RESUMO
A simple and reliable method was developed and validated to determine the endocannabinoids anandamide (AEA) and 2-arachidonoylglycerol (2-AG) in rat brain samples by micro salting-out assisted liquid-liquid extraction combined with ultra-high performance liquid chromatography tandem mass spectrometry (SALLLE/UHPLC-MS/MS). The SALLE parameters (brain homogenate volume, salting-out agent, salt concentration, salt solution volume, organic solvent, organic solvent volume, and centrifugation temperature) were optimized to improve sensitivity and selectivity of the method. The SALLE/UHPLC-MS/MS method presented linear ranges from 2.00 to 20.00 ng mL-1 for AEA and from 0.300 to 10.00 µg mL-1 for 2-AG, no significant matrix effect, and inter- and intra-assay precision and accuracy with CV and RSE values lower than 15%, respectively. This innovative method was successfully applied to determine AEA and 2-AG in brain hemispheres from a 6-OHDA animal model of Parkinson's disease (PD).
Assuntos
Ácidos Araquidônicos/análise , Química Encefálica/fisiologia , Endocanabinoides/análise , Glicerídeos/análise , Extração Líquido-Líquido/métodos , Alcamidas Poli-Insaturadas/análise , Animais , Ácidos Araquidônicos/isolamento & purificação , Cromatografia Líquida de Alta Pressão/métodos , Modelos Animais de Doenças , Endocanabinoides/isolamento & purificação , Glicerídeos/isolamento & purificação , Limite de Detecção , Modelos Lineares , Masculino , Doença de Parkinson/metabolismo , Alcamidas Poli-Insaturadas/isolamento & purificação , Ratos , Ratos Wistar , Reprodutibilidade dos Testes , Espectrometria de Massas em Tandem/métodosRESUMO
OBJECTIVE: The aim of the present paper was to assess whether lipopolysaccharide (LPS)-induced inhibition of salivary secretion involves the activation of the endocannabinoid system and the participation of tumor necrosis factor (TNF)alpha in the submandibular gland. DESIGN: Pharmacological approaches were performed by using CB1 and/or CB2 cannabinoid receptor antagonists, AM251 and AM630, respectively, injected into the submandibular gland, to study the participation of the endocannabinoid system in LPS inhibitory effects on metacholine-induced salivary secretion. To assess the participation of TNFalpha on LPS inhibitory effects, salivary secretion was studied in LPS treated rats after the intraglandular injection of etanercept, a soluble form of TNF receptor which blocks TNFalpha action. Finally, to evaluate the possible interplay between endocannabinoids and TNFalpha on the submandibular gland function reduced during LPS challenge, the salivary secretion was studied after the intraglandular injection of this cytokine alone or concomitantly with AM251 and AM630. RESULTS: AM251 and AM630, injected separately or concomitantly, partially prevented LPS-induced inhibition of salivation. Also, anandamide synthase activity was increased in submandibular glands extracted from rats 3h after LPS injection, suggesting that the endocannabinoid system was activated in response to this challenge. On the other hand, etanercept, prevented the inhibitory effect of LPS on salivary secretion and moreover, TNFalpha injected intraglandularly inhibited salivary secretion, being this effect prevented by AM251 and AM630 injected concomitantly. CONCLUSION: The present results demonstrate the participation of the endocannabinoid system and TNFalpha on salivary responses during systemic inflammation induced by LPS.
Assuntos
Moduladores de Receptores de Canabinoides/fisiologia , Endocanabinoides , Lipopolissacarídeos/farmacologia , Saliva/efeitos dos fármacos , Glândula Submandibular/efeitos dos fármacos , Amidoidrolases/farmacologia , Animais , Ácido Araquidônico/análise , Ácidos Araquidônicos/análise , Colforsina/farmacologia , AMP Cíclico/análise , AMP Cíclico/antagonistas & inibidores , Relação Dose-Resposta a Droga , Etanercepte , Imunoglobulina G/farmacologia , Fatores Imunológicos/farmacologia , Técnicas In Vitro , Indóis/farmacologia , Masculino , Cloreto de Metacolina/farmacologia , Agonistas Muscarínicos/farmacologia , Piperidinas/farmacologia , Alcamidas Poli-Insaturadas/análise , Pirazóis/farmacologia , Ratos , Ratos Wistar , Receptor CB1 de Canabinoide/antagonistas & inibidores , Receptor CB2 de Canabinoide/antagonistas & inibidores , Receptores do Fator de Necrose Tumoral , Proteínas Recombinantes de Fusão/farmacologia , Saliva/metabolismo , Salivação/efeitos dos fármacos , Taxa Secretória/efeitos dos fármacos , Glândula Submandibular/metabolismo , Fator de Necrose Tumoral alfa/antagonistas & inibidores , Fator de Necrose Tumoral alfa/farmacologiaRESUMO
The fungi strains were tested in Bioscreen automated system to select the best nutritional source. Following, shaking submserse cultures were studied in media containing sole carbon or nitrogen source. The growth of these strains improved in media containing vegetable oil, with high concentration of lipids. The high concentration of γ-linolenic acid was obtained with M. circinelloides in culture containing sesame oil.
