RESUMO
INTRODUCTION: The marula fruit is an important indigenous African fruit since various commercial products are produced from the pulp and the seed oil. The increased demand requires methods for authentication, quality control and determination of geographical origin. OBJECTIVE: The study aimed to establish a fast and reliable method for characterisation and authentication of marula seed oil. Furthermore, to identify marker compounds that can distinguish marula seed oils from other commercial oils and indicate regional differences. MATERIALS AND METHODS: Metabolic profiling of 44 commercial marula seed oils was performed using proton nuclear magnetic resonance (1 H NMR). For rapid classification similarity calculations were compared with principal component analysis. Differential NMR was used to determine marker compounds. RESULTS: Marula seed oil was found to be similar to macadamia and olive oils and was distinguished from these oils by the detection of minor components. Marula seed oil is differentiated from the other two oils by the absence of α-linolenic acid, relatively high levels of monoglycerides and diglycerides, and an approximately 1:1 ratio of 1,2- and 1,3-diglycerides. When comparing marula seed oils from various regions using hierarchical cluster analysis, clustering of the marula seed oils from Namibia and Zimbabwe was observed and was related to the quantities of linoleic acid and monoglycerides and diglycerides. Some samples displayed deviations in their composition which might indicate adulteration or contamination during the production process. CONCLUSION: The study demonstrates the potential of NMR as a tool in the quality control of marula seed oil. This technique requires very little sample preparation, circumvents derivatisation of the oil components with fast run-times. In addition, samples with chemical profiles that differ from the general signature profile can easily be identified.
Assuntos
Anacardiaceae , Óleos de Plantas , Óleos de Plantas/química , Diglicerídeos/análise , Monoglicerídeos , Azeite de Oliva , Espectroscopia de Ressonância Magnética/métodos , Análise por ConglomeradosRESUMO
Diacylglycerols (DAGs) are anabolic precursors to membrane lipid and storage triacylglycerol biosynthesis, metabolic intermediates of lipid catabolism, and potent cellular signaling molecules. The different DAG molecular species that accumulate over development or in different tissues reflect the changing aspects of cellular lipid metabolism. Consequently, an accurate determination of DAG molecular species in biological samples is essential to understand various metabolic processes and their diagnostic relevance. However, quantification of DAG molecular species in various biological samples represents a challenging task because of their low abundance, hydrophobicity, and instability. This chapter describes the most common chromatographic (TLC and HPLC) and mass spectrometry (MS) methods used to analyze DAG molecular species. In addition, we directly compared the three methods using DAG obtained by phospholipase C hydrolysis of phosphatidylcholine purified from a Nicotiana benthamiana leaf extract. We conclude that each method identified similar major molecular species, however, the exact levels of those varied mainly due to sensitivity of the technique, differences in sample preparation, and processing. This chapter provides three different methods to analyze DAG molecular species, and the discussion of the benefits and challenges of each technique will aid in choosing the right method for your analysis.
Assuntos
Diglicerídeos , Espectrometria de Massas por Ionização por Electrospray , Diglicerídeos/análise , Diglicerídeos/química , Diglicerídeos/metabolismo , Cromatografia Líquida de Alta Pressão/métodos , Espectrometria de Massas por Ionização por Electrospray/métodos , FosfatidilcolinasRESUMO
BACKGROUND: The high drug resistance and metabolic reprogramming of clear cell renal cell carcinoma (ccRCC) are considered responsible for poor prognosis. In-depth research at multiple levels is urgently warranted to illustrate the lipid composition, distribution, and metabolic pathways of clinical ccRCC specimens. METHODS: In this project, a leading-edge targeted quantitative lipidomic study was conducted using 10 pairs of cancerous and adjacent normal tissues obtained from ccRCC patients. Accurate lipid quantification was performed according to a linear equation calculated using internal standards. Qualitative and quantitative analyses of lipids were performed with multiple reaction monitoring analysis based on ultra-performance liquid chromatography (UPLC) and mass spectrometry (MS). Additionally, a multivariate statistical analysis was performed using data obtained on lipids. RESULTS: A total of 28 lipid classes were identified. Among them, the most abundant were triacylglycerol (TG), diacylglycerol (DG), phosphatidylcholine (PC), and phosphatidylethanolamine (PE). Cholesteryl ester (CE) was the lipid exhibiting the most considerable difference between normal samples and tumor samples. Lipid content, chain length, and chain unsaturation of acylcarnitine (CAR), CE, and DG were found to be significantly increased. Based on screening for variable importance in projection scores ≥1, as well as fold change limits between 0.5 and 2, 160 differentially expressed lipids were identified. CE was found to be the most significantly upregulated lipid, while TG was observed to be the most significantly downregulated lipid. CONCLUSION: Based on the absolute quantitative analysis of lipids in ccRCC specimens, it was observed that the content and change trends varied in different lipid classes. Upregulation of CAR, CE, and DG was observed, and analysis of changes in the distribution helped clarify the causes of lipid accumulation in ccRCC and possible carcinogenic molecular mechanisms. The results and methods described herein provide a comprehensive analysis of ccRCC lipid metabolism and lay a theoretical foundation for cancer treatment.
