Charles University Faculty of Pharmacy in Hradec Králové Department of Biophysics and Physical Chemistry Candidate: Monika Novotná Supervisor of Diploma Thesis: Mgr. Monika Kuchařová, Ph.D. Title of Diploma Thesis: The determination of fatty acid levels in the tissues of healthy, suddenly deceased persons and polymorbid patients The aim of the thesis was to determine the levels of 14 fatty acids in tissues of suddenly deceased, otherwise healthy individuals and in the group of polymorbid, chronic patients. It was a comparison of fatty acid levels in seven tissues of the human body: subendocardial left ventricular tissue, liver parenchyma tissue, kidney cortex, adrenal tissue, skeletal muscle, abdominal subcutaneous adipose tissue, and brain tissue. Each group included 10 deceased patients. The theoretical part incudes fatty acids as the main component of lipids. It deals mainly with the group of polyunsaturated fatty acids and their relation to pathologies in the human body. Gas chromatography, thanks to which we analyzed the fatty acid spectrum, is also described. The experimental part consists of the basic characteristics of the research groups, the description of the workflow and the results. Statistically processed data are divided into categories by individual fatty acids and other monitored...

Les dernières données de consommation alimentaire de la population française démontrent que les apports nutritionnels en acide eicosapentaénoïque (EPA) et en acide docosahexaénoïque (DHA) sont nettement inférieurs aux recommandations fixées par l’Agence nationale de sécurité sanitaire, de l’alimentation, de l’environnement et du travail (ANSES). Compte tenu de leurs rôles physiologiques, l’amélioration de leur biodisponibilité est devenue une priorité nutritionnelle. La maitrise des formes de vectorisation des acides gras polyinsaturés à longue chaine de la série n-3 (AGPI-LC n-3) représente une piste d’intérêt afin de répondre à cette problématique. C’est dans ce contexte que s’inscrit ce travail de thèse, visant à étudier l’influence de la forme moléculaire (phospholipides (PL) vs triglycérides (TG)) et/ou supramoléculaire (phase continue, émulsion, liposomes) de vectorisation des AGPI-LC n-3 sur leur devenir métabolique.La démarche expérimentale adoptée a consisté, dans un premier temps, à caractériser les différentes formes de vectorisation des AGPI-LC n-3 par un couplage de méthodes d’analyse physico-chimiques (microscopie optique, granulométrie, mesure des produits primaires et secondaires d’oxydation, lipolyse enzymatique). Les données obtenues ont permis de poser des hypothèses mécanistiques pour l’étude de la biodisponibilité chez le rat. Ainsi, dans un deuxième temps, l’étude in vivo a permis d’aborder (1) l’absorption intestinale des acides gras (AG) suite à un apport en bolus de lipides et (2) l’accrétion tissulaire suite à un régime de 8 semaines.Les résultats indiquent que la forme moléculaire de vectorisation des AGPI-LC n-3 (TG marins vs PL marins, en phase continue) n’influence pas leur absorption intestinale mais plutôt leur incorporation dans les lipides lymphatiques, principaux transporteurs des AG alimentaires dans l’organisme. A l’inverse, la structuration des PL marins en milieu aqueux (émulsion ou liposomes), permet d’améliorer l’absorption de l’EPA et du DHA, de façon spécifique à la nature de la structuration supramoléculaire. Les résultats de l’accrétion tissulaire montrent qu’au terme des 8 semaines de régime, l’enrichissement tissulaire en AGPI-LC n-3 s’effectue de façon différente selon leur forme moléculaire de vectorisation. Ainsi, la consommation de TG marins favorise l’incorporation des AGPI-LC n-3 dans les lipides hépatiques, alors que la supplémentation des régimes en PL marins permet d’orienter le DHA vers le cerveau et la rétine.L’ensemble de ces résultats montre que le choix de la forme moléculaire de vectorisation des AGPI-LC n-3 (PL vs TG) peut être un moyen d'orienter leur incorporation tissulaire, alors que leur structuration supramoléculaire (émulsion ou liposomes), une stratégie pour moduler leur absorption intestinale. / The latest data from the cross-sectional national French dietary study show that dietary intakes of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) are significantly lower than those recommended by the French Agency for Food, Environmental and Occupational Health & Safety (ANSES). Considering their physiological roles, improving their dietary intake has became a nutritional priority. The control of the different forms of n-3 long chain polyunsaturated fatty (n-3 LC-PUFA) intake represents an interesting way to address this issue. In this context, this thesis aims at studying the influence of the molecular form (phospholipids (PL) vs triglycerides (TG)) and/or supramolecular form (continuous phase, emulsion, liposomes) of n-3 LC-PUFA towards their bioavailability.The experimental approach consisted in characterizing the different forms of n-3 LC-PUFAs by coupling various physicochemical methods (optical microscopy, granulometry, measurement of primary and secondary oxidation products, enzymatic lipolysis). The data obtained made it possible to propose some mechanistic hypothesis for the bioavailability studies conducted in rat. Thus, in a second step, the in vivo study made addresses (1) the intestinal absorption of fatty acids following a single dose of lipids and (2) the lipid tissue accretion following an 8-week diet.The data obtained from the intestinal absorption study indicate that the molecular form of n-3 LC-PUFA targeting (marine TG vs marine PL, in continuous phase) does not influence their intestinal absorption but rather their incorporation into lymphatic lipids, which are the main carriers for dietary fatty acids in the organism. Conversely, structuring marine PL (emulsion or liposomes) improves the EPA and DHA absorption, but differently according to the supramolecular form. The results concerning tissue accretion show that, after 8-week diet, the tissue enrichment in n-3 LC-PUFA is different according to the molecular form of targeting. The consumption of marine TG favours the incorporation of n-3 LC-PUFA into hepatic lipids, while the supplementation of diets with marine PL targets DHA preferentially to brain and retina.All these results show that the molecular form of targeting (PL vs TG) of n-3 LC-PUFA can be a way for managing their tissue incorporation, whereas the supramolecular organization (emulsion or liposomes) can be envisaged to monitor their intestinal absorption.