Linhagens de fungos foram testadas em sistema automatizado Bioscreen para selecionar melhor fonte nutricional. Em seguida, foram estudadas culturas submersas em meios contendo uma única fonte de carbono e de nitrogênio. As linhagens contendo alta concentração de lipídeos tiveram melhor crescimento em meio contendo óleos de gergelim ou de dendê. Maior concentração de ácido γ-linolênico foi obtida com M. circinelloides nas culturas em óleo de gergelim.
Assuntos
Ácidos Araquidônicos/análise , Ácidos Linolênicos/análise , Lipídeos/análise , Mucorales/crescimento & desenvolvimento , Óleos de Plantas/análise , Rhizopus/crescimento & desenvolvimento , Zigomicose , Microbiologia Industrial , Métodos , MétodosRESUMO
Recently we and other groups have shown that molecular iodine (I(2)) exhibits potent antiproliferative and apoptotic effects in mammary cancer models. In the human breast cancer cell line MCF-7, I(2) treatment generates iodine-containing lipids similar to 6-iodo-5-hydroxy-eicosatrienoic acid and the 6-iodolactone (6-IL) derivative of arachidonic acid (AA), and it significantly decreases cellular proliferation and induces caspase-dependent apoptosis. Several studies have shown that AA is a natural ligand of the peroxisome proliferator-activated receptors (PPARs), which are nuclear transcription factors thought to participate in regulating cancer cell proliferation. Our results show that in MCF-7 cells: (1) 6-IL binds specifically and with high affinity to PPAR proteins (EMSA assays), (2) 6-IL activates both transfected (by transactivation assays) and endogenous (by lipid accumulation) peroxisome proliferator response elements, and (3) 6-IL supplementation increases PPAR gamma and decreases PPAR alpha expression. These results implicate PPARs in a molecular mechanism by which I(2), through formation of 6-IL, inhibits the growth of human breast cancer cells.
Assuntos
Antineoplásicos/farmacologia , Ácidos Araquidônicos/metabolismo , Neoplasias da Mama/tratamento farmacológico , Iodo/farmacologia , PPAR gama/metabolismo , Animais , Antineoplásicos/uso terapêutico , Ácido Araquidônico/análise , Ácido Araquidônico/química , Ácidos Araquidônicos/análise , Ácidos Araquidônicos/química , Linhagem Celular Tumoral , Biologia Computacional , Ensaio de Desvio de Mobilidade Eletroforética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Iodo/uso terapêutico , Radioisótopos do Iodo/química , PPAR alfa/metabolismo , PPAR gama/química , PPAR gama/genética , Ligação Proteica , Multimerização Proteica , Estrutura Quaternária de Proteína , Elementos de Resposta , Receptores X de Retinoides/química , Receptores X de Retinoides/metabolismo , Coloração e RotulagemRESUMO
OBJECTIVE: To determine the relationship between fatty acid ethyl esters (FAEE) in meconium and neurodevelopment in infants exposed to alcohol in utero at 6.5 months, 1 year, and 2 years of age. STUDY DESIGN: A secondary analysis of a prospective cohort of mothers at high risk and their infants recruited after admission to a labor and delivery unit. Mothers were screened for drug and alcohol use during pregnancy by clinical interview and urine screening. Meconium was analyzed for FAEE in 216 newborn infants. Outcome measures included the Bayley Scales of Infant Development Mental (MDI) and Psychomotor (PDI) Developmental Index scores in infants at 6.5 months, 1 year, and 2 years of age. RESULTS: After controlling for prenatal visits and maternal factors, increasing concentrations of FAEE were significantly associated with poorer mental and psychomotor development (beta +/- standard error) at all follow-up visits: ethyl myristate (MDI -2.46 +/- 1.24, P = .05; PDI -3.88 +/- 1.67, P = .02), ethyl oleate (MDI -1.94 +/- 0.65, P < .01; PDI -2.60 +/- 0.93, P < .01), ethyl linoleate (MDI -1.92 +/- 0.60, P < .01; PDI -2.28 +/- 0.84, P < .01), ethyl linolenate (MDI -1.99 +/- 0.74, P < .01; PDI -2.98 +/- 1.04, P < .01), and ethyl arachidonate (MDI -2.40 +/- 1.11, P = .03; PDI -3.32 +/- 1.51, P = .03). CONCLUSION: FAEE in meconium may be a marker for identifying newborns at risk for neurodevelopmental delay from alcohol exposure in utero.
