Thesis (Ph.D.)--University of Western Australia, 2003.

Thesis (M.S.)--Ohio State University, 2009. / Title from first page of PDF file. Includes bibliographical references (p. 85-94).

Thesis (PhD.(Physiology)--Faculty of Health Sciences)-University of Pretoria, 2005. / Summary in English and Afrikaans. Also available on the Internet via the World Wide Web.

Conselho Nacional de Desenvolvimento CientÃfico e TecnolÃgico / This work aimed to develop refining processes, chemical alcoholysis followed by separation of fatty acids using the complexation with urea technique for the synthesis of poly-unsaturated esters from waste of fish oils. The special crude fish oil was purchased from Company Campestre - SÃo Paulo. Initially this oil has undergone a process of physical and chemical refining. From the refined oil, an alcoholysis process was carried out to obtain the mixture of free fatty acids. From the hydrolyzed material were obtained 32.78% p/p of PUFAs against 19.73% p/p of ω-3 concentrates. The free fatty acids were separated using the complexation with urea technique. The best operating conditions for separation of the fatty acids was: ratio 7:1 (urea / oil) and the crystallization temperature at -23ÂC for a time of 20 hours. After treatment of the material, the total PUFAs production was 47.87%, a ω-3 concentration of 27.59% with a saturated fraction of 4.48%. When the temperature was raised to -10ÂC, the PUFAs production was halved, reaching the value of 28.08% and 25.49% of ω-3 which was slightly altered and a saturated fraction of 42.44%. For the ester synthesis was mounted a statistical factor of two levels in order to determine the parameters which optimized the process. In the synthesis phase, the combination of temperature, glycerin concentration and catalyst was significant, and it was observed a greater influence of the glycerin concentration due to the excessive use of glycerin to favor the formation of the ester. After the analysis of the kinetic results was observed that the interactions temperature-glycerin and temperature-glycerin-catalyst were not significant (below 95%). The response interaction graphic showed the least free fatty acids index after one hour of reaction, and that the greater interaction was glycerin (5%)-catalyst (3%). It was concluded that the yields to obtain the polyunsaturated ω-3 and ω -6 from the waste of fish oil were satisfactory (85,3%). Therefore, it is concluded that it is feasible the synthesis of polyunsaturated esters of marine oils from fish waste, because this technology provides important results to avoid environmental impacts, reduce imports of fish oils and, consequently, reduce improper fishing. The aquaculture industry may be stocked with diets enriched with EPA and DHA for shrimp and fish farming, besides contributing to supply ω-3 for nutraceutical purposes. / Este trabalho teve o objetivo de desenvolver os processos de refino, alcoÃlise quÃmica seguida da separaÃÃo dos Ãcidos graxos utilizando a tÃcnica da complexaÃÃo com urÃia para a sÃntese de Ãsteres poli-insaturados a partir de resÃduos de Ãleos de pescado. O Ãleo bruto especial de peixe foi adquirido da Empresa Campestre â SÃo Paulo. Inicialmente este Ãleo sofreu um processo de refino fÃsico e quÃmico. A partir do Ãleo refinado, foi realizado um processo de alcoÃlise para se obter a mistura de Ãcidos graxos livres. Do material hidrolisado, foram obtidos 32,78% p/p de PUFAs contra 19,73% p/p de concentrados de ω-3. Os Ãcidos graxos livres foram separados utilizando-se a tÃcnica da complexaÃÃo com urÃia. As melhores condiÃÃes operacionais para separaÃÃo dos Ãcidos graxos foram: a relaÃÃo 7:1 (urÃia/Ãleo) e a temperatura de cristalizaÃÃo a -23ÂC por um tempo de 20 horas. ApÃs o tratamento do material, a produÃÃo total de PUFAs foi de 47,87%, uma concentraÃÃo de ω-3 de 27,59% com uma fraÃÃo saturada de 4,48%. Quando se elevou a temperatura para -10ÂC, a produÃÃo de PUFAs reduziu pela metade, atingindo o valor de 28,08% e 25,49% de ω-3, que pouco foi alterada e uma fraÃÃo de saturados de 42,44%. Para a sÃntese do Ãster de glicerina foi montado um fatorial estatÃstico de dois nÃveis a fim de se determinar os parÃmetros que otimizaram o processo. Na fase de sÃntese, a conjugaÃÃo de temperatura, concentraÃÃo de glicerina e catalisador foram significantes, tendo sido observado uma maior influÃncia da concentraÃÃo de glicerina, em virtude do uso excessivo de glicerina para favorecer a formaÃÃo do Ãster. ApÃs as anÃlises dos resultados cinÃticos, foi observado que as interaÃÃes temperatura-glicerina e temperatura-glicerina-catalisador nÃo foram significantes (abaixo de 95%). O grÃfico da interaÃÃo para resposta mostrou o menor Ãndice de Ãcidos graxos livres apÃs uma hora de reaÃÃo, e que a maior interaÃÃo foi glicerina (5%)-catalisador (3%). Foi concluÃdo que os rendimentos para obtenÃÃo dos poli-insaturados ω-3 e ω -6 dos resÃduos de Ãleo de pescado foram satisfatÃrios (85,3%). Conclui-se, portanto, que à viÃvel a sÃntese de Ãsteres poli-insaturados de Ãleos marinhos a partir de rejeitos de pescados, pois esta tecnologia proporciona resultados importantes para evitar impactos ambientais, diminuir as importaÃÃes de Ãleos de peixe e, consequentemente, reduzir a pesca indevida. O setor aquÃcola poderà ser abastecido com raÃÃes enriquecidas com EPA e DHA para camarÃes e peixes de cultivo, alÃm de contribuir para oferta de ω-3 para fins nutracÃuticos.

