RESUMO
Macrophage activation plays a central role in the development of atherosclerotic plaques. Interaction with oxidized low-density lipoprotein (oxLDL) leads to macrophage differentiation into foam cells and oxylipin production, contributing to plaque formation. 7-Ketocholesterol (7KC) is an oxidative byproduct of cholesterol found in oxLDL particles and is considered a factor contributing to plaque progression. During atherosclerotic lesion regression or stabilization, macrophages undergo a transformation from a pro-inflammatory phenotype to a reparative anti-inflammatory state. Interleukin-10 (IL-10) and PGE1 appear to be crucial in resolving both acute and chronic inflammatory processes. After coffee consumption, the gut microbiota processes non-absorbed chlorogenic acids producing various lower size phenolic acids. These colonic catabolites, including dihydroferulic acid (DHFA), may exert various local and systemic effects. We focused on DHFA's impact on inflammation and oxidative stress in THP-1 macrophages exposed to oxLDL, 7KC, and lipopolysaccharides (LPS). Our findings reveal that DHFA inhibits the release of several pro-inflammatory mediators induced by LPS in macrophages, such as CCL-2, CCL-3, CCL-5, TNF-α, IL-6, and IL-17. Furthermore, DHFA reduces IL-18 and IL-1ß secretion in an inflammasome-like model. DHFA demonstrated additional benefits: it decreased oxLDL uptake and CD36 expression induced by oxLDL, regulated reactive oxygen species (ROS) and 8-isoprostane secretion (indicating oxidative stress modulation), and selectively increased IL-10 and PGE1 levels in the presence of inflammatory stimuli (LPS and 7KC). Finally, our study highlights the pivotal role of PGE1 in foam cell inhibition and inflammation regulation within activated macrophages. This study highlights DHFA's potential as an antioxidant and anti-inflammatory agent, particularly due to its ability to induce PGE1 and IL-10.
Assuntos
Ácidos Cumáricos , Cetocolesteróis , Lipopolissacarídeos , Lipoproteínas LDL , Macrófagos , Humanos , Macrófagos/efeitos dos fármacos , Macrófagos/metabolismo , Lipoproteínas LDL/metabolismo , Lipopolissacarídeos/farmacologia , Cetocolesteróis/farmacologia , Ácidos Cumáricos/farmacologia , Anti-Inflamatórios/farmacologia , Polifenóis/farmacologia , Ativação de Macrófagos/efeitos dos fármacos , Colo/metabolismo , Colo/efeitos dos fármacos , Interleucina-10/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Mediadores da Inflamação/metabolismoRESUMO
7-Ketocholesterol (7-KC) is a cholesterol oxidation product with several biological functions. 7-KC has the capacity to cause cell death depending on the concentration and specific cell type. Mesenchymal stem cells (MSCs) are multipotent cells with the ability to differentiate into various types of cells, such as osteoblasts and adipocytes, among others. MSCs contribute to the development of a suitable niche for hematopoietic stem cells, and are involved in the development of diseases, such as leukemia, to a yet unknown extent. Here, we describe the effect of 7-KC on the death of bone marrow MSCs from patients with acute myeloid leukemia (LMSCs). LMSCs were less susceptible to the death-promoting effect of 7-KC than other cell types. 7-KC exposure triggered the extrinsic pathway of apoptosis with an increase in activated caspase-8 and caspase-3 activity. Mechanisms other than caspase-dependent pathways were involved. 7-KC increased ROS generation by LMSCs, which was related to decreased cell viability. 7-KC also led to disruption of the cytoskeleton of LMSCs, increased the number of cells in S phase, and decreased the number of cells in the G1/S transition. Autophagosome accumulation was also observed. 7-KC downregulated the SHh protein in LMSCs but did not change the expression of SMO. In conclusion, oxiapoptophagy (OXIdative stress + APOPTOsis + autophagy) seems to be activated by 7-KC in LMSCs. More studies are needed to better understand the role of 7-KC in the death of LMSCs and the possible effects on the SHh pathway.