Assuntos
Deficiências do Desenvolvimento/epidemiologia , Mecônio/química , Ácidos Araquidônicos/análise , Pré-Escolar , Seguimentos , Humanos , Lactente , Ácidos Linoleicos/análise , Ácidos Linolênicos/análise , Miristatos/análise , Ácidos Oleicos/análise , Ácidos Palmíticos/análise , Prognóstico , Estudos Prospectivos , Desempenho PsicomotorRESUMO
The effects of red wine and ethanol on plasma lipoproteins and the fatty acid composition of kidney lipids and erythrocytes phospholipids were studied. Lipid peroxidation is one of the main deleterious effects of oxidant attack on biomolecules, due to the disruption of the structural integrity of membranes. The vulnerability of the kidney to oxidative damage has been partly attributed to its high content of long-chain polyunsaturated fatty acids. Antioxidants, such as flavonoids, would be a means of reducing the risk of oxidative damage to membranes. Nutritional sources rich in antioxidants, including those provided by wine, are expected to attenuate the effects of oxidative challenges. Adult rats were fed red wine rich in flavonols, ethanol (125 ml/l), or alcohol-free red wine. The control group drank water. After 10 weeks, blood samples served to measure plasma lipoproteins and antioxidant capacity. Kidney lipids and erythrocyte phospholipids were extracted. The samples were assayed by GLC. Energy intake did not differ between all the groups, but the weight gain of the ethanol group was less than the other three groups. Blood HDL and triacylglycerols were increased by both ethanol and red wine. Ethanol decreased arachidonic and docosahexaenoic acids in both kidney lipids and erythrocyte phospholipids, as compared with either water, red wine or alcohol-free red wine groups. These results indicate that non-alcoholic components of red wine could contribute to avoiding the unfavourable effects of ethanol on plasma lipoproteins, kidney lipids and membrane erythrocyte phospholipids.
Assuntos
HDL-Colesterol/sangue , Eritrócitos/metabolismo , Ácidos Graxos Insaturados/metabolismo , Rim/metabolismo , Vinho , Animais , Antioxidantes/farmacologia , Ácidos Araquidônicos/análise , Ácidos Docosa-Hexaenoicos/análise , Etanol/farmacologia , Flavonoides/farmacologia , Flavonóis , Lipídeos/química , Masculino , Fosfolipídeos/química , Ratos , Ratos Wistar , Triglicerídeos/sangue , Aumento de PesoRESUMO
El presente trabajo tuvo por objeto estudiar el origen de los ácidos grasos poliinsaturados de cadena larga (AGPI-CL) necesarios para el desarrollo postnatal del cerebro. Ratas normales recién nacidas de 1,5,10 y 20 días de edad fueron decapitadas, inmediatamente después los hígados fueron procesados para obtener la fracción microsomal y determinar la compossición de ácidos grasos y la actividad de la enzima delta6 desaturasa. También se nalizaron: la composición de ácidos grasos en plasma, homogenizado de hemisferios cerebrales y en la leche tomada del estómago de las ratas recién nacidas. Nosotros encontramos que la actividad de la enzima delta6 desaturada en el hígado fue muy baja en comparación a la observada en ratas adultas. Durante el desarrollo postnatal se observaron cantidades apreciables de DHA y AA en la lehe, microsomas hepáticos, lípidos de plasma y hemisferios cerebrales. En conclusión proponemos que durante el desarrollo postnatal, la síntesis hepática contribuye poco en el aporte de DHA y AA para el crecimiento del cerebro, pero tiene la capacidad de concentrar estos ácidos grasos provenientes de la leche, en los microsomas hepáticos y secretarlos hacia el plasma para finalmente llegar al cerebro.
