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The coronavirus disease 2019 (Covid-19), which caused respiratory problems in many patients worldwide, led to more than 5 million deaths by the end of 2021. Experienced symptoms vary from mild to severe illness. Understanding the infection severity to reach a better prognosis could be useful to the clinics, and one study area to fulfill one piece of this biological puzzle is metabolomics. The metabolite profile and/or levels being monitored can help predict phenotype properties. Therefore, this study evaluated plasma metabolomes of 110 individual samples, 57 from control patients and 53 from recent positive cases of Covid-19 (IgM 98% reagent), representing mild to severe symptoms, before any clinical intervention. Polar metabolites from plasma samples were analyzed by quantitative 1H NMR. Glycerol, 3-aminoisobutyrate, formate, and glucuronate levels showed alterations in Covid-19 patients compared to those in the control group (Tukey's HSD p-value cutoff = 0.05), affecting the lactate, phenylalanine, tyrosine, and tryptophan biosynthesis and d-glutamine, d-glutamate, and glycerolipid metabolisms. These metabolic alterations show that SARS-CoV-2 infection led to disturbance in the energetic system, supporting the viral replication and corroborating with the severe clinical conditions of patients. Six polar metabolites (glycerol, acetate, 3-aminoisobutyrate, formate, glucuronate, and lactate) were revealed by PLS-DA and predicted by ROC curves and ANOVA to be potential prognostic metabolite panels for Covid-19 and considered clinically relevant for predicting infection severity due to their straight roles in the lipid and energy metabolism. Thus, metabolomics from samples of Covid-19 patients is a powerful tool for a better understanding of the disease mechanism of action and metabolic consequences of the infection in the human body and may corroborate allowing clinicians to intervene quickly according to the needs of Covid-19 patients.
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COVID-19 , Aminoácidos , COVID-19/diagnóstico , Formiatos , Glucuronatos , Glicerol , Humanos , Lactatos , Metabolómica , SARS-CoV-2RESUMEN
Introduction: Cri-du-chat syndrome is generally diagnosed when patients present a high-pitched cry at birth, microcephaly, ocular hypertelorism, and prominent nasal bridge. The karyotype is useful to confirm deletions in the short arm of chromosome 5 (5p-) greater than 10 Mb. In cases of smaller deletions, it is necessary to resort to other molecular techniques such as fluorescence in situ hybridization, multiplex ligation-dependent probe amplification (MLPA) or genomic array. Case Presentation: We report a family with an atypical deletion in 5p (mother and 2 children) and variable phenotypes compared with the literature. We applied a P064 MLPA kit to evaluate 5p- in the mother and the 2 children, and we used the Infinium CytoSNP-850K BeadChip genomic array to evaluate the siblings, an 11-year-old boy and a 13-year-old girl, to better define the 5p breakpoints. Both children presented a high-pitched cry at birth, but they did not present any of the typical physical features of 5p- syndrome. The MLPA technique with 5 probes for the 5p region revealed that the patients and their mother presented an atypical deletion with only 4 probes deleted (TERT_ex2, TERT_ex13, CLPTM1L, and IRX4). The genomic array performed in the siblings' samples revealed a 6.2-Mb terminal deletion in 5p15.33p15.32, which was likely inherited from their mother, who presented similar molecular features, seen in MLPA. Discussion: The sparing of the CTNND2 gene, which is associated with cerebral development, in both siblings may explain why these 2 patients had features such as better communication skills which most patients with larger 5p deletions usually do not present. In addition, both patients had smaller deletions than those found in patients with a typical 5p- phenotype. This report demonstrates the utility of genomic arrays as a diagnostic tool to better characterize atypical deletions in known syndromes such as 5p- syndrome, which will allow a better understanding of the genotype-phenotype correlations.
