Study of Docosahexaenoic acid non-enzymatic oxidation products as biomarkers for neurodegenerative diseases

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 patient

Comparison of different laboratory tests in the evaluation of hemorrhagic risk of patients using rivaroxaban in the critical care setting: diagnostic accuracy study

Abstract Background Rivaroxaban is a direct oral anticoagulant designed to dispense with the necessity of laboratory monitoring. However, monitoring rivaroxaban levels is necessary in certain clinical conditions, especially in the critical care setting. Methods This is a diagnostic accuracy study evaluating sensitivity and specificity of prothrombin time (PT), activated partial thromboplastin time (aPTT), and Dilute Russell viper venom time (dRVVT), to evaluate the hemorrhagic risk in patients taking rivaroxaban. The study used a convenience sample of 40 clinically stable patients using rivaroxaban to treat deep vein thrombosis or atrial fibrillation admitted in a private hospital in Brazil, compared to a group of 60 healthy controls. The samples from patients were collected two hours after the use of the medication (peak) and two hours before the next dose (trough). Results The correlation with the plasmatic concentration measured by anti-FXa assay was higher for PT and dRVVTS. The PT and aPTT tests presented higher specificity, while dRVVT was 100% sensible. Conclusions There was a strong correlation between the tests and the plasma concentration of the drug. Additionally, our results demonstrated the potential use of dRVVT as a screening test in the emergency room and the need of a second test to improve specificity

Antioxidant, cytotoxic and antimutagenic activities of 7-epi-clusianone obtained from pericarp of Garcinia brasiliensis.

This paper describes the investigation of the cytotoxic and antioxidant activities and in vivo mutagenic/ antimutagenic potential of different concentrations of the hexane extract (EHP) and isolated molecule 7-epi-clusianone (MI) of Rheedia brasiliensis. The in vitro antioxidant activity of MI was investigated by monitoring the reduction of radical scavenging and metal chelating activity of DPPH (1,1-diphenyl-2-picrylhydrazil). Cytotoxic activity was assessed by measuring the mortality of brine shrimp in the presence and absence of the compounds. The mutagenic, antimutagenic and cytotoxic effects of these compounds were evaluated by a micronucleus test. During the antioxidant activity assessment, the 7-epi-clusianone was significantly higher than that of EHP at all concentrations in three assays. From the results obtained with the assessment of cytotoxic activity, all samples had a mortality rate (LC50b100 mg/mL) lower than the positive control (thymol). The results of the micronucleus test revealed that MI at 5, 10 and 15 mg/kg b.w. is antimutagenic. In conclusion, these results suggest that in the future, the EHP and MI could be used as prophylactic agents in cancer prevention

Palmitoleic acid (n-7) increases white adipocyte lipolysis and lipase content in a PPARα-dependent manner

We investigated whether palmitoleic acid, a fatty acid that enhances whole body glucose disposal and suppresses hepatic steatosis, modulates triacylglycerol (TAG) metabolism in adipocytes. For this, both differentiated 3T3-L1 cells treated with either palmitoleic acid (16:1n7, 200 μM) or palmitic acid (16:0, 200 μM) for 24 h and primary adipocytes from wild-type or PPARα-deficient mice treated with 16:1n7 (300 mg•kg(-1)•day(-1)) or oleic acid (18:1n9, 300 mg•kg(-1)•day(-1)) by gavage for 10 days were evaluated for lipolysis, TAG, and glycerol 3-phosphate synthesis and gene and protein expression profile. Treatment of differentiated 3T3-L1 cells with 16:1n7, but not 16:0, increased basal and isoproterenol-stimulated lipolysis, mRNA levels of adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL) and protein content of ATGL and pSer(660)-HSL. Such increase in lipolysis induced by 16:1n7, which can be prevented by pharmacological inhibition of PPARα, was associated with higher rates of PPARα binding to DNA. In contrast to lipolysis, both 16:1n7 and 16:0 increased fatty acid incorporation into TAG and glycerol 3-phosphate synthesis from glucose without affecting glyceroneogenesis and glycerokinase expression. Corroborating in vitro findings, treatment of wild-type but not PPARα-deficient mice with 16:1n7 increased primary adipocyte basal and stimulated lipolysis and ATGL and HSL mRNA levels. In contrast to lipolysis, however, 16:1n7 treatment increased fatty acid incorporation into TAG and glycerol 3-phosphate synthesis from glucose in both wild-type and PPARα-deficient mice. In conclusion, palmitoleic acid increases adipocyte lipolysis and lipases by a mechanism that requires a functional PPARαSão Paulo State Research Foundation (FAPESP, 2011/51627-8)São Paulo State Research Foundation (FAPESP, 2009/15354-7)São Paulo State Research Foundation (FAPESP, 2010/10909-8)São Paulo State Research Foundation (FAPESP, 2009/53964-1

Determination of rivaroxaban in patient’s plasma samples by anti-Xa chromogenic test associated to High Performance Liquid Chromatography tandem Mass Spectrometry (HPLC-MS/MS) - Fig 4

<p>Method comparison HPLC-MS/MS assay and the anti-Xa assay from external laboratory (n = 19): (A) Spearman correlation of rivaroxaban results obtained by the HPLC-MS/MS assay and the anti-Xa assay used for rivaroxaban measurement from external laboratory; (B) Bland-Altman analyses.</p

Number of samples according to sample type.

<p>Number of samples according to sample type.</p

Mass spectrometry parameters for rivaroxaban monitoring.

<p>Mass spectrometry parameters for rivaroxaban monitoring.</p

Intra-day and inter-day precision and accuracy values of plasmatic rivaroxaban.

<p>Intra-day and inter-day precision and accuracy values of plasmatic rivaroxaban.</p

The matrix effect by infusion method.

<p>The chromatogram shows in blue, red and green the multiple reaction monitoring (MRM) for quantitative and qualitative detection of rivaroxaban and Internal Standard (IS), respectively. The orange dashed line highlight the rivaroxaban retention time expected.</p