Assuntos
Carcinoma de Células Renais/metabolismo , Neoplasias Renais/metabolismo , Lipidômica/métodos , Lipídeos/análise , Adulto , Biomarcadores Tumorais/análise , Biomarcadores Tumorais/metabolismo , Carcinoma de Células Renais/patologia , Carcinoma de Células Renais/cirurgia , Carnitina/análogos & derivados , Carnitina/análise , Carnitina/metabolismo , Ésteres do Colesterol/análise , Ésteres do Colesterol/metabolismo , Cromatografia Líquida de Alta Pressão , Diglicerídeos/análise , Diglicerídeos/metabolismo , Feminino , Humanos , Neoplasias Renais/patologia , Neoplasias Renais/cirurgia , Lipídeos/química , Masculino , Pessoa de Meia-Idade , Espectrometria de Massas em TandemRESUMO
This study investigated whether oxidative and glycolytic rat skeletal muscles respond differently to a high-fat (HF) sucrose-enriched diet with respect to diacylglycerol (DAG) and ceramides accumulation, protein kinase C (PKC) activation, glucose metabolism, and the expression of inflammatory genes. HF diet (8 weeks) suppressed insulin-stimulated glycogen synthesis and glucose oxidation in soleus (Sol), extensor digitorum longus (EDL) and epitrochlearis (Epit) muscles. However, DAG and ceramides levels increased in Sol and EDL, but not in Epit muscles of HF-fed rats. Additionally, membrane-bound PKC-delta and PKC-theta increased in Sol and EDL, whereas in Epit muscles both PKC isoforms were reduced by HF diet. In Epit muscles, HF diet also increased the expression of tumor necrosis factor-α (TNF-α) receptors (CD40 and FAS), toll-like receptor 4 (TLR4), and nuclear factor kappa light polypeptide gene enhancer in B cells (NF-kB), whereas in Sol and EDL muscles the expression of these inflammatory genes remained unchanged upon HF feeding. In conclusion, HF diet caused DAG and ceramides accumulation, PKC activation, and the induction of inflammatory pathways in a fiber type-specific manner. These findings help explain why oxidative and glycolytic muscles similarly develop insulin resistance, despite major differences in their metabolic characteristics and responsiveness to dietary lipid abundance.
Assuntos
Glicólise/imunologia , Resistência à Insulina/imunologia , Músculo Esquelético/metabolismo , Obesidade/metabolismo , Animais , Ceramidas/análise , Ceramidas/metabolismo , Dieta Hiperlipídica/efeitos adversos , Sacarose Alimentar/efeitos adversos , Diglicerídeos/análise , Diglicerídeos/metabolismo , Modelos Animais de Doenças , Humanos , Inflamação/diagnóstico , Inflamação/imunologia , Inflamação/metabolismo , Insulina/metabolismo , Masculino , Músculo Esquelético/imunologia , Obesidade/etiologia , Obesidade/imunologia , Estresse Oxidativo/imunologia , RatosRESUMO
BACKGROUND: Accumulating evidence indicates alterations in lipid metabolism and lipid composition in neoplastic tissue. Earlier nuclear magnetic resonance studies showed that the contents of major lipid groups, such as triacylglycerols, phospholipids and cholesterol, are changed in colon cancer tissue. METHODS: In this study, a more detailed analysis of lipids in cancer and tumor adjacent tissues from colorectal cancer patients, using liquid chromatography-mass spectrometry, allowed for comparison of 199 different lipids between cancer tissue and tumor adjacent tissue using principal component analysis. RESULTS: Significant differences were found in 67 lipid compounds between the two types of tissue; many of these lipid compounds are bioactive lipids such as ceramides, lysophospholipids or sterols and can influence the development of cancer. Additionally, increased levels of phospholipids and sphingolipids were present, which are major components of the cell membrane, and increases in these lipids can lead to changes in cell membrane properties. CONCLUSIONS: This study showed that many complex lipids are significantly increased or decreased in colon cancer tissue, reflecting significant alterations in lipid metabolism. This knowledge can be used for the selection of potential molecular targets of novel anticancer strategies based on the modulation of lipid metabolism and the composition of the cell membrane in colorectal cancer cells.
