1,4-Diamino-2-butanone, a wide-spectrum microbicide, yields reactive species by metal-catalyzed oxidation

The alpha-aminoketone 1,4-diamino-2-butanone (DAB), a putrescine analogue, is highly toxic to various microorganisms, including Trypanosoma cruzi. However, little is known about the molecular mechanisms underlying DAB's cytotoxic properties. We report here that DAB (pK(a) 7.5 and 9.5) undergoes aerobic oxidation in phosphate buffer, pH 7.4, at 37 degrees C, catalyzed by Fe(II) and Cu(II) ions yielding NH(4)(+) ion, H(2)O(2), and 4-amino-2-oxobutanal (oxoDAB). OxoDAB, like methylglyoxal and other alpha-oxoaldehydes, is expected to cause protein aggregation and nucleobase lesions. Propagation of DAB oxidation by superoxide radical was confirmed by the inhibitory effect of added SOD (50 U ml(-1)) and stimulatory effect of xanthine/xanthine oxidase, a source of superoxide radical. EPR spin trapping studies with 5,5-dimethyl-1-pyrroline-1-oxide (DMPO) revealed an adduct attributable to DMPO-HO(center dot), and those with alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone or 3,5-dibromo-4-nitrosobenzenesulfonic acid, a six-line adduct assignable to a DAB(center dot) resonant enoyl radical adduct. Added horse spleen ferritin (HoSF) and bovine apo-transferrin underwent oxidative changes in tryptophan residues in the presence of 1.0-10 mM DAB. Iron release from HoSF was observed as well. Assays performed with fluorescein-encapsulated liposomes of cardiolipin and phosphatidylcholine (20:80) incubated with DAB resulted in extensive lipid peroxidation and consequent vesicle permeabilization. DAB (0-10 mM) administration to cultured LLC-MK2 epithelial cells caused a decline in cell viability, which was inhibited by preaddition of either catalase (4.5 mu M) or aminoguanidine (25 mM). Our findings support the hypothesis that DAB toxicity to several pathogenic microorganisms previously described may involve not only reported inhibition of polyamine metabolism but also DAB pro-oxidant activity. (C) 2011 Elsevier Inc. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)INCT Processos Redox em Biomedicina-RedoxomaUniv São Paulo, Inst Quim, Dept Bioquim, BR-05508900 São Paulo, BrazilUniversidade Federal de São Paulo, Inst Ciencias Ambientais Quim & Farmaceut, Dept Ciencias Exatas & Terra, Diadema, SP, BrazilUniversidade Federal de São Paulo, Inst Ciencias Ambientais Quim & Farmaceut, Dept Ciencias Exatas & Terra, Diadema, SP, BrazilWeb of Scienc

Neurotoxicity and aggressiveness triggered by low-level lead in children: a review

Lead-induced neurotoxicity acquired by low-level long-term exposure has special relevance for children. A plethora of recent reports has demonstrated a direct link between low-level lead exposure and deficits in the neurobehavioral-cognitive performance manifested from childhood through adolescence. In many studies, aggressiveness and delinquency have also been suggested as symptoms of lead poisoning. Several environmental, occupational and domestic sources of contaminant lead and consequent health risks are largely identified and understood, but the occurrences of lead poisoning remain numerous. There is an urgent need for public health policies to prevent lead poisoning so as to reduce individual and societal damages and losses. In this paper we describe unsuspected sources of contaminant lead, discuss the economic losses and urban violence possibly associated with lead contamination and review the molecular basis of lead-induced neurotoxicity, emphasizing its effects on the social behavior, delinquency and IQ of children and adolescent

Aumento do estresse oxidativo após hipotermia em ratos com pancreatite induzida por ceruleína