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The neurodegeneration that occurs in methylmalonic acidemia is proposed to be associated with impairment of mitochondrial oxidative metabolism resulting from methylmalonate (MMA) accumulation. The present study evaluated the effects of MMA on oxygen consumption by isolated rat brain mitochondria in the presence of NADH-linked substrates (α-ketoglutarate, citrate, isocitrate, glutamate, malate, and pyruvate). Respiration supported either by glutamate or glutamate plus malate was significantly inhibited by MMA (1-10 mM), whereas no inhibition was observed when a cocktail of NADH-linked substrates was used. Measurements of glutamate transport revealed that the inhibitory effect of MMA on respiration maintained by this substrate is not due to inhibition of its mitochondrial uptake. In light of this result, the effect of MMA on the activity of relevant enzymes involved in mitochondrial glutamate metabolism was investigated. MMA had minor inhibitory effects on glutamate dehydrogenase and aspartate aminotransferase, whereas α-ketoglutarate dehydrogenase was significantly inhibited by this metabolite (K(i) = 3.65 mM). Moreover, measurements of α-ketoglutarate transport and mitochondrial MMA accumulation indicated that MMA/α-ketoglutarate exchange depletes mitochondria from this substrate, which may further contribute to the inhibition of glutamate-sustained respiration. To study the effect of chronic in vivo MMA treatment on mitochondrial function, young rats were intraperitoneally injected with MMA. No significant difference was observed in respiration between isolated brain mitochondria from control and MMA-treated rats, indicating that in vivo MMA treatment did not lead to permanent mitochondrial respiratory defects. Taken together, these findings indicate that the inhibitory effect of MMA on mitochondrial oxidative metabolism can be ascribed to concurrent inhibition of specific enzymes and lower availability of respiratory substrates.
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Ácido Glutámico/metabolismo , Ácido Metilmalónico/farmacología , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Complejos Multienzimáticos/metabolismo , Sistema de Transporte de Aminoácidos X-AG/metabolismo , Análisis de Varianza , Animales , Animales Recién Nacidos , Carboxiliasas/metabolismo , Citrato (si)-Sintasa/metabolismo , Relación Dosis-Respuesta a Droga , Glutamato Deshidrogenasa/metabolismo , Complejo Cetoglutarato Deshidrogenasa/metabolismo , Ácidos Cetoglutáricos/metabolismo , Ácido Metilmalónico/metabolismo , Consumo de Oxígeno/efectos de los fármacos , Prosencéfalo/efectos de los fármacos , Prosencéfalo/ultraestructura , Ratas , Ratas WistarRESUMEN
Diacetyl, methylglyoxal, and glyoxal are α-dicarbonyl catabolites prone to nucleophilic additions of amino groups of proteins and nucleobases, thereby triggering adverse biological responses. Because of their electrophilicity, in aqueous medium, they exist in a phosphate-catalyzed dynamic equilibrium with their hydrate forms. Diacetyl and methylglyoxal can be attacked by peroxynitrite (k(2) ≈ 1.0 × 10(4) M(-1) s(-1) and k(2) ≈ 1.0 × 10(5) M(-1) s(-1), respectively), a potent biological nucleophile and oxidant, yielding the acetyl radical from the homolysis of peroxynitrosocarbonyl adducts, and acetate or formate ions, respectively. We report here that glyoxal also reacts with peroxynitrite, yielding formate ion at rates at least 1 order of magnitude greater than does methylglyoxal. A triplet EPR signal (1:2:1; a(H) = 0.78 mT) attributable to hydrated formyl radical was detected by direct flow experiments. In the presence of the spin trap 2-methyl-2-nitrosopropane, the EPR spectrum displays the di-tert-butyl nitroxide signal, another signal assignable to the spin trapping adduct with hydrogen radical (a(N) = a(H) = 1.44 mT), probably formed from formyl radical decarbonylation, and a third EPR signal assignable to the formyl radical adduct of the spin trap (a(N) = 0.71 mT and a(H) = 0.14 mT). The novelty here is the detection of singlet oxygen ((1)Δ(g)) monomol light emission at 1270 nm during the reaction, probably formed by subsequent dioxygen addition to formyl radical and a Russell reaction of nascent formylperoxyl radicals. Accordingly, the near-infrared emission increases upon raising the peroxynitrite concentration in D(2)O buffer and is suppressed upon addition of O(2) ((1)Δ(g)) quenchers (NaN(3), l-His, H(2)O). Unequivocal evidence of O(2) ((1)Δ(g)) generation was also obtained by chemical trapping of (18)O(2) ((1)Δ(g)) with anthracene-9,10-divinylsulfonate, using HPLC/MS/MS for detection of the corresponding 9,10-endoperoxide derivative. Our studies add insights into the molecular events underlying nitrosative, oxidative, and carbonyl stress in inflammatory processes and aging-associated maladies.