Les récepteurs nucléaires LXRα et LXRβ sont activés par la fixation de dérivés oxygénés du cholestérol. Ils régulent l’expression de nombreux gènes appartenant au métabolisme du cholestérol et des acides gras, et jouent un rôle important dans l’inflammation et l’immunité innée. L’activation de LXR inhibe le développement de l’athérosclérose, en augmentant l’efflux de cholestérol des macrophages ainsi que le transport inverse jusqu’au foie et l’excrétion biliaire. De plus, LXR diminue la biosynthèse et la captation du cholestérol dans les tissus périphériques. Enfin, les agonistes synthétiques de LXR administrés à des souris diminuent significativement l’inflammation dans les lésions athérosclérotiques, notamment en inhibant la sécrétion de certaines cytokines inflammatoires. Néanmoins LXR régule également la lipogenèse et la synthèse d’acides gras mono-insaturés, et l’administration d’agonistes de LXR s’accompagne également d’effets indésirables liés à cette régulation, comme une accumulation dérégulée d’acides gras dans le foie et une augmentation du taux de LDLs circulantes. Plusieurs autres mécanismes restent encore à être explorés, comme la synthèse d’acides gras polyinsaturés et les conséquences sur le métabolisme cellulaire. Nos travaux identifient une nouvelle voie régulée entièrement par LXR, le métabolisme des acides gras polyinsaturés. Le récepteur nucléaire LXR régule l’ensemble des enzymes FADS1, FADS2 et ELOVL5, responsables de la synthèse d’acides gras polyinsaturés oméga-6 et oméga-3. Cette régulation s’accompagne d’une incorporation d’acide arachidonique dans les phospholipides, via la régulation de LPCAT3, ce qui prépare les macrophages à une synthèse accrue de dérivés inflammatoires issus de l’acide arachidonique, comme la Prostaglandine E2, suite à une stimulation au lipopolysaccharide. La régulation de cette voie par LXR a également un effet sur le développement de l’athérosclérose, augmentant les taux d’acides gras polyinsaturés oméga-6 et oméga-3 dans les plaques d’athérome. Nos résultats montrent donc que LXR régule la synthèse des acides gras polyinsaturés en plus des acides gras mono-insaturés et de la lipogenèse et que cette régulation a des conséquences sur le profil lipidique des macrophages in vitro et in vivo ainsi que sur leur réponse inflammatoire. / The nuclear receptors LXRα and LXRβ are activated by oxygenated metabolites of cholesterol. They regulate the expression of numerous genes belonging to cholesterol and fatty acids metabolism, and play a central role in inflammation and innate immunity. LXR activation inhibits atherosclerosis development, by increasing cholesterol efflux from macrophages as well as reverse cholesterol transport and biliary excretion. In addition, LXR decreases cholesterol uptake and biosynthesis. Synthetic LXR agonists fed to mice significantly decrease inflammation in atherosclerotic lesions, by inhibiting several inflammatory cytokines. However, LXR also regulate lipogenesis and monounsaturated fatty acids synthesis, and LXR agonists supplementation is accompanied by side effects due to this regulation, such as a deregulated accumulation of fatty acids in the liver and an increase in circulating LDLs. Other mecanisms still need to be characterized, such as polyunsaturated fatty acids synthesis and the consequences on cell metabolism. Our work identify a new pathway regulated by LXR, the metabolism of polyunsaturated fatty acids. The nuclear receptor LXR regulates all enzymes responsible for omega-6 and omega-3 polyunsaturated fatty acids synthesis, FADS1, FADS2 and ELOVL5. This regulation is accompanied by an increase in arachidonic acid incorporation in phospholipids, via LPCAT3 regulation, which subsequently primes human macrophages for an increased inflammatory metabolites secretion derived from arachidonic acid, such as Protaglandin E2, following a LPS stimulation. The regulation of this pathway by LXR has an effect on atherosclerosis, increasing omega-6 and omega-3 ployunsaturated fatty acids in atheroma plaques. Our results show therefore that LXR regulates polyunsaturated fatty acids synthesis in addition to monounsaturated fatty acids and lipogenesis, and that this regulation has direct consequences on lipid profile of macrophages in vitro and in vivo as well as on their inflammatory response.