BACKGROUND: Hypothermia is a frequent event in severe acute pancreatitis (AP) and its real effects on the normal pancreas have not been well demonstrated. Moreover, neither have its effects on the outcome of acute pancreatitis been fully investigated. One hypothesis is that oxidative stress may be implicated in lesions caused or treated by hypothermia. AIM OF THE STUDY: To investigate the effect of hypothermia in cerulein-induced acute pancreatitis (CIAP) in rats and the role played by oxidative stress in this process. METHODS: Male Wistar rats were divided into hypothermic and normothermic groups. Hypothermia was induced with a cold mattress and rectal temperature was kept at 30ºC for one hour. Acute pancreatitis was induced with 2 doses of cerulein (20 ìg/kg) administered at a one-hour interval. Serum amylase, pancreas vascular permeability by Evan's blue method, pancreas wet-to-dry weight ratio and histopathology were analyzed in each group. RESULTS: When compared with normothermic rats, hypothermic animals, with cerulein-induced acute pancreatitis, showed higher levels of pancreatic vascular permeability (p < 0.05), pancreas wet-to-dry weight ratio (p = 0.03), and histologically verified edema (p < 0.05), but similar serum amylase levels. The hypothermic group showed a higher oxidized-reduced glutathione ratio than the normothermic group. CONCLUSION: Moderate hypothermia produced a greater inflammatory response in established acute pancreatitis induced by cerulein in rats. Moreover, this study suggests that oxidative stress may be one of the mechanisms responsible for the worse outcome in hypothermic rats with cerulein-induced acute pancreatitis.BACKGROUND: Hipotermia é um evento freqüente em episódios de pancreatite aguda, contudo seu efeito real sobre pâncreas normal ainda não esta bem demonstrado. Além do mais, o efeito da hipotermia no decorrer da pancreatite aguda também não está completamente esclarecido. Uma das hipóteses sobre as causas das lesões causadas ou tratadas por hipotermia aventa a implicação de estresse oxidativo. OBJETIVOS: Investigar o efeito da hipotermia em ratos com pancreatite aguda induzida por ceruleína e o papel do estresse oxidativo neste processo. MÉTODOS: Ratos Wistar machos foram divididos em grupos hipotérmicos e normotérmicos. Hipotermia foi induzida com uma bolsa gelada de forma que a temperatura retal permanecesse em 30ºC por uma hora. Pancreatite aguda foi induzida com duas aplicações de ceruleína (20 ìg/kg) administradas com intervalo de uma hora. A amilase sérica, a permeabilidade vascular do pâncreas, a razão peso seco/peso úmido do pâncreas, a histopatologia e os níveis de glutationa foram analisados em cada grupo. RESULTADOS: Ratos hipotérmicos, com pancreatite aguda induzida por ceruleína, apresentaram maiores níveis de permeabilidade vascular no pâncreas (p < 0.05), razão peso seco/peso úmido do pâncreas (p = 0.03), e edema histológico (p < 0.05), mas os níveis de amilase sérica permaneceram iguais aos níveis apresentados pelos ratos normotérmicos. O grupo hipotérmico apresentou maior relação glutationa oxidada/glutationa reduzida em relação ao grupo normotérmico. CONCLUSÃO: Hipotermia moderada produziu uma maior resposta inflamatória em ratos com pancreatite aguda estabelecida, induzida por ceruleína, sugerindo que este efeito pode estar ligado a um maior índice de estresse oxidativo em ratos com pancreatite aguda