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Glioxal/química , Oxidantes/química , Ácido Peroxinitroso/química , Oxígeno Singlete/químicaRESUMEN
Several conditions have been used in the coupling reaction of stepwise SPPS at elevated temperature (SPPS-ET), but we have elected the following as our first choice: 2.5-fold molar excess of 0.04-0.08 M Boc or Fmoc-amino acid derivative, equimolar amount of DIC/HOBt (1:1) or TBTU/DIPEA (1:3), 25% DMSO/toluene, 60 degrees C, conventional heating. In this study, aimed to further examine enantiomerization under such condition and study the applicability of our protocols to microwave-SPPS, peptides containing L-Ser, L-His, L-Cys and/or L-Met were manually synthesized traditionally, at 60 degrees C using conventional heating and at 60 degrees C using microwave heating. Detailed assessment of all crude peptides (in their intact and/or fully hydrolyzed forms) revealed that, except for the microwave-assisted coupling of L-Cys, all other reactions occurred with low levels of amino acid enantiomerization (<2%). Therefore, herein we (i) provide new evidences that our protocols for SPPS at 60 degrees C using conventional heating are suitable for routine use, (ii) demonstrate their appropriateness for microwave-assisted SPPS by Boc and Fmoc chemistries, (iii) disclose advantages and limitations of the three synthetic approaches employed. Thus, this study complements our past research on SPPS-ET and suggests alternative conditions for microwave-assisted SPPS.
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Microondas , Péptidos/química , Péptidos/síntesis química , Secuencia de Aminoácidos , Aminoácidos/química , Colecistoquinina/química , Colecistoquinina/genética , Cromatografía Liquida , Electroforesis Capilar , Cromatografía de Gases y Espectrometría de Masas , Calefacción , Datos de Secuencia Molecular , Péptidos/genética , Péptidos/aislamiento & purificación , Estereoisomerismo , Espectrometría de Masas en Tándem , TemperaturaRESUMEN
INTRODUCTION: The risk of systemic bupivacaine toxicity is a persistent problem, which makes its pharmacokinetic study fundamental for regional anesthesia safety. There is little evidence of its influence on plasma peak at different concentrations. The present study compares two bupivacaine concentrations to establish how the concentration affects this drug plasma peak in axillary brachial plexus block. Postoperative latency and analgesia were also compared. METHODS: 30 patients were randomized. In the 0.25% Group, 0.25% bupivacaine (10mL) was injected per nerve. In the 0.5% Group, 0.5% bupivacaine (5mL) was injected per nerve. Peripheral blood samples were collected during the first 2hours after the blockade. For sample analyses, high performance liquid chromatography mass spectrometry was used. RESULTS: Plasma peak occurred 45minutes after the blockade, with no difference between groups at the assessed time-points. Plasma peak was 933.97 ± 328.03 ng.mL-1 (mean ± SD) in 0.25% Group and 1022.79 ± 253.81 ng.mL-1 in 0.5% Group (p = 0.414). Latency was lower in 0.5% Group than in 0.25% Group (10.67 ± 3.71 × 17.33min ± 5.30, respectively, p = 0.004). No patient had pain within the first 4hours after the blockade. CONCLUSION: For axillary brachial plexus block, there was no difference in bupivacaine plasma peak despite the use of different concentrations with the same local anesthetic mass. The concentration inversely influenced latency.
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Anestésicos Locales/farmacocinética , Plexo Braquial , Bupivacaína/administración & dosificación , Bupivacaína/farmacocinética , Bloqueo Nervioso , Adulto , Anestésicos Locales/farmacología , Axila , Bupivacaína/farmacología , Femenino , Humanos , Masculino , Bloqueo Nervioso/métodos , Estudios ProspectivosRESUMEN
BACKGROUND: Acetylation alters several protein properties including molecular weight, stability, enzymatic activity, protein-protein interactions, and other biological functions. Our previous findings demonstrating that diacetyl/peroxynitrite can acetylate L-lysine, L-histidine, and albumin in vitro led us to investigate whether diacetyl-treated rats suffer protein acetylation as well. METHODS: Wistar rats were administered diacetyl daily for four weeks, after which they were sacrificed, and their lung proteins were extracted to be analysed by Nano-LC-MS/MS (Q-TOF). A C18 reversed-phase column and gradient elution with formic acid/acetonitrile solutions from 2 to 50% over 150 min were used to separate the proteins. Protein detection was performed using a microTOF-Q II (QTOF) equipped with captive source and an electrospray-ionization source. The data from mass spectrometry were processed using a Compass 1.7 and analyzed using Protein Scape, software that uses Mascot algorithms to perform protein searches. RESULTS: A set of 3,162 acetylated peptides derived from 351 acetylated proteins in the diacetyl-treated group was identified. Among them, 23 targeted proteins were significantly more acetylated in the diacetyl-treated group than in the PBS control. Protein acetylation of the group treated with 540 mg/kg/day of diacetyl was corroborated by Western blotting analysis. CONCLUSIONS: These data support our hypothesis that diacetyl exposure in animals may lead to the generation of acetyl radicals, compounds that attach to proteins, affecting their functions and triggering adverse health problems.
