Effect of changes in the deuterium content of drinking water on the hydrogen isotope ratio of urinary steroids in the context of sports drug testing

The hydrogen isotope ratio (HIR) of body water and, therefore, of all endogenously synthesized compounds in humans, is mainly affected by the HIR of ingested drinking water. As a consequence, the entire organism and all of its synthesized substrates will reflect alterations in the isotope ratio of drinking water, which depends on the duration of exposure. To investigate the effect of this change on endogenous urinary steroids relevant to doping-control analysis the hydrogen isotope composition of potable water was suddenly enriched from -50 to 200 ‰ and maintained at this level for two weeks for two individuals. The steroids under investigation were 5β-pregnane-3α,20α-diol, 5α-androst-16-en-3α-ol, 3α-hydroxy-5α-androstan-17-one (ANDRO), 3α-hydroxy-5β-androstan-17-one (ETIO), 5α-androstane-3α,17β-diol, and 5β-androstane-3α,17β-diol (excreted as glucuronides) and ETIO, ANDRO and 3β-hydroxyandrost-5-en-17-one (excreted as sulfates). The HIR of body water was estimated by determination of the HIR of total native urine, to trace the induced changes. The hydrogen in steroids is partly derived from the total amount of body water and cholesterol-enrichment could be calculated by use of these data. Although the sum of changes in the isotopic composition of body water was 150 ‰, shifts of approximately 30 ‰ were observed for urinary steroids. Parallel enrichment in their HIR was observed for most of the steroids, and none of the differences between the HIR of individual steroids was elevated beyond recently established thresholds. This finding is important to sports drug testing because it supports the intended use of this novel and complementary methodology even in cases where athletes have drunk water of different HIR, a plausible and, presumably, inevitable scenario while traveling.     

The optimal evolution of the free energy of interacting gases and its applications

We establish an inequality for the relative total – internal, potential and interactive – energy of two arbitrary probability densities, their Wasserstein distance, their barycenters and their generalized relative Fisher information. This inequality leads to many known and powerful geometric inequalities, as well as to a remarkable correspondence between ground state solutions of certain quasilinear (or semi-linear) equations and stationary solutions of (non-linear) Fokker–Planck type equations. It also yields the HWBI inequalities – which extend the HWI inequalities in Otto and Villani [J. Funct. Anal. 173 (2) (2000) 361–400], and in Carrillo et al. [Rev. Math. Iberoamericana (2003)], with the additional ‘B’ referring to the new barycentric term – from which most known Gaussian inequalities can be derived. To cite this article: M. Agueh et al., C. R. Acad. Sci. Paris, Ser. I 337 (2003).     

Recombinant immobilized rhizopuspepsin as a new tool for protein digestion in hydrogen/deuterium exchange mass spectrometry

Hydrogen/deuterium (H/D) exchange coupled to mass spectrometry is nowadays routinely used to probe protein interactions or conformational changes. The method has many advantages, e.g. very low sample consumption, but offers limited spatial resolution. One way to higher resolution leads through the use of different proteases or their combinations. In the present work we describe recombinant production, purification and use of aspartic protease zymogen from Rhizopus chimensis, protease type XVIII (EC 3.4.23.6), commonly referred to as rhizopuspepsinogen (Rpg). The enzyme was expressed in Escherichia coli, refolded and purified to homogeneity. A typical yield was approximately 100 mg of pure enzyme per 1 L of original bacterial culture. The kinetics of protease activation, i.e. removal of the propeptide achieved by autolysis in an acidic environment, was followed by mass spectrometry. The digestion efficiency was tested for the protease in solution as well as for the immobilized enzyme. Apomyoglobin was successfully digested under all conditions tested and the protease displayed very low or no autodigestion. The results outperformed those obtained with commercial protease where the digestion of apomyoglobin was incomplete and accompanied by many contaminating peptides. Taken together, the recombinant protease type XVIII can be considered as a new and highly efficient tool for H/D exchange followed by mass spectrometry. Copyright © 2009 John Wiley & Sons, Ltd.     

Excited States of Conjugated Hydrocarbons Using the Molecular Mechanics - Valence Bond (MMVB) Method: Conical Intersections and Dynamics

We developed the molecular mechanics—valence bond (MMVB) method following an original suggestion of Jean-Paul Malrieu and coworkers. By coupling a parameterized Heisenberg Hamiltonian to a standard classical force field (MM2), reliable ground and excited state geometries of conjugated hydrocarbons can be rapidly optimized. The MMVB method was central to our development of algorithms for locating conical intersections and calculating their associated decay dynamics. Here, we briefly review the chemical applications of MMVB to date, and present two new studies using the photostability of pyracylene and the excited state decay dynamics of the photochromic dihydroazulene/vinylheptafulvene (DHA/VHF) reaction.     

