Anne Regazzetti

Université René Descartes - Paris 5, Lutetia Parisorum, Île-de-France, France

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Publications (10)32.45 Total impact

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    ABSTRACT: The adsorption of Rb+, Cs+, Mn2+, Co2+ and Yb3+ onto the positively charged hen egg-white lysozyme (HEWL) has been investigated by solving 13 X-ray structures of HEWL crystallized with their chlorides and by applying electrospray ionization mass spectrometry (ESI-MS) first to dissolved protein crystals and then to the protein in buffered salt solutions. The number of bound cations follows the order Cs+ < Mn2+ [asymptotically equal to] Co2+ < Yb3+ at 293 K. HEWL binds less Rb+ (qtot = 0.7) than Cs+ (qtot = 3.9) at 100 K. Crystal flash-cooling drastically increases the binding of Cs+, but poorly affects that of Yb3+, suggesting different interactions. The addition of glycerol increases the number of bound Yb3+ cations, but only slightly increases that of Rb+. HEWL titrations with the same chlorides, followed by ESI-MS analysis, show that only about 10% of HEWL binds Cs+ and about 40% binds 1-2 Yb3+ cations, while the highest binding reaches 60-70% for protein binding 1-3 Mn2+ or Co2+ cations. The binding sites identified by X-ray crystallography show that the monovalent Rb+ and Cs+ preferentially bind to carbonyl groups, whereas the multivalent Mn2+, Co2+ and Yb3+ interact with carboxylic groups. This work elucidates the basis of the effect of the Hofmeister cation series on protein solubility.
    Acta Crystallographica Section D Biological Crystallography 08/2014; D70:2217-2231. · 12.67 Impact Factor
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    ABSTRACT: Cholesterol and oxysterols are involved as key compounds in the development of severe neurodegenerative diseases and in neuroinflammation processes. Monitoring their concentration changes under pathological conditions is of interest to get insights into the role of lipids in diseases. For numerous years, liquid chromatography coupled to mass spectrometry has been the method of choice for metabolites identification and quantification in biological samples. However, sterols and oxysterols are relatively apolar molecules poorly adapted to electrospray ionization (ESI). To circumvent this drawback, we developed a novel and robust analytical method involving derivatization of these analytes in cholesteryl N-4-(N,N-dimethylamino)phenyl carbamates under alkaline conditions followed by ultra-performance liquid chromatography-high resolution mass spectrometry analysis (UPLC-HRMS). Optimized UPLC conditions led to the separation of a mixture of 11 derivatized sterols and oxysterols especially side chain substituted derivatives after 6 min of chromatographic run. High sensitivity time-of-flight mass analysis allowed analytes to be detected in the nanomolar range. The method was validated for the analysis of oxysterols and sterols in mice brain in respect of linearity, limits of quantification, accuracy, precision, analyte stability, and recovery according to the Food and Drug Administration (FDA) guidelines. The developed method was successfully applied to investigate the impact of lipopolysaccharide (LPS) treatment on the rat cerebral steroidome.
    Analytical and Bioanalytical Chemistry 09/2012; · 3.66 Impact Factor
