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Fernando Ramos - One of the best experts on this subject based on the ideXlab platform.
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development optimization and application of an analytical methodology by ultra performance liquid chromatography tandem mass spectrometry for determination of Amanitins in urine and liver samples
Analytica Chimica Acta, 2013Co-Authors: Marta Leite, Andreia Freitas, Anabela Marisa Azul, Jorge Barbosa, Saul C Costa, Fernando RamosAbstract:Abstract Amanitins, highly toxic cyclopeptides isolated from various Amanita species, are the most potent poisons accounting for the hazardous effects on intestinal epithelium cells and hepatocytes, and probably the sole cause of fatal human poisoning. The present study was focused on the development, optimization and application of an analytical methodology by ultra performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS), following urine and liver sample preparation by protein precipitation with organic solvents, and solid phase extraction (SPE) procedure, for the determination of the amatoxins, α- and β-Amanitin. Linearity, detection and quantification limits, selectivity, sensitivity, intra and inter-assay precision and recovery were studied, in order to guarantee reliability in the analytical results. The developed method proved to be specific and selective, with LOD (Limit of Detection) values for α- and β-Amanitin of 0.22 and 0.20 ng mL −1 in urine and 10.9 and 9.7 ng g −1 in liver, respectively. LOQ (Limit of Quantification) values ranged from 0.46 to 0.57 ng mL −1 in urine and 12.3–14.7 ng g −1 in tissue, for both Amanitins. Linearity, in the range of 10.0–200.0 ng mL −1 or ng g −1 , shows that coefficients of correlation were greater than 0.997 for α-Amanitin and 0.993 for β-Amanitin. Precision was checked at three levels during three consecutive days with intra-day and inter-day coefficients of variation not greater than 15.2%. The extraction recovery presents good results for the concentrations analyzed, with values ranging from 90.2 to 112.9% for both matrices. Thus, the proposed analytical method is innovative, presents a high potential in the identification, detection and determination of α- and β-Amanitins in urine and tissue samples, as well as in other biological samples, such as kidney and mushrooms.
Akira Ishii - One of the best experts on this subject based on the ideXlab platform.
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simple and sensitive determination of α and β Amanitin by liquid chromatography quadrupole time of flight mass spectrometry
Forensic Toxicology, 2014Co-Authors: Akira Ishii, Tadashi Ogawa, Miho Tada, Maiko Kusano, Hideki Hattori, Hiroshi Seno, Kei ZaitsuAbstract:Amatoxins, including aand b-Amanitins, are highly toxic cyclic octapeptide compounds belonging to the Amanita species; these toxins exert gastroenteric symptoms in the early stage, and lead to hepatic failure and renal damage by inhibiting RNA polymerase activity in hepatocytes [1–3]. The Amanita species are considered to be responsible for more than 90 % of the lethal cases of mushroom toxin poisoning [4, 5]. It is thus important to detect amatoxins with high sensitivity. Recently, liquid chromatography (LC)–mass spectrometry (MS) [6–13] and tandem mass spectrometry (MS/MS) [14–18] or matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI–TOFMS) [19] have been increasingly applied to detect and identify these compounds from body fluids or mushroom specimens. In this Letter, we present a new method for detecting and identifying aand b-Amanitin using a novel LC–quadrupole time-of-flight mass spectrometer (Q-TOFMS) with high sensitivity and specificity. Amanitins were purchased from Sigma (St. Louis, MO, USA); other chemicals used were of the highest purity commercially available. Urine samples were collected from a healthy volunteer after obtaining informed consent. Analyses by Q-TOFMS were performed on an AB SCIEX Triple TOF 5600 system (AB SCIEX, Framingham, MA, USA) coupled to a Shimadzu Prominence XR LC system (Shimadzu, Kyoto, Japan). For LC separation, a Scherzo SM-C18 column (2 9 100 mm, particle size 3 lm, Imtakt, Kyoto, Japan) was used; the column temperature was kept at 40 C and the flow rate was 0.5 ml/ min. The gradient program was started at 90 % mobile phase A (5 mM ammonium formate in distilled water) and 10 % mobile phase B (methanol) for 1 min; it was changed linearly to 5 % A and 95 % B over 4 min and maintained with a 2-min hold. The MS conditions were as follows: ionization mode, electrospray ionization (ESI) positive mode; turbo gas temperature, 500 C; spray