Assuntos
Apoptose/efeitos dos fármacos , Autofagia/efeitos dos fármacos , Cetocolesteróis/farmacologia , Leucemia Mieloide Aguda/patologia , Células-Tronco Mesenquimais/metabolismo , Estresse Oxidativo/efeitos dos fármacos , Autofagossomos/metabolismo , Caspase 3/metabolismo , Caspase 8/metabolismo , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Proteínas Hedgehog/metabolismo , Humanos , Potencial da Membrana Mitocondrial/efeitos dos fármacos , Espécies Reativas de Oxigênio/metabolismo , Receptor Smoothened/metabolismoRESUMO
Oxysterol-binding proteins (OSBPs) comprise a family of sterol-binding proteins. In this study, we focused on AoOSBP1, one of the five OSBP proteins identified from the industrial fungus Aspergillus oryzae. The temporal expression pattern analysis showed that the expression of AoOSBP1, in both gene and protein levels, was stably expressed throughout the developmental stages, while was upregulated during the accelerated growth stage. The immunofluorescence observation revealed that AoOSBP1 protein was mainly distributed in the conidiophore, indicating its underlying role in spore formation. The ligand-binding domain of AoOSBP1, namely OSBP-related domain (ORD), was heterologously expressed in Escherichia coli and purified. The binding assay carried out using microscale thermophoresis showed that the recombinant AoORD protein exhibited binding affinity for ergosterol, and exhibited much higher affinity to oxysterols (25-hydroxycholesterol and 7-ketocholesterol) and phytosterols (ß-sitosterol and stigmasterol). By contrast, MBP tag as the negative control showed no binding affinity for sterols. The present work demonstrates that AoORD domain in AoOSBP1 is capable of binding sterols, plays an underlying role in sterols transportation, and may participate in spore formation.
Assuntos
Aspergillus oryzae/metabolismo , Transporte Biológico/fisiologia , Proteínas de Transporte/metabolismo , Receptores de Esteroides/metabolismo , Esporos Fúngicos/crescimento & desenvolvimento , Ergosterol/metabolismo , Expressão Gênica , Hidroxicolesteróis/metabolismo , Cetocolesteróis/metabolismo , Ligação Proteica/fisiologia , Domínios Proteicos/fisiologia , Estigmasterol/metabolismoRESUMO
Oxysterols are cholesterol oxidation products formed through enzymatic or autoxidation mechanisms. 7-ketocholeterol (7KC) is one of most abundant oxysterols found in atherosclerotic lesions. Its role in atherosclerosis pathogenesis has been broadly studied in a variety of models. The arterial microenvironment is a multicellular dynamic compartment that, among other systemic factors, is continuously stimulated by 7KC. Endothelial cells have a key role on that environment, being in intimate contact with both the blood stream and the vessel wall, the site of disease origin. 7KC has been shown to promote endothelial cell death and/or dysfunction, depending on its concentration. However, its contribution to the cell microenvironment through cell stimulation has not received much attention. Here we applied mass spectrometry-based proteomics followed by bioinformatics workflow to analyze the effect of a non-toxic 7KC concentration on endothelial cell protein expression and secretion in vitro. Trypsin digests were prepared from the secretome of the endothelial cells and from the total cell pellet after 24h exposure to 7KC. All samples were analyzed by high resolution and accurate mass nano-LC MS/MS. After database search and statistical analysis, differentially expressed proteins were selected for further studies. Our workflow identified 1805 secreted proteins and 2203 intracellular proteins, and of these, 48 and 53, respectively, were regulated. Regulated proteins upon 7KC exposure are involved in unfolded protein response, vascular homeostasis, and reduced control of angiogenesis. Moreover, blood coagulation was another main pathway regulated through Tissue Factor Pathway Inhibitor (TFPI), an antithrombotic agent associated with coronary disease that we found to be more than 2 times downregulated. Taken together, these data show differential endothelial protein regulation and secretion upon 7KC exposure for short time periods under non-toxic conditions. Herewith, these data support the role of 7KC in atherosclerosis pathophysiology and thus reinforce the deleterious effect of endothelial cells stress in the arterial microenvironment.
Assuntos
Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Cetocolesteróis/farmacologia , Estresse Oxidativo/efeitos dos fármacos , Proteômica , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Biologia Computacional , Relação Dose-Resposta a Droga , Humanos , Espectrometria de Massas , Agregação Plaquetária/efeitos dos fármacos , Relação Estrutura-AtividadeRESUMO
Mesenchymal stem cells (MSCs) are multipotent cells characterized by self-renewal and cellular differentiation capabilities. Oxysterols comprise a very heterogeneous group derived from cholesterol through enzymatic and non-enzymatic oxidation. Potent effects in cell death processes, including cytoxicity and apoptosis induction, were described in several cell lines. Very little is known about the effects of oxysterols in MSCs. 7-ketocholesterol (7-KC), one of the most important oxysterols, was shown to be cytotoxic to human adipose tissue-derived MSCs. Here, we describe the short-term (24h) cytotoxic effects of cholestan-3α-5ß-6α-triol, 3,5 cholestan-7-one, (3α-5ß-6α)- cholestane-3,6-diol, 7-oxocholest-5-en-3ß-yl acetate, and 5ß-6ß epoxy-cholesterol, on MSCs derived from human adipose tissue. MSCs were isolated from adipose tissue obtained from three young, healthy women. Oxysterols, with the exception of 3,5 cholestan-7-one and 7-oxocholest-5-en-3ß-yl acetate, led to a complex mode of cell death that include apoptosis, necrosis and autophagy, depending on the type of oxysterol and concentration, being cholestan-3α-5ß-6α-triol the most effective. Inhibition of proliferation was also promoted by these oxysterols, but no changes in cell cycle were observed.