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    ABSTRACT: Cholesterol and oxysterols are involved as key compounds in the development of severe neurodegenerative diseases and in neuroinflammation processes. Monitoring their concentration changes under pathological conditions is of interest to get insights into the role of lipids in diseases. For numerous years, liquid chromatography coupled to mass spectrometry has been the method of choice for metab-olites identification and quantification in biological samples. However, sterols and oxysterols are relatively apolar mole-cules poorly adapted to electrospray ionization (ESI). To circumvent this drawback, we developed a novel and robust analytical method involving derivatization of these analytes in cholesteryl N-4-(N,N-dimethylamino)phenyl carbamates under alkaline conditions followed by ultra-performance liquid chromatography–high resolution mass spectrometry analysis (UPLC-HRMS). Optimized UPLC conditions led to the separation of a mixture of 11 derivatized sterols and oxysterols especially side chain substituted derivatives after 6 min of chromatographic run. High sensitivity time-of-flight mass analysis allowed analytes to be detected in the nanomolar range. The method was validated for the analysis of oxysterols and sterols in mice brain in respect of linearity, limits of quantification, accuracy, precision, analyte stabili-ty, and recovery according to the Food and Drug Adminis-tration (FDA) guidelines. The developed method was successfully applied to investigate the impact of lipopoly-saccharide (LPS) treatment on the rat cerebral steroidome. Keywords UPLC/ESI/HRMS . Cholesterol . Oxysterols . Derivatization . Quantification
    Analytical and Bioanalytical Chemistry 01/2012; 404(10):3049. · 3.66 Impact Factor
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    ABSTRACT: Although the natural flavonoid fisetin (3,3',4',7-tetrahydroxyflavone) has been recently identified as an anticancer agent with antiangiogenic properties in mice, its in vivo pharmacokinetics and metabolism are presently not characterized. Our purpose was to determine the pharmacokinetics and metabolism of fisetin in mice and determine the biological activity of a detected fisetin metabolite. After fisetin administration of an efficacious dose of 223 mg/kg i.p. in mice, the maximum fisetin concentration reached 2.5 μg/ml at 15 min and the plasma concentration declined biphasically with a rapid half-life of 0.09 h and a terminal half-life of 3.1h. Three metabolites were detected, one of which was a glucuronide of fisetin (M1), whereas another glucuronide (M2) was a glucuronide of a previously unknown fisetin metabolite (M3). HPLC-MS/MS analysis indicated that M3 was a methoxylated metabolite of fisetin (MW=300 Da). The UV spectrum of M3 was identical to that of fisetin and standard 3,4',7-trihydroxy-3'-methoxyflavone (geraldol). In addition, because M3 co-eluted with standard geraldol in 4 different chromatographic ternary gradient conditions, M3 was therefore assigned to geraldol. Of interest, this metabolite was shown to achieve higher concentrations than fisetin in Lewis lung tumors. We also compared the cytotoxic and antiangiogenic activities of fisetin and geraldol in vitro and it was found that the latter was more cytotoxic than the parent compound toward tumor cells, and that it could also inhibit endothelial cells migration and proliferation. In conclusion, these results suggest that fisetin metabolism plays an important role in its in vivo anticancer activities.
    Biochemical pharmacology 08/2011; 82(11):1731-9. · 4.25 Impact Factor
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    ABSTRACT: The metabolism of flavone-8-acetic acid (FAA) has been hypothesized to be partly responsible for its potent anticancer activity in mice. The purpose of this study was to identify the mouse enzymes involved in FAA Phase I metabolism and evaluate their possible induction in vivo by FAA. Mouse microsomes metabolized FAA into 6 metabolites: 3',4'-dihydrodiol-FAA, 5,6-epoxy-FAA, 4'-OH-FAA, 3'-OH-FAA, 3',4'-epoxy-FAA and 6-OH-FAA. Using Cyp-specific inhibitors (furafylline, Cyp1a2; α-naphthoflavone, Cyp1b1; tranylcypromine, Cyp2b9; quercetin, Cyp2c29; quinidine, 