Liver fatty acid binding protein (L-FABP) binds and translocates many lipophilic substrates within the cytoplasm including long chain fatty acids. Moreover it was reported that L-FABP possesses antioxidative properties within hepatocytes. However, the mechanism of L-FABP’s antioxidative activity remains to be determined. Peroxisome proliferator activated receptor (PPAR) agonists and antagonists can regulate L-FABP levels. However, it needs to be investigated how PPAR agonists and antagonists regulate L-FABP expression. And whether the altered expression of L-FABP by these agents will affect its antioxidative properties within hepatocytes remains unclear. In this thesis we employed clofibrate (PPARα agonist), MK886 (PPARα antagonist), and GW9662 (PPARγ antagonist) to elucidate the mechanism whereby PPAR regulate L-FABP expression and what effect such expression has on the antioxidant activity of L-FABP in CRL-1548 hepatoma cells. Clofibrate served to upregulate L-FABP expression while MK886 and GW9662 were employed to inhibit L-FABP expression. The principal findings revealed that clofibrate treatment enhanced L-FABP mRNA stability and transcription, which resulted in increased L-FABP levels, while MK866 and GW9662 reduced these levels. We also demonstrated that increases in L-FABP levels were associated with reduced cytosolic reactive oxygen species (ROS), while L-FABP siRNA knockdown resulted in a decrease in L-FABP expression and an associated increase in ROS levels. The antioxidant mechanism of recombinant rat L-FABP in the presence of a hydrophilic (AAPH) and lipophilic (AMVN) free radical generators was also evaluated. Recombinant rat L-FABP was produced in E. coli and its amino acid sequence was confirmed by MALDI QqTOF MS. Antioxidant activity was assayed using the thiobarbituric acid method. Ascorbic acid served as a positive control for the AAPH reaction while α-tocopherol was used as a positive control for the AMVN reaction. The antioxidant activity of recombinant L-FABP was greater when free radicals were generated with AAPH than AMVN. Oxidative modification of L-FABP included up to five methionine oxidative peptides with a total of 80 Da mass shift compared to native L-FABP. These findings suggest that the mechanism of L-FABP’s antioxidant activity involved the reaction of methionine with free radicals. In conclusion, L-FABP expression is regulated by PPAR agonists and antagonists through transcription and mRNA stability. Moreover, methionine residues appear to play an important role in the antioxidative activity of L-FABP.

This study was carried out to investigate the fatty acid composition of tumoral, marginal and normal breast tissue in female breast cancer patients. Patients were recruited from St. Boniface General Hospital. A pre-operative blood sample was drawn. After surgery, sections were obtained from tumoral, marginal and normal breast tissues for histology and biochemical analysis. Extracted lipids from marginal tissue were significantly higher than those in normal or tumoral tissue. The lipid profile in tumoral tissue was significantly different in terms of fatty acid composition compared to normal and marginal tissue with less linoleic and alpha linolenic acid and more long chain polyunsaturated fatty acid of omega-3 and omega-6 series. Marginal tissue showed significantly less alpha linolenic acid compared to normal tissue. An inverse correlation existed between plasma level of 22:6 n-3 and breast cancer stage. We found different lipid profile in tumoral tissue compared to normal and marginal tissue.

The triglyceride-free fatty acid (TG-FFA) cycle was studied in white adipose tissue. The major aims of the study were 1) to see if the rate of TG-FFA cycling (i.e. FFA reesterification) and the sensitivity properties (see Newsholme and Crabtree, 1976, Biochem. Soc. Symp. 41, 61-109) were affected by various treatments, and 2) to measure the rate of cycling in vivo and assess its contribution to the metabolic rate of an animal. There are two ways of estimating the rate of TG-FFA cycling; the first is based on the release of glycerol and FFA from the tissue, and the second on the synthesis of the glycerol and FFA moieties of triglyceride. Experimental agreement between the two methods is very good. It is shown that the rate of TG glycerol synthesis can be estimated by measuring the incorporation of tritium from tritiated water into the TG-glycerol moiety; this method is used to study the TG-FFA cycle in vivo. Experimental results indicated that the rate of TG-FFA cycling in white adipose tissue in vitro and in vivo is affected by various short- and long-term treatments. However, the reesterification of FFA in adipose tissue can only account for perhaps ~1% of the basal metabolic rate of a mouse, and perhaps 4% of the increase in osygen consumption observed in fenoterol-treated mice. The equations of Newsholme and Crabtree (1976) describing the sensitivity properties of substrate cycles are extended and used to show that the TG-FFA cycle increases the sensitivity of control of FFA release from adipose tissue. The degree of sensitivity attainable is variable depending on the treatment used. The use of tritiated water for estimating TG-FFA cycling is tentatively extended to brown adipose tissue. It is suggested that the rate of cycling could be used as an indicator of sympathetic activity in brown and white adipose tissue.