Adipose tissue plays a crucial role in nutrient and energy homeostasis. At the time of worldwide pandemy of obesity and consequent metabolic syndrome, a great effort is made to find new treatments with potential to preserve insulin sensitivity, or even counteract development of obesity and type 2 diabetes. There are three principal possibilities how the adipose tissue biology can contribute to this goal: 1) induction of UCP1-dependent energy dissipation in brown adipose tissue; 2) conversion of white adipose depots to brown-like tissue (i.e. "browning"); and 3) stimulation of UCP1-independent thermogenesis in white adipose tissue. This thesis is based on two published works and one article under preparation. Generaly, it is focused on three different approaches targeting the above mentioned processes in adipose tissue of laboratory mouse: 1) diet supplementation with bile acids; 2) combination treatment of ω-3 polyunsaturated fatty acids and calorie restriction; and 3) cold exposure. In the experiments with administration of bile (specifically chenodeoxycholic) acid to mice, we confirm specific induction of UCP1 in both brown and subcutaneous white adipose tissue, as well as reversion of obesity in the response to the treatment. Nevertheless, most of the acute beneficial effects are mediated by...

Uma das razões da menor fertilidade dos touros europeus criados em regiões tropicais em relação a touros Bos indicus é uma maior índice de estresse oxidativo provocado por uma maior produção de espécies reativas de oxigênio (ROS), não compensada pela proteção antioxidante. Por outro lado, sabe-se que a célula espermática é extremamente susceptível ao estresse oxidativo devido à alta quantidade de ácidos graxos poli-insaturados (PUFA) em sua membrana plasmática, o que, no entanto, é muito importante para que o espermatozoide seja fértil e resistente ao choque frio. Sendo assim, tratamentos que interfiram no processo oxidativo, podem ser importantes para aumentar a produtividade destes animais, tanto à campo como em centrais de inseminação artificial. O presente experimento objetivou avaliar qual ROS seria a mais lesiva para touros europeus submetidos ao estresse térmico visando determinar um possível tratamento antioxidante direcionado para estes animais. Em um segundo momento visou então verificar a eficiência da interação entre uma dieta rica em PUFAs e o tratamento antioxidante sistêmico direcionado na qualidade espermática do sêmen ejaculado e epididimário (fresco e criopreservado) de touros europeus submetidos ao estresse térmico. Para isso, quatro touros Bos taurus adultos foram submetidos a insulação testicular (bolsa escrotal por 5 dias). Sessenta dias após a insulação o sêmen foi coletado por eletroejaculação. O sêmen de cada animal foi dividido em 4 alíquotas s submetidas à indução com quatro sistemas geradores de ROS: Ânion Superóxido (xantina/xantina oxidase), peróxido de hidrogênio, radical hidroxila (Ascorbato + Sulfato de Ferro) e malondialdeído (MDA; produto da peroxidação lipídica). As amostras foram incubadas por 1 hora e avaliadas através da análise espermática computadorizada (CASA), eoxina/nigrosina (integridade da membrana plasmática), fast-green/rosa bengala (integridade de acrossomo), 3, 3´ diaminobenzidina (atividade mitocondrial), ensaio da cromatina espermática (fragmentação de DNA) e substâncias reativas ao ácido tiobarbitúrico (TBARS; peroxidação lipídica). Os resultados indicaram ser o MDA, a substância mais deletéria aos animais Bos taurus submetidos ao estresse térmico aguda. Desta forma, 16 touros foram então submetidos á insulação testicular e divididos em 4 grupos: Controle (n=4; aplicação de óleo mineral; placebo); Grupo Vitamina E: (n=4; 5 ml de Monovin® E a cada 13 dias); Grupo PUFA: (n=4; 4 kg/dia Megalac® E1), Grupo PUFA+Vitamina E: (n=4; combinação entre os tratamentos dos grupos PUFA e Vitamina E). O sêmen destes animais foi coletado no dia da inserção da bolsa escrotal, no dia da retirada, 30 e 60 dias após. Os resultados indicaram que a vitamina E foi eficiente para a melhora nos danos causados pelo estresse térmico no DNA espermático e na mitocôndria, mas apenas nas amostras coletadas do epidídidmo. Da mesma forma, foi possível observar que a combinação entre a Vitamina E e a suplementação com PUFA foi eficiente na melhora dos padrões de motilidade espermática. Os resultados do presente estudo indicam que a combinação entre um tratamento antioxidante com Vitamina E e a suplementação com PUFA pode ser uma alternativa interessante para evitar os danos causados pelo estresse térmico agudo em touros europeus. No entanto, possivelmente este tratamento poderia ser ainda mais eficiente caso seja administrado de forma preventiva. / One reason for the lower fertility of European bulls bred in tropical regions is a higher rate of oxidative stress caused by increased production of reactive oxygen species (ROS) not compensated by antioxidant protection . On the other hand , it is known that the sperm cell is extremely susceptible to oxidative stress due to the high amount of polyunsaturated ( PUFA) on their plasma membrane. However the presence of these PUFAs is fundamental for the sperm to be fertile and resistant to cold shock. Thus, treatments that interfere with the oxidation process may be important to increase the productivity of these animals. This study aimed to evaluate which would be the most damaging ROS for European bulls subjected to heat stress to determine a possible antioxidant targeted treatment for these animals. In a second step we sought to verify the efficiency of the interaction between a diet rich in PUFAs and targeted-antioxidant treatment on sperm quality of ejaculated and epididymal sperm quality in European bulls subjected to heat stress. Four Bos