Background: Foods rich in polyunsaturated fatty acids (PUFA) are susceptible to oxidation through heating or storage. Oxidized lipids are known to act as ligands for a transcription factor (PPAR-gamma) that affects adipocyte differentiation and insulin sensitivity. Objective: The purpose of this study was to determine the amounts of oxidation products of a variety of PUFA containing foods over time, and to determine whether extracted fats from these foods act as ligands for PPAR-gamma. Method: To study the effect of room-temperature storage on oxidation, 5 foods (walnuts, sunflower seeds, ground flax, fish oil capsules, and infant formula) were purchased and stored at room temperature for 1, 2, and 3 months. To determine oxidation levels in fried foods, French fries and chicken nuggets were used. Fat was extracted from each food and the levels of oxidation products were analyzed by spectrophotometry and kits designed to measure oxidation products. Using a fluorescence polarization-based ligand screening assay kit, fat extracted from foods was analyzed for its binding affinity for PPAR-gamma. Results: Among foods stored at room temperature, the levels of oxidation products did not change significantly with time. Most foods exhibited the highest levels of oxidation at the purchase date. Infant formula and ground flax demonstrated higher levels of oxidation products than did other foods. In preliminary ligand binding assays, extracted fat from French fries showed the greatest binding affinity for PPAR-gamma; a select few other oils showed slight affinity. Discussion: Surprisingly, storage time did not affect oxidation levels. The greatest amount of oxidation may occur during pre-purchase storage conditions. The processing of formula and ground flax may be the cause of the relatively higher oxidation levels in those foods. The binding affinity for PPAR-gamma demonstrated by French fries needs further investigation. Conclusion: Certain oxidized lipids from foods may act as ligands for PPAR-gamma. Further research is required not only to determine which component of these PUFA-containing foods activates PPAR-gamma but also to determine whether that component acts as an agonist or antagonist for PPAR-gamma.