2d9; diethyldithiocarbamate, Cyp2e1; ketoconazole, Cyp3a11), the formation of 5,6-epoxy-FAA was mainly attributed to Cyps 1a2, 1b1, 2b9, 2c29 and 2e1, whereas the 3',4'-epoxy-FAA was formed by Cyps 2b9 and 3a11. The 4'-OH-FAA was generated by Cyps 1a2, 1b1, 2b9 and 2e1, and the 6-OH-FAA was formed by Cyps 1b1 and 2c9. Using the epoxide scavenger N-acetyl cysteine, 4'-OH-FAA, 3'-OH-FAA and 6-OH-FAA were shown to derive partly from non enzymatic isomerisation of their corresponding epoxides. The specific epoxide hydrolase inhibitor elaidamide allowed the confirmation that 3',4'-dihydrodiol-FAA was formed via the epoxide hydrolase. FAA treatment in vivo in mice led to a significant increase in the hepatic expression of Cyp1a2 (1.9-fold), 2e1 (2.1-fold), 2b10 (3.2-fold), 2d9 (2.3-fold) and 3a11 (2.2-fold), as evaluated by qRT-PCR. In conclusion, several Cyps were shown to be involved in FAA metabolism, particularly Cyps 3a11 and 2b9 which were responsible for the formation of the principal metabolites (5,6-epoxy-FAA, 3',4'-epoxy-FAA), and that FAA could induce the expression of several Cyps after in vivo administration. The possible implication of these enzymes in the in vivo anticancer activity of FAA in mice is discussed.
    Drug metabolism letters. 04/2011; 5(2):73-84.
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    ABSTRACT: The metabolism of flavone-8-acetic acid (FAA) has been hypothesized to be partly responsible for its potent anticancer activity in mice. The purpose of this study was to identify the mouse enzymes involved in FAA Phase I metabolism and evaluate their possible induction in vivo by FAA. Mouse microsomes metabolized FAA into 6 metabolites: 3',4'-dihydrodiol-FAA, 5,6-epoxy-FAA, 4'-OH-FAA, 3'-OH-FAA, 3',4'-epoxy-FAA and 6-OH-FAA. Using Cyp-specific inhibitors (furafylline, Cyp1a2; α-naphthoflavone, Cyp1b1; tranylcypromine, Cyp2b9; quercetin, Cyp2c29; quinidine, 2d9; diethyldithiocarbamate, Cyp2e1; ketoconazole, Cyp3a11), the formation of 5,6-epoxy-FAA was mainly attributed to Cyps 1a2, 1b1, 2b9, 2c29 and 2e1, whereas the 3',4'-epoxy-FAA was formed by Cyps 2b9 and 3a11. The 4'-OH-FAA was generated by Cyps 1a2, 1b1, 2b9 and 2e1, and the 6-OH-FAA was formed by Cyps 1b1 and 2c9. Using the epoxide scavenger N-acetyl cysteine, 4'-OH-FAA, 3'-OH-FAA and 6-OH-FAA were shown to derive partly from non enzymatic isomerisation of their corresponding epoxides. The specific epoxide hydrolase inhibitor elaidamide allowed the confirmation that 3',4'-dihydrodiol-FAA was formed via the epoxide hydrolase. FAA treatment in vivo in mice led to a significant increase in the hepatic expression of Cyp1a2 (1.9-fold), 2e1 (2.1-fold), 2b10 (3.2-fold), 2d9 (2.3-fold) and 3a11 (2.2-fold), as evaluated by qRT-PCR. In conclusion, several Cyps were shown to be involved in FAA metabolism, particularly Cyps 3a11 and 2b9 which were responsible for the formation of the principal metabolites (5,6-epoxy-FAA, 3',4'-epoxy-FAA), and that FAA could induce the expression of several Cyps after in vivo administration. The possible implication of these enzymes in the in vivo anticancer activity of FAA in mice is discussed.
    Drug Metabolism Letters 03/2011; 5(2):73-84.
  • Fuel and Energy Abstracts 01/2011; 205.