This Thesis investigates the effect of membrane-active agents, such as synthetic ether lipids (SEL), local anaesthetics and polyunsaturated fatty acids (PUFAs) on human leukaemia cells. The two cell lines used were human acute myeloblastic leukaemia (HL60) cells and human myelogenous leukaemia (K562) cells. SEL, local anaesthetics and PUFAs were found to be cytotoxic to both cell lines at certain concentrations. The SEL ET-18-OCH(_3) was found to be cytotoxic to both cell lines but the HL60 cells were found to be the more sensitive cell line. HL60 cells were found to be so sensitive to the action of the local anaesthetic dibucaine that a subtoxic concentration that killed ≤10% was not determined. However, in K562 cells the combination of a subtoxic dibucaine concentration together with a range of ET-I8-OCH(_3) concentrations increased the cytotoxicity over that of ether lipid alone. PUFAs were shown to incorporate into plasma membrane phospholipids at concentrations as low as 1 μM after an incubation of 48 hours. PUFAs were shown to be cytotoxic, but the addition of vitamin E reduced the cytotoxicity of arachidonic acid, eicosapentaenoic acid and docosahexaenoic acid in HL60 cells, and of docosahexaenoic acid in K562 cells. This implied that lipid peroxidation was involved in PUFA cytotoxicity. This was, however, not confirmed. PUFA in combination with ET-I8-OCH3 resulted in a slight decrease in cytotoxicity. PUFA combined with dibucaine did not alter cytotoxicity. Cells were also treated with a combination of PUFA and 1-β-D- arabinofliranosylcytosine (ara-C), which is an agent known to induce cell differentiation. Onset of differentiation was determined by following haemoglobin accumulation in K562 cells. PUFA on their own were found to promote accumulation of haemoglobin. The greatest accumulation of haemoglobin was observed with K562 cells treated with PUFA and ara-C.

No description available.

No description available.

Non-alcoholic fatty liver disease (NAFLD) includes simple hepatic steatosis (SS) and non-alcoholic steatohepatitis (NASH). NAFLD is tightly linked to obesity and is thought to be secondary to various noxious signals, some of which may originate from the intestinal microbiota (IM). Despite a growing body of evidence supporting a link between obesity and altered IM, there are no studies assessing the IM of patients with NAFLD. In this cross-sectional study we aimed at comparing fecal levels of total bacteria, Bacteroidetes, C. coccoides, C. leptum, Bifidobacteria, E. coli, and Archaea between healthy controls (HC) and patients with SS or NASH. We found higher C. coccoides levels in NASH compared to SS and lower percentage Bacteroidetes in NASH compared to SS and HC. Controlling for body mass index and fat intake we found an association between presence of NASH and percentage Bacteroidetes. The latter inversely correlated with insulin resistance.