Ultraviolet radiation (UVR) in solar light is important for skin biology. It is involved in the development acute and chronic skin inflammation, aging and cancer, causing erythema, tanning and local or systemic immunosuppression. Omega-3 polyunsaturated fatty acids (n-3 PUFA) are considered anti- inflammatory and could reduce the damage caused by overexposure to UVR. Although, n-3 PUFA have been considered as photoprotective agents, their exact mechanisms of action is not completely understood. The aim of the work is to determine the effect of UVR and the n-3 PUFA eicosapentaenoic acid (EPA), or docosahexaenoic acid (DHA) on human skin cells (in vitro study), specifically on: cell viability, apoptosis and their metabolism through the cyclooxygenase and lipoxygenase pathways. Also, to study the cellular incorporation and effect of n-3 PUFA on the fatty acid profile of skin cells. A clinical study was undertaken to assess the incorporation of n-3 PUFA supplements in human skin. A clinical study was performed in 40 healthy women (active group) supplemented with 4g/day of EPA (70%) and DHA (10%) and 40 healthy women (placebo group) supplemented with 4g/day of glyceryl tricoprylate coprate (GTCC). After 3 months, both blood samples and skin punch biopsies were collected and analysed for fatty acids by gas chromatography (GC). HaCaT keratinocytes and 46BR.1N fibroblasts were cultured and treated with 10 and 50μM of either EPA, or DHA or oleic acid (OA) for 72h and exposed to 15 and 50 mJ/cm2. Cell viability was measured by the MTT assay and cell apoptosis by a colorimetric method, at 24h post UVR. Cells and culture media were analysed by GC and liquid chromatography tandem mass spectrometry (LC/ESI-MS/MS) to assess cellular fatty acids and production of eicosanoids. The clinical a study showed that in RBC saturated fatty acids (SFA) (44.27±7.43%) were the main fatty acid group followed by n-6 PUFA (29.61±5.53%). While in dermal tissue monounsaturated fatty acids (MUFA) (58.90±9.80%) was the main fatty acid group followed by SFA (27.06±6.78%). A significant increase in EPA, DHA and docosapentaenoic acid (DPA) was observed in RBC but only EPA was significantly increased in the dermis post n-3 PUFA supplementation. . The viability of HaCaT keratinocytes and 46BR.1N fibroblasts decreased post UVR and this was further reduced post PUFA treatment. Cell apoptosis increased when cells were exposed to UVR and further increased when cells were treated with EPA and DHA. . In HaCaT keratinocytes MUFA (54.22±8.82%) was the main fatty acid group followed by FAS (37.11±.9.16%), while SFA (51.94±8.68%) was the main group followed by MUFA (27.07±4.79) in 46BR.1N. Treated both cells with EPA and DHA showed significant increased in cellular EPA, DPA and DHA. 46BR.1N fibroblasts produced higher levels of prostaglandins (PG) compared to HaCaT keratinocytes: PGE2 and PGD2 were the main PG in both HaCaT (7.96±3.18 and 1.48±1.19 pg/million cell; respectively) and 46BR.1N with (44.2±23.00 and 17.1±9.71 pg/million cell; respectively). Significant increase in PGE1 and PGE2 occurred when cells were exposed to 15mJ/cm2 UVR. Treatment with n-3 PUFA decreased the level of PGE1 and PGE2, and increase production PGE3 at the baseline and post UVR. Both cell lines produced hydroxy fatty acids and the concentration of these mediators was higher in 46BR.1N than HaCaT. The concentrations of these mediators were significant increased post UVR: treatment with n-3 PUFA decreased the level of HODE and HETE, and increase production of HEPE and HDHA at baseline and post UVR. Overall, n-3PUFA treatment led to increases in the content of EPA and DHA on RBC, dermal tissue and human skin cell lines. EPA and DHA in skin cell lines appear to offer protection by increasing cellular apoptosis, decreasing inflammatory mediators specifically PGE2 and 12-HETE, and increasing anti-inflammatory mediators such as PGE3, 15-HEPE and 17-HDHA.