Assuntos
Tecido Adiposo/citologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Oxisteróis/farmacologia , Actinas/metabolismo , Adulto , Apoptose , Autofagia , Caspase 3/metabolismo , Caspase 7/metabolismo , Ciclo Celular , Proliferação de Células , Sobrevivência Celular , Colestanos/farmacologia , Feminino , Técnica Indireta de Fluorescência para Anticorpo , Humanos , Cetocolesteróis/farmacologia , Potenciais da Membrana , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Necrose , OxirreduçãoRESUMO
Chronic myeloid leukemia (CML) is a myeloproliferative disease with a characteristic BCR-ABL tyrosine kinase (TK) fusion protein. Despite the clinical efficacy accomplished by TKIs therapies, disease progression may affect patient response rate to these inhibitors due to a multitude of factors that could lead to development of a mechanism known as multidrug resistance (MDR). 7-Ketocholesterol (7KC) is an oxidized cholesterol derivative that has been extensively reported to cause cell death in a variety of cancer models. In this study, we showed the in vitro efficacy of 7KC against MDR leukemia cell line, Lucena. 7KC treatment induced reduction in cell viability, together with apoptosis-mediated cell death. Moreover, downregulation of MDR protein caused intracellular drug accumulation and 7KC co-incubation with either Daunorubicin or Vincristine reduced cell viability compared to the use of each drug alone. Additionally, quantitative label-free mass spectrometry-based protein quantification showed alteration of different molecular pathways involved in cell cycle arrest, induction of apoptosis and misfolded protein response. Conclusively, this study highlights the effect of 7KC as a sensitizing agent of multidrug resistance CML and elucidates its molecular mechanisms. SIGNIFICANCE: CML patients treated with tyrosine kinase inhibitors (TKIs) have showed a 5-year estimated overall survival of 89%, with cumulative complete cytogenetic response of 87%. However, development of drug resistance is a common feature of the disease progression. This study aimed at showing the effect of 7KC as a cytotoxic and sensitizing agent of multidrug resistance CML cell lines. The cellular and molecular basis of this compound were elucidated using a comprehensive strategy based on quantitative proteomic and cell biology assays. We showed that 7KC induced cell death and overcomes drug resistance in CML through mechanisms that go beyond the classical MDR1 pathways.
Assuntos
Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Cetocolesteróis/farmacologia , Leucemia Mielogênica Crônica BCR-ABL Positiva/tratamento farmacológico , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Apoptose/efeitos dos fármacos , Pontos de Checagem do Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Daunorrubicina/uso terapêutico , Sinergismo Farmacológico , Humanos , Cetocolesteróis/uso terapêutico , Proteômica/métodos , Deficiências na Proteostase/metabolismo , Vincristina/uso terapêuticoRESUMO
Oxysterols comprise a very heterogeneous group derived from cholesterol through enzymatic and non-enzymatic oxidation. Among them, 7-ketocholesterol (7-KC) is one of the most important. It has potent effects in cell death processes, including cytoxicity and apoptosis induction. Mesenchymal stem cells (MSCs) are multipotent cells characterized by self-renewal and cellular differentiation capabilities. Very little is known about the effects of oxysterols in MSCs. Here, we describe the short-term cytotoxic effect of 7-ketocholesterol on MSCs derived from human adipose tissue. MSCs were isolated from adipose tissue obtained from two young, healthy women. After 24 h incubation with 7-KC, mitochondrial hyperpolarization was observed, followed by a slight increase in the level of apoptosis and changes in actin organization. Finally, the IC50 of 7-KC was higher in these cells than has been observed or described in other normal or cancer cell lines.
Assuntos
Tecido Adiposo/citologia , Cetocolesteróis/farmacologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Actinas/metabolismo , Tecido Adiposo/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Células Cultivadas , Relação Dose-Resposta a Droga , Feminino , Humanos , Adulto JovemRESUMO
Oxysterols are structurally similar to cholesterol, but are characterized by one or more additional oxygen-containing functional groups. These compounds are implicated in inflammation given their ability to cause irreversible damage to vascular cells. The aim of this study was to study the alteration of some inflammatory biomarkers in Wistar rats in response to dietary oxysterols. Eighteen rats were randomly divided into three groups of six rats each. A standard diet supplemented with 1% (w/w) pure cholesterol (Chol group) or 1% (w/w) of an oxidized cholesterol mixture (COPs group) was fed for 8 weeks. Blood serum was separated; abdominal, pericardial, and epididymal adipose tissue was removed carefully. The COPs subjects exhibited significant increase in blood pressure and serum triacylgycerols as well as increased body fat index and pericardic, abdominal, and epididymal adipose tissue. These effects were accompanied by elevated circulating levels of plasma high-sensitivity C-reactive protein, tumor necrosis factor alpha, and resistin. We suggest that dietary oxysterols have an important pro-inflammatory effect.
