Inhibition of LDL oxidation and inflammasome assembly by nitroaliphatic derivatives. Potential use as anti-inflammatory and anti-atherogenic agents.

We have previously shown the antioxidant and anti-inflammatory properties of several para-substituted arylnitroalkenes. Since oxidative stress and inflammation are key processes that drive the initiation and progression of atherosclerosis, in the present work the antioxidant, anti-inflammatory and anti-atherogenic properties of an extended library of aryl-nitroaliphatic derivatives, including several newly designed nitroalkanes, was explored. The antioxidant capacity of the nitroaliphatic compounds, measured using the oxygen radical absorbance capacity assay (ORAC) showed that the p-methylthiophenyl-derivatives were about three times more effective than Trolox to prevent fluorescein oxidation, independently of the presence or the absence of the double bond next to the nitro group. The peroxyl radical scavenger capacity of the p-dimethylaminophenyl-derivatives was even higher, being the reduced form of these compounds even more active. In fact, while the antioxidant capacity of 1-dimethylamino-4-(2-nitro-1Z-ethenyl)benzene and 1-dimethylamino-4-(2-nitro-1Z-propenyl)benzene was 4.2 ±â€¯0.1 and 5.4 ±â€¯0.1 Trolox Eq/mol, respectively; ORAC values obtained with the ethyl and the propyl derivatives were 10 ±â€¯1 and 13 ±â€¯2 Trolox Eq/mol, respectively. The p-dimethylamino-derivatives, especially the nitroalkanes, were also able to prevent LDL oxidation mediated by peroxyl radicals. Oxygen consumption due to the oxidation of fatty acids was delayed in the presence of the dimethylamino substituted compounds, only the alkanes interrupted the chain of lipid oxidations decreasing the rate of oxygen consumption. Although the formation of foam cells in the presence of oxidized-LDL (oxLDL) remained unaffected, the molecules containing the dimethylamino moiety were able to decrease the expression of IL-1ß in LPS/INF-γ challenged macrophages.

Nivel de calprotectina fecal en niños sanos menores de 4 años

Introducción: la calprotectina es una proteína del citoplasma de neutrófilos, con propiedades bacteriostáticas. Aumenta en materia fecal en procesos inflamatorios de la mucosa intestinal, siendo un marcador de inflamación. Su cuantificación es un método no invasivo y estable en su procesamiento. Presenta una alta sensibilidad para diferenciar entre enfermedad orgánica y funcional y tiene alto valor predictivo positivo para hallazgo de lesiones a nivel endoscópico. Presenta baja especificidad para diferenciar la etiología de la inflamación. El nivel de calprotectina fecal (CF) es variable de acuerdo a la edad. En niños menores de 4 años se han encontrado concentraciones mayores que en otras edades. Esto se puede explicar por factores como: mayor migración de neutrófilos en la mucosa durante el desarrollo de tolerancia oral, regulación de la microbiota intestinal, inmadurez de la barrera epitelial y el ambiente que rodea al niño con permanente estímulo para su sistema inmunológico. Por lo que no se ha podido establecer el cut off en esta franja etaria. Objetivo: conocer los valores de la CF en niños sanos menores de 4 años para determinar valor normal o cut off. Determinarlo según grupos etarios y por sexo. Método: estudio descriptivo con componente analítico. Se obtiene la muestra de los niños que realizan el coproparasitario para el ingreso escolar. Catalogados como sanos: sin antecedentes personales de enfermedad crónica, sin síntomas por lo menos cuatro semanas anteriores a la toma de la muestra. Buen crecimiento según las curvas OMS. Distribuidos en diferentes regiones geográficas de la zona metropolitana de Montevideo. Se utilizó el kit RIDASCREEN®. Resultados: fueron analizadas 155 muestras; 73 de sexo femenino y 82 sexo masculino. Se distribuyeron en grupos etarios: menor de 1 año (n=11); 1 a 2 años (n=36); 2 a 3 años (n=45); 3 a 4 años (n=63). La mediana fue de 152,2 mg/kg (percentil 50). Rango de 4,1 a 1944 mg/kg. Nuestro cut off fue de 954 mg/kg (percentil 95). Se aplica Wilcoxon test p=0,68 y test Kruskal Wallis p=0,06, sin diferencias significativas entre sexo y grupos etarios respectivamente. Conclusiones: es el primer estudio de CF en una población de niños sanos menores de 4 años en Latinoamérica. La mediana encontrada es mayor que la habitual en niños mayores de 4 años y adultos. Obtuvimos un límite normal de 954 mg/kg. No se encontró diferencia en la concentración según sexo o rango etario. Limitaciones de nuestro estudio: el tamaño y la obtención de la muestra corresponde solamente al departamento de Montevideo.

