The detection and analysis of lipids in biological matrices for clinical applications poses many challenges, but rapid and reliable detection will prove invaluable for clinical diagnosis. Herein, we report the application of drop-casted Ag nanoplatelets as surface enhanced Raman scattering (SERS) substrates for qualitative detection of 20-hydroxyeicosatetraenoic acid (20-HETE), which is a potential biomarker for diagnosis of hypertensive disorders. Biomarker peaks of 20-HETE can be reliably detected and differentiated from those of the structurally similar lipids (arachidonic acid, eicosapentaenoic acid, and docosahexaenoic acid) commonly found in human blood, even 1 pM concentrations. Additionally, one study mixed 20-HETE with three structurally similar lipids at concentrations several orders of magnitude greater than the target lipid and 20-HETE could still be detected under these conditions. These experiments demonstrate the viability of SERS for the rapid and reliable detection of endogenous bioactive lipids, which has significant clinical impact in enabling point of care diagnostics.

Submitted by Luciana Ferreira (lucgeral@gmail.com) on 2018-11-19T11:23:59Z No. of bitstreams: 2 Dissertação - Kamila Lauany Lucas Lima - 2018.pdf: 1815959 bytes, checksum: bc2167df3f3d8cab7e32728d28ff5a43 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Approved for entry into archive by Luciana Ferreira (lucgeral@gmail.com) on 2018-11-19T11:32:54Z (GMT) No. of bitstreams: 2 Dissertação - Kamila Lauany Lucas Lima - 2018.pdf: 1815959 bytes, checksum: bc2167df3f3d8cab7e32728d28ff5a43 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) / Made available in DSpace on 2018-11-19T11:32:54Z (GMT). No. of bitstreams: 2 Dissertação - Kamila Lauany Lucas Lima - 2018.pdf: 1815959 bytes, checksum: bc2167df3f3d8cab7e32728d28ff5a43 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2018-10-26 / Conselho Nacional de Pesquisa e Desenvolvimento Científico e Tecnológico - CNPq / The present study aimed to test whether: 1) the high fat diet can induce T2D without the administration of streptozotocin (STZ); 2) the high fat caloric intake causes metabolic, behavioral and morphological changes, similar to t2D and; 3) the practice of moderate physical exercise is effective to reverse the metabolic changes caused by T2D and of the high fat diet. To test these hypotheses, we used 57 Wistar 8 week old rats. They were splited into the five experimental groups: group D (N = 25) who received a single intraperitoneal dose of streptozotocin (STZ diluted in citrate buffer, pH 4.5-25mg / kg body weight of the animal) and were fed with high fat diet. To test the effect only of the high fat diet, the HL group (N = 8) was fed with high fat diet but did not receive the STZ injection. Animals with similar ages were used as control (CO, N = 24) and fed with a balanced diet. At the end of the third week, animals of CO and D and HL groups were subdivided into two other groups: sedentary (SE) and exercised (EF). EF groups were submitted to swimming for 6 weeks. The D-SE, D-EF and HL-SE groups presented significant changes in body weight gain, abdominal fat gain, caloric intake, food efficiency, lipid profile and fasting and capillary glucose levels, when compared to control groups. There was no significant difference in the concentrations of arachidonic acid products and the renal tissue did not present morphological alterations. Physical exercise increased high density lipoproteins (HDL), but did not change others evaluated parameters. We conclude that consumption of the high fat diet when associated with sedentary behavior may cause metabolic alterations similar to T2D and that physical exercise was efficient to improve the lipid profile. / Os objetivos do presente estudo foram investigar as seguintes hipóteses: 1) a dieta hiperlipídica pode induzir o Diabetes Mellitus tipo 2 (DM2) sem a administração de streptozotocina (STZ); a ingestão de dieta hiperlipídica e hipercalórica causam alterações metabólicas, comportamentais e morfológicas, similares ao DM2 e; 3) a prática de exercício físico moderado tem potencial sobre as alterações metabólicas causadas pelo DM2 e pela dietahiperlipídica. Para testarmos essas hipóteses foram utilizados 57 ratos Wistar com 8 semanas de idade, distribuídos nos seguintes grupos experimentais: grupo D (N=25) que receberam uma única injeção intraperitoneal de estreptozotocina (STZ diluído em tampão de citrato, pH 4,5 - 25mg/kg de peso corporal do animal) e foram alimentados com dieta hiperlipídica. Para testar o efeito somente da dieta hiperlipídica, o grupo HL (N=8), recebeu uma dieta hiperlipídica, mas não recebeu a injeção de STZ. Animais com idades similares foram utilizados como controle (CO, N=24) e alimentados com dieta balanceada. No final da terceira semana, animais dos grupos CO e D e HL foram subdivididos em dois grupos: sedentário (SE) e exercitado (EF). Os grupos EF foram submetidos a um protocolo de natação durante 6 semanas. Após o protocolo experimental, os grupos D-SE, D-EF e HL-SE apresentaram alterações significativas no ganho de massa, gordura abdominal, ingestão calórica, eficiência alimentar, perfil lipídico e glicemia em jejum e capilar quando comparados aos animais controles. Não houve diferença significativa nas concentrações dos produtos do ácido araquidônico e o tecido renal não apresentou alterações morfológicas. O exercício físico aumentou as lipoproteínas de alta densidade (HDL), mas não alterou os demais parâmetros avaliados. Concluímos que o consumo da dieta hiperlipídica quando associada ao comportamento sedentário pode causar alterações metabólicas similares ao DM2 e que o exercício físico foi eficiente para melhorar o perfil lipídico.

