Superoxide dismutase: a review and a modified protocol for activities measurements in rat livers.

Superoxide dismutase (SOD) enzymes are considered the first line of Defence against reactive oxygen species. Among the intracellular isoforms of SOD, Cu,ZnSOD is widely distributed, and the MnSOD is localised in the mitochondria. The SOD activities have been measured indirectly by inhibiting oxidation reactions. Most techniques assessing the hepatic SOD activity are adaptations of classical methods and differ significantly from each other. This work assessed the hepatic Cu,ZnSOD activity in the supernatants of two different centrifugations, using two isotonic medium and the pyrogallol method. In most studies conducted in rat liver, only the Cu,Zn or total SOD activities were assessed and sometimes the Cu,ZnSOD activity was calculated based on the inhibition by cyanide. But, as demonstrated here, this inhibition is not complete. Besides, the novelty of this work is that we presented a method for the evaluating the Cu,ZnSOD and MnSOD activities separately and from the same liver.

A Long-term Estrogen Deficiency in Ovariectomized Mice is Associated with Disturbances in Fatty Acid Oxidation and Oxidative Stress.

OBJECTIVE: The aim of this work was to evaluate the changes caused by estrogen deficiency in lipid metabolism. METHODS: This study encompassed direct measurements of plasma biochemical analyses, liver lipid contents, and assessments of the mitochondrial ß-oxidation capacity as well as an evaluation of the liver redox status in an animal model of estrogen deficiency. RESULTS: When compared with control mice, the livers of ovariectomized (OVX) mice presented considerable accretions in their lipid contents, which were accompanied by increased levels of lipid peroxidation in liver homogenates and mitochondria from OVX groups and decreased reduced glutathione (GSH) contents. In isolated mitochondria, estrogen deficiency inhibited mitochondrial ß-oxidation of fatty acids irrespective of their chain length. The liver mitochondrial and peroxisomal H2O2 generations in OVX mice were increased. Additionally, the activities of all antioxidant enzymes assessed were decreased. CONCLUSION: These data provide one potential explanation for the increased susceptibility to metabolic diseases observed after menopause.

OBJETIVO: O objetivo desse trabalho foi avaliar as alterações causadas pela deficiência estrogênica no metabolismo de lipídeos. MéTODOS: Este estudo abrangeu análises bioquímicas plasmáticas, verificação de conteúdo lipídico do fígado e avaliações da capacidade de ß-oxidação mitocondrial e do estado redox do fígado em um modelo animal de deficiência estrogênica. RESULTADOS: Os fígados das camundongas ovariectomizadas (OVXs) apresentaram acréscimos consideráveis no conteúdo de lipídeos, que foram acompanhados por aumento de peroxidação lipídica em homogenatos e mitocôndrias de fígado e diminuição do conteúdo de glutationa reduzida (GSH) quando comparadas as camundongas do grupo controle. Nas mitocôndrias isoladas, a deficiência estrogênica causou a inibição da ß-oxidação mitocondrial independentemente do comprimento da cadeia dos ácidos graxos. A geração mitocondrial e peroxissomal de H2O2 apresentou-se aumentada em camundongas OVXs. Além disso, as atividades de todas as enzimas antioxidantes avaliadas foram diminuídas. CONCLUSãO: Esses dados fornecem uma explicação potencial para o aumento da suscetibilidade às doenças metabólicas observadas após a menopausa.

A long-term estrogen deficiency in ovariectomized mice is associated with disturbances in fatty acid oxidation and oxidative stress / Deficiência de estrogênio em longo prazo em camundongas ovarietcomizadas está associada a distúrbios na oxidação de ácidos graxos e estresse oxidativo

Abstract Objective The aim of this work was to evaluate the changes caused by estrogen deficiency in lipid metabolism. Methods This study encompassed direct measurements of plasma biochemical analyses, liver lipid contents, and assessments of the mitochondrial β-oxidation capacity as well as an evaluation of the liver redox status in an animal model of estrogen deficiency. Results When compared with control mice, the livers of ovariectomized (OVX) mice presented considerable accretions in their lipid contents, which were accompanied by increased levels of lipid peroxidation in liver homogenates andmitochondria from OVX groups and decreased reduced glutathione (GSH) contents. In isolated mitochondria, estrogen deficiency inhibited mitochondrial β-oxidation of fatty acids irrespective of their chain length. The liver mitochondrial and peroxisomal H2O2 generations in OVX mice were increased. Additionally, the activities of all antioxidant enzymes assessed were decreased. Conclusion These data provide one potential explanation for the increased susceptibility to metabolic diseases observed after menopause.

