Tesi sul tema "Lipids in human nutrition"

Medium chain triglycerides (MCT) have long been advocated as potential weight-lowering agents or potential tools in the treatment and prevention of human obesity. These statements have been made after findings from human and animal trials that consumption of MCT increases energy expenditure and fat oxidation compared to long chain triglycerides (LCT). In addition, animal studies have resulted in lower body weight gain and smaller fat depots when animals were fed MCT compared to those fed LCT. However, long-term controlled trials studying the effects of consumption of MCT in humans have not been conducted and the longest trial to date, 14 d of duration, has shown that the effect of MCT on energy expenditure may be transient. Therefore, we aimed to determine whether, in controlled feeding conditions, consumption of MCT for 4 wk would lead to differences in energy expenditure and substrate oxidation versus consumption of an isocaloric diet rich in LCT. Furthermore, our aim was to establish whether consumption of MCT for 4 wk would lead to greater changes in body composition than would LCT consumption. We conducted two randomized, controlled, crossover feeding trials involving overweight women and men to test our objectives. A secondary objective was to examine the potential satiating effect of MCT, and this was tested in men. Finally, a third objective was to determine whether, when combined with phytosterols and flaxseed oil, MCT consumption would result in different blood lipid profile compared to LCT. Nineteen healthy overweight women and 24 healthy overweight men participated in two separate randomized controlled trials to test these objectives. Energy expenditure and body composition were assessed at the beginning and end of each experimental phases, which differed only in the type of fat included in the controlled diets. Blood samples were also taken at baseline and endpoint of each phase to determine plasma lipid concentrations. Result

Lifestyle and dietary predictors of total fat intake (%kcal) were examined, to identify groups potentially at risk of hypercholesterolemia. In 1987, 301 Montreal adults were sampled by random digit dialing. A diet history and lifestyle habits were recorded. Mean total fat intake (%kcal) was 34.75% ($ pm$6.23). Principal Components Analysis identified specific dietary patterns. Regression analyses indicated that the first 4 food factors were associated with total fat intake (%kcal p $<$ 0.01). Household size was inversely related to total fat (%kcal); 0.8% less kcal from fat for each additional member of the household (p $<$ 0.01). Females had 2.8% more kcal from fat than males (p $<$ 0.01). The number of fast food meals consumed per week was positively associated with outcome when age was considered (p $<$ 0.05). BMI and education level were not associated with total fat (%kcal). Smoking was associated with intake of higher fat foods (p $<$ 0.01). These predictors can identify groups in the population who may be at increased cardiovascular risk.

The objective of the research was to carry out the biogeneration of lipophenols by enzymatic esterification of tricaprylin and caprylic acid with catechin and catechol in a model hexane system. Commercial lipases, including Lipase N from Rhizopus niveus, Lipozyme IM from Mucor miehei and Novozym 435 from Candida antarctica were used throughout this study. The effects of reaction time, incubation temperatures and agitation speeds on enzymatic hydrolytic activity were investigated to determine the optimal conditions for biocatalysis. The optimal temperatures for biocatalysis were determined to be 37.5°C for Lipase N, and 55°C for Lipozyme IM and Novozym 435; the optimum agitation speed was 100 rpm. Using Lipase N, maximum hydrolysis of 1.66 mumol free fatty acids/mL was obtained after 1.5 days of incubation, while with Lipozyme IM, maximum hydrolysis of 8.1 and 8.5 mumol free fatty acids/mL was obtained after 1 and 4 days, respectively. With Novozym 435, the highest hydrolysis of 4.0 and 6.1 mumol free fatty acids/mL were found after 2 and 9 days, respectively.