voltage, 5,000 V. In the MS mode, ions were scanned in the range from m/z 850 to 1,000; the declustering potential (DP) and collision energy (CE) were 80 V and 10 V, respectively. In the MS/MS mode, four conditions for production ion monitoring were settled as follows: precursor ions were 919.4 for a-Amanitin and 920.4 for b-Amanitin, and product ions were scanned in the range of m/z 200–1,000 (DP, 80 V; CE, 50 V); and precursor ions were 919.4 for a-Amanitin and 920.4 for b-Amanitin, and product ions were scanned in the range of m/z 900–1,600 (DP, 80 V; CE, 10 V). Measurements in MS and MS/MS modes were employed alternatively, for 100 ms in each condition; the total cycle time was 550 ms. Authentic Amanitins were dissolved in methanol or added to purified urine samples to assess the sensitivity of the instrument and evaluate matrix effects. In the samples diluted in methanol, the concentrations of both Amanitins were in the range of 0.001–5 ng/ll. In urine samples, 200 ll of urine was mixed with 100 ll of acetonitrile and centrifuged at 5,000 rpm for 10 min. The supernatant was mixed with 700 ll of distilled water and A. Ishii (&) M. Kusano K. Zaitsu Department of Legal Medicine and Bioethics, Nagoya University Graduate School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan e-mail: akishii@med.nagoya-u.ac.jp
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Simple analysis of α-Amanitin and β-Amanitin in human plasma by liquid chromatography-mass spectrometry
Forensic Toxicology, 2010Co-Authors: Tanahashi Masakazu, Rina Kaneko, Yukari Hirata, Makoto Hamajima, Tetsuya Arinobu, Tadashi Ogawa, Akira IshiiAbstract:A number of reports are available in the literature that describe liquid chromatography-mass spectrometry (LC-MS) and LC-tandem mass spectrometry (LC-MS-MS) analysis of Amanitins, very toxic mushroom toxins, in biological samples. However, the extractive pretreatment methods and LC separation column materials vary remarkably according to the different reports. This communication presents a very simple and suffi ciently sensitive method for LC-MS analysis of Amanitins. A plasma sample was diluted with distilled water and buffer solution, and applied to a Discovery DSC 18 column (500 mg packing material), followed by washing with distilled water and elution with methanol. The extract, after evaporation and reconstitution in mobile phase solution, was subjected to LC-MS analysis with a conventional octadecyl LC separation column. The selected ion monitoring of α-Amanitin and β-Amanitin at m/z 919–921 and m/z 920–922, respectively, gave symmetrical peaks and good separation of both Amanitin peaks. Using an external calibration method, linearity, detection limits, recovery rates, and precision were tested; they were all satisfactory. To our knowledge, the present method gives the simplest LC-MS analysis for Amanitins among those so far reported. We recommend the method for use in actual forensic and clinical toxicological analysis of Amanitins in biological samples.
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SHORT COMMUNICATION for Forensic Toxicology Simple analysis of α−Amanitin and β−Amanitin in human plasma by LC−MS
2010Co-Authors: Tanahashi Masakazu, Rina Kaneko, Yukari Hirata, Makoto Hamajima, Tadashi Ogawa, Akira Ishii, M. Tanahashi, Yoshihiro HirataAbstract:Although some reports dealing with liquid chromatography (LC)– mass spectrometry (MS) (−MS) analysis of Amanitins, very toxic mushroom toxins, in biological samples are available, the extractive pretreatment methods and also LC separation column materials remarkably vary according to the different reports. In this communication, we present a very simple and sufficiently sensitive method for LC−MS analysis of Amanitins. A plasma sample was diluted with distilled water and buffer solution, and applied to a Discovery DSC 18 column (500 mg packing material), followed by washing with distilled water and elution with methanol. The extract, after evaporation and reconstitution in mobile phase solution, was subjected to LC−MS analysis with the conventional octadecyl LC separation column. The selected ion monitoring of α−Amanitin and β−Amanitin at m/z 919−921 and m/z 920−922, respectively, gave symmetrical peaks and good separation of both Amanitin peaks. Using the external calibration method, linearity, detection limits, recovery rates and precision were tested; they were all satisfactory. To our knowledge, the present method seems to be the simpliest LC−MS analysis for Amanitins among those so far reported. We can recommend it to be widely used in actual forensic and clinical toxicological analysis of Amanitins in biological samples.