Assuntos
Neoplasias Colorretais/metabolismo , Metabolismo dos Lipídeos , Idoso , Idoso de 80 Anos ou mais , Neoplasias Colorretais/química , Diglicerídeos/análise , Diglicerídeos/metabolismo , Feminino , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Lipídeos/análise , Lisofosfolipídeos/análise , Lisofosfolipídeos/metabolismo , Espectroscopia de Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Fosfolipídeos/química , Fosfolipídeos/metabolismo , Esfingolipídeos/análise , Esfingolipídeos/metabolismo , Triglicerídeos/química , Triglicerídeos/metabolismoRESUMO
Single-cell analysis is critical to revealing cell-to-cell heterogeneity that would otherwise be lost in ensemble analysis. Detailed lipidome characterization for single cells is still far from mature, especially when considering the highly complex structural diversity of lipids and the limited sample amounts available from a single cell. We report the development of a general strategy enabling single-cell lipidomic analysis with high structural specificity. Cell fixation is applied to retain lipids in the cell during batch treatments prior to single-cell analysis. In addition to tandem mass spectrometry analysis revealing the class and fatty acyl-chain for lipids, batch photochemical derivatization and single-cell droplet treatment are performed to identify the C=C locations and sn-positions of lipids, respectively. Electro-migration combined with droplet-assisted electrospray ionization enables single-cell mass spectrometry analysis with easy operation but high efficiency in sample usage. Four subtypes of human breast cancer cells are correctly classified through quantitative analysis of lipid C=C location or sn-position isomers in ~160 cells. Most importantly, the single-cell deep lipidomics strategy successfully discriminates gefitinib-resistant cells from a population of wild-type human lung cancer cells (HCC827), highlighting its unique capability to promote precision medicine.
Assuntos
Lipidômica/métodos , Lipídeos/análise , Análise de Célula Única/métodos , Espectrometria de Massas em Tandem/métodos , Linhagem Celular Tumoral , Ésteres do Colesterol/análise , Ésteres do Colesterol/química , Diglicerídeos/análise , Diglicerídeos/química , Ácidos Graxos/análise , Ácidos Graxos/química , Humanos , Isomerismo , Lipídeos/química , Células MCF-7 , Estrutura Molecular , Reprodutibilidade dos Testes , Triglicerídeos/análise , Triglicerídeos/químicaRESUMO
Approximately 900 tonne of crude palm oil (CPO) underwent washing using 5 to 10% hot water (90 to 95°C) at a palm oil mill. The aim of the CPO washing was to eliminate and/or reduce total chlorine content present in the conventional CPO, as it is known as the main precursor for the formation of 3-monochloropropane-1, 2-diol esters (3-MCPDE). By a simple hot water washing, more than 85% of the total chlorine was removed. However, washing did not have significant (p > 0.05) effect on other oil quality parameters such as the deterioration of bleachability index (DOBI), free fatty acid (FFA) content and diacylglycerol (DAG) content of the oil. The latter has been established as the main precursor for glycidyl esters (GE) formation. The treated CPO was then transported using tankers and further refined at a commercial refinery. Refining of washed CPO resulted in significantly (p < 0.05) lower formation of 3-MCPDE, but GE content remained slightly high. Post-treatment of refined oil significantly reduced the GE content (p < 0.05) to an acceptable level whilst almost maintaining the low 3-MCPDE level. The study has proven that water washing of CPO prior to refining and subsequent post-refining is so far the most effective way to produce good quality refined oil with considerably low 3-MCPDE and GE contents. Dry fractionation of refined palm oil showed these contaminants partitioned more into the liquid olein fraction compared to the stearin fraction.
Assuntos
Fracionamento Químico/métodos , Cloro/isolamento & purificação , Ésteres/isolamento & purificação , Contaminação de Alimentos/análise , Contaminação de Alimentos/prevenção & controle , Manipulação de Alimentos/métodos , Temperatura Alta , Óleo de Palmeira/química , Água , alfa-Cloridrina/isolamento & purificação , Diglicerídeos/análise , Ácidos Graxos não Esterificados/análise , Qualidade dos AlimentosRESUMO
Diacylglycerols (DAG) and ceramides have been suggested as early predictors of insulin resistance. This study was aimed to examine the combined effects of fish oil (FO) and grape seed extract (GSE) on hepatic endogenous antioxidants, DAG and ceramides in diet-induced early stages of insulin resistance. Thirty-five rats were fed one of the following diets: (1) a standard diet (STD group), (2) a high-fat high-sucrose diet (HFHS group), (3) an HFHS diet enriched with FO (FO group), (4) an HFHS diet enriched with GSE (GSE group) or (5) an HFHS diet enriched with FO and GSE (FO + GSE group). In the liver, endogenous antioxidants were measured using spectrophotometric and fluorometric techniques, and non-targeted lipidomics was conducted for the assessment of DAG and ceramides. After 24 weeks, the FO + GSE group showed increased glutathione peroxidase activity, as well as monounsaturated fatty acid and polyunsaturated fatty acid-containing DAG, and long-chain fatty acid-containing ceramides abundances compared to the STD group. The FO and GSE combination induced similar activation of the antioxidant system and bioactive lipid accumulation in the liver than the HFHS diet without supplementation. In addition, the FO and GSE combination increased the abundances of polyunsaturated fatty acid-containing DAG in the liver.