A series of experiments was conducted to test the overall hypothesis that reducing the omega-6 (n-6) to omega-3 (n-3) fatty acid (FA) ratio in sow diets would improve sow reproductive performance (characterized by increases in numbers and body weight of piglets born alive and weaned) and would lessen the inflammatory responses of their offspring post weaning. Diets were wheat/barley based and consisted of a control (tallow based, similar to a standard production diet), 3 diets with plant oil based n-6:n-3 ratios (9:1P, 5:1P, and 1:1P) and a 5:1 fish oil diet (5:1F). The control diet had a ratio of 8:1, but contained approximately half the polyunsaturated FA content of the other diets. Sows were randomly assigned to a treatment diet on d 80 of gestation, and remained on that treatment for three consecutive reproductive cycles (gestation/lactation 1 = P1, gestation/lactation 2 = P2, gestation/lactation 3 = P3). Experiment 1 was designed to test the hypothesis that reducing the n-6:n-3 FA ratio in sow diets would increase circulating concentrations of n-3 FA’s in sows and in their offspring, and the passive immune status of piglets would be improved. Performance data was collected throughout P1 and P2 on 150 sows (n = 30/diet). Sow and piglet serum, colostrum and milk were analyzed for FA profiles, and colostrum and piglet serum were analyzed for immunoglobulin (Ig) A and IgG. In P1, birth weights were unaffected by diet (P > 0.05). Average piglet weaning weights (P = 0.02) and ADG (P = 0.01) however, were highest for piglets born to sows consuming the 9:1P and 5:1P diets. During P2, 5:1F sows consumed 10% less feed (P = 0.04), their piglets had reduced birth weights (P = 0.05), and average weaning weight was reduced by 0.8 kg (P = 0.04) relative to control or 5:1P sows. Colostral and piglet plasma IgA and IgG were unaffected by diet (P > 0.05). Colostrum FA profile patterns were similar to that of the sow diets. Serum n-3 FA’s were greatest in sows (P < 0.01) and piglets (P < 0.01) consuming 1:1P or 5:1F diets. Serum α-linolenic acid (ALA) was highest in the 1:1P sows and eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were highest in the 5:1F sows. In piglet serum obtained prior to suckling, ALA and DHA did not differ among treatments (P > 0.05) but EPA was 2.5 times greater in the 1:1P group and 4 times greater in the fish group (P < 0.01) compared to those from the control diet. In post-suckle samples, ALA was highest in serum from 1:1P diet piglets (P < 0.01), and EPA and DHA were highest in piglet serum from the 5:1F sows (P < 0.01). Omega-3 FA’s can perturb lipid metabolism, specifically increasing the lipolytic activity of adipose tissue and thus the second experiment tested the hypothesis that high producing sows, consuming reduced n-6:n-3 ratios would have increased body fat mobilization. Twenty sows per diet, farrowing ≥ 11 piglets and nursing ≥ 10 piglets during P3, were used. Performance data on sows and piglets (such as weights, numbers, backfat changes) was collected throughout lactation and milk samples obtained on d 4 and d 16 of lactation. Jugular catheters were inserted into 8 sows from each of the 9:1P and 1:1P groups on d 5 of lactation and sows were challenged with a single injection of epinephrine followed by serial blood collections. Feed intake was highest for sows consuming the control (8.4 kg/d) and 5:1P (8.2 kg/d) diets and lowest for the sows fed the 1:1P (7.4 kg/d) and 5:1F (7.7 kg/d) diets (P = 0.05). Altering the n-6:n-3 FA ratio did not affect sow BW, piglet ADG, milk DM and N content or the total output of milk (P > 0.2). Sows consuming the 1:1P diet had greater backfat thickness (P < 0.05) and numerically higher plasma NEFA at baseline compared with the 9:1P sows (240 vs 93 uM; P = 0.16). When given epinephrine, 9:1P fed sows tended to have lower net incremental area under the curve (niAUC) glucose (P = 0.08) and numerically higher niAUC NEFA (P = 0.17) and glycerol (P = 0.15). A third experiment was conducted to test the hypothesis that piglets raised by sows consuming reduced n-6:n-3 ratios would have reduced inflammatory responses post-weaning. Piglets (n = 20/diet) raised by sows consuming the treatment diets described above for 2 gestation/lactation cycles (P2) were selected at weaning. Within diet group, pigs were randomized to either a challenge control group (saline injected) or to a lipopolysaccharide (LPS) injected group (n=10/challenge•diet-1). Piglets were fed a common starter diet for 6 days followed by saline or LPS injections on d 7. Rectal temperatures were recorded for 24 hrs and blood samples were collected at 0, 2, 6 and 12 hrs post injection for pro-inflammatory cytokine and blood urea nitrogen (BUN) analysis. Injecting LPS caused decreased feed intake and reduced ADG (P < 0.01), and increased temperature and cytokine production (P < 0.05). Piglets raised by sows consuming the 1:1P diet had elevated temperatures (P = 0.01; diet x challenge P > 0.05). Overall, circulating plasma ALA and EPA were increased in sows and piglets when sows were fed a 1:1 plant based ratio compared to the control or high n-6:n-3 ratio groups. Sows fed a ratio of 1:1 mobilized more body fat relative to those consuming the 9:1 ratio; there were no treatment effects on piglet growth. Reducing maternal n-6:n-3 FA ratios below 5:1 increased piglet body temperature prior to and during an LPS induced inflammatory challenge,. Reducing the sow dietary n-6:n-3 FA ratio below 5:1 may have detrimental effects on piglets due to over-stimulation of inflammatory responses.