Orientador: Fernanda Klein Marcondes
Tese (doutorado) - Universidade Estadual de Campinas, Faculdade de Odontologia de Piracicaba
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Resumo: O estresse crônico é um fator de risco para doenças cardiovasculares e metabólicas e tem sido relacionado ao desenvolvimento de distúrbios alimentares. O objetivo deste trabalho foi avaliar o efeito, a longo prazo, do estresse crônico moderado e imprevisível (ECMI) e da ingestão de dieta hipercalórica (DH) sobre o peso corporal e metabolismo de ratos. No capítulo 1, foi avaliado o efeito da associação entre ECMI e DH sobre o peso corporal, adiposidade, teste de tolerância à glicose (TTG) e perfil lipídico de ratos Sprague-Dawley, divididos em 4 grupos: dieta padrão (DP), dieta padrão+ECMI (DPE), DH e DH+ECMI (DHE), analisados durante sete semanas. Duas semanas após a aplicação do ECMI, os grupos DPE e DHE apresentaram aumento significativo na concentração plasmática de corticosterona que os grupos DP (2,09±0,41 vs. 19,42±2,85ng/mL) e DH (3,34±0,66 vs. 18,72±3,18ng/mL), respectivamente. Os grupos DH e DHE apresentaram aumento significativo no peso corporal final que os grupos DP (435±3 vs 463±8g) e DPE (425±5 vs 444±8g), respectivamente. O estresse induziu redução significativa no ganho de peso e na ingestão alimentar, na primeira semana do protocolo de ECMI. Os grupos ECMI e DH apresentaram aumentos significativos nas concentrações plasmáticas (mmol/L) de colesterol total (DP: 1,44±0,05; DPE: 1,54±0,05; DH: 1,53±0,09; DHE: 1,88±0,08), triglicerídeos (DP: 1,41±0,09; DPE: 1,75±0,16; DH: 1,67±0,13; DHE: 2,42±0,28) e LDL (DP: 0,46±0,06; DPE: 0,73±0,08; DH: 0,77±0,08; DHE: 0,87±0,10). No TTG, os grupos DPE e DHE apresentaram área sob a curva significantemente maior comparado aos grupos DP (13549±387 vs. 14267±344) e DH (15852±270 vs. 16476±559mg x min./dL), respectivamente. No capítulo 2 avaliamos a relação entre redução do ganho de peso corporal induzida pelo ECMI e os períodos de restrição alimentar do protocolo de estresse. Ratos Sprague- Dawley (2 meses de idade) foram divididos em três grupos: Controle, ECMI e Alimentação-Pareada (AP: alimentados com a mesma quantidade de ração ingerida pelo grupo ECMI). Os grupos ECMI e AP apresentaram redução significativa de 12 e 15% na ingestão alimentar durante o protocolo de ECMI, comparado ao controle. Imediatamente após o ECMI, ratos estressados e AP apresentaram redução significativa de 6 e 10% no peso corporal e de 19 e 14% na gordura epididimal, respectivamente, comparados ao grupo controle. O grupo AP, mas não o grupo ECMI, apresentou redução nas gorduras mesentérica (41%), inguinal (28%) e perirrenal (40%), menor proporção gordura total/peso corporal final (0,02±0,001 vs. 0,03±0,001) e menor porcentagem de gordura na carcaça (3,39±0,44 vs. 6,29±0,51%) comparado ao controle, sem diferença entre controle e ECMI. O efeito redutor do ECMI sobre o peso corporal não pode ser totalmente explicado pela restrição alimentar durante o estresse. A longo prazo, o ECMI e o tratamento com dieta hipercalórica têm efeitos semelhantes sobre a dislipidemia em ratos. Tais efeitos são potencializados quando combinados. O efeito redutor no peso corporal, promovido pelo ECMI, sofre influência da dieta empregada, e é revertido após o estresse.
Abstract: Chronic stress is a risk factor for cardiovascular and metabolic diseases and has been associated to development of eating disorders. The purpose of this study was to investigate the long-term effect of chronic mild and unpredictable stress (CMS) and hypercaloric diet (HD) on body weight and metabolism of rats. In Chapter 1, we studied the effect of the association of CMS and HD, fifteen days after the end of CMS on body weight, adiposity, oral glucose tolerance test (OGTT) and lipid profile of Sprague-Dawley rats. The rats were divided into 4 groups: standard diet (SD), SD + CMS (CMS), hypercaloric diet (HD) and HD + CMS, evaluated during seven weeks. The data were analyzed by two-way ANOVA (P<0.05). Two weeks after the end of CMS, both the groups SD+CMS (2,09±0,41 vs. 19,42±2,85ng/mL) and HD + CMS (3.34±0.66 vs 18.72±3.18ng/mL) had higher plasmatic corticosterone concentration than SD groups and HD, respectively. The groups HD and HD + CMS had higher final body weight that SD groups (435±3 vs 463±8g) and SD+CMS (425±5 vs 444±8g), respectively. CMS induced lower body weight gain and lower food intake only in the first week of CMS protocol. The SD+CMS and HD groups showed increased plasma concentrations (mmol/L) of total cholesterol (SD: 1.44±0.05; SD+CMS: 1.54±0.05; HD: 1.53±0.09, HD+CMS: 1.88±0,08), triglycerides (SD: 1.41±0.09; SD+CMS: 1.75±0.16, HD: 1.67±0.13; HD+CMS: 2.42±0.28) and LDL (DC: 0.46±0.06; SD+CMS: 0.73±0.08; HD: 0.77±0.08; HD+CMS: 0.87±0.10). SD+CMS and HD+CMS groups had higher area under the curve of the OGTT than SD groups (13,549±387 vs. 14267±344 mg x min/dL) and HD (16476±559 vs. 15,852±270 mg x min/dL), respectively. In Chapter 2, we investigated the association between CMS-induced body weight loss and food restriction, utilized in the protocol of stress. Sprague-Dawley rats (2 months old) were divided into three groups: Control, CMS and pair-fed (PF: the rats were fed with the same amount of food as the CMS group ate voluntarily during the corresponding period of stress protocol). The data were analyzed by One-way ANOVA (P<0.05). CMS and PF groups showed reduction of 12 and 15% in food intake during the protocol of CMS, compared to control. After CMS, PF and stressed rats showed a reduction of 6 and 10% in the final body weight and had reduction of 19 and 14% in epididymal fat compared to the control group, respectively. The PF group, but not the group CMS, showed lower mesenteric (41%), inguinal (28%) and perirenal (40%) fat mass, lower total fat / final body weight (0.02±0001 vs. 0,03±0001) and lower percentage of fat in the carcass analysis (3.39±0.44 vs. 6.29±0.51%) compared to the control, without difference between control and CMS. The lower body weight of CMS cannot be fully explained by food restriction during the stress. The CMS and hypercaloric diet did have similar long-term effects on dyslipidemia in rats. These effects are enhanced when combined. The reduction in body weight promoted by CMS is influenced by diet employed, and is reversed after the stress.
Doutorado
Fisiologia Oral
Doutor em Odontologia

An evaluation of the impact of the nutritional program "Mangeons en coeur" was conducted with 64 participants in this program. The two study objectives were to measure any changes in the dietary intake of program participants and to evaluate the performance of a food habits questionnaire in evaluating change in fat intake at the individual level. The dietary evaluation by the diet history indicated no change in the total intake of lipids (31% of energy as lipids) over the course of the program. Comparisons of the total lipid intake as measured by the diet history and food habits questionnaire indicated significant correlations at both baseline and at the end of the program.

Despite the recognition that culturally diverse groups of children in low income areas are at elevated risk for future health problems, no dietary/nutritional studies have been reported on this population. Children aged 9-12 were sampled from schools selected on the basis of a poverty index and ethnicity. Nutritional information (24-hour recall, heights and weights) indicated children were growing well but a high proportion were overweight. A comparison of reported energy intake to calculated BMR indicated that overweight children under-estimated their intakes. Mean intakes met the Canadian RNIs except for calcium in 10-12 year old girls. Nutrient intake was related to family size, income and ethnic origin. Validation of two alternative tools to measure fat intake proved unsuccessful. In conclusion, dietary intake for these school children was adequate for all nutrients except calcium in 10-12 year old girls, but for approximately 40% of children, intake exceeded energy requirements.

The aim of the present study was to determine the effect of vitamin D supplementation (4000 IU or 6000 IU of cholecalciferol daily for 6 months) on fasting plasma glucose, fasting insulin, glycated hemoglobin, and lipid profile in a sample of African-Americans and Hispanics with T2D and vitamin D insufficiency. Seventy five participants were recruited by community outreach. Plasma glucose concentration was measured by hexokinase enzymatic method. Glycated hemoglobin was measured by the DCA2000+ system. Insulin in fasting blood was determined by radioimmunoassay. Plasma total cholesterol, triglycerides, low-density lipoprotein, and high-density lipoprotein cholesterol were assayed by enzymatic methods. Serum vitamin D concentrations were measured with an enzyme-immunoassay kit. Mixed model was used to compare treatment effects and Bonferroni multiple comparison tests was used to detect significant changes from baseline, 3 months, and 6 months on the outcome variables. Significant improvements in serum 25(OH)D levels were seen from baseline to 3 month and 6 months respectively in both treatments (from 22.25 ± 7.19 to 37.34 ± 12.31 and 37.99 ± 13.22, PPP=0.040) was observed. Similarly, a significant change in serum triglycerides was observed at 6 months in the 6000 IU group (from 201.44 ± 91.35 to 172.92 ± 76.87 mg/dl, P=0.037). However, when the model was adjusted for confounders, significance was lost. Vitamin D supplementation did not improve glucose homeostasis in this sample. The positive effect of vitamin D supplementation on lipid profile may be mediated by other cofactors related to vitamin D metabolism.