Taurus bulls were submitted to testicular insulation (5 days). Sixty days after insulation semen was collected by electroejaculation. Semen from each animal was divided into 4 aliquots and submitted to the induction with four ROS generating systems: superoxide anion (xanthine / xanthine oxidase), hydrogen peroxide, hydroxyl radical (ascorbate + Ferrous Sulfate) and malondialdehyde (MDA; lipid peroxidation product). The samples were incubated for 1 h and assessed by computerized sperm analysis (CASA), eoxina / nigrosin (membrane integrity), fast-green/Bengal rose (acrosome integrity) , 3 , 3 \' diaminobenzidine (mitochondrial activity), sperm chromatin structure assay (DNA fragmentation) and thiobarbituric acid reactive substances (TBARS, lipid peroxidation). The results indicated that the MDA was the most deleterious substance to Bos Taurus semen subjected to an acute heat stress. Thus, 16 bulls were then subjected to testicular insulation and divided into 4 groups: control (n= 4; application of mineral oil; placebo); Group Vitamin E: (n= 4, 5 ml of Monovin ® E every 13 days); PUFA group: (n= 4; 4 kg/day Megalac ® E1 ); Group PUFA+Vitamin E (n=4; combination of groups PUFA and Vitamin E treatments). Semen was collected from these animals on the day of insertion of the thermal bag, on the day of withdrawal, 30 and 60 days after. The results indicated that vitamin E was effective for the improvement in damages caused by heat stress in sperm DNA and mitochondria, but only in samples collected from epididymis. Similarly, the combination of vitamin E and PUFA supplementation was effective in improving sperm motility patterns . The results of this study indicate that the combination of an antioxidant treatment with vitamin E and PUFA supplementation may be an interesting alternative to avoid the damage caused by acute heat stress in European bulls. However, possibly, this treatment would be more effective if performed preventively.

Introdução Os recém-nascidos de muito baixo peso (<1.500g) necessitam de aporte adequado de nutrientes para desenvolver-se satisfatoriamente. O leite humano é fundamental na sua alimentação, já que possui uma composição nutricional balanceada que inclui nutrientes essenciais, tais como os ácidos graxos poli-insaturados, incluindo os ácidos ômega 3 e 6, de extrema importância para o desenvolvimento do sistema nervoso central e retina. No entanto, apenas o leite materno pode não ser suficiente para suprir essas necessidades, necessitando, portanto, de suplementação. Atualmente, surgiu a possibilidade de adequação do leite humano acrescido de um concentrado com liofilizado do próprio leite humano, que foi desenvolvido neste trabalho. Objetivos determinar a quantidade de lipídios totais e o perfil lipídico do Leite Humano Baseline (LHB) e dos Concentrados com o liofilizado de Leite Humano em diferentes momentos (Leite Humano concentrado no período imediato - LHCI, Leite Humano concentrado no período de 3 meses - LHC3m e Leite Humano concentrado no período de 6 meses - LHC6m). Métodos Foram obtidas 50 amostras de leite humano de mulheres doadoras do Banco de Leite Humano da FMRP-USP que aceitaram participar do estudo. A quantificação de lipídeos ocorreu pelo Analisador de Leite Humano MIRIS® e o perfil de ácidos graxos foi determinado por CG-FID, SHIMADZU®. Resultados Houve uma concentração de lipídios totais no LHCI em relação ao LHB; a concentração (% em relação ao total de ácidos graxos) dos ácidos foi de: palmítico (C16:0) 22,30% LHB, 21,46% LHCI, 21,54% LHC3m e 21,95% LHC6m (p<0,01); oleico (C18:1n-9) 30,41% LHB, 30,47% LHCI, 30,55% LHC3m e 29,79% LHC6m (p=0,46); linoléico (C18:2 n-6) 19,62% LHB, 19,88% LHCI, 19,49% LHC3m e 19,45% LHC6m (p=0,58); araquidônico (C20:4 n-6) 0,35% LHB, 0,16% LHCI, 0,13% LHC3m e 0,15% LHC6m (p<0,01); ?- linolênico (C18:3 n-3) 1,32% LHB, 1,37% LHCI, 1,34% LHC3m e 1,34% LHC6m (p=0,14); docosahexaenóico (C22:6 n- 3) 0,10% LHB, 0,06% LHCI, 0,05% LHC3m e 0,06% LHC6m (p<0,01). Há modificações específicas em alguns ácidos graxos, no entanto a somatória de ácidos graxos por suas classes estruturais não apresentam diferenças. Conclusão Apesar das pequenas diferenças encontradas no perfil lipídico, o concentrado com leite humano liofilizado é um alimento que pode vir a atender as necessidades do recém-nascido, já que houve aumento dos lipídios totaise preservação dos nutrientes essenciais presentes apenas no leite materno, mas estudos clínicos ainda precisam ser feitos para avaliar a segurança e eficácia do concentrado como alimentação dos recém-nascidos de muito baixo peso. / Introduction Very low birth weight infants (<1,500 g) require adequate nutrient intake to develop satisfactorily. Human milk is essential in its diet, since it has a balanced nutritional composition that includes essential nutrients, such as polyunsaturated fatty acids, including omega 3 and 6 acids, of extreme importance for the development of the central nervous system and retina. However, only breast milk may not be sufficient to meet these needs, thus requiring supplementation. Nowadays, the possibility of adapting the human milk plus a concentrate with lyophilized from the human milk itself, which was developed in this work, has arisen. Objectives To determine the amount of total lipids and the lipid profile of Human Baseline Milk (LHB) and Concentrates with human milk lyophilized at different times (Human Milk Concentrated in the immediate period - LHCI, Concentrated Human Milk in the 3-month period - LHC3m and Human Milk Concentrated in the 6-month period - LHC6m). Methods Fifty samples of human milk from women donors of the Human Milk Bank of FMRP-USP who were accepted to participate in the study were obtained. Lipid quantification was performed by the MIRIS® Human Milk Analyzer and the fatty acid profile was determined by CG-FID, SHIMADZU®. Results There was a total lipid concentration in LHCI relative to LHB; the concentration (% of total fatty acids) of the acids was: palmitic (C16: 0) 22.30% LHB, 21.46% LHCI, 21.54% LHC3m and 21.95% LHC6m (p <0) , 01); oleic acid (C18: 1n-9) 30.41% LHB, 30.47% LHCl, 30.55% LHC3m and 29.79% LHC6m (p = 0.46); linoleic acid (C18: 2 n-6) 19.62% LHB, 19.88% LHCI, 19.49% LHC3m and 19.45% LHC6m (p = 0.58); arachidonic (C20: 4 n-6) 0.35% LHB, 0.16% LHCl, 0.13% LHC3m and 0.15% LHC6m (p <0.01); ?