Félix Carvalho - One of the best experts on this subject based on the ideXlab platform.
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In vitro mechanistic studies on α-Amanitin and its putative antidotes
Archives of Toxicology, 2020Co-Authors: Daniela Ferreira Rodrigues, Ricardo Pires Das Neves, Alexandra T. P. Carvalho, Maria Bastos, Vera M. Costa, Félix CarvalhoAbstract:α-Amanitin plays a key role in Amanita phalloides intoxications. The liver is a major target of α-Amanitin toxicity, and while RNA polymerase II (RNA Pol II) transcription inhibition is a well-acknowledged mechanism of α-Amanitin toxicity, other possible toxicological pathways remain to be elucidated. This study aimed to assess the mechanisms of α-Amanitin hepatotoxicity in HepG2 cells. The putative protective effects of postulated antidotes were also tested in this cell model and in permeabilized HeLa cells. α-Amanitin (0.1–20 µM) displayed time- and concentration-dependent cytotoxicity, when evaluated through the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) reduction and neutral red uptake assays. Additionally, α-Amanitin decreased nascent RNA synthesis in a concentration- and time-dependent manner. While α-Amanitin did not induce changes in mitochondrial membrane potential, it caused a significant increase in intracellular ATP levels, which was not prevented by incubation with oligomycin, an ATP synthetase inhibitor. Concerning the cell redox status, α-Amanitin did not increase reactive species production, but caused a significant increase in total and reduced glutathione, which was abolished by pre-incubation with the inhibitor of gamma-glutamylcysteine synthase, buthionine sulfoximine. None of the tested antidotes [ N -acetyl cysteine, silibinin, benzylpenicillin, and polymyxin B (PolB)] conferred any protection against α-Amanitin-induced cytotoxicity in HepG2 cells or reversed the inhibition of nascent RNA caused by the toxin in permeabilized HeLa cells. Still, PolB interfered with RNA Pol II activity at high concentrations, though not impacting on α-Amanitin observed cytotoxicity. New hepatotoxic mechanisms of α-Amanitin were described herein, but the lack of protection observed in clinically used antidotes may reflect the lack of knowledge on their true protection mechanisms and may explain their relatively low clinical efficacy.
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An effective antidotal combination of polymyxin B and methylprednisolone for α-Amanitin intoxication
Archives of Toxicology, 2019Co-Authors: Juliana Garcia, Vera Marisa Costa, Antonio Bovolini, José Alberto Duarte, Daniela Ferreira Rodrigues, Maria Lourdes Bastos, Félix CarvalhoAbstract:Amanita phalloides is one of the most toxic mushrooms worldwide, and it is involved in the majority of human fatal cases of mushroom poisoning. α-Amanitin, the most deleterious toxin of A. phalloides to humans, inhibits RNA polymerase II (RNAPII), causing hepatic and renal failure. Previously, we have shown that polymyxin B (polB) reverts α-Amanitin inhibition of RNAPII, although it was not able to guarantee the full survival of α-Amanitin-intoxicated mice or prevent α-Amanitin pro-inflammatory effects. α-Amanitin is also a substrate of the organic-anion-transporting polypeptide 1B3 (OATP1B3) and Na(+)-taurocholate cotransporter polypeptide (NTCP) transporters. Therefore, in the present work, we used a combination of polB [(2.5 mg/kg intraperitoneal (i.p.)] with the anti-inflammatory and NTCP inhibitor drug, methylprednisolone (MP) (10 mg/kg i.p.), as an attempt to fully revert α-Amanitin-induced toxicity (0.33 mg/kg i.p.) in CD-1 mice. Results showed that the administration of the polB + MP combination, 4 h after α-Amanitin, led to the full survival of the intoxicated animals, with a significant attenuation of α-Amanitin-induced renal and hepatic necrosis. Also, the combination polB + MP led to a decrease of aminotransferase plasma levels, of the renal myeloperoxidase activity and of renal inflammatory cell infiltrate promoted by α-Amanitin, although not preventing any of the hepatic pro-inflammatory effect of the toxin. The obtained results indicate that this combination may represent an important and valuable therapeutic approach to be used against α-Amanitin intoxication.
