Thesis (M.Sc.)--University of Alberta, 2010. / Title from PDF file main screen (viewed on July 29, 2010). A thesis submitted to the Faculty of Graduate Studies and Research in partial fulfillment of the requirements for the degree of Master of Science in Plant Science, Department of Agricultural, Food and Nutritional Science, University of Alberta. Includes bibliographical references.

Docosahexaenoic acid (22:6n-3) and arachidonic acid (20:4n-6) are deposited in large amounts in membrane phospholipids of the developing central nervous system (CNS). High levels of 22:6n-3 are found in synaptic terminals and retina, and are important for normal visual development and function. 20:4n-6 and22:6n-3 are supplied in human milk. In contrast, infants fed formula rely completely on endogenous synthesis of 20:4n-6 and 22:6n-3 from linoleic (18:2n-6) and a-linolenic (18:3n-3) acid, respectively. Levels of 22:6n-3 in the blood lipids of infants fed formula are lower than in infants fed human milk. Concern over the supply of 22:6n-3 led to clinical trials in which premature infants were fed formulas containing fish oils as a source of 22:6n-3. Piglets, which have a similar lipid metabolism and perinatal timing of the brain growth spurt to humans, have a lower percentage of 22:6n-3 in blood, liver and CNS tissues when fed formula with 30% of fatty acids as18:2n-6 and 0.8% 18:3n-3, compared to sow milk. It was hypothesized that the low blood and tissue 22:6n-3 in formula-fed piglets was due to inappropriate quantities and/or ratios of dietary 18:2n-6 and 18:3n-3 limiting the synthesis of 22:6n-3. Thus, the main objectives of this thesis were to determine. (1) if 22:6n-3 is an essential dietary nutrient for the term gestation piglet, (2) if appropriate quantities and ratios of 18:2n-6 and 18:3n-3 in formula will support CNS membrane accretion of 20:4n-6 and 22:6n-3, comparable to piglets fed varying amounts of 22:6n-3 in natural milk, and (3) if lower blood phospholipid 22:6n-3 consistently reflects reduced 22:6n-3 in the CNS. Initial studies (Experiment I) showed that formula with 4% 18:3n-3 supported a similar percentage of22:6n-3 in piglet liver and CNS membrane lipids to sow milk, but was associated with lower brain weight. Deposition of 22:6n-3 in brain was influenced by the formula 18:3n-3 content. The 18:2n-6:18:3n-3 ratio (22:1and 37:1) seemed to be important, however, when formulas contained 1% 18:3n-3. Low levels of fish oil in formula, similar to those used in clinical trials, were effective in supplying 22:6n-3 to the developing piglet brain (Experiment II). The efficacy of 18:3n-3 in supporting the deposition of 22:6n-3 in the brain was estimated to be at least 20% that of dietary 20:5n-3 plus 22:6n-3. With increasing dietary fish oil, however, levels of eicosapentaenoic acid (20:5n-3) increased and 20:4n-6decreased in plasma, liver and retina, but not brain (Experiment III). This suggests regulatory mechanisms may exist to maintain relatively constant levels of 20:4n-6 and 20:5n-3 in brain. Milk 22:6n-3 varies with maternal intake of 22:6n-3. The effect of milk 22:6n-3 content was studied in piglets fed milk with 0.1% or 1.5% 22:6n-3 obtained from sows fed usual pig diets containing vegetable fats without or with fish oil, respectively (Experiment IV). Consumption of 1.5 vs 0.1% 22:6n-3 from sow milk resulted in 300% higher 22:6n-3 in liver and blood phospholipids and 11% higher 22:6n-3 in cerebrum of nursing piglets. Despite similar milk 20:4n-6, the % 20:4n-6 in tissues other than the brain was lower in piglets fed high22:6n-3 sow milk. Thus, high intakes of n-3 fatty acids decrease 20:4n-6 in piglet liver and blood lipids. The blood phospholipid % 22:6n-3 in piglets fed formulas containing 18:2n-6 and 18:3n-3 but not their long-chain derivatives, was lower than in piglets fed 22:6n-3 in natural milk, consistent with published findings in formula-fed infants. However, in contrast to circulating lipids, formulas with 4% 18:3n-3 maintained similar levels of 22:6n-3in the piglet CNS compared to milk. These studies show that blood phospholipid 22:6n-3 and 20:4n-6 are not specific indices of effects in CNS lipids. This thesis has shown (1) 22:6n-3 is not essential in the diet of the term piglet, if adequate 18:3n-3 is given, (2) fish oils are an effective source of 22:6n-3 for deposition in the developing brain, (3) high dietary n-3fatty acids interfere with 20:4n-6 metabolism, and (4) blood lipid 20:4n-6 and 22:6n-3 do not accurately reflect CNS fatty acids. / Land and Food Systems, Faculty of / Graduate

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Colon cancer is a leading cause of cancer related deaths worldwide. Lifestyle factors such as diet and exercise have been implicated as important agents in colon cancer development and progression. Epidemiological, in vivo and in vitro studies have found that n-3 polyunsaturated fatty acids (PUFAs) reduce colon carcinoma. The role of n-6 PUFAs remains a controversial topic, with studies indicating both promoting and preventing capabilities published. In order to better understand the effects of PUFAs on colon carcinoma, it is important to have an understanding of how they will be broken down in the body. During this study, in silico metabolism of eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA) and arachidonic acid (AA) predicted the formation of hydroxy-, di-hydroxy- and epoxy-FAs. A gas chromatography-mass spectrometry method was developed and validated for the detection of these PUFAs and their cytochrome P450 (CYP) metabolites. A human liver microsomal system for the in vitro metabolism of EPA, DHA and AA was optimised in terms of microsomal and PUFA concentration. The system resulted in the metabolism of the positive control, lauric acid, to 12-hydroxy-lauric acid but was unable to metabolise the PUFAs of interest. EPA, DHA and AA reduced cell viability in the colon carcinoma cell lines SW480 and SW620 in the micromolar concentration range (25 – 200 μM). The CYP epoxidation metabolite of EPA, 17, 18-epoxyeicosatetraenoic acid (17, 18-EpETE) resulted in a significant reduction in SW480 cell viability relative to the parent compound at lower concentrations (25 and 50 μM). Annexin V apoptosis analysis revealed that EPA and 17, 18- EpETE did not result in apoptosis in SW480 cells at a concentration of 25 μM and over an incubation period of 24 hours. A significant reduction in reactive oxygen species production was seen in SW480 cells after incubation with 25 μM 17, 18-EpETE for 24 hours. EPA and 17, 18-EpETE were implicated in the reduction of colon cancer metastasis since they were able to reduce SW480 migration and anchorage independent cell growth. These results indicate that the dietary intake of EPA, DHA and AA may be beneficial to one’s health due to the negative effects that these PUFAs had on colon carcinoma. Future studies are needed to confirm these benefits and compare the effects of the PUFAs to their CYP-metabolites.