To examine the effects of fat composition and supplemental vitamin E (Vit E) and selenium (Se) on in vivo lipid peroxidation, diet-induced hypercholesterolemia, and glutathione (GSH) metabolism, male Syrian hamsters were fed for three weeks butter fat (BF-) or fish oil- (FO-)based diets supplemented with Vit E and/or Se. The effect of supplemental Vit E and Se on tissue lipid peroxidation (LPO), glutathione peroxidase (GSH-Px) activity and GSH concentrations differed between heart and liver and also was affected by dietary fat. The reduced glutathione/oxidized glutathione (GSH/GSSG) ratio was more consistently associated with tissue lipid peroxidation than was tissue Vit E content. Plasma lipids were lowered with supplemental Se and Vit E. Se supplementation, however, exerted a more potent hypolipidemic effect than Vit E. A pro-oxidative action of Se in hearts of FO-fed hamsters was noted, which was inhibited by supplemental Vit E. Hence, the combination of Vit E and Se may offer the most benefit against diet-induced oxidative stress and hyperlipidemia.

The present study explored whether African American women's level of dietary fat intake could be predicted by the variables of food preferences and preparation methods, support for healthy eating from family and friends, attitudes toward health, and acculturation. The present study also explored whether African American women's level of dietary fat intake could be predicted by the variables of food preferences and preparation methods, support for healthy eating from family and friends, and attitudes toward health, as moderated by acculturation.Information was obtained from five hundred and nintey nine African American females between the ages of eighteen and forty four. The women were recruited from a Midwestern univeristy, an undergraduate and graduate chapter of an African American sorority, two African American professional organizations, and through the snowball method. Particpants completed a Demographic Questionnaire, The Eating Behavior Patterns Questionnaire, The African American Acculturation Scale Short Form, The Health Attitudes Scale, The Social Support Scale, and The Eating Patterns Subscale on the Eating Habits Questionnaire. The data was collected via hardcopy and InQsit, an online survey program.Numerous preliminary tests were run to screen the data for outliers, linearity, and multicollinearity. Then, two forced entry multiple regressions were performed. In the first analysis, the overall model was a significant predictor of dietary fat intake. African American women's preferred foods, positive and negative support from friends, overallconcern for health, and intentions to adopt positive health practices are significant predictors of their level of dietary fat intake. In fact, these variables acccounted for almost half of the amount of variance in dietary fat intake. The second model was not significant and acculturation was not a significant predictor or moderator of dietary fat intake. Although acculturation was proven to be influential to dietary behaviors in African Americans in previous literature, the findings were not confirmed in this study.Multiple possibilies may explain the lack of significant findings between level of acculturation and dietary fat intake. The women in the current study were of a higher educational and income status than women in previous studies indicating significant findings. This difference in education and income, among other factors, may account for the difference in significant findings. The information gained in this study can be used to develop pschoeducational and treatment programs aimed at helping African American women prevent or treat health problems associated with poor eating habits. Several research implications are also noted.
Department of Counseling Psychology and Guidance Services

Fish oil sparing and replacement is a major focus in the fields of aquaculture and aquaculture nutrition. Most of the commercial fish oil production is consumed by the aquafeed industry due to its highly digestible energy and elevated content of long-chain polyunsaturated fatty acids (LC-PUFAs; ARA – 20:4n-6, EPA – 20:5n-3, and DHA – 22:6n-3), being a valuable ingredient. Given the finite supply and the growing demand for fish oil its price has increased quite drastically, leading to the search for alternative lipid sources. Generally, vegetable- and terrestrial animal-origin alternatives lack LC-PUFAs, which are physiologically important nutrients for all fish, and considered essential fatty acids for carnivorous species. When fish oil is spared or replaced by alternative lipids fish survival, growth performance, and fish health are commonly impaired if adequate levels of essential fatty acids are not provided within feeds. Additionally, fish oil sparing typically distorts fillet fatty acid profile and associated nutritional value compared to a fish oil-based diet reflecting the composition of the alternative lipid used. It is clear that to address the fish oil bottleneck in aquafeed manufacturing, researchers must understand the essential fatty acid requirements of the key commercial fish species. Fatty acid essentiality in fish has been investigated, and there is preliminary evidence that not all LC-PUFAs may be equally required, with DHA being more important, and EPA being more expendable. Whereas ARA has not been investigated in the same extent as n-3 LC-PUFAs. Additionally, certain fatty acids groupings such as saturated fatty acids (SFAs) and monounsaturated fatty acids (MUFAs) may influence LC-PUFAs bioavailability, and in some cases maintain or enhance LC-PUFAs deposition. The current dissertation sought to provide new knowledge regarding LC-PUFA requirements of marine carnivorous fish (White Seabass Atractoscion nobilis, California Yellowtail Seriola lalandi and Florida Pompano Trachinotus carolinus) in the context of C18 PUFA-rich (i.e. polyunsaturated fatty acid with chain length of 18 carbon atoms) and SFA- and MUFA-rich alternative lipids. Determine if all LC-PUFAs (ARA, EPA, DHA) are equally important in meeting fatty acids requirements and also determine the effects of dietary SFA, MUFA, and C18 PUFA content in fish oil sparing and tissue deposition of LC-PUFAs. The overall findings highlighted that DHA and ARA appear to be the primary drivers of fatty acid essentiality, whereas EPA is likely required in minor amounts. It was also demonstrated that DHA/EPA ratio had little-to-no effect on fish performance. Additionally, LC-PUFA requirements seem to be more flexible than previously assumed being influenced by dietary fatty acid profile. LC-PUFAs in marine finfish are more bioavailable in the context of SFA-/MUFA-rich alternative lipids, thus, reducing the requirements for these nutrients and allowing the fish’s physiological demand to be met with dietary levels below the minimum levels recommended. Finally, these findings suggest that although marine fish accept a variety of alternative lipids, those rich in SFAs and/or MUFAs seem advantageous in terms of limiting the effects of fish oil sparing on tissue fatty acid profiles.