-linolenic acid (C18: 3 n-3) 1.32% LHB, 1.37% LHCI, 1.34% LHC3m and 1.34% LHC6m (p = 0.14); (C22: 6 n-3) 0.10% LHB, 0.06% LHCl, 0.05% LHC3m and 0.06% LHC6m (p <0.01). There are specific modifications in some fatty acids, however the sum of fatty acids by their structural classes do not present differences. Conclusion Despite the small differences found in the lipid profile, the concentrate with freeze-dried human milk is a food that can meet the needs of the newborn, since there was an increase in total lipids and preservation of essential nutrients present only in breast milk, butclinical studies still need to be done to evaluate the safety and efficacy of concentrate as a feed for very low birth weight infants.

Os ácidos graxos polinsaturados atuam no organismo humano em diversos processos fisiológicos e metabólicos, além de serem importantes na nutrição infantil. A quantificação dos ácidos graxos polinsaturados, devido à presença de vários sítios reativos na molécula, deve envolver processos de extração da gordura em condições amenas. Este trabalho teve como objetivos a comparação dos métodos analíticos para determinação de lipídios totais e ácidos graxos polinsaturados (ácido linoléico, ácido &#945;-linolênico, ácido araquidônico e ácido docosahexaenóico) em fórmula infantil, a quantificação dos ácidos graxos polinsaturados nas fórmulas infantis comerciais e o acompanhamento da estabilidade destes ácidos graxos neste alimento. Foram analisadas 15 amostras, sendo uma amostra da Nacional Institute of Standards and Techonology (NIST 1849) e 14 fórmulas infantis comercializadas no Estado de São Paulo. Os métodos analíticos comparados para extração de lipídios foram: gravimétricos (Bligh Dyer, Roese Gottlieb e hidrólise ácida AOAC 963.15) e por cálculo (AOAC 996.06 e método direto adaptado de Golay et al. (2006)). Para a preparação dos ésteres metílicos de ácidos graxos utilizaram-se metodologias descritas pela IUPAC, Hartman e Lago e método direto adaptado de Golay et al. (2006). Compararam-se diferentes padrões interno, sendo estes dos ésteres metílicos de ácidos graxos 13:0, 21:0 e 23:0 e fatores de resposta para quantificação dos ácidos graxos polinsaturados em relação aos ácidos graxos 16:0, 18:0 e 23:0. O estudo de estabilidade durou 8 meses, e as análises foram realizadas nos meses de março, abril, julho e outubro de 2008. Os melhores resultados, para gordura total e para ácidos graxos polinsaturados, foram obtidos pelo método oficial (Roese Gottilieb). Quanto ao cálculo dos ácidos graxos polinsaturados, o uso do padrão interno 23:0 e o fator de resposta de correção teórico em relação ao 23:0 revelam resultados mais satisfatórios. Das fórmulas infantis comerciais analisadas, 85,7% apresentaram pelo menos um analito em desacordo com a informação nutricional e 100% com relação à legislação brasileira e o Codex Alimentarius. Em relação à estabilidade dos ácidos graxos polinsaturados, apenas três fórmulas infantis não apresentaram diferença estatisticamente significativa (p>0,05) nos teores de ácido graxos no período de 8 meses. / Long chain polyunsaturated fatty acids are involved in several physiological and metabolic process of human organism. The quantification of polyunsaturated fatty acid, must involve the fat extraction in mild conditions, due the reactive sites in the molecule. This work had as objective the comparison of the analytical methods for determination of polyunsaturated fatty acid (linoleic acid, &#945;-linolenic acid, arachidonic acid and docosahexaenoic acid) and lipids in infant formula, quantification of polyunsaturated fatty acids in commercial infant formulas and polyunsaturated fatty acids stability evaluation in these foods. A 15 samples of infant formulas were analyzed, these one from NIST and 14 commercial infant formulas. The analytical methods to lipids extraction were: Bligh and Dyer, Roese Gottlieb, acid hydrolyze AOAC 963.15, acid hydrolyze AOAC 996.06 and direct method adapted from Golay et al. (2006). To prepared esters metilics fatty acids utilized the methods: IUPAC, Hartman and Lago and direct method. The quantization of polyunsaturated fatty acid was realized with different standards internal, like fatty acid methyl ester 13:0, 21:0 and 23:0, and flame ionization detector response factors of correction in relation fatty acids 16:0, 18:0 and 23:0. The stability study during 8 months and analysis were analyzed in the months March, April, July and October. The best results, for lipids and polyunsaturated fatty acid, were obtained by the official method, Roese Gottlieb and metilation followed Hartman and Lago. In accordance with the present study the internal standard 23:0, and the theoretical correction factors with relation 23:0 showed satisfactory trueness and precision for calculation of polyunsaturated fatty acid. Among of commercial infant formula analyzed, 85.7% had at least one analyte in disagreement with the nutritional label facts and 100% with respect to Brazilian Legislation and the Codex Alimentarius. Regarding polyunsaturated fatty acids stability, three infant formulas showed no statistically significant difference (p > 0.05) in levels of these fatty acids during the period of analysis (8 months).