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Calor , Péptidos/síntesis química , Animales , Bovinos , Cromatografía Líquida de Alta Presión , Electroforesis Capilar , HumanosRESUMEN
Abstract Introduction: The risk of systemic bupivacaine toxicity is a persistent problem, which makes its pharmacokinetic study fundamental for regional anesthesia safety. There is little evidence of its influence on plasma peak at different concentrations. The present study compares two bupivacaine concentrations to establish how the concentration affects this drug plasma peak in axillary brachial plexus block. Postoperative latency and analgesia were also compared. Methods: 30 patients were randomized. In the 0.25% Group, 0.25% bupivacaine (10 mL) was injected per nerve. In the 0.5% Group, 0.5% bupivacaine (5 mL) was injected per nerve. Peripheral blood samples were collected during the first 2 h after the blockade. For sample analyses, high performance liquid chromatography mass spectrometry was used. Results: Plasma peak occurred 45 min after the blockade, with no difference between groups at the assessed time-points. Plasma peak was 933.97 ± 328.03 ng.mL−1 (mean ± SD) in 0.25% Group and 1022.79 ± 253.81 ng.mL−1 in 0.5% Group (p = 0.414). Latency was lower in 0.5% Group than in 0.25% Group (10.67 ± 3.71 × 17.33 min ± 5.30, respectively, p = 0.004). No patient had pain within the first 4 h after the blockade. Conclusion: For axillary brachial plexus block, there was no difference in bupivacaine plasma peak despite the use of different concentrations with the same local anesthetic mass. The concentration inversely influenced latency.
Resumo Introdução: O risco de intoxicação sistêmica pelo uso da bupivacaína é um problema persistente e torna seu estudo farmacocinético fundamental para a segurança da anestesia regional. São escassas as evidências sobre a influência de diferentes concentrações no pico plasmático desse fármaco. O presente estudo compara duas concentrações de bupivacaína para estabelecer como a concentração afeta o pico plasmático desse fármaco no bloqueio do plexo braquial via axilar. Também se compararam latência e analgesia pós-operatória. Métodos: Foram randomizados 30 pacientes. No Grupo 0,25%, injetaram-se 10 mL de bupivacaína 0,25% por nervo. No Grupo 0,5%, injetaram-se 5 mL de bupivacaína 0,5% por nervo. Amostras de sangue periférico foram colhidas durante as duas primeiras horas após o bloqueio. Para análise das amostras, usou-se a cromatografia líquida de alta frequência acoplada ao espectrômetro de massas. Resultados: O pico plasmático ocorreu 45 minutos após o bloqueio, sem diferença entre os grupos nos tempos avaliados. O pico plasmático (média ± DP) foi 933,97 ± 328,03 ng.mL−1 no Grupo 0,25% e 1.022,79 ± 253,81 ng.mL−1 no Grupo 0,5% (p = 0,414). O Grupo 0,5% apresentou menor latência com relação ao Grupo 0,25% (10,67 ± 3,71 × 17,33 min ± 5,30; respectivamente; p = 0,004). Nenhum paciente apresentou dor nas primeiras quatro horas após o bloqueio. Conclusão: Para o bloqueio do plexo braquial via axilar, não foi detectada diferença no pico plasmático de bupivacaína apesar do uso de diferentes concentrações, com a mesma massa de anestésico local. A concentração influenciou inversamente a latência.
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Humanos , Masculino , Femenino , Adulto , Plexo Braquial , Bupivacaína/administración & dosificación , Bupivacaína/farmacocinética , Anestésicos Locales/farmacocinética , Bloqueo Nervioso/métodos , Axila , Bupivacaína/farmacología , Estudios Prospectivos , Anestésicos Locales/farmacologíaRESUMEN
Diacetyl, like other alpha-dicarbonyl compounds, is reportedly cytotoxic and genotoxic. A food and cigarette contaminant, it is related with alcohol hepatotoxicity and lung disease. Peroxynitrite is a potent oxidant formed in vivo by the diffusion-controlled reaction of the superoxide radical anion with nitric oxide, which is able to form adducts with carbon dioxide and carbonyl compounds. Here, we investigate the nucleophilic addition of peroxynitrite to diacetyl forming acetyl radicals, whose reaction with molecular oxygen leads to acetate. Peroxynitrite is shown to react with diacetyl in phosphate buffer (bell-shaped pH profile with maximum at 7.2) at a very high rate constant ( k 2 = 1.0 x 10 (4) M (-1) s (-1)) when compared with monocarbonyl substrates ( k 2 < 10 (3) M (-1) s (-1)). Phosphate ions (100-500 mM) do not affect the rate of spontaneous peroxynitrite decay, but the H 2PO 4 (-) anion catalyzes the nucleophilic addition of the peroxynitrite anion to diacetyl. The intermediacy of acetyl radicals is suggested by a three-line spectrum ( a N = a H = 0.83 mT) obtained by EPR spin trapping of the reaction mixture with 2-methyl-2-nitrosopropane. The peroxynitrite reaction is accompanied by concentration-dependent oxygen uptake. Stoichiometric amounts of acetate from millimolar amounts of peroxynitrite and diacetyl were obtained under nonlimiting conditions of dissolved oxygen. In the presence of either l-histidine or 2'-deoxyguanosine, the peroxynitrite/diacetyl system afforded the corresponding acetylated molecules identified by HPLC-MS ( n ). These studies provide evidence that the peroxynitrite/diacetyl reaction yields acetyl radicals and raise the hypothesis that protein and DNA nonenzymatic acetylation may occur in cells and be implicated in aging and metabolic disorders in which oxygen and nitrogen reactive species are putatively involved.