Thesis (M. Tech.) -- Central University of Technology, Free State, 2007
Cerebrovascular disease and coronary heart disease (CHD) are of the most important causes of morbidity and mortality amongst South Africans. The risk factor prevalence for stroke and CHD becomes altered by changes in lifestyle, including diet. In general it is suggested that lifestyle management should be the first choice when having to treat patients with increased cardiovascular risk. The prudent low-fat, high-fibre diet is regarded as an apparently healthy diet. It is suspected that this diet is effective for the control of known coronary risk factors as well as raised clotting factors. Research studies have shown the addition of dietary fibre to the diet as a promising therapeutic agent for the limited control of known coronary risk factors. The physiological effects of dietary fibre in humans are significantly influenced by the degree to which fibre is fermented in the colon. Fermentation results in the production of short-chain fatty acids (SCFAs); acetate, propionate and butyrate. The aim of this study was to examine the possible effects of different combinations of short-chain fatty acids on some metabolic risk markers. In this study a group of westernised African male volunteers was recruited and randomly assigned to three groups. Group one received a placebo. Group two received a supplement containing 50% acetate and 50% propionate. Group three received a SCFA supplement in the ratio of 70% acetate, 15% propionate and 15% butyrate. Supplementation was sustained for a period of six weeks. Blood samples were drawn during the different visits. At baseline the study group represented a group of black African men without any apparent metabolic or physical abnormalities. All measured variables fell within the normal range. In the placebo group, there was a statistically significant decrease in plasma fibrinogen levels from baseline to the end of supplementation. In the acetatepropionate supplement study group a statistically significant decrease in factor VIII (from 91.1 ± 11.2 to 90.9 ± 8.3%, respectively), and ATIII (from 114.3 ± 13.1 to 108.34 ± 9.5%), as well as a statistically significant decrease in low-density lipoprotein cholesterol (LDL-C) from 3.10 ± 0.79 to 2.64 ± 0.73 mmol/L. The significant increase in %HDL-C from 26.3 ± 6.5 to 30.2 ± 9.3% should also be noted. Both triglycerides (8%) and plasma fibrinogen (2%) showed a statistically significant increase. However, these changes are of no clinical significance. For the high-acetate supplement study group (with the addition of butyrate), a statistically significant decrease in factor VII (from 102.5 ± 13.7 to 101.1 ± 6.4%), VIII (from 92.6 ± 12.8 to 87.6 ± 6.0%), ATIII (from 109.2 ± 16.0 to 103.0 ± 9.9%) as well as fibrin monomer concentration (from 13.9 ± 2.2 to 12.1 ± 3.6 mg/L), were measured. Fibrin network compaction increased significantly from 14.2 ± 4.6 to 13.7 ± 4.0%. Other changes include a statistically significant increase in the serum-TC of 4.2%. From the results it is evident that the acetate-propionate supplement, with exclusion of butyrate, has a beneficial effect on metabolic parameters when compared to a highacetate- propionate supplement. The results do provide evidence of a possible therapeutic application for the propionate-acetate containing supplement. The specific mechanism should, however, still be investigated. It can be concluded from this study that acetate, propionate and butyrate each have different effects on human metabolism. It is evident that the use of a mixture of acetate and propionate may have a beneficial effect on patients at risk of developing CVD. Further studies that investigate the optimum ratio of these two products may lead to the development of a naturally derived therapeutic product for the prevention or treatment of CVD in black African men, as well as the population at large.

Numerous diseases are associated with reduced antioxidant defence and oxidative stress. The antioxidant defence includes dietary and endogenous antioxidants and involves complex interactions between them. The effects of dietary factors on antioxidant status and oxidative stress of healthy humans were investigated in the studies described in this thesis. Assays of plasma antioxidant capacity encompass interactions between various antioxidants. Although uric acid has an unclear function as an antioxidant, it is a major determinant of antioxidant capacity. We measured antioxidant capacity in the presence and absence of uric acid to provide more information on the application of measures of antioxidant capacity. Individuals with high dietary intakes of various antioxidants and antioxidant rich foods, especially when combined, had higher plasma antioxidant capacities than those with lower antioxidant intakes. However, there were no associations between dietary intake of antioxidants or antioxidant rich foods and the plasma concentration of F₂-isoprostanes, which is considered a reliable biomarker for oxidative stress. Intakes of various doses of a mixture of bilberry juice and black tea, rich in flavonoids for four weeks, increased antioxidant capacity in some groups, but urine levels of F₂-isoprostanes were not affected. There were substantial individual variations in responses to the drinks related to baseline antioxidant capacity. Supplementation with eicosapentaenoic acid and docosahexaenoic acid decreased the plasma levels of F₂-isoprostanes, but not prostaglandin F_2α formation or antioxidant capacity.

It was concluded that a high intake of foods rich in antioxidants is related to improved antioxidant status. After intake of foods rich in antioxidants, the antioxidant status may increase, but with considerable individual variation in the responses, which warrants further investigation. Lipid peroxidation in vivo is not easily affected by dietary antioxidants in healthy humans. Although n-3 fatty acids are highly unsaturated, they reduce nonenzymatic free radical-catalyzed lipid peroxidation, but not enzymatic lipid peroxidation.

En els darrers anys s’està produint un increment en l’interès i nombre de participants en proves de resistència i ultra resistència. Aquest exercici d’alta intensitat i durada té uns grans requeriments de substrats energètics i també presenta conseqüències importants a nivell inflamatori. S’han revisat els estudis previs que han estudiat diferents estratègies nutricionals per augmentar el rendiment. Algunes d’aquestes estratègies han explorat la utilització de suplementació amb greixos amb l’objectiu d’estalviar les reserves de glucogen muscular, ja que aquests són el principal substrat energètic en aquests esports. La hipòtesi d’aquesta tesi és que una suplementació aguda amb greixos prèvia i durant la pràctica d’esports de residència millora el rendiment i disminueix l’impacte inflamatori produït en exercicis amb grans requeriments energètics. L’objectiu principal és l’avaluació de la influència dels suplements rics en greixos en esforç de llarga durada i elevada intensitat. S’han realitzat quatre experiments. El primer, és un estudi de camp que avalua les repercussions sobre la funció cardiovascular d’una cursa de muntanya de llarga distància. A partir de proves de laboratori, els tres estudis restants avaluen l’efecte de la suplementació rica en greixos i la seva influència en el rendiment, així com el seu efecte antiinflamatori en esforços d’alta intensitat i llarga durada. Realitzats els citats experiments, es demostra la transcendència del metabolisme dels lípids en esforços d’alta intensitat i llarga durada, així com la possibilitat d’utilitzar suplements naturals rics en greixos, ben tolerats i amb una clara influència positiva en el rendiment i en els efectes inflamatoris d’aquests tipus d’exercicis.
In recent years, interest in endurance and ultra-endurance challenges has been growing, as has the number of people taking part in them. Such high-intensity, long-duration exercise has substantial energy substrate requirements and important inflammatory consequences. Previous studies on different nutritional strategies aimed at enhancing performance have been reviewed. Some of those studies explored the use of fat supplementation to save muscle glycogen stores, since the latter are the main energy substrate in these sports. The hypothesis of this thesis is that acute fat supplementation before and during endurance sports improves performance and reduces the inflammatory impact caused by exercises having substantial energy requirements. The aim is to evaluate the influence of supplements rich in fats on long-duration, high-intensity effort. Four experiments were conducted. The first was a field study evaluating the repercussions of a long-distance mountain race on cardiovascular function. Based on laboratory tests, the three other studies evaluated the effect of supplementation rich in fats and its influence on performance, as well as its anti-inflammatory effect in high-intensity, long-duration efforts. Having conducted the aforementioned experiments, the relevance of lipid metabolism in high-intensity, long-duration efforts is demonstrated, as is the possibility of using natural supplements rich in fats, which are well tolerated and have a clear positive impact on performance and on the inflammatory effects of these types of exercises.