Young and ovariectomized animals eating diets rich in omega-3 polyunsaturated fatty acids (n-3 PUFAs) exhibit enhanced bone formation and decrease bone loss, respectively. Eicosapentaenoic acid, an n-3 PUFA found in fish oil, competes with arachidonic acid, an n-6 PUFA, for the cyclooxygenase enzyme, modulating prostaglandin E2, a mediator of bone mechanotransduction. Whether this diet affects bone gains during exercise is not well defined. We hypothesized rats consuming a high n-3 PUFA diet would gain more bone mass with increased bone formation compared to the rats consuming a high n-6 PUFA diet in response to exercise. Virgin Sprague-Dawley rats (5-mo-old, n=18) were assigned to one of two groups: diet rich in corn oil with a n-6:n-3 dietary ratio of 23:1 (O6) or a diet rich in fish oil with an n-6:n-3 dietary ratio of 2:1 (O3). After acclimation, rats completed 9 sessions on alternate days of stimulated muscle contractions at 75% peak isometric strength. Structural and densitometric properties of proximal tibia were measured using in vivo peripheral quantitative CT. Bone formation rate was quantified on the periosteal the surface by standard bone histomorphometry after intraperitoneal injections of calcein. There was a significant main effect due to diet on total volumetric bone mineral density. The diet rich in n-3 PUFAs also allowed for increases in cancellous volumetric bone mineral density at the proximal tibia independent from exercise, as high as 28%. However, proximal tibia metaphysis bone size and shape was not modified due to changes in diet. The training protocol resulted in a robust increase in bone formation, mass, and area at the midshaft tibia. Mineral apposition rate and bone formation rate were significantly greater in the O3 group compared to the O6 group with exercise at the midshaft tibia, ~36% and ~38% respectively. However, the greater bone formation seen in the O3 groups did not translate over to significantly greater bone mass and size as noted by the pQCT results at the same bone site, because there were no detectable differences between groups. In summary, our data demonstrate that a diet high in n-3 PUFAs independently increases bone density at the proximal tibia. In addition, there was enhanced BFR due to a diet high in n-3 PUFAs with exercise, but those increases did not translate over to increased cortical bone mass or size. These data provide evidence that fish oil consumption with and without simulated resistance training exercise can be beneficial to bone outcomes.

The cholesterol-lowering efficacy of the original sugarcane-derived Cuban policosanol (OPC) supplement has been attributed to an exclusive policosanol purity and composition. The first objective of the following study was to compare the purity and composition of the OPC and alternative sugarcane derived policosanol (APC) products. Second, to measure blood lipids and policosanol levels in tissues, plasma and feces in hamsters receiving diet fortified with no policosanols, OPC, or an APC (APC1) product. Results indicated that the policosanol purity and composition of the OPC and APC formulations are similar. Lipid levels were not significantly different between groups. Policosanols were undetectable in the plasma and tissues of any animals following policosanol supplementation. Policosanols were excreted at a higher rate in animals consuming APC1. Sugarcane-derived policosanols are not an efficacious cholesterol-lowering therapy. The purity and relative percent composition of the OPC supplement cannot account for its efficacy as a lipid lowering agent.

Four studies were conducted to investigate some factors affecting milk fatty acid composition of dairy ewes. The first study was performed to determine the influence of freezing temperatures and storage time on ovine milk composition and cheese making. The other three experiments were conducted to examine the effects of dietary added fats with different profiles of fatty acids from canola, sunflower and flaxseeds on animal performance, nutrient utilization, milk yield and cheese making. The main emphasis was on fatty acid composition, particularly CLA and Omega-3 fatty acids, in milk and cheese. Results showed that: (1) feeding up to 8% of canola, sunflower and flaxseed had no adverse effects on dry matter intake and total tract digestibilities of dry matter, neutral detergent fiber, acid detergent fiber and crude protein, while the digestibilities of fatty acids and gross energy were increased with oilseed supplementation; (2) feeding flaxseed to lactating ewes up to 260g/ewe/d increased milk yield by up to 8.4%, and fat content by up to 14.3% without adversely affecting other milk components or cheese yield and composition; (3) Oilseed supplementation increased milk concentrations of long-chain and unsaturated fatty acids in the expenses of short-chain, medium-chain and saturated fatty acids. The concentrations of CLA and Omega-3 fatty acids were also increased by oilseed supplementation; (4) the manipulated fatty acids profiles can be reflected in cheese; (5) good quality cheese can be produced from ovine milk frozen at -15°C and -25°C for up to 6 months without influencing cheese content of fat and protein and fatty acid composition. / In conclusion, ovine milk with nutritionally healthy characteristics can be produced by feeding ewes diets with oilseeds, and freezing storage of milk for up to 6 months at -15°C or -25°C does not significantly affects milk composition including fatty acid composition. The characteristics of milk can be reflected in cheese.