Introdução: As doenças cardiovasculares são a principal causa de morbimortalidade mundial, tendo o Brasil um perfil semelhante. Embora diversos fatores de risco sejam associados com os eventos cardiovasculares, esses não conseguem justificar a manifestação de todos os eventos clínicos, indicando que outros componentes estão envolvidos. Diante desse cenário, a dieta ocupa papel de destaque na modulação dos fatores de risco modificáveis e por isso tem sido foco de inúmeros programas de políticas públicas. Objetivo: Avaliar o efeito da suplementação de ômega-3 sobre os fatores de risco cardiometabólicos clássicos e emergentes em indivíduos com diferentes níveis de risco cardiovascular. Métodos: Este estudo foi do tipo clínico baseado em intervenção nutricional, prospectivo, randomizado, duplo cego e placebo controlado. Foram selecionados indivíduos adultos e idosos (n=146), de ambos os sexos, dentre os quais 77 faziam parte do grupo intervenção w-3 e 69 eram do grupo placebo. O risco cardiovascular foi estimado por meio do escore de risco de Framingham (ERF), os grupos foram subdivididos em escore de risco baixo, intermediário e alto. Nos tempos basal e após 4 e 8 semanas de intervenção [3,0 g/dia w-3, contendo 60% de ácido eicosapentaenoico-docosahexaenoico (EPA-DHA) ou placebo] foram coletadas informações socioeconômicas e clínicas, antropométricas, consumo alimentar, nível de atividade física e coleta de sangue após jejum de 12-14h. A partir do plasma foram analisadas as concentrações dos marcadores lipídicos [Colesterol Total, lipoproteínas de baixa densidade (LDL) e alta densidade (HDL), Triacilglicerol (TG), Apolipoproteínas AI e B, ácidos graxos não esterificados (NEFAs) e LDL eletronegativa (-)], ácidos graxos plasmático e aspectos fisicoquímicos de lipopartículas (tamanho e concentração). Os resultados foram analisados por meio do programa SPSS 16.0, sendo adotado o nível de significância de p < 0,05. Resultados: Houve incorporação de 4% do DHA plasmático e consequente redução de 34% do TG em indivíduos com ERF intermediário e de 0,2% da APO AI, 20% de NEFAs e de 0,6% de HDLINTERMEDIÁRIA em indivíduos com alto ERF. Ao longo da intervenção, houve diminuição do CT (p = 0,002), da LDL (p = 0,003), do TG (p < 0,001), do colesterol não HDL (p < 0,001), da HDLPEQUENA (p = 0,011) e da LDL (-) (p < 0,001). Houve aumento da HDL-c (p = 0,003), das partículas de HDLGRANDE (p < 0,001) e HDLINTERMEDIÁRIA (p < 0,001) Conclusão: A suplementação com w-3 promoveu redução dos biomarcadores de risco cardiovascular clássicos e emergentes. Esse conjunto de resultados amplia o papel cardioprotetor do w-3. / Introduction: Cardiovascular diseases are the leading cause of morbidity and mortality worldwide, with Brazil a similar profile. Although many risk factors are associated with cardiovascular events, these can not justify the manifestation of all clinical events, indicating that other components are involved. Given this scenario, the diet takes prominent role in the modulation of modifiable risk factors and so has been the focus of numerous programs of public policies Objective: To evaluate the effect of supplementation of omega3 (n-3) on cardiometabolic risk factors and emerging classics in individuals with different levels cardiovascular risk. Methods: This study was based on clinical type of nutrition intervention, prospective, randomized, double-blind and placebo controlled. We selected adult and elderly subjects (n=146), both sexes, among which 77 were in the intervention group n - 3 and 69 were in the placebo group. Cardiovascular risk was estimed using the Framingham risk score (FRS), the groups were divided into low-risk score, intermediate and high. At baseline and after 4 and 8 weeks of intervention [3.0 g/ day n-3 containing 60% eicosapentaenoic + docosahexaenoic acid (EPA + DHA) or placebo] socioeconomic and clinical, anthropometric data, dietary intake, physical activity level and blood samples were collected after fasting for 12 -14h. Were analyzed from the plasma concentrations [total cholesterol (TC), low-density lipoprotein (LDL), high-density lipoprotein (HDL), triglycerides (TG), apolipoprotein (Apo) AI and B, non-esterified fatty acids (NEFAs) and electronegative LDL (-)], plasma fatty acids and physicochemical aspects of lipoparticles (size and concentration). The results were analyzed using the SPSS 16.0 program, adopting the level significance p < 0.05. Results: There was incresed of 4 % of plasma DHA and consequent 34% reduction of TG in individuals with intermediate FRS and 0.2% of the APO AI, 20 % of NEFAs and 0.6 % HDLINTERMEDIATE in subjects with high FRS. During the intervention, CT decreased (p = 0.002), LDL (p = 0.003), TG (p < 0.001), non-HDL cholesterol (p < 0.001) of the HDLSMALL (p = 0.011) and LDL (-) (p < 0.001). There was an increase in HDL-c (p = 0.003), HDLLARGE particles (p < 0.001) and HDLINTERMEDIATE (p < 0.001). Conclusion: Supplementation with n-3 promoted reduction of c1assic and emerging biomarkers on cardiovascular risk. This set of results extends the cardioprotective role of n-3.