Chez l'Homme, le vieillissement de la rétine peut aboutir à des pathologies telles que la dégénérescence maculaire liée à l'âge (DMLA) ou la rétinopathie diabétique (RD). Il semble qu'une alimentation riche en acides gras polyinsaturés à longue chaîne (AGPI-LC), notamment en oméga-3 comme l'EPA et le DHA, soit potentiellement protecteur vis-à-vis du développement de la DMLA et de l'insulinorésistance (IR), principal facteur de risque de la RD. Dans ce contexte, nous avons tenter d'évalué 1- l'impact de facteurs endogènes et environnementaux générateurs de stress oxydatif, de produits terminaux de glycation (PTG) ou d'insulinorésistance sur la fonction et le vieillissement de la rétine, et 2- l'adaptation de la rétine à un régime riche en AGPI-LC oméga-3 dans modèle murin de vieillissement de la rétine humaine, la souris ApoB100,LDLR-/-.Les animaux soumis à un stress oxydatif et à des PTG présentent une altération de la fonction rétinienne associée à une accumulation de cellules microgliales et/ou macrophages dans la rétine externe. L'IR induit une modulation de gènes impliqués dans le métabolisme des lipides, l'inflammation et dans la synthèse de facteurs nucléaires. Une alimentation riche en AGPI-LC oméga-3 induit une amélioration de l'incorporation d'acides gras oméga-3 dans la rétine et la modulation du gène codant le récepteur aux LDL dans la rétine neurosensorielle.En conclusion, nos travaux montrent une adaptation de la rétine d'une part à des conditions propices au vieillissement de la rétine et l'insulinorésistance, et d'autre part à un régime alimentaire riche en acides gras oméga-3 et pauvre en oméga-6, reconnu comme protecteur du vieillissement de la rétine

Current feed formulations within the aquaculture industry have tended to rely on high dietary lipid thus offsetting relatively expensive protein as a source of energy. In this way, protein can be ‘spared’ for synthesis of new tissue and the high lipid content can also fulfil both fish and consumer essential fatty acid (EFA) requirements. However, the main disadvantage of feeding high lipid levels to farmed fish is a surplus of fat deposition in the flesh and other important tissues, which can detrimentally impact on quality characteristics central to the human consumer. However, based on previous work in other animal models, it is entirely feasible that supplementation of the diet with bioactive fatty acids such as conjugated linoleic acid (CLA) and tetradecylthioacetic acid (TTA) may mitigate the deleterious effects of feeding farmed fish high fat diets by reducing fat deposition in particular. The general objective of this research work was to test the hypothesis that CLA and/or TTA could augment growth, reduce fat deposition and enhance fatty acid composition via incorporation of these bioactive fatty acids, and increase n-3 highly unsaturated fatty acid (HUFA) levels in the flesh of commercially important fish species such as Atlantic salmon (Salmo salar), Atlantic cod (Gadus morhua L.) and rainbow trout (Oncorhynchus mykiss). This project also considered the influence of CLA and TTA on enzymes and transcription factors thought to be pivotal in lipid metabolism and fatty acid oxidation in particular. A subsidiary aim of this research work was to investigate the immunological impact of dietary CLA and TTA administration in these fish. The results of this project have revealed that the hypothesis was only partly proved. There was no effect in growth or biometry after either CLA or TTA supplementation in any of the fish species investigated. Additionally, there were few physiologically significant effects on fat levels on fish as a result of TTA or CLA administration. However, there were a number of effects on fatty acid metabolism including inhibition of steroyl coenzyme desaturase (SCD) in cod and trout in particular and also enhancement of hepatic n-3 HUFA levels in trout. Importantly, it was determined that both TTA and CLA could be incorporated into the flesh thus providing a vehicle through which these bioactive fatty acids can be delivered to the consumer. There were also a number of beneficial effects on activity and gene expression of a number of enzymes and transcription factors thought to be fundamental to the modulation of fatty acid oxidation in particular. However, the effects on gene transcription and biochemistry had little impact at the whole body level. This research work also showed that there were no detrimental effects on immune status after supplementation with dietary CLA or TTA. Conclusively, this thesis has contributed to the overall understanding of the influence of dietary CLA and TTA in farmed fish.

Fish meal (FM) and fish oil (FO) have traditionally been central in aquaculture feed formulation but the finite global supply situation limiting future use along with issues of contaminant levels in these feed ingredients have become critical issues. The objectives of the present study were to investigate alternative feed ingredients as substitutes for both FM and FO in feeds for Atlantic salmon (Salmo salar) to ensure optimal growth, feed efficiency and health of the fish as well as maintaining the nutritional quality of the fish product to the human consumer, especially the levels of n-3 highly unsaturated fatty acid (HUFA), eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), in the flesh. The results of the present study revealed that there were no negative effects on growth performance, feed utilisation and apparent digestibility in Atlantic salmon when FO was substituted with vegetable oil (VO) but these parameters were affected when FM was replaced with alternative protein sources from plants and animals at high levels, despite dietary supplementation with crystalline amino acids and lecithin. Reduction in feed intake was a factor affecting growth retardation when FM inclusion decreased. However, replacing FM with alternative plant and animal proteins along with partial replacement of FO had no major effect on nutritional quality, particularly n-3 HUFA content of salmon tissues. Replacing Northern FO with decontaminated FO or blends of southern hemisphere FO and VOs strategies to reduce POP contaminants and retain high nutritional values in flesh were very successful. Dietary treatments and genetic origin of fish both had effects on tissue compositions and gene expression. All fish groups (strain/family), consist of CAL, LEAN and FAT strains, fed a diet containing VO showed significant differential expression of lipid metabolism-related genes compared to fish fed a FO diet with LEAN strain appearing to adapt to VO inclusion better than FAT strain. This thesis has demonstrated dual replacement of FM and FO with alternative raw materials in salmon feeds without a major negative impact on nutritional quality.

As beta-carotene is transported via micelles across the intestinal lumen for absorption, the factors that regulate uptake into micelles could also regulate at least in part beta-carotene absorption in humans. Thus, we have studied the effect of different dietary lipids on beta-carotene uptake into mixed micelles in vitro. Using this method, the effects of fatty acids of increasing degrees of unsaturation, vitamin A, cholesterol, and vitamin E on beta-carotene uptake into micelles were studied. Uptake of beta-carotene into micelles was stimulated by cholesterol and unsaturated fatty acids (uptake correlated with degree of unsaturation). beta-Carotene solubilization into micelles was inhibited by vitamin A and vitamin E. These results suggest that the absorption of beta-carotene may be partly regulated at the level of formation of intestinal micelles by the presence of different dietary components.