Assuntos
Antioxidantes/administração & dosagem , Suplementos Nutricionais , Óleos de Peixe/administração & dosagem , Extrato de Sementes de Uva/administração & dosagem , Resistência à Insulina , Fígado/efeitos dos fármacos , Animais , Ceramidas/análise , Ceramidas/metabolismo , Dieta Hiperlipídica/efeitos adversos , Diglicerídeos/análise , Diglicerídeos/metabolismo , Modelos Animais de Doenças , Ácidos Graxos Insaturados , Feminino , Humanos , Metabolismo dos Lipídeos/efeitos dos fármacos , Lipidômica , Fígado/metabolismo , RatosRESUMO
The detection of soft deodorized olive oils in extra virgin olive oil (EVOO) has become a challenging task ever since it was demonstrated that: 1. The process does not form the typical refining markers, e.g. stigmastadienes, and 2. The determination of the fatty acid alkyl esters renders useful only when the deodorized matrix comes from oils with fermentative defects. Recently researchers have developed strategies to detect such kind of blends, being one of them based on the fact that both diacylglycerol (DAG) and free fatty acids are not interdependent after mild refining activities. Presently, we propose two factors to confirm the absence of soft deodorized oils in EVOO: R1 (10 × free acidity/DAGexp) ≥ 0.23 and R2 (DAGexp-DAGtheor) < 0, in genuine EVOO. We demonstrate that such approach is useful to detect the presence of soft deodorized olive oil when this is at least at 30% in the mixture.
Assuntos
Diglicerídeos/análise , Azeite de Oliva/química , Diglicerídeos/química , Ácidos Graxos não Esterificados/química , Extração em Fase SólidaRESUMO
Coconut oil (CO) from fifteen different varieties of coconuts (Cocos nucifera L.) and one CO processed on an industrial scale were analyzed by electrospray ionization mass spectrometry (ESI-MS) and the data processed using the chemometric tools principal component analysis and independent component analysis. ESI-MS fingerprinting of lipid compounds showed predominance of diacylglycerols and triacylglycerols, as confirmed by high-resolution MS measurements. Chemometric processing of the ESI-MS data differentiated the coconut oil samples, showing that different coconut varieties/cultivars produce oils with distinguishable abundances of lipidic compounds. Thus ESI-MS analysis followed by data treatment using chemometric tools offers a tool able to classify the industrial coconut oils in a fast, simple and effective way, as well as serving as a potential method to identify the coconut varieties by the CO origin, and the occurrence of any adulteration. The procedure may also be applied for quality control of the industrial processes.
Assuntos
Cocos/química , Análise de Componente Principal , Espectrometria de Massas por Ionização por Electrospray , Óleo de Coco , Diglicerídeos/análise , Análise de Alimentos , Triglicerídeos/análiseRESUMO
Deterioration of frying oil greatly affects the nutrition of fried foods. In this study, influence of fried food and oil type on the distribution of polar compounds in oils at total polar compound (TPC) of 24%, 25% and 27% was evaluated. Both food and oil significantly affected the composition of frying oil (Pâ¯<â¯0.01) and the effect of oil was relatively more significant than that of food. Five groups of polar compounds were presented: triglyceride oligomer (TGO), triglyceride dimer (TGD), oxidized triglyceride monomer (oxTGM), diglyceride (DG) and free fatty acid (FFA). TGD and DG had the highest contents, up to 6.9%-7.5% and 8.1%-8.9% respectively, while FFA had the lowest content with 1.4%-1.7%. The compound of the greatest concern in frying oil, oxTGM, varied from 4.6% to 4.8%, when TPC reached 24%-27%. Data will be expected to provide the evidence for toxicological evaluation of fried oils and foods in restaurants.
Assuntos
Culinária , Análise de Alimentos , Óleos de Plantas/química , Cromatografia Gasosa , Diglicerídeos/análise , Diglicerídeos/química , Ácidos Graxos não Esterificados/análise , Ácidos Graxos não Esterificados/química , Óleo de Palmeira/química , Óleos de Plantas/análise , Óleo de Brassica napus/química , Óleo de Girassol/química , Triglicerídeos/análise , Triglicerídeos/químicaRESUMO
Unlike the synthetic surfactants, mono- and diacylglycerols have the advantage to be biodegradable and non-toxic. In the present work, the hydrolysis of lipid fraction by-products of refined vegetable oils was performed by Serratia sp. W3 lipase immobilized on CaCO3 by combined adsorption and precipitation. This support was selected out of four carriers as it exhibited the finest activity support (950 U/g) and the most satisfactory behavior at use. The immobilized preparation with CaCO3 was stable and active in the whole range of pH (4 to 9) and temperature (37 to 55°C), yielding a 75% degree of hydrolysis at optimal environmental conditions of pH 8.5 and temperature 55°C. Thin-layer chromatography, gas chromatography, and liquid chromatography methods were evaluated to determine the analytical characterization of hydrolysis products. For monoacylglycerols and diacylglycerol fractions identified in the samples, a novel approach by liquid chromatography method was employed, through a homemade linear retention index database and a dedicated software. The adopted approach allowed the use of basic instrumentation set-ups, without the need of sophisticated detectors, such as mass spectrometers. Thus, it could be an effective alternative to produce emulsifiers from cheap vegetable oils.