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Quantification of alpha-Amanitin in biological samples by HPLC using simultaneous UV- diode array and electrochemical detection.
Journal of chromatography. B Analytical technologies in the biomedical and life sciences, 2015Co-Authors: Juliana Garcia, Vera Marisa Costa, Maria Lourdes Bastos, Paula Baptista, Félix CarvalhoAbstract:Abstract α-Amanitin is a natural bicyclic octapeptide, from the family of amatoxins, present in the deadly mushroom species Amanita phalloides . The toxicological and clinical interests raised by this toxin, require highly sensitive, accurate and reproducible quantification methods for pharmacokinetic studies. In the present work, a high-performance liquid chromatographic (HPLC) method with in-line connected diode-array (DAD) and electrochemical (EC) detection was developed and validated to quantify α-Amanitin in biological samples (namely liver and kidney). Sample pre-treatment consisted of a simple and unique deproteinization step with 5% perchloric acid followed by centrifugation at 16,000 × g , 4 °C, for 20 min. The high recovery found for α-Amanitin (≥96.8%) makes this procedure suitable for extracting α-Amanitin from liver and kidney homogenates. The resulting supernatant was collected and injected into the HPLC. Mobile phase was composed by 20% methanol in 50 mM citric acid, and 0.46 mM octanessulfonic acid, adjusted to pH 5.5. The chromatographic runs took less than 22 min and no significant endogenous interferences were observed at the α-Amanitin retention time. Calibration curves were linear with regression coefficients higher than 0.994. The overall inter- and intra-assay precision did not exceed 15.3%. The present method has low interferences with simple and fast processing steps, being a suitable procedure to support in vivo toxicokinetic studies involving α-Amanitin. In fact, the validated method was successfully applied to quantify α-Amanitin in biological samples following intraperitoneal α-Amanitin administration to rats. Moreover, human plasma was also used as matrix and the purposed method was adequate for detection of α-Amanitin in that matrix. The results clearly indicate that the proposed method is suitable to investigate the pharmacokinetic and tissue distribution of α-Amanitin. Additionally, the method will be very useful in the development of novel and potent antidotes against amatoxins poisoning and to improve the knowledge of α-Amanitin toxicity.
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new in silico insights into the inhibition of rnap ii by α Amanitin and the protective effect mediated by effective antidotes
Journal of Molecular Graphics & Modelling, 2014Co-Authors: Juliana Garcia, Maria Lourdes Bastos, Alexandra T. P. Carvalho, Daniel F. A. R. Dourado, Paula Baptista, Félix CarvalhoAbstract:Poisonous α-Amanitin-containing mushrooms are responsible for the major cases of fatalities after mushroom ingestion. α-Amanitin is known to inhibit the RNA polymerase II (RNAP II), although the underlying mechanisms are not fully understood. Benzylpenicillin, ceftazidime and silybin have been the most frequently used drugs in the management of α-Amanitin poisoning, mostly based on empirical rationale. The present study provides an in silico insight into the inhibition of RNAP II by α-Amanitin and also on the interaction of the antidotes on the active site of this enzyme. Docking and molecular dynamics (MD) simulations combined with molecular mechanics-generalized Born surface area method (MM-GBSA) were carried out to investigate the binding of α-Amanitin and three antidotes benzylpenicillin, ceftazidime and silybin to RNAP II. Our results reveal that α-Amanitin should affects RNAP II transcription by compromising trigger loop (TL) function. The observed direct interactions between α-Amanitin and TL residues Leu1081, Asn1082, Thr1083, His1085 and Gly1088 alters the elongation process and thus contribute to the inhibition of RNAP II. We also present evidences that α-Amanitin can interact directly with the bridge helix residues Gly819, Gly820 and Glu822, and indirectly with His816 and Phe815. This destabilizes the bridge helix, possibly causing RNAP II activity loss. We demonstrate that benzylpenicillin, ceftazidime and silybin are able to bind to the same site as α-Amanitin, although not replicating the unique α-Amanitin binding mode. They establish considerably less intermolecular interactions and the ones existing are essential confine to the bridge helix and adjacent residues. Therefore, the therapeutic effect of these antidotes does not seem to be directly related with binding to RNAP II. RNAP II α-Amanitin binding site can be divided into specific zones with different properties providing a reliable platform for the structure-based drug design of novel antidotes for α-amatoxin poisoning. An ideal drug candidate should be a competitive RNAP II binder that interacts with Arg726, Ile756, Ala759, Gln760 and Gln767, but not with TL and bridge helix residues.