The hypothesis that antioxidant vitamins (ascorbate and tocopherols) along with urate protect blood plasma lipids from oxidation was tested. Dietary fat is also an important factor influencing plasma lipid peroxidation. The purpose of this study was to investigate the role of plasma antioxidants and dietary fat on low density lipoprotein (LDL) and plasma lipid oxidation. In the first part of this study, we compared the ability of urate and ascorbate to protect human LDL from in vitro oxidation. LDL oxidation was initiated by 15 mM of a water soluble azo-initiator in the presence or absence of ascorbate or urate. The rate of lipid hydroperoxide (LOOH) formation was increased after the LDL tocopherols were totally consumed, i.e., after the lag phase. Urate (50 $\mu$M) was more effective than ascorbate (50 $\mu$M) in extending the lag phase. Moreover, urate was consumed more slowly than ascorbate under identical oxidation conditions. The combination af 25 $\mu$M ascorbate and 25 $\mu$M urate was more effective in extending the lag phase than ascorbate alone but less effective than urate alone. An empirical mathematical model was developed to describe the oxidation kinetics of LDL tocopherols. In the second part of this study, we studied the role of dietary fat and dietary $\alpha$-tocopherol ($\alpha$-toc) levels on rat plasma oxidation. The fatty acid composition of plasma was found to be modulated by the type of dietary fat. Neither dietary fat nor $\alpha$-toc influenced the plasma levels of water soluble antioxidants (ascorbate, urate and sulfhydryl content). Rat plasma was oxidized either by a water soluble azo-initiator (25 mM) or a lipid soluble azo-initiator (10 mM). In both cases, the rate of LOOH formation in plasma from rats fed butter oil diets was markedly suppressed compared to the plasma from rats fed corn oil diets. When oxidation was initiated by a lipid soluble azo-initiator, plasma from rats fed $\alpha$-toc supplemented diets showed higher LOOH levels than plasma from rats fed $\alpha$-toc deficient diets. Surprisingly, when oxidation was initiated by water soluble azo-initiator, tocopherol appeared to act as a pro-oxidant. The results suggest that urate may be more significant than ascorbate in delaying the consumption of tocopherols in human LDL and that low dietary PUFAs levels are more important in preventing the in vitro oxidation of plasma lipids than high dietary levels of $\alpha$-tocopherol.

Endogenous cholesterol (Ch) fractional synthesis rate (FSR) is inversely related to infant dietary Ch at 4 months (mo) of age. The objective of the present study was to determine whether level of infant dietary Ch induced changes in FSR that persisted at 18 mo. Forty-seven infants received, human milk (HM) from birth until weaned (n=15), or were randomized to receive modified cow-milk formula (MCF) with added Ch (n=15), or cow-milk formula (CF, n=17), for 12 mo. Ch contents of HM, MCF, and CF were 120, 80, and 40 mg/L, respectively. At 4 mo, FSR in the HM group was lower than in CF, but at 18 mo, there were no differences between groups. Therefore, while Ch intake prior to weaning affects FSR, the differences do not persist after weaning to unrestricted diet. These data provide further evidence that there is no imprinting of FSR in infancy by differing dietary levels of Ch.
Le taux de synthèse fractionée (TSF) du cholestérol (Ch) endogène est inversement proportionnel à la prise alimentaire du Ch à 4 mois (mo). L’objectif de cette étude était de verifier si ces différences du TSF persistent à 18 mo. Quarante-sept enfants ont reçu, dès la naissance, soit lait maternel (HM), jusqu’au sevrage (n=15), ou ont reçu de façon aléatoire soit du lait maternisé (CF), à base lait de vache (n=17), ou une formule modifiée par l’addition de Ch (MCF, n=15) durant 12 mo. Les HM, CF, et MCF contenaient respectivement 120, 80, 40 mg/L de Ch. À 4 mois, le TSF dans HM était plus faible que chez CF, mais cette différence avait disparue à 18 mois avec le sevrage au régime sans restriction. Ces données confirment que le Ch alimentaire chez le nourrisson n’affecte pas le TSF de façon permanente. fr