Assuntos
Diglicerídeos/biossíntese , Lipase/metabolismo , Monoglicerídeos/biossíntese , Óleos de Plantas/química , Serratia/enzimologia , Produtos Vegetais/análise , Adsorção , Carbonato de Cálcio/química , Carbonato de Cálcio/metabolismo , Diglicerídeos/análise , Enzimas Imobilizadas/química , Enzimas Imobilizadas/metabolismo , Concentração de Íons de Hidrogênio , Hidrólise , Lipase/química , Monoglicerídeos/análise , Tamanho da Partícula , Óleos de Plantas/metabolismo , Software , Propriedades de Superfície , TemperaturaRESUMO
BACKGROUND: Accumulation of diacylglycerol (DAG) and sphingolipids is thought to promote skeletal muscle insulin resistance by altering cellular signaling specific to their location. However,the subcellular localization of bioactive lipids in human skeletal muscle is largely unknown. METHODS: We evaluated subcellular localization of skeletal muscle DAGs and sphingolipids in lean individuals (n = 15), endurance-trained athletes (n = 16), and obese men and women with (n = 12) and without type 2 diabetes (n = 15). Muscle biopsies were fractionated into sarcolemmal, cytosolic, mitochondrial/ER, and nuclear compartments. Lipids were measured using liquid chromatography tandem mass spectrometry, and insulin sensitivity was measured using hyperinsulinemic-euglycemic clamp. RESULTS: Sarcolemmal 1,2-DAGs were not significantly related to insulin sensitivity. Sarcolemmal ceramides were inversely related to insulin sensitivity, with a significant relationship found for the C18:0 species. Sarcolemmal sphingomyelins were also inversely related to insulin sensitivity, with the strongest relationships found for the C18:1, C18:0, and C18:2 species. In the mitochondrial/ER and nuclear fractions, 1,2-DAGs were positively related to, while ceramides were inversely related to, insulin sensitivity. Cytosolic lipids as well as 1,3-DAG, dihydroceramides, and glucosylceramides in any compartment were not related to insulin sensitivity. All sphingolipids but only specific DAGs administered to isolated mitochondria decreased mitochondrial state 3 respiration. CONCLUSION: These data reveal previously unknown differences in subcellular localization of skeletal muscle DAGs and sphingolipids that relate to whole-body insulin sensitivity and mitochondrial function in humans. These data suggest that whole-cell concentrations of lipids obscure meaningful differences in compartmentalization and suggest that subcellular localization of lipids should be considered when developing therapeutic interventions to treat insulin resistance. FUNDING: National Institutes of Health General Clinical Research Center (RR-00036), National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK) (R01DK089170), NIDDK (T32 DK07658), and Colorado Nutrition Obesity Research Center (P30DK048520).
Assuntos
Diglicerídeos/metabolismo , Resistência à Insulina/fisiologia , Músculo Esquelético/metabolismo , Esfingolipídeos/metabolismo , Adulto , Biópsia , Glicemia/análise , Estudos Transversais , Citosol/metabolismo , Diabetes Mellitus Tipo 2/sangue , Diabetes Mellitus Tipo 2/metabolismo , Diglicerídeos/análise , Retículo Endoplasmático/metabolismo , Feminino , Técnica Clamp de Glucose , Teste de Tolerância a Glucose , Humanos , Masculino , Pessoa de Meia-Idade , Mitocôndrias/metabolismo , Músculo Esquelético/citologia , Músculo Esquelético/patologia , Obesidade/sangue , Obesidade/metabolismo , Sarcolema/metabolismo , Esfingolipídeos/análiseRESUMO
The performance of two new commercial low-cost lipases Eversa® Transform and Eversa® Transform 2.0 immobilized in different supports was investigated. The two lipases were adsorbed on four different hydrophobic supports. Interesting results were obtained for both lipases and for the four supports. However, the most active derivative was prepared by immobilization of Eversa® Transform 2.0 on Sepabeads C-18. Ninety-nine percent of fatty acid ethyl ester was obtained, in 3 h at 40 °C, by using hexane as solvent, a molar ratio of 4:1 (ethanol/oil), and 10 wt% of immobilized biocatalyst. The final reaction mixture contained traces of monoacylglycerols but was completely free of diacylglycerols. After four reaction cycles, the immobilized biocatalyst preserved 75% of activity. Both lipases immobilized in Sepabeads C-18 were very active with ethanol and methanol as acceptors, but they were much more stable in the presence of ethanol.