Assuntos
Colesterol na Dieta/análogos & derivados , Colesterol na Dieta/administração & dosagem , Hidroxicolesteróis/administração & dosagem , Inflamação/induzido quimicamente , Cetocolesteróis/administração & dosagem , Tecido Adiposo/efeitos dos fármacos , Tecido Adiposo/metabolismo , Animais , Biomarcadores/análise , Pressão Sanguínea/efeitos dos fármacos , Proteína C-Reativa/análise , Colesterol na Dieta/sangue , Distribuição Aleatória , Ratos , Ratos Wistar , Resistina/sangue , Triglicerídeos/sangue , Fator de Necrose Tumoral alfa/sangueRESUMO
BACKGROUND: Advanced glycation end products (AGE) alter lipid metabolism and reduce the macrophage expression of ABCA-1 and ABCG-1 which impairs the reverse cholesterol transport, a system that drives cholesterol from arterial wall macrophages to the liver, allowing its excretion into the bile and feces. Oxysterols favors lipid homeostasis in macrophages and drive the reverse cholesterol transport, although the accumulation of 7-ketocholesterol, 7alpha- hydroxycholesterol and 7beta- hydroxycholesterol is related to atherogenesis and cell death. We evaluated the effect of glycolaldehyde treatment (GAD; oxoaldehyde that induces a fast formation of intracellular AGE) in macrophages overloaded with oxidized LDL and incubated with HDL alone or HDL plus LXR agonist (T0901317) in: 1) the intracellular content of oxysterols and total sterols and 2) the contents of ABCA-1 and ABCG-1. METHODS: Total cholesterol and oxysterol subspecies were determined by gas chromatography/mass spectrometry and HDL receptors content by immunoblot. RESULTS: In control macrophages (C), incubation with HDL or HDL + T0901317 reduced the intracellular content of total sterols (total cholesterol + oxysterols), cholesterol and 7-ketocholesterol, which was not observed in GAD macrophages. In all experimental conditions no changes were found in the intracellular content of other oxysterol subspecies comparing C and GAD macrophages. GAD macrophages presented a 45% reduction in ABCA-1 protein level as compared to C cells, even after the addition of HDL or HDL + T0901317. The content of ABCG-1 was 36.6% reduced in GAD macrophages in the presence of HDL as compared to C macrophages. CONCLUSION: In macrophages overloaded with oxidized LDL, glycolaldehyde treatment reduces the HDL-mediated cholesterol and 7-ketocholesterol efflux which is ascribed to the reduction in ABCA-1 and ABCG-1 protein level. This may contribute to atherosclerosis in diabetes mellitus.
Assuntos
Transportadores de Cassetes de Ligação de ATP/metabolismo , Regulação para Baixo , Produtos Finais de Glicação Avançada/metabolismo , Cetocolesteróis/metabolismo , Lipoproteínas/metabolismo , Macrófagos/metabolismo , Esteróis/metabolismo , Transportador 1 de Cassete de Ligação de ATP , Membro 1 da Subfamília G de Transportadores de Cassetes de Ligação de ATP , Acetaldeído/análogos & derivados , Acetaldeído/farmacologia , Animais , Anticolesterolemiantes/farmacologia , Linhagem Celular , Angiopatias Diabéticas/imunologia , Angiopatias Diabéticas/metabolismo , Regulação para Baixo/efeitos dos fármacos , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/metabolismo , Receptores X do Fígado , Macrófagos/efeitos dos fármacos , Macrófagos/imunologia , Camundongos , Receptores Nucleares Órfãos/agonistas , Oxidantes/farmacologia , Estresse OxidativoRESUMO
UNLABELLED: The effects of the addition of sage and garlic in chicken meat on lipid and cholesterol oxidation, having as prooxidant factors the addition of salt, thermal treatment, and frozen storage, were evaluated. The content of unsaturated fatty acids did not change in the presence of sage; on the contrary, with garlic, the content of these fatty acids decreased after cooking and storage. Hexanal and pentanal contents were lower in patties containing sage, and higher in those with garlic. The 7-ketocholesterol was the cholesterol oxide found in higher amount in raw chicken on day 0, while the formation of 7ß- and 7α-hydroxycholesterol was verified only from day 30 on. Cooking and storage resulted in increase of total cholesterol oxides and decrease of α- and γ-tocopherol. Sage was effective in controlling lipid and cholesterol oxidation, minimizing the prooxidant effects of salt, cooking, and storage. However, garlic presented no effect as antioxidant and accelerated lipid oxidation. PRACTICAL APPLICATION: The addition of sage to chicken meat (0.1 g/100 g) is a good alternative to prevent and delay the formation of compounds derived from lipid oxidation that are responsible for off-flavors and loss of nutritional quality during long-term frozen storage. Care must be taken when using garlic to seasoning chicken meat products, such as hamburgers and meatballs, especially cooked or precooked due to its potential to promote lipid oxidation and consequently raising the risk of having the product rejected by the consumer.