The effect of different blanching treatments and packaging materials on the enzymatic (lipoxygenase and peroxidase) activity and fatty acid stability of two different varieties of sweet corn on the cob (Jubilee and GH 2684) was evaluated during nine months of frozen storage at -23.3°C. The initial moisture content in the kernels of the two sweet corn varieties averaged 72.5%. After nine months of frozen storage the moisture content in the kernels of corn depended greatly on the packaging material used. The ears stored in Cryovac B and E bags showed the best moisture retention (72.2% final moisture content), followed by the polyethylene bags (71.4%) while the ears stored without packaging material showed severe dehydration (70.1%). The peroxidase and lipoxygenase activities were determined using spectrophotometric assays on a crude extract obtained from liquid nitrogen powdered corn. Both unblanched varieties of sweet corn showed similar initial peroxidase specific activity and general behavior during the nine months of frozen storage. The presence of lipoxygenase isozymes with different thermal stabilities in both varieties was suggested by the higher lipoxygenase specific activity found in Jubilee after freezing and nine months of frozen storage (0.135 units/mg protein) compared with the GH 2684 variety (0.115 units/mg protein). Complete inactivation of lipoxygenase was obtained after 9 minutes steam blanching at 100°C. Peroxidase was more heat resistant showing some remaining specific activity after 9 minutes steam blanching with a complete inactivation after 15 minutes steam blanching. No regeneration of either enzyme was observed during the nine months of frozen storage suggesting a permanent disruption of the active site of both enzymes. Relative fatty acid content was determined by gas chromatographic analysis of fatty acids methyl esters. The major fatty acids present in both varieties were palmitic (14.93%), stearic (2.79%), oleic (31.54%), linoleic (46.87%) and linolenic (1.89%) acids. Good stability of the polyunsaturated fatty acids was observed during the nine months storage at -23.3°C, with autoxidation as the main mechanism responsible for the decrease in the relative percent of polyunsaturated fatty acids. Some enzymatic oxidation also occurred, decreasing the linolenic acid content. The control of the degradation of polyunsaturated fatty acids depended mostly on the frozen storage temperature (-23.3°C) and not on the oxygen permeability of the different packaging materials. The results obtained in our study suggested that blanching of the ears of sweet corn had an important effect on reducing the enzyme activity but little effect on the polyunsaturated fatty acid degradation after 9 months of storage at -23.3°C. / Graduation date: 1994

Preliminary data from studies in rodents suggests time-dependent associations between dietary LC n-3 PUFA and hepatic levels of the enzyme soluble epoxide hydrolase (sEH), which regulates the metabolism and availability of epoxyeicosatrienoic acids (EET).  EET are cytochrome P450 epoxygenase products of arachidonic acid associated with  lower blood pressure, decreased inflammatory response and inhibition of blood coagulation. To further investigate the association between LC n-3 PUFA and sEH, ApoE^-/^- mice were fed a high-fat high-cholesterol diet supplemented with either fish oil (EPA + DHA) or DHA or HOSF (all 2% w/w) for 10 weeks and livers and aortic roots were collected on day 2 and weeks 1, 2, 4 and 10.  Proteomics analysis showed an overall decreasing effect of fish oil (but not DHA) supplementation on hepatic protein levels of sEH compared to the control throughout the intervention period (<i>P</i> &lt; 0.05).  Neither fish oil nor DHA intervention affected atherosclerotic plaque size in the aortic root. We also examined how dietary supplementation with 1 g/day EPA or 1 g/day DHA for 10 days affects platelet sEH levels and platelet aggregation compared to 1 g/day HOSF (control) in healthy volunteers in a double-blind, placebo-controlled, cross-over trial.  We found that DHA decreased platelet aggregation by 10% (<i>P =</i> 0.04) and EPA also inhibited ADP (5 μM)-induced platelet aggregation by 14% compared to the control group but this effect did not reach statistical significance due to high variability between subjects.  EPA decreased platelet sEH levels by 25% (not significant), whereas DHA had no effect.  We also attempted to optimize a method for measuring EET in plasma and platelets.  However, the rapid conversion of EET to other compounds and their low concentration in tissues prevented us from optimizing such a method within the time limits of the project.