Assuntos
Enzimas Imobilizadas/química , Etanol/química , Ácidos Graxos/biossíntese , Lipase/química , Óleo de Girassol/química , Biocatálise , Cromatografia Líquida de Alta Pressão , Diglicerídeos/análise , Esterificação , Ésteres , Hexanos/química , Interações Hidrofóbicas e Hidrofílicas , Monoglicerídeos/análise , Polímeros/químicaRESUMO
Lipids are the main component of the cell membrane. They not only provide structural support of cells but also directly participate in complex cellular metabolic processes. Lipid signaling is an important part of cell signaling. Evidence showed that abnormal cellular metabolism may induce lipids changes. Besides, owing to single cell heterogeneity, it is necessary to distinguish different behaviors of individual cells. Time-of-flight secondary ion mass spectrometry (ToF-SIMS) is a sensitive surface analysis technique with high spatial resolution, which is useful in single cell surface analysis. Herein, we used ToF-SIMS to investigate silver nanoparticle induced lipids changes on the surface of single macrophage cells. Delayed extraction mode of ToF-SIMS was used to simultaneously obtain high mass resolution of mass spectra and high spatial resolution of single cell chemical imaging. Principle component analysis (PCA) results showed good agreement with the cytotoxicity assay results. Clear distinctions were observed between the cell groups treated with high or low dose of silver nanoparticles. The loadings plots revealed that the separation was mainly due to changes of cholesterol and diacylglycerol (DAG) as well as monoacylglycerol (MAG). Meanwhile, the chemical mapping of single cell components showed that cholesterol and DAG tend to migrate to the surrounding of the cells after high dose silver nanoparticles (Ag NPs) treatment. Our results demonstrated the feasibility of ToF-SIMS for characterizing the changes of the lipids on a single cell surface, providing a better understanding of the mechanism of cell-nanoparticle interactions at the molecular level.
Assuntos
Metabolismo dos Lipídeos , Macrófagos/metabolismo , Nanopartículas/metabolismo , Prata/metabolismo , Análise de Célula Única/métodos , Espectrometria de Massa de Íon Secundário/métodos , Animais , Colesterol/análise , Colesterol/metabolismo , Diglicerídeos/análise , Diglicerídeos/metabolismo , Lipídeos/análise , Camundongos , Monoglicerídeos/análise , Monoglicerídeos/metabolismo , Células RAW 264.7RESUMO
Monoacylglycerol (MAG) and diacylglycerol (DAG) are minor components of edible fats and oils, and they relate to the quality of these foods. The AOCS official method Cd 11b-91 has been used to determine MAG and DAG contents in fats and oils. There are, however, difficulties in the determination of MAG and DAG using this analytical procedure. Therefore, we improved this method by modifying the trimethylsilyl derivatization procedure and replacing the internal standard (IS) material. In our modified method, TMS-HT (mixture of hexamethyldisilazane and trimethylchlorosilane) was used for derivatization of MAG and DAG, which was followed by liquid-liquid extraction with water and n-hexane solution containing the IS, tricaprin. Using the modified method, we demonstrated superior repeatability in comparison with that of the AOCS method by reducing procedural difficulties. The relative standard deviation of distearin peak areas was 1.8% or 2.9% in the modified method, while it was 5.6% in the AOCS method. In addition, capillary columns, such as DB-1ht and DB-5ht could be used in this method.
Assuntos
Cromatografia Gasosa/métodos , Gorduras Insaturadas na Dieta/análise , Gorduras na Dieta/análise , Diglicerídeos/análise , Monoglicerídeos/análise , Qualidade dos Alimentos , Hexanos , Extração Líquido-Líquido/métodos , Compostos de Organossilício , Reprodutibilidade dos Testes , Soluções , Triglicerídeos , Compostos de Trimetilsilil , ÁguaRESUMO
Alpha-linolenic acid (ALA)-enriched diacylglycerol (DAG) oil is an edible oil enriched with DAG (>80%) and ALA (>50%). The present study investigated whether ALA-DAG oil promotes tumorigenesis in the tongue and gastrointestinal tract, using a rat medium-term multi-organ carcinogenesis bioassay model. Rats were treated with five genotoxic carcinogens to induce multi-organ tumorigenesis until week 4, and from 1 week after withdrawal, fed a semi-synthetic diet (AIN-93G) containing ALA-DAG oil at concentrations of 0, 13,750, 27,500, and 55,000 ppm. Rats fed AIN-93G containing 55,000 ppm ALA-triacylglycerol or a standard basal diet served as reference and negative control groups, respectively. Animals were euthanized at week 30. ALA-DAG oil was shown to have no effects on survival, general condition, body weight, food consumption, or organ weight. More discolored spots were observed in the stomachs of the 13,750- and 55,000-ppm ALA-DAG groups than in those of the control groups; however, there were no differences in the frequency of histopathological findings across groups. There were no meaningful increases in the incidence of pre-neoplastic and neoplastic lesions in the tongue and gastrointestinal tract among the groups. We therefore conclude that ALA-DAG oil does not promote tumor development in the digestive system.