Der globale Prävalenzanstieg allergischer Erkrankungen wird mit der westlichen Ernährung und einem sich ändernden Fettsäurespektrum assoziiert. Die omega-3 Fettsäure Docosahexaensäure (DHA) wurde bereits bei verschiedenen chronisch-entzündlichen Erkrankungen erfolgreich therapeutisch eingesetzt. Die dabei zugrunde liegenden Wirkmechanismen sind jedoch nicht vollständig geklärt. Deshalb wurde hier der molekulare Mechanismus der DHA-vermittelten Hemmung der IgE-Produktion in humanen B-Zellen sowie der verminderten Differenzierung IgE-produzierender Plasmazellen in vitro untersucht. Analysen der beteiligten Signaltransduktionswege offenbarten eine Reduktion der IL-4-abhängigen STAT6-Phosphorylierung und der CD40-vermittelten NFkappaB-Translokation, was zu einer Inhibition des IgE-Klassenwechsels auf dem Niveau des epsilon-Keimbahntranskriptes sowie der Aktivierungsinduzierten Desaminase führte. Weiterhin wurde in einer randomisierten, kontrollierten Doppelblindstudie die Supplementierung mit hochdosierter DHA bei Patienten mit atopischem Ekzem hinsichtlich klinischer und immunologischer Parameter geprüft. Dabei führte DHA zu einer Reduktion des Schweregrades der Erkrankung und zu einer verminderten IgE-Produktion anti-CD40/IL 4-stimulierter Blutzellen ohne Beeinflussung des Serum-IgE-Spiegels. Schließlich wurden die lokalen Prozesse nach DHA-Verabreichung in einem Mausmodell für proteininduzierte Dermatitis analysiert. Dabei war die Reduktion der klinischen Ekzemausprägung mit der verminderten Zahl dermaler CD8+ T-Zellen verbunden. Andere histologische Parameter und das Serum-IgE blieben jedoch unbeeinflusst. Durch die Fähigkeit von DHA, in den IgE-Klassenwechsel in B-Zellen einzugreifen, stellt die Supplementierung mit DHA somit eine mögliche präventive Maßnahme gegen allergische Erkrankungen dar. Ebenso ist DHA in der Lage, den Schweregrad des atopischen Ekzems durch die positive Beeinflussung lokaler inflammatorischer Prozesse signifikant zu verbessern. / The prevalence of allergic diseases has increased worldwide. Westernised diet with its changing polyunsaturated fatty acid (PUFA) proportions is considered to contribute to this development. The omega-3 PUFA Docosahexaenoic acid (DHA) has been reported to be antiinflammatory, but its way of action is not completely understood. Initially, the molecular mechanisms of DHA impact on IgE production in human B cells were examined in vitro. Thereby, DHA inhibited IgE production and the differentiation of IgE secreting cells. This was mediated through direct inhibition of the immunoglobulin isotype switching process by decreasing epsilon germline transcript and activation induced desaminase transcription. Analysis of involved signalling pathways revealed an inhibition of IL-4 driven STAT6 phosphorylation and a reduced NFkappaB translocation into nucleus upon CD40 ligation. Next, in a randomised, double bind, controlled clinical study the efficacy of high-dose DHA supplementation in atopic eczema was determined by investigating the impact on clinical and immunological parameters. In the DHA, but not in control group a clinical improvement of atopic eczema and a reduction of CD40/IL-4 mediated IgE synthesis of peripheral blood cells were observed whereas serum IgE levels remained unchanged. Finally, in a mouse model the impact of oral DHA application on allergen induced dermatitis as well as the underlying local mechanisms were investigated. Thereby, the DHA mediated reduction of clinical skin score was associated with decreased dermal CD8+ T cell numbers, whereas other histological parameters or serum IgE values were not affected. In summary the results indicate that dietary DHA may be effective in prevention of allergic diseases by interference with the IgE switching process and improve the clinical outcome of atopic eczema by its positive impact on local inflammatory processes.