Assuntos
Antioxidantes/química , Galinhas , Gorduras na Dieta/análise , Conservantes de Alimentos/química , Peroxidação de Lipídeos , Produtos da Carne/análise , Salvia officinalis/química , Aldeídos/análise , Animais , Gorduras Insaturadas na Dieta/análise , Alimentos Congelados/análise , Alho/química , Temperatura Alta/efeitos adversos , Hidroxicolesteróis/análise , Hidroxicolesteróis/química , Cetocolesteróis/análise , Cloreto de Sódio na Dieta/análise , Especiarias/efeitos adversos , Especiarias/análise , Estereoisomerismo , Tocoferóis/análise , Tocoferóis/químicaRESUMO
Produtos de glicação avançada (AGE) alteram o metabolismo de lípides e, em especial, o efluxo de colesterol de macrófagos, por meio da redução dos receptores ABCA-1 e ABCG-1. Isto prejudica o transporte reverso de colesterol, sistema que favorece o fluxo de colesterol de macrófagos arteriais ao fígado, permitindo sua excreção na bile e eliminação fecal. Óxidos de colesterol modulam favoravelmente a homeostase lipídica em macrófagos e favorecem o transporte reverso de colesterol, embora o acúmulo de 7-cetocolesterol, 7-hidroxicolesterol e 7-hidroxicolesterol associe-se à aterogênese e morte celular. Neste estudo, avaliou-se o efeito do tratamento com glicolaldeído (GAD; oxoaldeído que induz rápida geração intracelular de AGE), em macrófagos sobrecarregados com LDL oxidada e incubados com HDL ou HDL e indutor de LXR (T0901317) sobre: 1) a distribuição seletiva de óxidos de colesterol e o conteúdo total de esteróis intracelulares e 2) o conteúdo de ABCA-1 e ABCG-1. Colesterol total e os diversos subtipos de óxidos de colesterol foram determinados por cromatografia a gás acoplada à espectrômetro de massa. O conteúdo dos receptores de HDL (ABCA-1 e ABCG-1) foi avaliado por imunoblot. Em macrófagos controles (C), a adição de HDL ou HDL + T0901317 promoveu redução no conteúdo de esteróis totais (colesterol + óxidos de colesterol), colesterol e 7-cetocolesterol. No entanto, isto não foi observado em macrófagos GAD. Nas diversas condições experimentais, não houve diferença no conteúdo intracelular dos outros subtipos de óxidos de colesterol, em células C e GAD. Macrófagos GAD apresentaram menor conteúdo de ABCA-1 (45%), quando comparados aos macrófagos C, mesmo após adição de HDL ou HDL + T0901317. O conteúdo de ABCG-1 foi 36,6% menor em macrófagos GAD, na presença de HDL, em comparação às células C. Em conclusão, em macrófagos sobrecarregados com LDL oxidada, o tratamento com glicolaldeído diminui a exportação celular de colesterol e 7-cetocolesterol...
Advanced glycation end products (AGE) alter lipid metabolism and reduce the macrophage expression of ABCA-1 and ABCG-1 which impairs the reverse cholesterol transport, a system that drives cholesterol from arterial wall macrophages to the liver, allowing its excretion into the bile and feces. Oxysterols favors lipid homeostasis in macrophages and drive the reverse cholesterol transport, although the accumulation of 7-ketocholesterol, 7- hydroxycholesterol and 7- hydroxycholesterol is related to atherogenesis and cell death. We evaluated the effect of glycolaldehyde treatment (GAD; oxoaldehyde that induces a fast formation of intracellular AGE) in macrophages overloaded with oxidized LDL and incubated with HDL alone or HDL plus LXR agonist (T0901317) in: 1) the intracellular content of oxysterols and total sterols and 2) the contents of ABCA-1 and ABCG-1. Total cholesterol and oxysterol subspecies were determined by gas chromatography/mass spectrometry and HDL receptors content by immunoblot. In control macrophages (C), incubation with HDL or HDL + T0901317 reduced the intracellular content of total sterols (total cholesterol + oxysterols), cholesterol and 7-ketocholesterol, which was not observed in GAD macrophages. In all experimental conditions no changes were found in the intracellular content of other oxysterol subspecies comparing C and GAD macrophages. GAD macrophages presented a 45% reduction in ABCA-1 protein level as compared to C cells, even after the addition of HDL or HDL + T0901317. The content of ABCG-1 was 36.6% reduced in GAD macrophages in the presence of HDL as compared to C macrophages. In conclusion, in macrophages overloaded with oxidized LDL, glycolaldehyde treatment reduces the HDL-mediated cholesterol and 7-ketocholesterol efflux which is ascribed to the reduction in ABCA-1 and ABCG-1 protein level. This may contribute to atherosclerosis in diabetes mellitus...