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    ABSTRACT: Flavone-8-acetic acid (FAA) is a potent anticancer agent in mouse but has not shown activity in humans. Because FAA metabolism could play a role in this interspecies difference, our aim was to identify the metabolites formed in vitro using mouse microsomes compared with those in human microsomes. Mouse microsomes produced six metabolites as detected by reversed-phase high-performance liquid chromatography-mass spectrometry (MS). Three metabolites were identified as the 3'-, 4'-, or 6-hydroxy-FAA, by comparison with retention times and UV and MS spectra of standards. Two metabolites presented a molecular weight of 296 (FAA = 280) indicating the presence of one oxygen but did not correspond to any monohydroxylated FAA derivative. These two metabolites were identified as epoxides because they were sensitive to epoxide hydrolase. The position of the oxygen was determined by the formation of the corresponding phenols under soft acidic conditions: one epoxide yielded the 3'- and 4'-hydroxy-FAA, thus corresponding to the 3',4'-epoxy-FAA, whereas the other epoxide yielded 5- and 6-hydroxy-FAA, thus identifying the 5,6-epoxy-FAA. The last metabolite was assigned to the 3',4'-dihydrodiol-FAA because of its molecular weight (314) and sulfuric acid dehydration that indicated that the 3'- and 4'-positions were involved. Compared with mouse microsomes, human microsomes (2 pools and 15 individual microsomes) were unable to metabolize FAA to a significant extent. In conclusion, we have identified six new FAA metabolites formed by mouse microsomes, whereas human microsomes could not metabolize this flavonoid to a significant extent. The biological importance of the new metabolites identified herein remains to be evaluated.
    Drug Metabolism and Disposition 12/2007; 35(11):2023-34. · 3.36 Impact Factor
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    ABSTRACT: Cyclic peptides were obtained, on-resin, by the copper (I) catalysed 1,3-dipolar cycloaddition of azides and alkynes. The reaction led exclusively to the formation of the expected cyclomonomeric products which acted as ligands of the Vascular Endothelial Growth Factor receptor 1.
    Bioorganic & Medicinal Chemistry Letters 11/2007; 17(20):5590-4. · 2.34 Impact Factor
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    ABSTRACT: The experimental anticancer agent flavone-8-acetic acid (FAA) is metabolized into several monohydroxylated derivatives using mouse microsomes. Because these metabolites could be involved in the biological effects of FAA, the aim of this study was to characterize all its possible monohydroxylated derivatives. To do so, we have developed a methodology using reversed-phase high-performance liquid chromatography (RP-HPLC) coupled with ultraviolet (UV) detection and mass spectrometry (MS) to analyze and identify FAA derivatives hydroxylated at the 2', 3', 4', 3, 5, 6, or 7 position. In RP-HPLC, 4'-, 3'-, 2'-, 6-, and 7-OH-FAA eluted before FAA, whereas 3- and 5-OH-FAA eluted after FAA. UV spectra showed a bathochromic shift of band I for all derivatives and of band II for 5- and 6-OH-FAA. In addition, the position of the OH group could be determined by the presence of certain product ions in MS. Ions at m/z 133 and 151 were specific for 2'-, 3'-, 4'-, and 3-OH-FAA, whereas the ion at m/z 177 was specific for 3-OH-FAA only. The ions m/z 133, 151 and 167 were specific for 2'-OH-FAA. Ions at m/z 149 were specific for the presence of the OH group on cycle A only (i.e., 5-, 6- or 7-OH-FAA). The presence of both product ions m/z 149 and 179 were specific for 7-OH-FAA. Finally, ions at m/z 149 and several product ions of even m/z values were specific for 5-OH-FAA. In conclusion, the methodology described can be used to identify all possible monohydroxylated FAA derivatives.
    Rapid Communications in Mass Spectrometry 02/2007; 21(20):3373-86. · 2.51 Impact Factor

Publication Stats

36 Citations
32.45 Total Impact Points

Institutions

  • 2007–2014
    • Université René Descartes - Paris 5
      • • Laboratoire de Chimie et Toxicologie Analytique et Cellulaire (EA 4463)
      • • Faculté des Sciences Pharmaceutiques et Biologiques de Paris
      Lutetia Parisorum, Île-de-France, France
    • French Institute of Health and Medical Research
      Lutetia Parisorum, Île-de-France, France