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Desoxiguanosina/química , Diacetil/química , Histidina/química , Ácido Peroxinitroso/química , Acetilación , Oxígeno/químicaRESUMEN
Amino metabolites with potential prooxidant properties, particularly alpha-aminocarbonyls, are the focus of this review. Among them we emphasize 5-aminolevulinic acid (a heme precursor formed from succinyl-CoA and glycine), aminoacetone (a threonine and glycine metabolite), and hexosamines and hexosimines, formed by Schiff condensation of hexoses with basic amino acid residues of proteins. All these metabolites were shown, in vitro, to undergo enolization and subsequent aerobic oxidation, yielding oxyradicals and highly cyto- and genotoxic alpha-oxoaldehydes. Their metabolic roles in health and disease are examined here and compared in humans and experimental animals, including rats, quail, and octopus. In the past two decades, we have concentrated on two endogenous alpha-aminoketones: (i) 5-aminolevulinic acid (ALA), accumulated in acquired (e.g., lead poisoning) and inborn (e.g., intermittent acute porphyria) porphyric disorders, and (ii) aminoacetone (AA), putatively overproduced in diabetes mellitus and cri-du-chat syndrome. ALA and AA have been implicated as contributing sources of oxyradicals and oxidative stress in these diseases. The end product of ALA oxidation, 4,5-dioxovaleric acid (DOVA), is able to alkylate DNA guanine moieties, promote protein cross-linking, and damage GABAergic receptors of rat brain synaptosome preparations. In turn, methylglyoxal (MG), the end product of AA oxidation, is also highly cytotoxic and able to release iron from ferritin and copper from ceruloplasmin, and to aggregate proteins. This review covers chemical and biochemical aspects of these alpha-aminoketones and their putative roles in the oxidative stress associated with porphyrias, tyrosinosis, diabetes, and cri-du-chat. In addition, we comment briefly on a side prooxidant behaviour of hexosamines, that are known to constitute building blocks of several glycoproteins and to be involved in Schiff base-mediated enzymatic reactions.
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Acetona/análogos & derivados , Ácido Aminolevulínico/metabolismo , Hexosaminas/metabolismo , Iminoazúcares/metabolismo , Oxidantes/metabolismo , Acetona/metabolismo , Animales , Síndrome del Maullido del Gato/metabolismo , Diabetes Mellitus/metabolismo , Humanos , Intoxicación por Plomo/metabolismo , Oxidación-Reducción , Estrés Oxidativo/fisiología , Porfiria Intermitente Aguda/metabolismo , RatasRESUMEN
This work brings together some contributions for the use of nonaqueous media for proteomic analysis, for both capillary electrophoresis (CE) separation and the preparation of tryptic digests. First, a ternary nonaqueous buffer consisting of 60/30/10 v/v methanol/acetonitrile/acetic acid with 12.5 mmol/L ammonium acetate was optimized for CE separation of the tryptic digest of lysozyme. Lysozyme was chosen as a model system for the protein digestion, which has also been prepared in an organic-rich medium with methanol/50 mmol/L NH(4)HCO(3), pH 8.0 (60/40 v/v). The separation results were compared to in silico (PeptideCutter program) digestion conditions, and high-efficiency peak separation (18 peaks) was obtained in 20 min with an electric field of 350 V/cm. In addition, we have evaluated the stability of a coated capillary with poly-N,N-dimethylacrylamide (60/30 cm total/effective length and 75 microm ID) for over 100 runs of tryptic digest with the nonaqueous background electrolyte solvent system. The migration times for ten selected peptide peaks presented 3-7% relative standard deviation.