The effective acyl flux between phospholipids and neutral lipids is critical for a high level of the biosynthesis of very long chain polyunsaturated fatty acids (VLCPUFAs) such as arachidonic acid (ARA,20:4-5,8,11,14), eicosapentaenoic acid (EPA, 20:5-5,8,11,14,17) and docosahexaenoic acid (DHA, 22:6-4,7,10,13,16,19) which are essential for human health and wellbeing. Three membrane-bound enzymes, phosphatidylcholine:diacylglycerol cholinephosphotransferase (PDCT), cholinephosphotransferase (CPT) and ethanolaminephosphotransferase (EPT) from VLCPUFA-producing fungi were selected as candidates for my thesis research based on the hypothesis that these enzymes play important roles in acyl trafficking between phosphatidylcholine (PCs) and diacylglycerols (DAGs) during the biosynthesis of VLCPUFAs. Two putative PDCT cDNAs (PiPDCT1 and PiPDCT2) were cloned from Phytophthora infestans which encode polypeptides with two conserved domains and about 15% of amino acid identity to an Arabidopsis PDCT. However, in vitro assays in yeast Saccharomyces cerevisiae showed they did not have any PDCT activity. Four putative CPT and EPT cDNAs (PiCPT1, PiCPT2, PiEPT and TaCPT) were cloned from P. infestans and Thraustochytrium aureum which encode proteins with a conserved CDP-alcohol phosphotransferase motif and 22% to 26% of amino acid identity to the yeast CPT. In vitro assays indicated PiCPT1 and TaCPT had CPT activity, PiEPT had EPT activity and PiCPT2 did not have either activity. Substrate specificity assays showed that all the three functional CPT and EPT preferred VLCPUFA-containing DAGs as substrates with PiCPT1 being the most specific towards ARA-DAG and DHA-DAG. Real-time qPCR analysis revealed that the expression of PiCPT1 was up-regulated in P. infestans fed with exogenous VLCPUFAs. These results lead us to conclude that PiCPT1 is a VLCPUFAs-specific CPT which may play an important role in shuffling VLCPUFAs from phospholipids to storage neutral lipids, would thus have potential use in metabolic engineering of VLCPUFAs in heterologous systems including oilseed crops.

This study explored the effects of dietary unsaturated fatty acids on feline lipid metabolism. It was hypothesized that high dietary linoleic acid (18:2n-6, LA) would enhance conversion to arachidonic acid (20:4n-6), enrichment of dietary long chain n-3 FA (LCn-3FA) would affect lipid parameters, and n-3 FAs incorporation may blunt n-6 FA incorporation. Twenty-nine cats were randomized into groups (n = 9, 10, 10), and fed for 28 days with blood collections on days 0, 14, and 28. Experimental diets consisted of a commercial diet, supplemented with 8g oil/100g kibble. Oil supplements and subsequent diets were: high-oleic sunflower (H diet) with 82% oleic acid (18:1n-9), Menhaden fish (M diet) with LCn-3FA, and safflower (S diet) with 75% 18:2n-6. Dietary 20:4n-6 content was: 0.03 for H and S, and 0.09 for M (g FA/kg diet). Nonesterified fatty acid (NEFA), triacylglycerol (TG), total cholesterol (TC), lipoproteincholesterol (LP-C), plasma phospholipid (PL) FAs, red blood cell membrane (RBC) FAs, and ∆5 and ∆6 desaturase indices were measured. Statistical analyses were performed with SAS PROC MIXED with p < 0.05 determining significance. Neither TC nor NEFA showed significant effects. Diet M resulted in significant TG lowering, despite typically low feline TGs. Similarly, pre-β LP-C (i.e. TG-rich VLDL) was decreased in diet M. Plasma PL FAs revealed significant accumulations of the following: 18:1n-9 in diet H, 18:2n-6 in diet S, and LCn-3FA in diet M. Despite high dietary 18:2n-6, plasma PL 20:4n-6 was not increased in diet S over diets H or M. Increased docosadienoic acid (20:2n-6) in diet S demonstrated that 18:2n-6 chain elongation occurred in deference to its ∆6 desaturation further substantiating low feline ∆6 desaturase activity. Interestingly, no diet M blunting of 20:4n-6 incorporation occurred because fish oil supplementation provided additional 20:4n-6. Tissue 20:4n-6 content appears to be diet-dependent. Accumulation of eicosapentaenoic acid (20:5n-3), but low affinity for docosahexaenoic acid (22:6n-3) occurred in diet M RBC membranes. After 28 days, plasma PLs reflect dietary intake more readily than RBC membranes. Fish oil supplementation resulted in plasma PL LCn-3FA enrichment and lowered plasma TG concentrations, both of which may have physiological significance in cats.