Assuntos
Diglicerídeos/farmacologia , Trato Gastrointestinal/efeitos dos fármacos , Língua/efeitos dos fármacos , Ácido alfa-Linolênico/farmacologia , Animais , Bioensaio , Testes de Carcinogenicidade , Diglicerídeos/análise , Trato Gastrointestinal/patologia , Masculino , Ratos , Ratos Endogâmicos F344 , Língua/patologia , Ácido alfa-Linolênico/análiseRESUMO
Exosomes are membrane-bound extracellular vesicles involved in intercellular communication and tumor cell metastasis. In this study, flow field-flow fractionation (FlFFF) was utilized to separate urinary exosomes by size, demonstrating a significant difference in exosome sizes between healthy controls and patients with prostate cancer (PCa). Exosome fractions of different sizes were collected for microscopic analysis during an FlFFF run and evaluated with exosome marker proteins using Western blot analysis. The results indicated that exosomes of different sizes originated from different types of cells. Collected exosome fractions were further examined using nanoflow ultrahigh performance liquid chromatography-electrospray ionization-tandem mass spectrometry (nUPLC-ESI-MS/MS) for lipidomic analysis. A total of 162 lipids (from 286 identified) were quantified using a selected reaction monitoring (SRM) method. The overall amount of lipids increased by 1.5- to 2-fold in patients with PCa and degree of increase was more significant in the smaller fractions (diameter <150 nm) than in the larger ones (diameter >150 nm) some classes of lipids. In addition, neutral lipids like diacylglycerol (DAG) and triacylglycerol (TAG) decreased in all exosomes without size dependency. Moreover, a dramatic increase in 22:6/22:6-phosphatidylglycerol (PG) was observed and significant decrease in (16:0,16:0)- and (16:1, 18:1)-DAG species (nearly 5-fold) and high abundant TAG species (>2.5-fold) was observed in patients with PCa. The results of this study indicate that FlFFF can be employed for the high-speed screening of urinary exosome sizes in patients with PCa and lipidomic analysis of the fractionated exosomes has potential for developing and distinguishing biomarkers of PCa.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Exossomos/metabolismo , Lipídeos/análise , Neoplasias da Próstata/patologia , Espectrometria de Massas em Tandem , Adulto , Diglicerídeos/análise , Fracionamento por Campo e Fluxo , Humanos , Masculino , Nanotecnologia , Neoplasias da Próstata/metabolismo , Triglicerídeos/análiseRESUMO
Mono e diacilgliceróis são produtos empregados na indústria alimentícia, farmacêutica, cosmética e química como emulsificantes e melhoradores de viscosidade de produtos alimentícios, cosméticos e farmacêuticos. No entanto, a forma mais usual de obtê-los é por síntese química, o que acaba rendendo produtos finais caros e com atributos de qualidade, rendimento e de aplicabilidade tecnológica inferiores aos esperados. A busca por formas de obtenção mais racionais, eficientes e com melhor padrão de qualidade destes produtos foi o objetivo principal do trabalho, por meio de hidrólise parcial enzimática, que necessita de condições de reação mais brandas. Foram avaliadas a hidrólise enzimática descontína, empregando como substrato a trioleína técnica, e a hidrólise enzimática descontínua-alimentada, usando como substrato o óleo de girassol médio oléico. Foi utilizada, em ambos processsos, a lipase imobilizada sn-1,3 específica Lipozyme RM IM (de Rhizomucor miehei). A caracterização dos padrões e dos substrados, bem como o acompanhamento da formação dos produtos da hidrólise enzimática foram feitos por determinação da porcentagem de hidrólise, cromatografia em camada delgada (TLC), dos perfis das curvas de fusão e cristalização por calorimetria diferencial de varredura (DSC), cromatografia gasosa (CG) e cromatografia de exclusão de tamanho de alto desempenho (HPSEC). Os parâmetros de hidrólise descontínua foram o tempo de reação, a temperatura e a concentração inicial de substrato. Os parâmetros de hidrólise descontínua-alimentada foram tempo de enchimento e intervalo de alimentação de substrato. Para as respostas analíticas de porcentagem de hidrólise e de composição de frações lipídicas foi aplicado um modelo de regressão múltipla com base em metodologia de superfície de resposta. Os resultados experimentais observados nas reações de hidrólise enzimática descontínua de trioleína técnica mostraram de 24,7 a 34,2% de mono e diacilgliceróis (para 5% de óleo na emulsão) e de 21,4 a 33,6% de mono e diacilgliceróis (para 20% de óleo na emulsão). Os resultados experimentais observados nas reações de hidrólise enzimática descontínua-alimentada de óleo de girassol médio oléico (para 15% de óleo na emulsão), mostraram de 7,9 a 31,8% de mono e diacilgliceróis. Os modelos de superfície de resposta foram considerados significativos e preditivos. As hidrólises obtidas no formato descontínuo e descontínuo-alimentado permitiram efetivamente a obtenção de frações de mono/ diacilgliceróis com vários graus de eficiência de conversão e com corretas identificação e quantificação das frações de lipídios procuradas. As correlações feitas entre porcentagem de hidrólise e entalpias de cristalização e fusão, corroboradas com os resultados qualitativos e/ou quantitativos diretos obtidos na cromatografia de camada delgada (TLC) e de HPSEC, demonstraram que estes atributos podem positivamente indicar a ocorrência efetiva de reação de hidrólise, além de auferir uma escala de desempenho de reação alinhada com o previsto na literatura, à medida que são aumentadas a temperatura, o tempo de hidrólise e a porcentagem inicial de substrato oleoso, sob regime descontínuo, e que puderam ser melhoradas, de forma inovadora, sob parâmetros de tempo total de alimentação e de intervalo de alimentação, sob regime descontínuo-alimentado. A hidrólise parcial enzimática de triacilgliceróis utilizando lipase imobilizada sn-1,3 específica pode ser considerada uma alternativa às vias químicas para a produção de misturas de mono e diacilgliceróis para utilização como aditivos químicos.