Potential diagnostic of Branched-Chain Ketoaciduria by HPLC-DAD

A system of high performance liquid chromatography (HPLC) was used for the development and validation of efficient method for quantitative determination of three aminoacids involved in the inherited metabolic disease Branched-Chain Ketoaciduria (BCK), also called maple syrup urine disease. The analytical conditions were selected in order to obtain baseline separation profiles of the amino acids known to be altered in blood plasma of BCK patients, namely L-valine, L-isoleucine, and L-leucine. Most accurate data were obtained using HPLC/diode detector. As the analytes do not have chromophore groups, they were pre-derivatized with o-phthaldialdehyde (OPA), yielding an unsaturated adduct, making thus possible the detection of amino acids. The validation was conducted according to National Health Surveillance Agency (ANVISA) and Guidance for Industry (Bioanalytical Method Validation) United States Food and Drug Administration (U.S. FDA). The results were satisfactory, with high sensitivity, good linearity, precision and accuracy, limit of detection and quantification, all within the established parameters for bioanalytical methods, showing its applicability and low cost compared to other existing techniques such as sequential mass spectrometry. For the three amino acids, L-valine, L-isoleucine and L-leucine, the detection limits (LOD) found were: 1.61, 1.84 and 1.88 mmol L- 1 and the quantification limits (LOQ) 4.37, 6.13 and 6.27 mmol L- 1, respectively.Neste trabalho, um sistema de cromatografia líquida de alta eficiência (HPLC) foi usado para desenvolver e validar um eficiente método para determinar quantitativamente aminoácidos envolvidos na desordem rara conhecida como cetonúria de aminoácidos de cadeia ramificada ou doença do xarope de bordo. As condições analíticas foram desenvolvidas para obter os perfis dos aminoácidos de L-valina, L-isoleucina e L-leucina, sabidamente alterados no plasma sanguíneo dos pacientes. Empregou-se HPLC provido de um detector com arranjo de diodo. Os analitos não possuem grupos cromóforos e, por isso, foram pré-derivatizados com o-ftalaldeído (OPA) para tornar possível sua detecção. A validação foi conduzida de acordo com as normas da Agência Nacional de Vigilância Sanitária (ANVISA) (RDC No. 27, de 17 de maio de 2012) e secção de validação de bioanalítica da United States Food and Drug Administration (U.S. FDA). Os resultados foram satisfatórios, apresentando alta sensibilidade, boa linearidade, precisão e exatidão, limite de detecção e quantificação, todos parâmetros estabelecidos para métodos bioanalíticos, demonstrando a aplicabilidade e baixo custo do método comparado com outras técnicas como espectrometria de massas. Para os três aminoácidos, L-valina, L-isoleucina e L-leucina, os limites de detecção encontrados foram: 1,61, 1,84 e 1,88 mmol L 1 e limites de quantificação 4,37, 6,13 e 6,27 mmol L 1, respectivamente.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)INCT Redoxoma (National Institute of Science and Technology of Redox Processes in Biomedicine)Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Federal de São Paulo (UNIFESP) Instituto de Ciências Ambientais, Química e FarmacêuticasUniversidade de São Paulo Instituto de Química Departamento de Química FundamentalUNIFESP, Instituto de Ciências Ambientais, Química e Farmacêuticas2006/60245-3 e 2007/59039-2 e 2012/02514-9SciEL

Bioanalytical studies of porphyric disorders using HPLC with fluorescence detection

We describe here the development, validation, quantification and application of a method for determination of heme porphyrin precursors in the urine of porphyric patients. The isomers coproporphyrinogen I and III (COPRO I and III), uroporphyrinogen I (URO I), heptacarboxylporphyrinogen I (HEPTA I), pentacarboxylporphyrinogen (PENTA I), and hexacarboxylporphyrinogen I (HEXA I) were analyzed. These six urinary heme precursors were determined in urine samples collected from 24 patients by high-performance liquid chromatography (HPLC) equipped with a fluorescence detector. The inter- and intra-day precision (coefficient of variation < 5%) and accuracy (95-99%) were evaluated. The limits of detection and of quantification of the porphyrins, expressed in nmol L-1, were as follows: URO I, 0.62 and 2.05; HEPTA I, 0.59 and 1.96; HEXA I, 0.54 and 1.81; PENTA I, 0.52 and 1.73; COPRO I, 2.03 and 6.77; and COPRO III, 0.43 and 1.44. The method described here satisfactorily results in an acceptable cost-benefit ratio, precision and speed for determining the concentrations of heme precursors in the urine of latent or symptomatic acute intermittent porphyria individuals or porphyria cutanea tarda carriers. Since it was analytically validated, this method may be used for accurate and reliable diagnostic reports to follow-up the onset of acute crisis in porphyria carriers to adopt preventive pharmacological treatment.Neste artigo, desenvolvemos, validamos e aplicamos um método para separação e quantificação de porfirinas precursoras do grupo heme na urina de portadores de porfirias. Os isômeros coproporfirinogenio I e III (COPRO I e III), uroporfirinogenio I (URO I), heptacarboxilporfirinogenio I (HEPTA I), pentacarboxilporfirinogenio I (PENTA I) e hexacarboxilporfirinogenio (HEXA I) foram determinados em amostras coletadas de 24 pacientes de porfiria aguda intermitente e de porfiria cutânea tarda. Utilizou-se cromatografia líquida de alta eficiência (HPLC) e detector de fluorescência. As concentrações de porfirinas foram determinadas com precisão inter e intra-dias (< 5%) e exatidão dentro da faixa 95-99%. Os limites de detecção e quantificação das porfirinas, expressos em nmol L-1, foram os seguintes: URO I, 0,62 e 2,05; HEPTA I, 0,59 e 1,96; HEXA I, 0,54 e 1,81; PENTA I, 0,52 e 1,73; COPRO I, 2,03 e 6,77; e COPRO III, 0,43 e 1,44. O método descrito aqui obedece a parâmetros analíticos satisfatórios, com excelente relação custo-benefício, e foi aplicado a amostras de urina de portadores assintomáticos e pacientes de porfirias. Este método foi validado analiticamente e mostrou potencial para diagnóstico de portadores de diferentes tipos de porfirias, imediatamente antes ou durante crises, e até mesmo para monitorar um tratamento farmacológico.Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)INCT Redoxoma Redox Processes in BiomedicineFundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Universidade Federal de São Paulo (UNIFESP) Instituto de Ciências Ambientais, Químicas e Farmacêuticas Departamento de Ciências Exatas e da TerraUniversidade de São Paulo Instituto de Química Departamento de Química FundamentalUNIFESP, Instituto de Ciências Ambientais, Químicas e Farmacêuticas Depto. de Ciências Exatas e da Terra2006/60245-3 e 2006/56530-4SciEL