The aim of this research was to isolate and characterize novel strains of marine protists with potential to commercially produce PUFAs. Twelve trips were made visiting 10 different locations in the Canadian Maritime Provinces. Sixty-nine strains were isolated and screened for biomass and fatty acid production. Those meeting specific criteria were selected for further investigation including characterization by 18S rDNA sequencing. Isolate ONC-KTB-56 produced the greatest amount of biomass (1 807 mg L-1) and fatty acids (24.6% dry weight). Of the total fatty acids, ARA, EPA and DHA comprised 0.89, 1.22 and 4.7 percent, respectively. Isolate ONC-KTB-14 produced 1 704 mg L-1 dry biomass with 5.4 percent fatty acids including 1.44, 1.35 and 37.5 percent, ARA, EPA and DHA, respectively. Through optimization of culture conditions biomass, fatty acid content and the proportions of specific fatty acids can be increased. With such optimization, there is potential for isolates ONC-KTB-14 and ONC-KTB-56 to be grown at a commercial scale for production of PUFAs.

Cholesterol is critical to ensure proper functioning of a membrane. Despite this, the movement of cholesterol within the cell is not fully understood. The molecular order of binary and ternary mixtures of polyunsaturated fatty acids with varying degrees of hy- drocarbon chain unsaturation with 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) and/or cholesterol was studied using 2H NMR. The introduction of cholesterol into sam- ples of 18:1PC, 18:2PC (unsaturated lipid/DMPC-d54/CHOL, 75:5:20mol%) increased the C-2H bond order by ∼30%. Similar bond ordering was found for 20:4PC and 22:6PC samples, however, they were temperature dependent. A two-phase region (lo-ld) was found for 22:6PC:DMPC-d54/CHOL (75:5:20mol%) for temperatures below 286.7 K. The reorientation axis formed an angle of 78±4◦ with respect to the C3-2H bond vector re- gardless of the lipid. The order parameter of cholesterol was temperature independent, and ranged from 0.69±0.04 to 0.78±0.04 depending on the lipid unsaturation. The re- orientation axis of cholesterol was oriented at ∼25◦ to the bilayer normal.

Based on their highly bioactive properties in membrane phospholipids, there is growing recognition that dietary n-3 polyunsaturated fatty acids (PUFA) may be of significant benefit in the prevention and treatment of many lifestyle related pathologies, however direct evidence is lacking. The fat-1 transgenic mouse, a genetic model of n-3 PUFA enrichment, is a useful tool in nutritional research which has provided enhanced insight into the health effects of lifelong n-3 PUFA exposure. However, the influence of timing of n-3 PUFA exposure on health related outcomes remains unclear. This thesis describes the functional characterization of the novel Cre recombinase dependent inducible fat-1 (iFat-1) transgene. In the presence of Cre, the iFat-1 transgene was found to reduce phospholipid n-6/n-3 PUFA ratios both in vitro (100%) and in vivo (upwards of 70%), suggesting that the iFat-1 transgene has potential application to address temporal effects of n-3 PUFA in health and disease. / Canadian Institutes of Health Research - Frederick Banting and Charles Best Canada Graduate Scholarship, Sun Life Financial