Conoscenze preesistenti: Le transglutaminasi (TGase) sono una classe di enzimi ampiamente diffusa tra gli organismi procarioti ed eucarioti. Gli enzimi di questa famiglia catalizzano modifiche post-traduzionali in molte proteine attraverso reazioni di trasferimento dell’acile, reazioni di deaminazione e di crosslinking (polimerizzazione) tra residui peptidici di lisina (accettore di acile) e glutammina (donatore di acile) intra- o inter-catena proteica. A causa della sua facilità di espressione e di purificazione, l’unica TGase ampiamente usata per le applicazioni industriali è la TGase microbica estratta da Streptomyces mobaraensis (MTGase). Oggigiorno la MTGase è disponibile in commercio ed è ampiamente usata nell’industria dei biopolimeri, in cosmetica, per applicazioni cliniche, nell’industria tessile della lana e soprattutto nell’industria alimentare. La sua abilità di catalizzare legami crociati in molti substrati proteici differenti è sempre più usata non solo per la produzione di salsicce, prosciutti e formaggi ma, molto recentemente, anche per la detossificazione della farina, come possibile terapia alternativa alla dieta senza glutine. Ne consegue che oggigiorno le applicazioni industriali della MTGase stiano aumentando, coinvolgendo sempre più settori e producendo una ricerca scientifica su questo argomento sempre più fervente, allo scopo di tentare di rispondere a specifiche esigenze industriali, come l’implementazione di sistemi di purificazione della MTGase più efficienti, la ricerca di fonti alternative di transglutaminasi microbica, e di fonti sicure di enzimi ricombinanti. Scopo del progetto di dottorato: lo scopo principale del progetto è l’identificazione di nuove forme di transglutaminasi microbica che possano diventare un’alternativa a quella attualmente in uso. È stata eseguita un’analisi approfondita delle sequenze note allo scopo di ottenere una classificazione delle TGase microbiche attraverso la loro similarità a forme note. Per selezionare le migliori candidate che possano essere forme attive in appropriate condizioni, le sequenze selezionate sono state soggette di modellamento molecolare e simulazioni molecolari. Per testare l’attività enzimatica, sono stati effettuati dei saggi sperimentali su una nuova forma trovata ed un’ulteriore nuova forma è stata espressa. Risultati: il presente lavoro propone in primo luogo un’analisi, ad oggi assente, dell’ampio panorama delle transglutaminasi microbiche, sviluppando la prima classificazione delle TGase microbiche basata sulle loro caratteristiche di sequenza e sulle loro specifiche strutture secondarie predette. Al fine di classificare ed analizzare le caratteristiche strutturali di tutte le sequenze annotate come aventi un TGase core, sono state utilizzate tecniche computazionali che coinvolgono analisi di sequenza, studi comparativi, costruzione di alberi filogenetici, modellamento per omologia e simulazioni di dinamica molecolare. Tramite questo approccio, è stata effettuata una classificazione preliminare di queste sequenze dividendole in cinque gruppi principali. Ogni gruppo è stato studiato dal punto di vista delle sequenze per analizzare la presenza di motifs specifici. Per tre di questi cinque gruppi, sono state studiate anche le strutture secondarie e, da questa analisi, sono state rilevate caratteristiche specifiche per ogni gruppo. Inoltre, due nuove forme di TGase microbica (mTGase) sono state studiate in dettaglio: K. albida mTGase e l’ipotetica mTGase da SaNDy (organismo non rivelato per possibilità di brevetto). Per la prima, in comparazione con la MTGase, sono state effettuate analisi della tasca relativa al sito attivo e simulazioni di dinamica molecolare. Per la seconda, invece, sono state utilizzate tecniche sperimentali per purificare l’ipotetico enzima al fine di testarne l’attività su substrati alimentari. Saggi sperimentali su entrambe le proteine sono ancora in corso, al fine di trovare le migliori condizioni di attività enzimatica e i migliori substrati di reazione. Le simulazioni di dinamica molecolare eseguite sulla mTGase di K. albida hanno suggerito alcune spiegazioni alla maggiore specificità di questo enzima rispetto alla MTGase, dimostrata sperimentalmente da Steffan e colleghi, ed alcune indicazioni per variare le condizioni di attività usate per testarla. Inoltre, l’analisi dei substrati ha permesso di trovare nuovi possibili substrati, sui quali l’enzima potrebbe essere impiegato ai fini della riduzione delle allergenicità. D’altro canto, l’enzima estratto da SaNDy, mostrando una più alta somiglianza con la MTGase, potrebbe essere meno selettivo della mTGase da K. albida nei confronti di specifici substrati, pertanto potrebbe essere possibile una sua applicazione anche su substrati gliadinici, tuttavia, per provare ciò, sono necessari ulteriori esperimenti. Note: il presente lavoro di dottorato è stato principalmente svolto presso il Laboratorio di Bioinformatica del CNR di Avellino sotto la supervisione del Dr. Facchiano, tuttavia, tutte le simulazioni di dinamica molecolare sono state eseguite presso il Dipartimento di Biochimica dell’Università di Zurigo, nel laboratorio di biologia strutturale e computazionale sotto la supervisione del Prof. A. Caflisch e del suo gruppo di ricerca (periodo di formazione all’estero obbligatorio). I saggi di attività sperimentale sul substrato gliadinico sono stati effettuati dal laboratorio di spettrometria di massa CeSMA-ProBio presso il CNR di Avellino; e l’ipotetica mTGase da SaNDy è stata invece clonata, espressa e purificata durante la collaborazione con il laboratorio di Molecular Sensing presso il CNR of Avellino.
Background: transglutaminases (TGase) are a class of enzymes widely spread in eukaryotic and prokaryotic organisms. Enzymes of this family catalyze post-translational modifications in many proteins by acyl transfer reactions, deamidation and crosslinking (polymerisation) between protein intra- or inter-chain glutamine (acyl donor) and lysine (acyl acceptor) peptide residues. Due to its facility of expression and purification, the only TGase enzyme widely used for industrial applications is the microbial TGase extracted from Streptomyces mobaraensis (MTGase). Nowadays the MTGase is commercially available and widely used in biopolymers industry, in cosmetics, in clinical applications, in wool textiles, and above all in the food processing industry. Its ability to catalyze crosslinks on many different protein substrates is increasingly used not only for sausage, ham and cheese production but, very recently, also for flour detoxification, as a possible alternative therapy to the gluten free diet. It follows that nowadays the industrial applications of MTGase have increased, covering more and more fields producing a very active scientific research about this topic aimed at attempt to meet specific industrial needs, as the implementation of more efficient system for MTGase production, the research of alternative sources of microbial TGase, and safe source of recombinant enzymes. Aims of the doctorate project: the main aim of the project is the identification of novel forms of microbial TGases that could become an alternative to that in use. A depth screening of known sequences has been performed, with the aim of obtaining a classification of microbial TGases for their similarity to known forms. To select the best candidates to be active forms under appropriate conditions, molecular modelling and molecular simulations have been performed on selected sequences. To test the enzymatic activity, experimental assays have been performed with a novel form, and another novel form has been expressed. Results: the present work proposes at first an analysis, lacking so far, of the wide microbial transglutaminase world, developing the first classification of the microbial TGase based on their sequence features and their specific predicted secondary structures. In order to classify and analyze the structural features of all the sequences annotated as having a TGase core computational techniques involving sequence analyses, comparative studies, building of phylogenetic trees, homology models and molecular dynamic simulations have been used. From this approach, a preliminary classification of these sequences was done by dividing them in five main groups. Each group has been investigated from the sequence point of view to analyze the presence of specific motifs. For three of this five groups, also the secondary structures have been investigated and, from this analysis, features specific for each group have been detected. Moreover, two novel forms of microbial TGase (mTGase) have been investigated in the detail: K. albida mTGase and the hypothetical mTGase from SaNDy (organism not disclosed for patent opportunity). Molecular dynamics simulations and active site pocket analyses have been performed for the first, in comparison with MTGase. For the second, instead, experimental technique has been used to purify the hypothetical enzyme in order to test it on food related substrates. Experimental assays on both the proteins are still ongoing, to find the best enzymatic activity conditions and the best substrates of reaction. The molecular dynamic simulations performed on K. albida mTGase have suggested some explanations to the higher specificity of this enzyme than MTGase, experimentally demonstrated by Steffen et colleague, and several indications to change the activity conditions used to test it. Moreover, the substrates screening has allowed to find novel possible substrates, on which this enzyme could be employed for the allergenicity reduction. On the other hand, the enzyme extracted from SaNDy, showing a higher similarity with MTGase, could be less selective than K. albida mTGase for specific substrates, so it could be possible its application also on the gliadin substrate, but to prove it further experiments are necessary. Note: the present PhD work has been mainly performed in the Bioinformatics Laboratory at the CNR of Avellino under Dr. Facchiano’s supervision, however all the MD simulations have been performed at the Biochemistry Department of the University of Zurich, in the computational and structural biology laboratory under the supervision of Prof. A. Caflisch and his research group (compulsory abroad training period). Experimental activity assays on gliadin substrate have been performed by the spectrometry mass CeSMA-ProBio lab at the CNR of Avellino; and the hypothetical mTGase from SaNDy was instead cloned, expressed and purified in collaboration with the Laboratory for Molecular Sensing at the CNR of Avellino.

