RESUMO

Os n-3 e n-6 são duas famílias de ácidos graxos poli-insaturados. Os ácidos graxos de cadeia longa como o ácido araquidônico (AA) e docosahexaenoico (DHA) apresentam importantes funções no desenvolvimento e funcionamento do cérebro. Os produtos de oxidação dos ácidos graxos poli-insaturados estão presentes ou aumentados ao longo do desenvolvimento de doenças neurodegenerativas. A caracterização de tais produtos é crítica para o estudo que busca entender o seu papel fisiopatológico no desenvolvimento de tais doenças. No presente trabalho, buscou-se o desenvolvimento de uma ferramenta analítica sensível e específica para a detecção e quantificação dos hidroperóxidos e hidróxidos do AA (HpETE e HETE), do seu precursor, o ácido linoleico (HpODE e HODE) e do DHA (HpDoHE e HDoHE). Estes hidroperóxidos foram sintetizados por fotooxidação e os hidróxidos correspondentes foram obtidos através da redução com o NaBH4. Os isômeros isolados foram caracterizados por LC-MS/MS. Os íons produto específicos de cada isômero foram escolhidos para a construção do método de monitoramento de reação selecionada (selected reaction monitoring - SRM) para a realização da análise quantitativa dos analitos de interesse. Cabe salientar que os dados obtidos poderão ser utilizados em bibliotecas de análise lipidômica e oxi-lipidômica pois serão essenciais para a identificação e quantificação dos analítos de interesse do presente estudo em diversas doenças. Utilizando o método padronizado, buscamos investigar o papel dos hidroperóxidos e hidróxidos do DHA, LA e AA em um modelo animal para a esclerose lateral amiotrófica (ELA), uma doença neurodegenerativa que acomete neurônios motores. Foi observado um aumento nos níveis de 13-HpODE, 9-HpODE e 12-HETE no córtex motor dos animais avaliados. Adicionalmente, foram observadas alterações nas taxas lipólica e lipogênica no tecido adiposo para os animais ELA em relação aos respectivos controles. Em conjunto, os dados apresentados no presente trabalho corroboram com os trabalhos da literatura que associam alteração dos níveis dos produtos de oxidação dos ácidos graxos poli-insaturados em doenças neurodegenerativas e o metabolismo energético alterado em ELA. Futuramente é necessária uma investigação mais ampla dos níveis dos hidroperóxidos e hidróxidos lipídicos em diferentes tecidos e do metabolismo lipídico, e os conhecimentos gerados poderão ser uma importante fonte de novas opções terapêuticas para os pacientes portadores de ELA

The n-3 and n-6 are two olyunsaturated fatty acids families. The long chain fatty acids such as arachidonic (AA) and docosahexaenoic acid (DHA) have important roles in the development and function of the brain. Polyunsaturated fatty acids (PUFAs) oxidation products are present or increased during the progression of neurodegenerative diseases. The characterization of DHA oxidation products is critical to understand their roles in the development of such diseases. In the present study, we sought to develop a sensitive and specific analytical tool for the detection and quantification of AA hydroperoxides and hydroxides (HPETE and HETE), its precursor linoleic acid (HPODE and HODE) and DHA (HpDoHE and HDoHE). These hydroperoxides were synthesized by photooxidation and the corresponding hydroxides were obtained by reduction with NaBH4. The isolated isomers were characterized by LC-MS/MS, and unique and specific fragment ions were chosen to construct a selected reaction monitoring (SRM) method for the targeted quantitative analysis. It should be emphasized that the data obtained - in the form of lipidomics and oxy-lipidomics libraries - may be used to assist in several diseases. Using the standardized method, we investigated the role of hydroperoxides and hydroxides of DHA, LA and AA in an animal model of amyotrophic lateral sclerosis (ALS), a neurodegenerative disease that affects motor neurons. Increased levels of 13-HPODE, 9-HPODE and 12-HETE were observed in the animals motor cortex. Additionally, results show changes in lipogenic and lipolytic rates in adipose tissue for ALS animals when compared to their respective controls. Altogether, the data presented herein corroborate with the literature by linking altered levels of PUFAs oxidation products in neurodegenerative diseases with altered energetic metabolism in ALS. In the future, a more extensive investigation of the hydroperoxide and hydroxide level in different tissues as well as the lipid metabolism must be done, which could lead to new therapeutic options for ALS patients

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RESUMO

Alterations in the production of arachidonic acid metabolites by squamous epithelium of the upper aerodigestive tract may play a role in the pathogenesis of squamous cell carcinoma of the head and neck. The levels of cyclooxygenase and lipoxygenase metabolites of arachidonic acid metabolism were measured by radioimmunoassay in the saliva of 51 patients with squamous cell carcinoma of the head and neck and compared with a control group of 27 patients with no history of cancer. Levels of leukotriene B4 were significantly increased in cancer patients (p = 0.02), whereas there were no significant differences in levels of prostaglandin E2 or 15-hydroxy-eicosatetranoic acid. Levels of metabolites did not correlate with a history of tobacco use and did not change in a consistent manner after surgery. The significance of arachidonic acid metabolites in the saliva of patients with squamous cell carcinoma of the head and neck is unknown.

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RESUMO

The biosynthesis of leukotrienes is known to occur through a series of complex processes which, in part, can be influenced by cell-cell interactions. Several studies have suggested that arachidonic acid availability is a major limiting step for leukotriene biosynthesis and that its transfer between cells can represent a significant source of this precursor. Accordingly, effect of time and source of arachidonic acid on transcellular leukotriene synthesis was studied in mixed platelet/neutrophil populations challenged with the calcium ionophore A23187. A time-dependent contribution of platelet-derived as well as neutrophil-derived arachidonate was found in the selective formation of neutrophil 5-lipoxygenase metabolites. Utilization of platelet or neutrophil arachidonate was followed by incorporation of radiolabeled arachidonic acid into platelet or neutrophil phospholipids prior to stimulation. Specific activity of liberated arachidonic acid along with numerous 5-lipoxygenase products (including LTB4, 20-hydroxy-LTB4, 5-HETE and LTC4) was determined in order to follow mass and radiolabel. A large amount of platelet-derived arachidonic acid was released in the first 1.5 min, whereas 10 min platelet-derived arachidonate was much lower in amount but significantly higher in specific activity, suggesting different precursor pools. The platelet-derived arachidonate was heavily utilized by the neutrophils at the early time points for formation of 5-HETE and delta 6-trans-LTB4 isomers, but appeared to contribute only marginally to the constitutive metabolism of neutrophil arachidonate into LTB4. Results from these experiments suggest different pools of 5-lipoxygenase in the neutrophil and indicate a time and source dependent modulation of arachidonate metabolism in mixed cell interactions.

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