Astaxanthin Restrains Nitrative-Oxidative Peroxidation in Mitochondrial-Mimetic Liposomes: A Pre-Apoptosis Model

Astaxanthin (ASTA) is a ketocarotenoid found in many marine organisms and that affords many benefits to human health. ASTA is particularly effective against radical-mediated lipid peroxidation, and recent findings hypothesize a "mitochondrial-targeted" action of ASTA in cells. Therefore, we examined the protective effects of ASTA against lipid peroxidation in zwitterionic phosphatidylcholine liposomes (PCLs) and anionic phosphatidylcholine: phosphatidylglycerol liposomes (PCPGLs), at different pHs (6.2 to 8.0), which were challenged by oxidizing/nitrating conditions that mimic the regular and preapoptotic redox environment of active mitochondria. Pre-apoptotic conditions were created by oxidized/nitr(osyl) ated cytochrome c and resulted in the highest levels of lipoperoxidation in both PCL and PCPGLs (pH 7.4). ASTA was less protective at acidic conditions, especially in anionic PCPGLs. Our data demonstrated the ability of ASTA to hamper oxidative and nitrative events that lead to cytochrome c-peroxidase apoptosis and lipid peroxidation, although its efficiency changes with pH and lipid composition of membranes.Fundacao de Amparo a Pesquisa do Estado de Sao Paulo (FAPESPBPE fellowship)Conselho Nacional de Desenvolvimento Cientifico e Tecnologico (Bolsa Produtividade em Pesquisa, Nivel 2, CNPq, Brazil)Programa Iberoamericano de Ciencia y Tecnologia para el Desarollo (CYTEDRed iberoamericana para el estudio de nuevos carotenoides bioactivos como ingredientes de alimentos, Spain)Univ Sao Paulo IQUSP, Inst Quim, Dept Bioquim, BR-05508000 Sao Paulo, SP, BrazilUniv Cruzeiro Sul, ICAFE, BR-01506000 Sao Paulo, SP, BrazilSuperintendencia Policia Tecn Cient, BR-05507060 Sao Paulo, SP, BrazilLychnoflora Pesquisa & Dev Prod Nat LTDA, BR-14030090 Ribeirao Preto, SP, BrazilGrp Fleury, BR-04344070 Sao Paulo, SP, BrazilUniv Fed Sao Paulo, Dept Ciencias Exatas & Terra, UNIFESP, BR-09972270 Diadema, SP, BrazilUniv Sao Paulo IQUSP, Inst Quim, Dept Quim Fundamental, BR-05508000 Sao Paulo, SP, BrazilCSIC, IATA, Dept Ciencia Alimentos, Calle Catedrat Agustin Escardino 7, Paterna 46980, SpainUniv Fed Sao Paulo, Dept Ciencias Exatas & Terra, UNIFESP, BR-09972270 Diadema, SP, BrazilFAPESP: 017/06032-2CNPq: 304663/2015-8RIENCBI: 112RT0445Web of Scienc

Potential Diagnostic Assay for Cystinuria by Capillary Electrophoresis Coupled to Mass Spectrometry