Mono and diacylglycerols are products used in the food, pharmaceutical, cosmetic and chemical industries as emulsifiers and viscosity improvers for food products, cosmetics and pharmaceuticals. However, the most usual forms of obtaining them are by chemical synthesis, which ends up yielding expensive final products with attributes of quality, yield and technological applicability lower than expected. The search for more rational, efficient and better quality standards of these products was the aim of the work, through partial enzymatic hydrolysis, which requires milder reaction conditions. Discontinuous enzymatic hydrolysis was evaluated using technical triolein as substrate and discontinuous-fed enzymatic hydrolysis using as the substrate the mid oleic sunflower oil. In both processes, immobilized lipase sn-1,3 specific Lipozyme RM IM (from Rhizomucor miehei) was used. The characterization of the patterns and substrates, as well as the monitoring of the formation of the products from the enzymatic hydrolysis were made by determining the percentage of hydrolysis, thin layer chromatography (TLC), profiles of the melting and crystallization curves by differential scanning calorimetry ( DSC), gas chromatography (GC) and high performance size exclusion chromatography (HPSEC). The parameters of discontinuous hydrolysis were the reaction time, the temperature and the initial substrate concentration. The parameters of discontinuous-fed hydrolysis were filling time and substrate feed interval. For the analytical responses of hydrolysis percentage and composition of lipid fractions a multiple regression model was applied based on response surface methodology. The experimental results observed in the reactions of discontinuous enzymatic hydrolysis of technical triolein indicated amounts of mono- and diacylglycerols from 24.7 to 34.2% (for 5% of oil in the emulsion) and from 21.4 to 33.6% for mono and diacylglycerols with 20% oil in the emulsion. The experimental results observed in the reactions of discontinuous-fed enzymatic hydrolysis of mid oleic sunflower oil (for 15% oil in the emulsion), showed from 7.9 to 31.8% of mono and diacylglycerols. Response surface models were considered significant and viii predictive. The hydrolysis obtained in the discontinuous and discontinuous-fed form allowed to obtain fractions of mono / diacylglycerols with various degrees of conversion efficiency and with correct identification and quantification of the lipid fractions sought. The correlations between the percentage of hydrolysis and enthalpies of crystallization and fusion, corroborated with the qualitative and / or quantitative direct results obtained in thin layer chromatography (TLC) and HPSEC, showed that these attributes can positively indicate the effective occurrence of reaction of Hydrolysis, in addition to achieving a reaction performance scale in line with the literature, as the temperature rate, the hydrolysis time and the initial percentage of oily substrate are increased under a discontinuous regime and can be improved, in a innovative form, under parameters of total filling time and feeding interval, under a fed-batch regime. The partial enzymatic hydrolysis of triacylglycerols using specific sn-1,3-specific immobilized lipase may be considered an alternative to the chemical pathways for the production of mono- and diacylglycerol blends for use as chemical additives.
Assuntos
Diglicerídeos/análise , Hidrólise , Varredura Diferencial de Calorimetria , Óleos , Cromatografia em Camada Fina/métodos , Monoglicerídeos/classificação , LipaseRESUMO
This study investigated the effect of a single oral ingestion of alpha-linolenic acid-enriched diacylglycerol (ALA-DAG) on postprandial serum triglyceride (TG) levels. A randomized, double-blind, controlled, crossover study was performed in subjects with normal or moderately high fasting serum TG levels. Subjects ingested 0.00 g [control: triacylglycerol; TAG (rapeseed oil)], 1.25 g (1.25-g: mixture of 1.25 g ALA-DAG and 1.25 g TAG), or 2.50 g (2.50 g) of ALA-DAG in random order with a 6-d washout period. Serum TG levels were evaluated in the fasting state, and at 2, 3, 4, and 6 h after the test meal. Thirty-eight subjects completed the study and were defined as the per protocol set. As the primary outcome, postprandial serum TG levels were significantly lower in the 2.50-g treatment compared with the control. The TG level did not differ significantly between the 1.25-g and control. The suppressive effect of ALA-DAG on the serum TG level correlated significantly with the body mass index and fasting insulin level. ALA-DAG at a dose of 2.50 g had greater effects on serum TG and apolipoprotein B levels in subjects with a higher body mass index (≥25 kg/m2) and higher fasting serum insulin levels (>10 µU/mL). Our findings suggest that ingesting 2.50 g ALA-DAG suppresses the postprandial serum TG level in people with normal and moderately high fasting serum TG levels, presumably as a result of poor re-esterification of dietary fat into TG in the intestinal mucosa.