Receptor-Based Screening Assays: New Perspectives in Anti-Doping Control

The so-called “growth promoters”, steroid hormones and β-agonists, are currently controlled by using hyphenated analytical methods (chromatography coupled to mass spectrometry) or, sometimes for screening purposes, on immunoassays. These methods are often too specific to allow an effective multianalyte control. To develop more efficient assays, the use of hormonal receptors as detection tools (receptor-based binding assays and cell-based assays) is proposed. Receptor-based assays represent useful tools in screening of hormonal residues in food, but they could also be applied in doping control (to detect “new” hormonal substances). Furthermore, these assays could be used to monitor the human exposure to endocrine disruptors.     

Probing protein interactions with hydrogen/deuterium exchange and mass spectrometry—A review

Assessing the functional outcome of protein interactions in structural terms is a goal of structural biology, however most techniques have a limited capacity for making structure–function determinations with both high resolution and high throughput. Mass spectrometry can be applied as a reader of protein chemistries in order to fill this void, and enable methodologies whereby protein structure–function determinations may be made on a proteome-wide level. Protein hydrogen/deuterium exchange (H/DX) offers a chemical labeling strategy suitable for tracking changes in “dynamic topography” and thus represents a powerful means of monitoring protein structure–function relationships. This review presents the exchange method in the context of interaction analysis. Applications involving interface detection, quantitation of binding, and conformational responses to ligation are discussed, and commentary on recent analytical developments is provided.     

α-Mannosidases from the Digestive Fluid of <Emphasis Type="Italic">Rhynchophorus Palmarum</Emphasis> Larvae as Novel Biocatalysts for Transmannosylation Reactions

Two biological fluids, namely hemolymph and digestive fluid from the larval stage of Rhynchophorus palmarum Linneaus, a serious pest in agroecosystem exploiting oil palm, were screened for hydrolytic activities, by the use of synthetic and natural glycoside substrates. Several exo and endoglycosidase activities were observed but, the interesting α-mannosidase activity (0.41 ± 0.04 UI) had attracted our attention. So, we have previously demonstrated that this activity harbours four distinctive α-mannosidase isoforms named RpltM, RplM1, RplM2 and RplM3. We have extended this work to determine the ability of these enzymes to catalyze synthesis reactions. Finally, we have revealed that, α-mannosidases from the digestive fluid of R. palmarum larvae catalyze transmannosylation reactions. The stability of the enzymes and the optimization of the transfer product yield were studied as functions of pH, enzyme unit, starting concentration of donor or acceptor and time. It was shown that, in experimental optimum conditions, average yields of 12.34 ± 0.75, 12.15 ± 0.79, 5.59 ± 0.35 and 8.43 ± 0.50% were obtained for the α-mannosidases RpltM, RplM1, RplM2 and RplM3, respectively. On the basis of this work, α-mannosidases from the digestive fluid of Rhynchophorus palmarum larvae appear to be a valuable tool for the preparation of neoglycoconjugates.     

Photochemical reactivity of 2-vinylbiphenyl and 2-vinyl-1,3-terphenyl: the balance between nonadiabatic and adiabatic photocyclization

A mechanism for the photochemical conversion of 2-vinyl-1,3-terphenyl to 8,9a-dihydrophenanthrene (Lewis, F. D.; Zuo, X.; Gevorgyan, V.; Rubin, M. J. Am. Chem. Soc. 2002, 124, 13664-13665) is presented in this study, based on ab initio restricted active space self-consistent field calculations and a molecular mechanics-valence bond dynamics simulation of a model system: the syn isomer of 2-vinylbiphenyl. An extended crossing seam between the ground and first excited electronic states was found to be largely responsible for the efficient photocyclization of the photochemically active syn isomer. This mechanism is nonadiabatic in nature, with an excited-state reaction pathway approaching the crossing region during the initial stage of cyclization. Dynamics simulation shows that this seam is easily accessible by vibrational motion in the branching space, once a small barrier is passed on the S1 excited-state potential energy surface. Ultrafast radiationless decay to the ground state then follows, and the cyclization is completed on this surface. A second possible mechanism was identified, which involves complete adiabatic cyclization on the S1 surface, with decay to the ground state (at a different conical intersection) only taking place once the product is formed. Thus, there is a competition between these two mechanisms-nonadiabatic and adiabatic-governed by the dynamics of the system. A large quantum yield is predicted for the photocyclization of the syn isomer of 2-vinylbiphenyl and 2-vinyl-1,3-terphenyl, in agreement with experimental observations.