Cistinúria é uma alteração genética autossômica recessiva caracterizada por transporte intestinal e renal anormal tubular de L-cistina, assim como de L-lisina, L-arginina e L-ornitina. Esta alteração leva a excreção urinária excessiva destes aminoácidos com a formação de pedras nos rins provocados pela baixa solubilidade de L-cistina na urina. Neste trabalho, um método analítico para a determinação destes quatro aminoácidos por eletroforese capilar acoplada à espectrometria de massas com ionização por electrospray (CE-ESI-MS) foi desenvolvido e validado. Usando soluções padrão de L-cistina, L-lisina, L-arginina e L-ornitina, os limites de detecção dos aminoácidos por este método foram 114,2, 61,3, 72,7 e 86,7 µmol L -1 . Soluções padrão foram amostrados em um capilar de sílica (50 µm de diâmetro interno e 70 cm de comprimento total) e injeção de 2 psi de pressão por 10 s. A separação ocorreu a 300 V cm -1 , utilizando 1,0 mol L -1 de ácido fórmico em 10% de metanol em água como eletrólito de separação. Aplicação do método para a urina de um paciente diagnosticado clinicamente como portador de cistinúria revelou a presença de 900,5 ± 5, 600,0 ± 2, 700,2 ± 1 e 500,0 ± 3 µmol L -1 de aminoácidos, respectivamente, e 75,3 ± 1 µmol L -1 de creatinina. O método de CE-ESI-MS descrito neste trabalho para a análise de L-cistina e outros aminoácidos associados com cistinúria constitui uma ferramenta para diagnóstico sensível e confiável para caracterização e monitoramento desta doença. Cystinuria is an autosomal recessive genetic disorder characterized by abnormal intestinal and renal tubular transport of L-cystine as well as of L-lysine, L-arginine and L-ornithine. This leads to excessive urinary excretion of amino acids, with the formation of kidney stones caused by the low solubility of L-cystine in the urine. In this study, an analytical method for simultaneous determination of these four amino acids in urine by capillary electrophoresis coupled to electrospray ionization mass spectrometry (CE-ESI-MS) was developed and validated. Using standard solutions of L-cystine, L-lysine, L-arginine and L-ornithine, the amino acid detection limits by this method were 114.2, 61.3, 72.7 and 86.7 µmol L -1 . Standard solutions were injected in a silica capillary column (50 mm i.d. and 70 cm length) under 2 psi of pressure by 10 s. The separation occurred at 300 V cm -1 , using 1.0 mol L -1 formic acid in 10% methanol in water as the background electrolyte. The method was applied to the urine of a patient clinically diagnosed as a cystinuria carrier, which revealed the presence of 900.5 ± 5, 600.0 ± 2, 700.2 ± 1 and 500.0 ± 3 µmol L -1 of amino acid, respectively, and 75.3 ± 1 µmol L -1 of creatinine. The CE-ESI-MS method described here for analyzing L-cystine and other cystinuria-related amino acids is a sensitive and reliable diagnostic tool for characterizing and monitoring this disease. Keywords: cystinuria, CE/ESI-MS, inborn error of metabolism, clinical analysis Barbosa et al

Antioxidant Effects of Quercetin and Catechin Encapsulated into PLGA Nanoparticles

Polymeric nanoparticles (PLGA) have been developed for the encapsulation and controlled release of quercetin and catechin. Nanoparticles were fabricated using a solvent displacement method. Physicochemical properties were measured by light scattering, scanning electron microscopy and ζ-potential, X-ray diffraction, infrared spectroscopy and differential scanning calorimetry. Encapsulation efficiency and in vitro release profiles were obtained from differential pulse voltammetry experiments. Antioxidant properties of free and encapsulated flavonoids were determined by TBARS, fluorescence spectroscopy and standard chelating activity methods. Relatively small (d≈ 400 nm) polymeric nanoparticles were obtained containing quercetin or catechin in a non-crystalline form (EE ≈ 79%) and the main interactions between the polymer and each flavonoid were found to consist of hydrogen bonds. In vitro release profiles were pH-dependant, the more acidic pH, the faster release of each flavonoid from the polymeric nanoparticles. The inhibition of the action of free radicals and chelating properties, were also enhanced when quercetin and catechin were encapsulated within PLGA nanoparticles. The information obtained from this study will facilitate the design and fabrication of polymeric nanoparticles as possible oral delivery systems for encapsulation, protection and controlled release of flavonoids aimed to prevent oxidative stress in human body or food products