Ultraviolet radiation (UVR) in solar light is important for skin biology. It is involved in the development acute and chronic skin inflammation, aging and cancer, causing erythema, tanning and local or systemic immunosuppression. Omega-3 polyunsaturated fatty acids (n-3 PUFA) are considered anti- inflammatory and could reduce the damage caused by overexposure to UVR. Although, n-3 PUFA have been considered as photoprotective agents, their exact mechanisms of action is not completely understood. The aim of the work is to determine the effect of UVR and the n-3 PUFA eicosapentaenoic acid (EPA), or docosahexaenoic acid (DHA) on human skin cells (in vitro study), specifically on: cell viability, apoptosis and their metabolism through the cyclooxygenase and lipoxygenase pathways. Also, to study the cellular incorporation and effect of n-3 PUFA on the fatty acid profile of skin cells. A clinical study was undertaken to assess the incorporation of n-3 PUFA supplements in human skin. A clinical study was performed in 40 healthy women (active group) supplemented with 4g/day of EPA (70%) and DHA (10%) and 40 healthy women (placebo group) supplemented with 4g/day of glyceryl tricoprylate coprate (GTCC). After 3 months, both blood samples and skin punch biopsies were collected and analysed for fatty acids by gas chromatography (GC). HaCaT keratinocytes and 46BR.1N fibroblasts were cultured and treated with 10 and 50μM of either EPA, or DHA or oleic acid (OA) for 72h and exposed to 15 and 50 mJ/cm2. Cell viability was measured by the MTT assay and cell apoptosis by a colorimetric method, at 24h post UVR. Cells and culture media were analysed by GC and liquid chromatography tandem mass spectrometry (LC/ESI-MS/MS) to assess cellular fatty acids and production of eicosanoids. The clinical a study showed that in RBC saturated fatty acids (SFA) (44.27±7.43%) were the main fatty acid group followed by n-6 PUFA (29.61±5.53%). While in dermal tissue monounsaturated fatty acids (MUFA) (58.90±9.80%) was the main fatty acid group followed by SFA (27.06±6.78%). A significant increase in EPA, DHA and docosapentaenoic acid (DPA) was observed in RBC but only EPA was significantly increased in the dermis post n-3 PUFA supplementation. . The viability of HaCaT keratinocytes and 46BR.1N fibroblasts decreased post UVR and this was further reduced post PUFA treatment. Cell apoptosis increased when cells were exposed to UVR and further increased when cells were treated with EPA and DHA. . In HaCaT keratinocytes MUFA (54.22±8.82%) was the main fatty acid group followed by FAS (37.11±.9.16%), while SFA (51.94±8.68%) was the main group followed by MUFA (27.07±4.79) in 46BR.1N. Treated both cells with EPA and DHA showed significant increased in cellular EPA, DPA and DHA. 46BR.1N fibroblasts produced higher levels of prostaglandins (PG) compared to HaCaT keratinocytes: PGE2 and PGD2 were the main PG in both HaCaT (7.96±3.18 and 1.48±1.19 pg/million cell; respectively) and 46BR.1N with (44.2±23.00 and 17.1±9.71 pg/million cell; respectively). Significant increase in PGE1 and PGE2 occurred when cells were exposed to 15mJ/cm2 UVR. Treatment with n-3 PUFA decreased the level of PGE1 and PGE2, and increase production PGE3 at the baseline and post UVR. Both cell lines produced hydroxy fatty acids and the concentration of these mediators was higher in 46BR.1N than HaCaT. The concentrations of these mediators were significant increased post UVR: treatment with n-3 PUFA decreased the level of HODE and HETE, and increase production of HEPE and HDHA at baseline and post UVR. Overall, n-3PUFA treatment led to increases in the content of EPA and DHA on RBC, dermal tissue and human skin cell lines. EPA and DHA in skin cell lines appear to offer protection by increasing cellular apoptosis, decreasing inflammatory mediators specifically PGE2 and 12-HETE, and increasing anti-inflammatory mediators such as PGE3, 15-HEPE and 17-HDHA.