Assuntos
Transportadores de Cassetes de Ligação de ATP , Colesterol , Cetocolesteróis , Macrófagos , Produtos Finais de Glicação AvançadaRESUMO
7-ketocholesterol (7-KC) differs from cholesterol by a functional ketone group at C7. It is an oxygenated cholesterol derivative (oxysterol), commonly present in oxidized low-density lipoprotein (LDL). Oxysterols are generated and participate in several physiologic and pathophysiologic processes. For instance, the cytotoxic effects of oxidized LDL have been widely attributed to bioactive compounds like oxysterols. The toxicity is in part due to 7-KC. Here we aimed to demonstrate the possibility of incorporating 7-KC into the synthetic nanoemulsion LDE, which resembles LDL in composition and behavior. This would provide a suitable artificial particle resembling LDL to study 7-KC metabolism. We were able to incorporate 7-KC in several amounts into LDE. The incorporation was evaluated and confirmed by several methods, including gel filtration chromatography, using radiolabeled lipids. The incorporation did not change the main lipid composition characteristics of the new nanoparticle. Particle sizes were also evaluated and did not differ from LDE. In vivo studies were performed by injecting the nanoemulsion into mice. The plasma kinetics and the targeted organs were the same as described for LDE. Therefore, 7-KC-LDE maintains composition, size and some functional characteristics of LDE and could be used in experiments dealing with 7-ketocholesterol metabolism in lipoproteins.
Assuntos
Cetocolesteróis/química , Lipoproteínas LDL/química , Nanopartículas , Animais , Cromatografia em Gel , Emulsões , Cetocolesteróis/farmacocinética , Lipoproteínas LDL/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Modelos Biológicos , Nanopartículas/químicaRESUMO
7-ketocholesterol (7-KC) differs from cholesterol by a functional ketone group at C7. It is an oxygenated cholesterol derivative (oxysterol), commonly present in oxidized low-density lipoprotein (LDL). Oxysterols are generated and participate in several physiologic and pathophysiologic processes. For instance, the cytotoxic effects of oxidized LDL have been widely attributed to bioactive compounds like oxysterols. The toxicity is in part due to 7-KC. Here we aimed to demonstrate the possibility of incorporating 7-KC into the synthetic nanoemulsion LDE, which resembles LDL in composition and behavior. This would provide a suitable artificial particle resembling LDL to study 7-KC metabolism. We were able to incorpórate 7-KC in several amounts into LDE. The incorporation was evaluated and confirmed by several methods, including gel filtration chromatography, using radiolabeled lipids. The incorporation did not change the main lipid composition characteristics of the new nanoparticle. Particle sizes were also evaluated and did not differ from LDE. In vivo studies were performed by injecting the nanoemulsion into mice. The plasma kinetics and the targeted organs were the same as described for LDE. Therefore, 7-KC-LDE maintains composition, size and some functional characteristics of LDE and could be used in experiments dealing with 7-ketocholesterol metabolism in lipoproteins.
Assuntos
Animais , Camundongos , Cetocolesteróis/química , Lipoproteínas LDL/química , Nanopartículas , Cromatografia em Gel , Emulsões , Cetocolesteróis/farmacocinética , Lipoproteínas LDL/metabolismo , Modelos Biológicos , Nanopartículas/químicaRESUMO
Fresh fillets of Atlantic hake were stored at -18 degrees C for 120 days and changes in lipid composition and the formation of cholesterol oxidation products (COP) during storage and subsequent grilling were evaluated. Fresh hake showed low COP levels (8.0 microg/g, dry basis); however, a significant increase in COP (P < 0.02) and a concomitant decrease in the cholesterol and polyunsaturated fatty acids content during frozen storage and after grilling were observed. The main cholesterol oxides present in the analyzed samples were: 19-Hydroxycholesterol, 24(S)-hydroxycholesterol, 22(S)-hydroxycholesterol, 25-hydroxycholesterol, 25(R)-hydroxycholesterol and 7-Ketocholesterol. The oxides which were more influenced by the thermal treatment were 24(S)-OH and 25(R)-OH; however, after 120 days of storage 7-ketocholesterol was the main product formed. Frozen storage and subsequent grilling under domestic conditions are important factors in damage of cholesterol and unsaturated fatty acids levels, with consequent production of cholesterol oxides, although the mechanism of the formation of these compounds by the different processes is probably different.