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Three experiments were conducted to study the influence of dietary fatty acids on the production performance and immune response of chickens. In experiment I, forty day-old broiler chicks were fed diets containing 5% of either animal fat + conjugated linoleic acid (CLA) (Diet I), sunflower oil (Diet II), flax oil (Diet III) or fish oil (Diet IV). No significant differences (P>0.05) were observed between the live weight of birds. The liver tissue total fat content was lower (P<0.05) in treatment I and II. The fatty acid composition of breast and thigh muscle, liver, heart, pericardial fat, plasma, splenocytes and gut associated lymphoid tissue differed (P<0.05) between treatments. The thiobarbituric acid reactive substances (TBARS) of breast and thigh muscle, liver and heart tissue were lower (P<0.05) in Diet I fed birds. Serum antibody activity was decreased (P<0.05) in Diet II fed birds. In experiment II, 120 day-old broiler chicks were fed diets containing 3.5% of either animal fat + conjugated linoleic acid (CLA) (Diet I), sunflower oil (Diet II), linseed oil (Diet III) or fish oil (Diet IV). Body weight gain was higher (P<0.05) in Diets III and IV compared to Diets I and II fed birds. Feed intake was increased (P<0.05) in Diet IV fed birds. Birds fed Diets III and IV had higher (P<0.05) n-3 fatty acids in all tissues studied. A preferential incorporation of CLA was observed in spleen mononuclear cells. TBARS were higher (P<0.05) in the breast and thigh muscle of Diet IV fed birds. Serum anti-BSA antibody content was higher (P<0.05) in birds fed Diets III and IV. Delayed type hypersensitivity (DTH) response was increased (P<0.05) in Diets IV and III fed birds. Lymphocyte and spleen mononuclear cell CD4⁺, CD8⁺ and IgM⁺ cell population did not differ (P>0.05) among treatments. In experiment III, 120 layer birds were fed diets containing 3% of CLA+animal fat (Diet I), sunflower oil (Diet II), canola+flax oil (Diet III) or fish oil (Diet IV). Egg production, feed consumption and feed efficiency did not differ (P>0.05) among treatments. Birds fed Diets III and IV had higher content of n-3 fatty acids in eggs. Eggs from hens fed Diet I incorporated higher (P<0.05) CLA and saturated fatty acids with a concomitant reduction in (P<0.05) monounsaturated fatty acid content. A preferential incorporation of CLA was observed in eggs over other tissues. TBARS were higher (P<0.05) in breast and thigh muscle of Diet IV fed birds. Egg TBARS content did not differ (P>0.05) among treatments. Serum and yolk anti-BSA antibody contents were higher (P<0.05) in birds fed Diets III and IV. DTH response was increased (P<0.05) in Diets IV and III fed birds. Lymphocyte and spleen mononuclear cell CD4⁺, CD8⁺ and IgM⁺ cell population did not differ (P>0.05) among treatments. Feeding n-3 fatty acids increased antibody mediated immune response while n-6 fatty acids and CLA increased cell mediated immune response. / Graduation date: 2003

ABSTRACT Background: Polyunsaturated fatty acids (PUFA) are found in nuts and seeds, salad dressings and vegetable oil and are prone to oxidation during storage and food preparation. Evidence supports that consumption of oxidized lipids promotes atherosclerosis and glucose intolerance in animal models. However there is a dearth of evidence with regard to the amount of oxidized lipids consumed and its association with parameters of adiposity and glucose homeostasis in humans. Objective: The objective of this study is to estimate the amount of oxidized lipids in common foods and the oxidized lipid consumption in the US population using the data from National Health and Nutrition Examination Survey (NHANES) 2005-06. The second objective of this study is to investigate if there is an association between consumption of oxidized lipids with markers of adiposity and glucose tolerance. Methods- Foods with possible high oxidized lipid content were selected from the NHANES food frequency questionnaire. Oxidized lipid content /Peroxide Values (PV) of these foods were determined from published values in the literature. Oxidized lipid consumption was stratified into tertiles to determine the relationship between consumption of oxidized lipids and markers of adiposity. Regression analysis was used to explore to the extent to which body fat % and HOMA- IR scores could be attributed to oxidized lipid intake. Results- The estimated mean daily consumption of oxidized lipids was 0.625 meq/kg of fat for the US population. Estimated mean consumption of oxidized lipids was significantly greater in men compared to women, in children compared to adults and among African Americans compared to other races. In both men and women it was observed that the markers of adiposity like body fat%, waist circumference, triceps skinfold decreased significantly with increased consumption of oxidized lipids. However in women (below 18 years) there was a significant increase in HOMA-IR with increased consumption of oxidized lipids. Conclusion- Increased consumption of oxidized lipids is associated with decreased fat mass but increased glucose intolerance in women, but not in men.