[Truncated abstract] Arachidonic acid is oxidised in vivo by non-enzymatic (free radical) or enzymatic pathways (cyclooxygenase, lipoxygenase, and cytochrome P450) to form a range of biologically active eicosanoids. Specifically, arachidonic acid is metabolised by cytochrome P450 -hydroxylase to produce vasoactive 20-hydroxyeicosatetraenoic acid (20-HETE), and by 5-lipoxygenase to produce proinflammatory leukotriene B4 (LTB4), which can further be metabolised by -hydroxylase to from 20-OH-LTB4 and 20-COOH-LTB4. F2-Isoprostanes (F2-IsoPs) are produced through free radical attack on arachidonic acid and have been recognised as the most reliable markers of lipid peroxidation in vivo. The metabolic syndrome (MetS) is characterised by abdominal obesity, hypertension, insulin resistance, glucose intolerance, and dyslipidemia. It is associated with low-grade inflammation and oxidative stress and an increased risk of developing cardiovascular diseases. Dietary weight loss is strongly recommended for the management of the MetS and can potentially minimise the risk of cardiovascular diseases and diabetes in individuals with the MetS. Little is known regarding the role of these arachidonic acid metabolites in the MetS and the effect of weight loss on their metabolism. Chapter three comprised of three in vitro studies aimed to examine 20-HETE synthesis in human blood cells. 20-HETE acts as a second messenger for vasoconstrictor actions of angiotensin II (Ang II) and endothelin-1 (ET-1) in renal and mesenteric beds. Human neutrophils and platelets are integral to the inflammatory process. ... Production of LTB4 and 20-OH-LTB4 was significantly lower compared with controls (P<0.005) and remained so after adjustment for neutrophil count (P<0.05).The weight loss intervention resulted in a 4.6kg reduction in body weight and a 6.6cm decrease in waist circumference and a significant increase in LTB4 and 20-OH- LTB4 in the weight loss group. Chapter Five continued to investigate the role of other arachidonic acid metabolites, 20-HETE and F2-IsoPs in the MetS and the effect of weight loss. In the case-control study (Human study 1), plasma and urinary 20-HETE and F2-IsoPs were significantly elevated in the MetS group, but no significant difference was found in stimulated-neutrophil 20-HETE. A significant gender x group interaction was observed in that women with the MetS had higher urinary 20-HETE and F2-IsoPs compared to controls (P<0.0001). In a randomised controlled trial (Human study 2), relative to the weight- maintenance group, a 4.6 kg loss in weight resulted in a 2 mmHg fall in blood pressure but did not alter the production of 20-HETE or F2-IsoPs. No significant differences were shown in 20-HETE released from stimulated-neutrophils before and after weight loss. 20-HETE and oxidative stress may be important mediators of cardiovascular disease risk in the MetS. Although a 4% reduction in body weight reduced BP, there were no changes in plasma or urinary 20-HETE or F2-IsoPs. In summary, in vitro studies show that human neutrophils and platelets can produce 20-HETE in response to Ang II and ET-1, and human studies demonstrate that the presence of MetS has a significant impact on arachidonic acid metabolism and effective weight loss can restore leukocyte synthesis of LTB4.