Introdução Os recém-nascidos de muito baixo peso (<1.500g) necessitam de aporte adequado de nutrientes para desenvolver-se satisfatoriamente. O leite humano é fundamental na sua alimentação, já que possui uma composição nutricional balanceada que inclui nutrientes essenciais, tais como os ácidos graxos poli-insaturados, incluindo os ácidos ômega 3 e 6, de extrema importância para o desenvolvimento do sistema nervoso central e retina. No entanto, apenas o leite materno pode não ser suficiente para suprir essas necessidades, necessitando, portanto, de suplementação. Atualmente, surgiu a possibilidade de adequação do leite humano acrescido de um concentrado com liofilizado do próprio leite humano, que foi desenvolvido neste trabalho. Objetivos determinar a quantidade de lipídios totais e o perfil lipídico do Leite Humano Baseline (LHB) e dos Concentrados com o liofilizado de Leite Humano em diferentes momentos (Leite Humano concentrado no período imediato - LHCI, Leite Humano concentrado no período de 3 meses - LHC3m e Leite Humano concentrado no período de 6 meses - LHC6m). Métodos Foram obtidas 50 amostras de leite humano de mulheres doadoras do Banco de Leite Humano da FMRP-USP que aceitaram participar do estudo. A quantificação de lipídeos ocorreu pelo Analisador de Leite Humano MIRIS® e o perfil de ácidos graxos foi determinado por CG-FID, SHIMADZU®. Resultados Houve uma concentração de lipídios totais no LHCI em relação ao LHB; a concentração (% em relação ao total de ácidos graxos) dos ácidos foi de: palmítico (C16:0) 22,30% LHB, 21,46% LHCI, 21,54% LHC3m e 21,95% LHC6m (p<0,01); oleico (C18:1n-9) 30,41% LHB, 30,47% LHCI, 30,55% LHC3m e 29,79% LHC6m (p=0,46); linoléico (C18:2 n-6) 19,62% LHB, 19,88% LHCI, 19,49% LHC3m e 19,45% LHC6m (p=0,58); araquidônico (C20:4 n-6) 0,35% LHB, 0,16% LHCI, 0,13% LHC3m e 0,15% LHC6m (p<0,01); ?- linolênico (C18:3 n-3) 1,32% LHB, 1,37% LHCI, 1,34% LHC3m e 1,34% LHC6m (p=0,14); docosahexaenóico (C22:6 n- 3) 0,10% LHB, 0,06% LHCI, 0,05% LHC3m e 0,06% LHC6m (p<0,01). Há modificações específicas em alguns ácidos graxos, no entanto a somatória de ácidos graxos por suas classes estruturais não apresentam diferenças. Conclusão Apesar das pequenas diferenças encontradas no perfil lipídico, o concentrado com leite humano liofilizado é um alimento que pode vir a atender as necessidades do recém-nascido, já que houve aumento dos lipídios totaise preservação dos nutrientes essenciais presentes apenas no leite materno, mas estudos clínicos ainda precisam ser feitos para avaliar a segurança e eficácia do concentrado como alimentação dos recém-nascidos de muito baixo peso. / Introduction Very low birth weight infants (<1,500 g) require adequate nutrient intake to develop satisfactorily. Human milk is essential in its diet, since it has a balanced nutritional composition that includes essential nutrients, such as polyunsaturated fatty acids, including omega 3 and 6 acids, of extreme importance for the development of the central nervous system and retina. However, only breast milk may not be sufficient to meet these needs, thus requiring supplementation. Nowadays, the possibility of adapting the human milk plus a concentrate with lyophilized from the human milk itself, which was developed in this work, has arisen. Objectives To determine the amount of total lipids and the lipid profile of Human Baseline Milk (LHB) and Concentrates with human milk lyophilized at different times (Human Milk Concentrated in the immediate period - LHCI, Concentrated Human Milk in the 3-month period - LHC3m and Human Milk Concentrated in the 6-month period - LHC6m). Methods Fifty samples of human milk from women donors of the Human Milk Bank of FMRP-USP who were accepted to participate in the study were obtained. Lipid quantification was performed by the MIRIS® Human Milk Analyzer and the fatty acid profile was determined by CG-FID, SHIMADZU®. Results There was a total lipid concentration in LHCI relative to LHB; the concentration (% of total fatty acids) of the acids was: palmitic (C16: 0) 22.30% LHB, 21.46% LHCI, 21.54% LHC3m and 21.95% LHC6m (p <0) , 01); oleic acid (C18: 1n-9) 30.41% LHB, 30.47% LHCl, 30.55% LHC3m and 29.79% LHC6m (p = 0.46); linoleic acid (C18: 2 n-6) 19.62% LHB, 19.88% LHCI, 19.49% LHC3m and 19.45% LHC6m (p = 0.58); arachidonic (C20: 4 n-6) 0.35% LHB, 0.16% LHCl, 0.13% LHC3m and 0.15% LHC6m (p <0.01); ?-linolenic acid (C18: 3 n-3) 1.32% LHB, 1.37% LHCI, 1.34% LHC3m and 1.34% LHC6m (p = 0.14); (C22: 6 n-3) 0.10% LHB, 0.06% LHCl, 0.05% LHC3m and 0.06% LHC6m (p <0.01). There are specific modifications in some fatty acids, however the sum of fatty acids by their structural classes do not present differences. Conclusion Despite the small differences found in the lipid profile, the concentrate with freeze-dried human milk is a food that can meet the needs of the newborn, since there was an increase in total lipids and preservation of essential nutrients present only in breast milk, butclinical studies still need to be done to evaluate the safety and efficacy of concentrate as a feed for very low birth weight infants.