Summary: Introduction: calprotectin is a protein with bacteriostatic properties, found in cytoplasmic neutrophils. Concentration in fecal matter increases in inflammatory processes of the intestinal mucous membrane, being it an inflammation marker. Quantification involves a non-invasive method, stable during processing. Fecal calprotectin concentration is highly sensitive to differentiate between organic and functional disease and has a high positive predictive value for the finding of endoscopic lesions. It evidences low specificity to distinguish the inflammation's etiology. Fecal calprotectin concentration (FCC) varies according to age. Higher concentrations have been found in children younger than 4 years old, what may be explained by the following factors: greater migration of neutrophils in the mucous during the development of oral tolerance, regulation of gut microbiota, non-mature epithelial tissue and the context the child grows in as a permanent stimulation for his immune system. Thus, cut off has not been established yet for this age group. Objective: to learn about FCC in healthy children younger than 4 years old to determine normal values or cut off. To determine values according to age and sex. Method: descriptive study with an analytic component. A sample of the children who undergo a coproparsitary test upon entering school is taken. A healthy child is regarded to: have no personal history of chronic disease, no symptoms at least in the 4 weeks prior to the sample being taken. Good growth is defined as per the WHO curves. Samples included came from different regions in the Montevideo metropolitan area. The RIDASCREEN® kit was used. Results: 155 samples were analysed, 73 of them corresponding to boys and 82 to girls. As to age: younger than 1 year old (n= 11), from 1 to 2 years old (n= 36), from 2 to 3 years old (n= 45), from 3 to 4 years old (n= 63). Median concentration was 152.2 mg/kg (Percentile 50). Range 4.1 to 1944 mg/kg. Our cut off was 954 mg/kg (percentile 95). Wolcoxon test was applied (p= 0.68) and Kruskal Wallis test (p=0.06), no significant differences were found between sex and age groups respectively. We obtained a normal limit of 954 mg/Kg. No difference was found in the concentration for different sex or age. Limitations of our study result from the size of the sample and the fact that all samples correspond to Montevideo.