Resumo Objetivo O objetivo desse trabalho foi avaliar as alterações causadas pela deficiência estrogênica no metabolismo de lipídeos. Métodos Este estudo abrangeu análises bioquímicas plasmáticas, verificação de conteúdo lipídico do fígado e avaliações da capacidade de β-oxidação mitocondrial e do estado redox do fígado em um modelo animal de deficiência estrogênica. Resultados Os fígados das camundongas ovariectomizadas (OVXs) apresentaram acréscimos consideráveis no conteúdo de lipídeos, que foram acompanhados por aumento de peroxidação lipídica em homogenatos e mitocôndrias de fígado e diminuição do conteúdo de glutationa reduzida (GSH) quando comparadas as camundongas do grupo controle. Nas mitocôndrias isoladas, a deficiência estrogênica causou a inibição da β-oxidação mitocondrial independentemente do comprimento da cadeia dos ácidos graxos. A geração mitocondrial e peroxissomal de H2O2 apresentou-se aumentada em camundongas OVXs. Além disso, as atividades de todas as enzimas antioxidantes avaliadas foram diminuídas. Conclusão Esses dados fornecem uma explicação potencial para o aumento da suscetibilidade às doenças metabólicas observadas após a menopausa.

Analytical methods for evaluation of the fatty acid metabolism in rat liver / Métodos analíticos para avaliação do metabolismo em ratos hepáticos

The liver is an essential organ for body energy homeostasis, controlling the biosynthesis, uptake and the disposal of carbohydrates and lipids. The hepatic steatosis is a common condition frequently associated with metabolic diseases and is characterized by the excessive accumulation of triglycerides in the liver. In recent years, many efforts have been devoted to prevent and treat the hepatic steatosis, but it remains being pointed out as the major cause for chronic hepatic diseases in Western countries. A considerable part of the knowledge about the physiopathology of hepatic steatosis, the effects of diets and drugs on the metabolic capacity of the liver to metabolize fatty acids, as well as the potential therapeutic approaches for hepatic steatosis derived from experimental animal models using rodents. Here, in this article, we present the details of some of the most common techniques used to evaluate fatty acid metabolism in liver of rats, including quantification of total lipid content, measurement of fatty acid oxidation in isolated subcellular fractions and procedures to measure the activities of important lipogenic enzymes. Classical protocols previously described to be performed using samples from other tissues were adapted to liver samples and different techniques with equivalent aims were compared. The principles and the advantages in terms of reliability and costs were discussed and the procedures here described can be applied for a low-cost broad evaluation of the fatty acid metabolism in liver of rats submitted to different experimental conditions.

O fígado é um órgão essencial para a homeostase energética, controlando a biossíntese, a captação e a eliminação de carboidratos e lipídios. A esteatose hepática é uma condição frequentemente associada a doenças metabólicas e é caracterizada pelo acúmulo excessivo de triacilgliceróis no fígado. Nos últimos anos, muitos esforços têm sido dedicados para prevenir e tratar a esteatose hepática, mas essa condição continua sendo apontada como a principal causa de doenças hepáticas crônicas em países ocidentais. Uma parte considerável do conhecimento sobre a fisiopatologia da esteatose hepática, sobre os efeitos de dietas e drogas na capacidade metabólica do fígado em metabolizar ácidos graxos, bem como sobre as possíveis abordagens terapêuticas para a esteatose hepática, derivam de estudos com modelos animais experimentais usando roedores. Neste artigo, apresentamos os detalhes de algumas das técnicas que podem ser usadas para avaliar o metabolismo de ácidos graxos no fígado de ratos, incluindo a quantificação do conteúdo lipídico total, medida da oxidação de ácidos graxos em frações subcelulares isoladas e procedimentos para medir as atividades de importantes enzimas lipogênicas. Protocolos clássicos previamente descritos para serem realizados utilizando amostras de outros tecidos foram adaptados para amostras de fígado e diferentes técnicas com objetivos equivalentes foram comparadas. Os princípios e as vantagens em termos de confiabilidade e custos foram discutidos e os procedimentos aqui descritos podem ser aplicados para uma avaliação ampla e de baixo custo do metabolismo de ácidos graxos no fígado de ratos submetidos a diferentes condições experimentais.

Vitex agnus-castus L. (Verbenaceae) Improves the Liver Lipid Metabolism and Redox State of Ovariectomized Rats.

Vitex agnus-castus (VAC) is a plant that has recently been used to treat the symptoms of menopause, by its actions on the central nervous system. However, little is known about its actions on disturbances in lipid metabolism and nonalcoholic fat liver disease (NAFLD), frequently associated with menopause. Ovariectomized (OVX) rats exhibit increased adiposity and NAFLD 13 weeks after ovary removal and were used as animal models of estrogen deficiency. The rats were treated with crude extract (CE) and a butanolic fraction of VAC (ButF) and displayed the beneficial effects of a reduction in the adiposity index and a complete reversion of NAFLD. NAFLD reversion was accompanied by a general improvement in the liver redox status. The activities of some antioxidant enzymes were restored and the mitochondrial hydrogen peroxide production was significantly reduced in animals treated with CE and the ButF. It can be concluded that the CE and ButF from Vitex agnus-castus were effective in preventing NAFLD and oxidative stress, which are frequent causes of abnormal liver functions in the postmenopausal period.