Marta Leite - One of the best experts on this subject based on the ideXlab platform.
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development optimization and application of an analytical methodology by ultra performance liquid chromatography tandem mass spectrometry for determination of Amanitins in urine and liver samples
Analytica Chimica Acta, 2013Co-Authors: Marta Leite, Andreia Freitas, Anabela Marisa Azul, Jorge Barbosa, Saul C Costa, Fernando RamosAbstract:Abstract Amanitins, highly toxic cyclopeptides isolated from various Amanita species, are the most potent poisons accounting for the hazardous effects on intestinal epithelium cells and hepatocytes, and probably the sole cause of fatal human poisoning. The present study was focused on the development, optimization and application of an analytical methodology by ultra performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS), following urine and liver sample preparation by protein precipitation with organic solvents, and solid phase extraction (SPE) procedure, for the determination of the amatoxins, α- and β-Amanitin. Linearity, detection and quantification limits, selectivity, sensitivity, intra and inter-assay precision and recovery were studied, in order to guarantee reliability in the analytical results. The developed method proved to be specific and selective, with LOD (Limit of Detection) values for α- and β-Amanitin of 0.22 and 0.20 ng mL −1 in urine and 10.9 and 9.7 ng g −1 in liver, respectively. LOQ (Limit of Quantification) values ranged from 0.46 to 0.57 ng mL −1 in urine and 12.3–14.7 ng g −1 in tissue, for both Amanitins. Linearity, in the range of 10.0–200.0 ng mL −1 or ng g −1 , shows that coefficients of correlation were greater than 0.997 for α-Amanitin and 0.993 for β-Amanitin. Precision was checked at three levels during three consecutive days with intra-day and inter-day coefficients of variation not greater than 15.2%. The extraction recovery presents good results for the concentrations analyzed, with values ranging from 90.2 to 112.9% for both matrices. Thus, the proposed analytical method is innovative, presents a high potential in the identification, detection and determination of α- and β-Amanitins in urine and tissue samples, as well as in other biological samples, such as kidney and mushrooms.
David M. Perrin - One of the best experts on this subject based on the ideXlab platform.
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Synthesis of the Death-Cap Mushroom Toxin α-Amanitin.
Journal of the American Chemical Society, 2018Co-Authors: Kaveh Matinkhoo, Alla Pryyma, Mihajlo Todorovic, Brian O. Patrick, David M. PerrinAbstract:α-Amanitin is an extremely toxic bicyclic octapeptide isolated from the death-cap mushroom, Amanita phalloides. As a potent inhibitor of RNA polymerase II, α-Amanitin is toxic to eukaryotic cells. Recent interest in α-Amanitin arises from its promise as a payload for antibody–drug conjugates. For over 60 years, A. phalloides has been the only source of α-Amanitin. Here we report a synthesis of α-Amanitin, which surmounts the key challenges for installing the 6-hydroxy-tryptathionine sulfoxide bridge, enantioselective synthesis of (2S,3R,4R)-4,5-dihydroxy-isoleucine, and diastereoselective sulfoxidation.
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Synthesis of the Death-Cap Mushroom Toxin α‑Amanitin
2018Co-Authors: Kaveh Matinkhoo, Alla Pryyma, Mihajlo Todorovic, Brian O. Patrick, David M. PerrinAbstract:α-Amanitin is an extremely toxic bicyclic octapeptide isolated from the death-cap mushroom, Amanita phalloides. As a potent inhibitor of RNA polymerase II, α-Amanitin is toxic to eukaryotic cells. Recent interest in α-Amanitin arises from its promise as a payload for antibody–drug conjugates. For over 60 years, A. phalloides has been the only source of α-Amanitin. Here we report a synthesis of α-Amanitin, which surmounts the key challenges for installing the 6-hydroxy-tryptathionine sulfoxide bridge, enantioselective synthesis of (2S,3R,4R)-4,5-dihydroxy-isoleucine, and diastereoselective sulfoxidation