Assuntos
Animais , Ratos , Masculino , Feminino , Encéfalo/crescimento & desenvolvimento , Ácidos Graxos Essenciais/metabolismo , Fígado/metabolismo , Ácidos Araquidônicos/análise , Ácidos Araquidônicos/metabolismo , Ácidos Docosa-Hexaenoicos/análise , Ácidos Docosa-Hexaenoicos/metabolismo , Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos/análise , Fígado/enzimologia , Fígado/fisiologia , Microssomos Hepáticos/química , Leite/química , Fosfolipídeos/química , Ratos Sprague-DawleyRESUMO
We address here the issue concerning the origin of essential long chain polyunsaturated fatty acids (LC-PUFA) needed for early postnatal brain development. Normal newborn rats 1, 5, 10 and 20 day-old were decapitated, immediately after, the livers were processed to obtain the microsomal fraction and its delta 6 fatty acid desaturase activity and fatty acid composition were determined. Samples of plasma, whole homogenized brain hemispheres and milk contained in the gastric cavity of the suckling rats, were analyzed to obtained their fatty acid composition. We fond that the rat neonate has a very low delta 6 desaturase activity in liver microsomes as compared with the adult. In contrast, there is a considerable quantity of DHA an AA in the milk, liver microsomes, plasma lipids and brain. In conclusion, we propose that brain DHA and AA cannot be derived substantially through liver synthesis, but through the availability of these preformed fatty acids in the milk. These fatty acids are concentrated in liver microsomes and then secreted into plasma lipid pool to reach finally to brain structures.
Assuntos
Encéfalo/crescimento & desenvolvimento , Ácidos Graxos Essenciais/metabolismo , Fígado/metabolismo , Animais , Ácidos Araquidônicos/análise , Ácidos Araquidônicos/metabolismo , Ácidos Docosa-Hexaenoicos/análise , Ácidos Docosa-Hexaenoicos/metabolismo , Ácidos Graxos Dessaturases/metabolismo , Ácidos Graxos/análise , Feminino , Linoleoil-CoA Desaturase , Fígado/enzimologia , Fígado/fisiologia , Masculino , Microssomos Hepáticos/química , Leite/química , Fosfolipídeos/química , Ratos , Ratos Sprague-DawleyRESUMO
1. Colonic mucosa from 19 patients with ulcerative colitis, eight with Crohn's disease and 14 controls were analysed for arachidonic acid (C20:4), linoleic acid (C18:2), oleic acid (C18:1), stearic acid (C18:0) and palmitic acid (C16:0). 2. Gas-liquid chromatography of lipid extracts showed that arachidonic acid was significantly higher in ulcerative colitis (19 +/- 4) and Crohn's disease (20 +/- 3) than in controls (13 +/- 5 micrograms/mg of protein) (means +/- SD). Neither the degree of inflammation nor treatment with sulphasalazine or prednisolone appeared to influence the fatty acid concentrations. 3. Seventy-five to ninety-five per cent of the arachidonic acid was found in the phospholipid fraction after separation by thin-layer chromatography. There were no significant changes in the concentrations of the other fatty acids measured, although oleic acid was lower in inflammatory bowel disease. The ratios of oleic acid to stearic acid and to palmitic acid were lower in inflammatory bowel disease. 4. The alteration in the fatty acid profile may partly explain the increased synthesis of eicosanoids in colonic mucosa in inflammatory bowel disease.
Assuntos
Ácidos Araquidônicos/análise , Colite Ulcerativa/metabolismo , Doença de Crohn/metabolismo , Mucosa Intestinal/metabolismo , Fosfolipídeos/análise , Adolescente , Adulto , Idoso , Ácidos Graxos/análise , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Fosfolipídeos/metabolismoRESUMO
The level and proportion of lipids and their fatty acid composition were analyzed in highly purified transverse tubule membranes of amphibian skeletal muscle. Tubule membranes show (a) a higher content of lipids, (b) a higher phospholipid/cholesterol ratio and (c) a different phospholipid composition from other subcellular fractions, such as the light and heavy membranes from sarcoplasmic reticulum, which are similar in lipid profile. Transverse tubule membranes are characterized by a high percentage of phosphatidylserine and sphingomyelin and a low proportion of phosphatidylcholine compared with the other membranes. All three show a high proportion of ethanolamine plasmalogens (50% of the total ethanolamine glycerophospholipid). Transverse tubule membrane lipids contain a high proportion of 20- and 22-carbon polyunsaturated fatty acids, predominantly 20:4, 20:5, 22:5 and 22:6. Arachidonate predominates in phosphatidylinositol, eicosapentaenoate and docosahexaenoate in ethanolamine and serine glycerophospholipids.