Diss. (sammanfattning) Umeå : Umeå universitet, 2010.

Human skin has distinct lipid metabolism and production of bioactive lipid mediators that can be modulated by nutritional supplementation with omega-3 polyunsaturated fatty acids (n-3 PUFA), of which eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids exert anti-inflammatory effects. The aims of this project were to gain better understanding of their individual mechanisms in human epidermis and dermis. HaCaT keratinocytes, 46BR.1N fibroblasts, primary human epidermal keratinocytes and dermal fibroblasts were treated with EPA or DHA for 72h and then sham-irradiated or exposed to 15 mJ/cm2 ultraviolet radiation (UVR). Viability was measured by the MTT assay. The expression of cyclooxygenase-2 (COX-2), microsomal prostaglandin synthase-1 (mPGES-1) and 15-hydroxyprostaglandin dehydrogenase (15-PGDH) proteins was explored by western blotting. Human skin explants (n=4 donors) were cultured for 3 or 6 days and supplemented with EPA, DHA or vehicle. Culture media were collected to evaluate tissue damage and PUFA cytotoxicity (lactate dehydrogenase assay). Epidermal and dermal lipid profiles were assessed by gas chromatography and liquid chromatography coupled to tandem mass spectrometry. Primary keratinocytes were treated with fatty acids and various lipid mediators for 48h. Their effect was determined by the scratch assay and transepithelial electrical resistance. UVR upregulated COX-2 in HaCaT and primary epidermal keratinocytes, but did not affect mPGES-1 and 15-PGDH protein expression. UVR upregulated COX-2 and mPGES-1 in 46BR.1N fibroblasts but had no effect on 15-PGDH expression. The same UVR dose did not alter the expression of COX-2, mPGES-1 and 15-PGDH in primary dermal fibroblasts. Only EPA attenuated COX-2 expression in HaCaT and primary keratinocytes and either EPA or DHA had any effect in 46BR.1N and primary fibroblasts. Skin explants showed initial post-biopsy tissue damage. EPA and DHA supplementation augmented cellular levels of the corresponding fatty acids in both epidermis and dermis to a different extent. Increased uptake of DHA in the dermis was accompanied by reduced arachidonic acid levels. EPA treatment stimulated the production of PGE3 and various HEPE in epidermis, while DHA treatment caused high levels of HDHA species in dermis. N-3 PUFA and their derivatives delayed wound healing, cell migration and epidermal barrier permeability, while n-6 PUFA lipids showed the opposite effect. Overall, these findings suggest that EPA and DHA differently affect skin cells and skin, with EPA preference in epidermis and DHA in the dermis. These results highlight the importance of differential skin responses that could be important in skin health and disease.