Introdução: a calprotectina é uma proteína neutrofílica citoplasmática com propriedades bacteriostáticas. Ela aumenta na matéria fecal durante os processos inflamatórios da mucosa intestinal, e é um marcador de inflamação. Pode ser quantificada utilizando um método não invasivo, estável em seu processamento. Apresenta alta sensibilidade para diferenciar entre doença orgânica e funcional e tem alto valor preditivo positivo para encontrar lesões no nível endoscópico. Apresenta baixa especificidade para diferenciar a etiologia da inflamação. O nível de calprotectina fecal (CF) varia de acordo com a idade, e para o caso de crianças com menos de 4 anos ela teve concentrações mais elevadas, o que pode ser explicado por fatores tais como o aumento da migração de neutrófilos na mucosa durante o desenvolvimento de tolerância oral, regulação da microbiota intestinal, imaturidade da barreira epitelial e ambiente que envolve a criança como estímulo permanente do sistema imunológico. Portanto, não foi possível estabelecer o corte nesta faixa etária. Objetivo: conhecer os valores da FC em crianças saudáveis menores de 4 anos para determinar o valor normal ou o de corte de acordo com as faixas etárias e sexo. Método: estudo descritivo com componente analítico. A amostra foi obtida das crianças que realizaram o estudo coproparasitológico para a admissão escolar. Crianças catalogadas como saudáveis: sem histórico de doença crônica, sem sintomas durante pelo menos 4 semanas antes de experimentar a amostra. Bom crescimento: de acordo com as curvas da OMS. Distribuído em diferentes regiões geográficas da área metropolitana de Montevidéu. Se utilizou o kit RIDASCREEN®. Resultados: foram analisadas 155 amostras: 73 de mulheres e 82 de homens distribuídos em grupos etários: com menos de 1 ano (n = 11), 1 a 2 anos (n = 36), 2 a 3 anos (n = 45), 3 a 4 anos (n = 63). A mediana foi de 152,2 mg / kg (percentil 50). Faixa de 4,1 a 1944 mg / kg. Nosso corte foi 954mg / Kg (percentil 95). Aplicou-se o teste de Wilcoxon p = 0,68 e o teste de Kruskal Wallis p = 0,06, e não observamos diferenças significativas entre os grupos por sexo ou faixa etária, respectivamente. Conclusões: este foi o primeiro estudo de FC numa população de crianças saudáveis com menos de 4 anos de idade na América Latina. A mediana encontrada é maior que a usual em crianças com mais de 4 anos e em adultos. Obtivemos um limite normal de 954mg / Kg. Nenhuma diferença foi encontrada na concentração dependendo do sexo ou faixa etária. As limitações do nosso estudo são o tamanho da amostra e o fato de que a amostra foi obtida apenas de Montevidéu.

Arylnitroalkenes as scavengers of macrophage-generated oxidants.

Oxygen and nitrogen derived molecules mediated oxidation and nitration have been involved in several pathological conditions. Conversely, nitric oxide and hydrogen peroxide are important signalization intermediates, whose concentrations are tightly regulated by specialized enzyme repertoires and should remain undisturbed by the addition of exogenous antioxidant molecules, as already demonstrated by intervention studies with antioxidant vitamins. Our goal was to develop specific antioxidants able to scavenge peroxynitrite anion, as well the radicals derived from the homolytic decomposition of its conjugated acid, nitrogen dioxide and hydroxyl radical. Fourteen substituted nitroalkenes, seven 4-substituted 1-(2-nitro-1Z-ethenyl)benzene, and seven 4-substituted (2-nitro-1Z-propenyl)benzene, with different stereochemical and electronic characteristics were synthesized and tested. Compounds with the electron donor group N,N-dimethylamino showed the highest reaction rates against peroxynitrite, and also reacted with its homolytic decomposition products, OH and NO2. While 1,1-dimethylamino-4-(2-nitro-1Z-ethenyl)benzene came up as a lead for future developments without the risk of interfering with signalization pathways, since it was highly specific for peroxynitrite and peroxynitrite derived radicals, its methylated analogous 1,1-dimethylamino-4-(2-nitro-1Z-propenyl)benzene was less specific and also reacted with NO and O2(-), the biological precursor of H2O2.

Analysis of two methods to evaluate antioxidants.

This exercise is intended to introduce undergraduate biochemistry students to the analysis of antioxidants as a biotechnological tool. In addition, some statistical resources will also be used and discussed. Antioxidants play an important metabolic role, preventing oxidative stress-mediated cell and tissue injury. Knowing the antioxidant content of nutritional components can help make informed decisions about diet design, and increase the commercial value of antioxidant-rich natural products. As a reliable and convenient technique to evaluate the whole spectrum of antioxidants present in biological samples is lacking, the general consensus is to use more than one technique. We have chosen two widely used and inexpensive methods, Trolox-equivalent antioxidant capacity and the ferric reducing antioxidant power assays, to evaluate the antioxidant content of several fruits, and to compare and analyze the correlation between both assays.