiae was subjected to directed mol. evolution to generate mutants with increased activity against the classical peroxidase substrate, guaiacol, thus changing the substrate specificity of CCP from the protein, cytochrome c, to a small org. mol. After 3 rounds of DNA shuffling and screening, mutants were isolated which possessed a 300-fold increased activity against guaiacol and an up to 1000-fold increased specificity for this substrate relative to that for the natural substrate. In all of the selected mutants, the distal Arg-48 residue, which is fully conserved in the superfamily of peroxidases, was mutated to His, showing that this mutation plays a key role in the significant increase in activity against phenolic substrates. The results suggest that, in addn. to stabilizing the reactive intermediate compd. I, the distal Arg residue plays an important role as a gatekeeper in the active site of CCP, controlling the access to the ferryl O atom and the distal His residue. Other isolated mutations increased the general reactivity of CCP, or increased the intracellular concn. of the active holo form, allowing their selection under the employed screening conditions. The results illustrated the ability of directed mol. evolution technologies to deliver solns. to biochem. problems that would not be readily predicted by rational design. [on SciFinder (R)

    Directed Molecular Evolution of Cytochrome c

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    Ruthenium <i>Lewis</i> Acid-Catalyzed Asymmetric <i>Diels-Alder</i> Reactions: Reverse-Face Selectivity for <i>α</i>,<i>β</i>-Unsaturated Aldehydes and Ketones

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    Acrolein, methacrolein, methyl vinyl ketone, ethyl vinyl ketone, 3-methyl-3-en-2-one, and divinyl ketone were coordinated to a cationic cyclopentadienyl ruthenium(II) Lewis acid incorporating the electron-poor bidentate BIPHOP–F ligand. Analysis by NOESY and ROESY NMR techniques allowed the determination of conformations of enals and enones present in solution in CD2Cl2. The results were compared to solid-state structures and to the facial selectivities of catalytic asymmetric Diels–Alder reactions with cyclopentadiene. X-Ray structures of four Ru-enal and Ru-enone complexes show the α,β-unsaturated C=O compounds to adopt an anti-s-trans conformation. In solution, enals assume both anti-s-trans and anti-s-cis conformations. An additional conformation, syn-s-trans, is present in enone complexes. Enantioface selectivity in the cycloaddition reactions differs for enals and enones. Reaction products indicate enals to react exclusively in the anti-s-trans conformation, whereas with enones, the major product results from the syn-s-trans conformation. The alkene in s-cis conformations, while present in solution, is shielded and cannot undergo cycloaddition. A syn-s-trans conformation is found in the solid state of the bulky 6,6-dimethyl cyclohexanone-Ru(II) complex. The X-ray structure of divinyl ketone is unique in that the Ru(II) center binds the enone via a η2 bond to one of the alkene moieties. In solution, coordination to Ru–C=O oxygen is adopted. A comparison of facial preference is also made to the corresponding indenyl Lewis acids

    Denaturant-induced movement of the transition state of protein folding revealed by high-pressure stopped-flow measurements

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    The small all-β protein tendamistat folds and unfolds with two-state kinetics. We determined the volume changes associated with the folding process by performing kinetic and equilibrium measurements at variable pressure between 0.1 and 100 MPa (1 to 1,000 bar). GdmCl-induced equilibrium unfolding transitions reveal that the volume of the native state is increased by 41.4 ± 2.0 cm(3)/mol relative to the unfolded state. This value is virtually independent of denaturant concentration. The use of a high-pressure stopped-flow instrument enabled us to measure the activation volumes for the refolding (ΔV(f)(0‡)) and unfolding reaction (ΔV(u)(0‡)) over a broad range of GdmCl concentrations. The volume of the transition state is 60% native-like (ΔV(f)(0‡) = 25.0 ± 1.2 cm(3)/mol) in the absence of denaturant, indicating partial solvent accessibility of the core residues. The volume of the transition state increases linearly with denaturant concentration and exceeds the volume of the native state above 6 M GdmCl. This result argues for a largely desolvated transition state with packing deficiencies at high denaturant concentrations and shows that the structure of the transition state depends strongly on the experimental conditions
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