Assuntos
Colesterol/análise , Ácidos Graxos Insaturados/análise , Manipulação de Alimentos/métodos , Alimentos Congelados/análise , Carne/análise , Óxidos/análise , Animais , Colesterol/química , Temperatura Baixa , Culinária , Ácidos Graxos Insaturados/química , Peixes , Hidroxicolesteróis/análise , Hidroxicolesteróis/química , Cetocolesteróis/análise , Cetocolesteróis/química , OxirreduçãoRESUMO
BACKGROUND: Oxidative stress plays an important role in the pathophysiology of diabetes mellitus. The aim of this study was to evaluate the formation of cholesterol oxides (ChOx) as biomarkers of oxidative stress in subjects with impaired glucose tolerance (IGT) and diabetes. METHODS: Blood plasma levels of cholesterol oxidation products were determined in the following groups: type 1 diabetes mellitus (DM1), type 2 diabetes (DM2), impaired glucose tolerance (IGT), children without diabetes (C1) and adults without diabetes (C2). The serum levels of cholest-5-ene-3alpha,7alpha-diol (7alpha-hydroxycholesterol, 7alpha-OH), cholest-5-ene-3beta,7beta-diol (7beta-hydroxycholesterol, 7beta-OH), 3beta-hydroxycholest-5-7-one (7-ketocholesterol, 7-K), 5alpha-cholestane-3beta,5,6beta-triol (cholestanetriol), 5,6alpha-epoxy-5alpha-cholestan-3alpha-ol (cholesterol-5alpha,6alpha-epoxide,), 5,6beta-epoxy-5beta-cholestan-3beta-ol (cholesterol-5beta,6beta-epoxide) and cholest-5-eno-3beta,25-diol (25-hydroxycholesterol, 25-OH) (trivial name and abbreviations indicated in parentheses) were quantified by gas chromatography using flame ionization detection. RESULTS: The levels of total ChOx were elevated in the DM1 and DM2 groups compared to age-matched subjects without diabetes (p < 0.05). The concentrations of 7beta-hydroxycholesterol, cholesterol-alpha-epoxide and cholesterol-beta-epoxide were higher in the blood plasma of subjects in the DM2 group than in the blood plasma of subjects in the C2 and IGT groups (p < 0.05). Treatment of type 2 diabetic patients with oral hypoglycemic drugs associated with insulin resulted in lower concentrations of nitrotyrosine in the blood plasma without significant changes in the concentrations of glucose and glycated hemoglobin. Moreover, combination with statins in both treatments decreased the concentrations of ChOx. CONCLUSIONS: ChOx are suitable biomarkers of oxidative stress and may be useful in clinical studies to follow drug effects on lipid oxidative modifications in diabetic patients.
Assuntos
Colesterol/metabolismo , Diabetes Mellitus Tipo 1/metabolismo , Diabetes Mellitus Tipo 2/metabolismo , Estresse Oxidativo/fisiologia , Adolescente , Adulto , Idoso , Inibidores da Enzima Conversora de Angiotensina/uso terapêutico , Biomarcadores , Criança , Colestanóis/sangue , Colesterol/análogos & derivados , Colesterol/sangue , Diabetes Mellitus Tipo 1/tratamento farmacológico , Diabetes Mellitus Tipo 2/tratamento farmacológico , Feminino , Intolerância à Glucose/sangue , Humanos , Hidroxicolesteróis/sangue , Hipoglicemiantes/uso terapêutico , Insulina/uso terapêutico , Cetocolesteróis/sangue , Masculino , Pessoa de Meia-IdadeRESUMO
7-cetocoleterol (7KC) é um oxisterol conhecido por inibir a proliferação celular e por ser citotóxico. Uma nanoemulsão contendo 7KC (LDE/7KC) demonstrou efeito anti-proliferativo sobre as linhagens RPMI 8226 (mieloma) e melanoma (B16F10), in vitro. Sendo preferencialmente captada via receptores de LDL. No presente trabalho, avaliamos, in vivo, a cinética plasmática, biodistribuição e ação anti-tumoral em camundongos portadores de melanoma. A nanoemulsão dirigiu-se principalmente no fígado e ao tumor, demonstrando direcionamento a tecidos com alta expressão de receptores para LDL. LDE/7KC promoveu uma redução superior a cinqüenta por cento do tamanho do tumor, que apresentou maior área de necrose e menor quantidade de vasos e aumentou a sobrevida dos camundongos, sem causar toxicidade.
7-ketocholesterol (7KC) is an oxysterol known to inhibit cell proliferation and to be cytotoxic. A nanoemulsion containing-7KC (LDE/7KC) was shown to had antiproliferative effects on RPMI 8226 myeloma cell line and melanoma (B16F10), in vitro. This particle is taken up mainly by LDL receptors. Here we have evaluated the plasma kinetic, biodistribution and the anti-tumoral action of LDE/7KC in melanoma bearing mice. The nanoemulsion accumulated in the liver and tumor, tissues with a high expression of LDL receptors. LDE/7KC promoted a tumor size reduction over fifty percent. A increased necrosis area and a decreased amount of blood vessels was found. A increased survival rate was observed, together with a lack of toxicicity.