Cardiovascular disease remains one of the leading causes of death globally. Conflicting information regarding optimal nutritional intake and balance for cardiometabolic health has often led to confusion and lack of expert consensus. The precise nutritional profile and dietary patterns for optimal cardiovascular health are yet to be elucidated. This PhD covers a broad range of topics and aspects relating to cardiometabolic health. Chapter 1 explores maternal nutritional status during pregnancy, as it is a powerful contributor to the intrauterine environment and may alter offspring physiology and later life cardiometabolic risk. Therefore, we sought to determine whether maternal dietary carbohydrate quantity and/or quality during pregnancy are associated with newborn body composition and cardiac autonomic function. In chapter 2 we conducted a systematic review to determine the effect of medium-chain triglyceride oil on blood lipids. Dietary saturated fat raises total cholesterol and low-density lipoprotein cholesterol levels. However, it is unclear whether these effects differ by the fatty acid chain length of saturated fats. In chapter 3 we designed a pilot study aimed to investigate the acute postprandial effects of two different meals on flow-mediated dilation (FMD), using a randomised cross-over design. FMD is a non-invasive marker of arterial endothelial function, and the vascular endothelium is involved in early atherogenic events and regulating vascular tone. For chapter 5 we sought to determine if improved gum health resulting from periodontal intervention in Indigenous Australians with chronic kidney disease would increase consumption of healthier foods and have reciprocal benefits for carotid atherosclerosis. Collectively, our findings presented in this thesis provide a rational basis for appropriately powered studies investigating the impacts of dietary composition, and dietary patterns on the cardiometabolic health of individuals throughout the life course.

Dietary fats and animal proteins have been shown to exert different lipidemic responses in many animals, including humans. Oxidative stress has been associated with the development of several diseases including atherosclerosis. The hypotheses of this study were that: (1) the degree and type of dietary fatty acid unsaturation will influence the type of cholesterolemic responses via the induction of differential levels of oxidative stress; and (2) hyperlipidemia induced by casein in a butterfat-based diet is related to increased tissue lipid peroxidation. Male Golden Syrian hamsters were fed diets containing butterfat/casein (BF/CAS), butterfat/egg white (BF/EGG), safflower oil/egg white (SAFF/EGG) or menhaden oil/egg white (MHO/EGG) for 27 days. In comparison to the BF/EGG and SAFF/EGG diet groups, the MHO/EGG hamsters exhibited higher levels of total serum cholesterol, serum triglyceride, serum apolipoprotein B and serum lipid peroxides. These results suggest that MHO induced-hyperlipidemia could be exerted through increased concentrations of serum apolipoprotein B and increased serum lipid peroxidation. The BF/CAS diet, in comparison to the BF/EGG diet, increased serum total cholesterol and increased serum and hepatic levels of lipid peroxides. These results suggest that dietary casein-induced hypercholesterolemia could be related to increased oxidative stress. The protective effect of dietary egg white on lipid peroxidation may involve sulfur amino acids which are found in greater amounts in egg white than casein.

Primary cultures of human umbilical vein endothelial cells were incubated with n-3 and n-6 fatty acids for a maximum of 24 hours, to investigate their distribution in membrane neutral lipids and phospholipid. Furthermore, the release of labelled free fatty acids due to induction of PLA2 by cytokines and the further production of PGI2 in cells supplemented with different PUFA was investigated in the present study to elucidate the metabolism of lipids in these cells. In addition, the extent of lipid peroxidation and the potency of antioxidants in inhibiting the formation of lipid peroxides due to dietary modification in cells were also investigated in relation to their effects on essential fatty acid metabolism. Endothelial cells in the present study had a significant capacity to metabolise PUFA which was supplied exogenously. PUFA of n-3 and n-6 families behaved differently with respect to their incorporation into membrane lipids. ARA of n-6 family had the greatest incorporation into the total lipids compared to n-3 PUFA ie, EPA and DHA. About 80% of PUFA was incorporated into the phospholipid fraction and another 20% was found in neural lipids. [14C]-ARA and [14C]-DHA when co-incubated with endothelial cells for longer incubation periods were found mainly incorporated into TAG. In contrast, [14C]-EPA was distributed significantly into other fractions of neutral lipids with no significant increase in the TAG fraction. TAG is important as a fatty acid store in the cells including essential PUFA which can be metabolised and utilised for PG synthesis during the deficiency of eicosanoid precursor (PUFA) in the phospholipids of cell membranes. All the PUFA tested were incorporated into PC to a similar extent with time. Transfer between PC and PE during the longer incubation time was observed in the present study which indicated that PE may be important in the storage of fatty acids in phospholipids since PC and PI were the most preferred substrates for the phospholipases.

Thesis (Ph. D.)--Ohio State University, 2004.
Title from first page of PDF file. Document formatted into pages; contains xii, 113 p.; also includes graphics. Includes bibliographical references (p. 99-113).

Many foods, particularly fish, change in texture and become tough on frozen storage. In addition to ice crystal formation that causes protein denaturation, lipid oxidation products including aldehydes may induce protein cross-linking. In this study, Atlantic Mackerel was stored for up to 26 weeks at -80 and -10 °C. The formation of aldehydes was investigated by thiobarbituric (TEARS) test, HPLC and LC-MS. As expected, there was an increase in the production of aldehydes during storage at -10 °C compared with the control fillets at -80 °C. However, in addition to malonaldehyde (MDA) and hexanal (HEX), we report the formation of gluteraldehyde (GLA) and hydroxynonenal (4-HNE) in frozen fish for the first time. Frozen storage also decreased protein solubility, induced changes in protein conformation and increased formaldehyde and DMA formation. Changes in texture during storage, assessed by small deformation rheology showed increased elastic modulus (G') values. Aldehyde formation in fish and consequently the G' values were reduced in samples that were treated with antioxidant (green tea). A model study using BSA with various aldehydes showed a decrease in lysine availability, increased G' values and conformational changes including increased beta-sheet and tyrosine doublet ratio and decreased tryptophan and disulphide groups. Increased MW of BSA-aldehyde adducts as well as fragmentations were observed by LC-MS. Methyl linoleate (ML) and extracted mackerel oil were oxidised under ultraviolet (UV) radiation for 24, 48 and 72 hours and lipid peroxidation was assessed by measuring peroxide value (PV) and TEARS. To assess the cytotoxicity, oxidized lipids (0, 20, 40, 80 and 100 mug/ml) (ML or fish oil) were added to human colon cancer (Caco-2) cells (density 2 x 104 cells/well) and incubated for 24 h. Using the MTT assay, a decrease in cell viability was observed in all samples treated with UV oxidized ML or fish oil, at concentrations above 40 mug/ml. Caco-2 cells treated with 24 and 72 h oxidized mackerel oil were less damaged compared to cells treated with oxidized ML. Improved cell viability and decrease in lipid peroxidation were shown in cells pretreated with 50 muM EGCG. The mechanism of cell death either by necrosis or apoptosis was investigated. Oxidized 100 mug/ml ML or fish oil-treated caco-2 cell line showed characteristics of apoptosis. Morphological changes resulted from cellular membrane rupture, the formation of apoptotic bodies, DNA fragmentation and caspase-3 activation, detected by Western blotting; these features were visualized by bioimaging techniques such as light microscopy, fluorescence microscopy, Raman microspecfroscopy and atomic force microscopy.