Assuntos
Emulsões/farmacocinética , Cetocolesteróis , Neoplasias Cutâneas/imunologia , Lipoproteínas LDL , Melanoma Experimental/imunologia , Receptores de LDLRESUMO
A new method was developed for the simultaneous determination of cholesterol and its oxidation products in eggs, using HPLC with UV and refractive index (RI) detectors, and HPLC interfaced with atmospheric pressure chemical ionization coupled to MS (HPLC-APCI-MS). The best conditions for direct saponification of the sample and extraction of the non-saponifiable material were defined using complete factorial designs with central points. The method showed accuracy and precision with a detection limit between 0.002 and 0.079 microg/g. The oxides cholest-5-ene-3beta,20alpha-diol and cholest-5-ene-3beta,25-diol identified by HPLC-UV-RI were not confirmed by HPLC-APCI-MS.
Assuntos
Colesterol/análogos & derivados , Colesterol/química , Cromatografia Líquida de Alta Pressão/métodos , Ovos/análise , Ionização do Ar , Pressão Atmosférica , Colesterol/análise , Colesterol/isolamento & purificação , Colesterol/metabolismo , Cromatografia Líquida de Alta Pressão/instrumentação , Cromatografia Líquida , Desmosterol/análise , Desmosterol/isolamento & purificação , Análise de Alimentos , Hidroxicolesteróis/análise , Hidroxicolesteróis/isolamento & purificação , Cetocolesteróis/análise , Cetocolesteróis/isolamento & purificação , Espectrometria de Massas/métodos , Oxirredução , Sensibilidade e Especificidade , Espectrofotometria Ultravioleta , Raios UltravioletaRESUMO
Ovos são importantes como alimento e com matéria-prima industrial. Ao mesmo tempo, têm elevada concentração de colesterol, principalmente quando desidratado. O colesterol, por sua vez, está sujeito à oxidação durante o processamento e/ou estocagem, formando, em conseqüência, derivados oxidados com atividades tóxicas, entre as quais a aterogenicidade. Foi avaliada, neste trabalho, a estabilidade do colesterol em ovo integral em pó comercial, através da ocorrência do 7-cetocolesterol livre, quantificado por cromatografia líqüida de alta eficiência, depois da extração dos lípides totais e separação em coluna de Florisil. O 7-cetocolesterol livre ocorreu em todos os três lotes das cinco marcas analisadas de ovo integral em pó, em teor médio de 84,01±5,34 µg/g de lípides...
Assuntos
Cetocolesteróis/biossíntese , Colesterol , Conservação de Alimentos/métodos , Ovos , Cromatografia Líquida de Alta Pressão/métodos , Amostras de AlimentosRESUMO
The effect of agaric acid as inducer of mitochondrial permeability transition was studied. It was found that: (i) agaric acid (AA) promoted efflux of accumulated Ca2+, collapse of transmembrane potential, and mitochondrial swelling; (ii) these effects depend on membrane fluidity; (iii) ADP inhibited the effect of AA on Ca2+ efflux, and (iv) AA blocked binding of the sulfhydryl reagent, eosin-5-maleimide, to the adenine nucleotide translocase. It is proposed that AA induces pore opening through binding of the citrate moiety to the ADP/ATP carrier; this interaction must be stabilized by insertion of the alkyl chain in the lipid milieu of the membrane.
Assuntos
Ácido Cítrico/análogos & derivados , Membranas Intracelulares/fisiologia , Fluidez de Membrana/fisiologia , Mitocôndrias/fisiologia , Translocases Mitocondriais de ADP e ATP/efeitos dos fármacos , Difosfato de Adenosina/farmacologia , Animais , Cálcio/metabolismo , Ácido Cítrico/antagonistas & inibidores , Ácido Cítrico/farmacologia , Amarelo de Eosina-(YS)/análogos & derivados , Amarelo de Eosina-(YS)/metabolismo , Membranas Intracelulares/efeitos dos fármacos , Cetocolesteróis/farmacologia , Fluidez de Membrana/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/ultraestrutura , Translocases Mitocondriais de ADP e ATP/metabolismo , Dilatação Mitocondrial/efeitos dos fármacos , Ratos , Reagentes de Sulfidrila/metabolismo , TemperaturaRESUMO
O ovo é utilizado como matéria-prima na produção de alimentos devido suas propriedades funcionais e nutricionais. Existe, entretanto, a possibilidade de ocorrer a oxidação do colesterol constituinte do ovo em proporções significativas, durante o processamento, estocagem, ou preparo do produto para o consumo. Amostras comerciais de macarrão contendo ovos foram analisadas em relação ao colesterol e à sua estabilidade oxidativa, avaliada pela ocorrência de 7-cetocolesterol livre. A qualificação foi feita por Cromatografia Líquida de Alta Eficiência - CLAE. O colesterol variou bastante, de 11, 0 a 155,88mg/100g. Na maioria das amostras, os valores encontrados estavam abaixo dos declarados pelos fabricantes, indicando menor adição de ovo no produto que a recomendada pela legislação, caracterizando fraude...