<p>Several abnormal conditions, including some cancers, have been associated with changes in the membrane lipid and FA composition. Dietary fat serves as a major source of lipids and FA, particularly the polyunsaturated fatty acids (PUFA), n-6 and n-3. High intakes of n-6 PUFA have been linked to the development of colon cancer in association with low n-3 PUFA intake. Therefore understanding the differences in the lipid and FA profiles between cancer and normal cells in the colon, and the role diet plays in these factors may be invaluable in understanding their role in carcinogenesis. This study compares the lipid profile of azoxymethane (AOM) induced colon polyps to that of the surrounding mucosa tissue in rats fed a diet high in n-6 PUFA. Male Fischer rats were fed the AIN-76A diet containing sunflower oil that has high n-6 PUFA content for a period of nine months. Results indicate that the lipid and FA content of the colon polyps differs significantly from the surrounding mucosa. Colon polyps had an increase in membrane phopholipids phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Changes in membrane fluidity were indicated by the decrease (p&lt / 0.05) in the PC/PE and cholesterol/phospholipids (chol/PL) ratios, and increase (p&lt / 0.05) in the polyunsaturated FA/saturated FA (P/S) ratio. Metabolism of FA was significantly altered in the polyps favouring n-6 FA metabolism and the production of prostaglandin E2. No clear indication of impaired &Delta / 6-desauturase enzyme activity was noticed. Increases in the n-6 PUFA content could be a reflection of the dietary FA intake that increases FA incorporation in the polyps. Changes in the FA parameters of the polyps, particularly an increase in C20:4n-6 and the n6/n3 ratio have been shown to contribute to the rapid growth of cancer tissue. These lipid changes associated with the development of colon polyps could provide unique targets for developing strategies in chemoprevention by dietary manipulation.</p>

The present research examined the hypothesis that omega-3 polyunsaturated fatty acids would increase seizure threshold in rats in vivo, and reduce neuronal excitability in mouse hippocampal slices. Seizure thresholds were measured in rats using the maximal pentylenetetrazol and electrical stimulation seizure tests following α-linolenic acid (ALA) or docosahexaenoic acid administration. ALA raised seizure threshold in the maximal PTZ seizure test, but this effect probably occurred because ALA displaced DHA from liver to the brain. DHA itself was therefore tested in the PTZ and electrical stimulation seizure tests. Direct administration of DHA by subcutaneous injection raised seizure thresholds in the PTZ seizure test, which models tonic-clonic attacks in humans. Dietary enrichment with DHA raised afterdischarge seizure thresholds in the cortex and amygdala, which model simplex and complex partial seizures in humans, although this effect took some time to occur. In vitro, the application of DHA also reduced the incidence of excitatory sharp waves in mouse hippocampal slices. This effect did not appear to be due to either an increase in GABAergic inhibitory tone, nor to a decrease in glutamatergic drive. The fatty acid composition of phospholipids and unesterified fatty acids were measured in the brain following microwave fixation in order to determine whether the effects of DHA on seizure thresholds were due to its de-esterification from the phospholipid membrane. The assay surprisingly revealed that subcutaneous administration of DHA at a dose that raised seizure threshold, increased unesterified arachidonic acid, but not unesterified DHA concentrations during seizures. The results of these studies support the hypothesis that DHA raises seizure threshold in rats, and reduces neuronal excitability in vitro. The effects of DHA on seizure threshold are possibly mediated by the de-esterification of arachidonic acid, which is known to have effects on the voltage-dependent sodium channel.

Omega-3 fatty acids are increasingly recognised as playing an important role in human brain development and mental health. The polyunsaturated fatty acids (PUFAs) include omega-3 and omega-6 fats which are essential fatty acids (EFAs), consumed via the diet. Omega-3 fatty acids are particularly abundant in fish oils. The omega-3 fatty acids are being focused on for their role in depression, the main types being docosahexaenoic acid (DHA), which is abundant in neural tissue, and also eicosapentaenoic acid (EPA) which is biologically very active. There is an emerging literature in relation to omega-3 fatty acid blood levels in depression and the effects of treatment with omega-3. Strong epidemiological evidence has also been published indicating an association between a population's fish intake and depression rates. A specific research focus on omega-3 as a treatment for depression in the perinatal period is also starting to emerge. The importance of this particular area is enhanced by the knowledge that omega-3 depletion occurs during the perinatal period due to fetal diversion for neurodevelopment. In view of the lay public promotion of omega-3 and its appeal to women as a 'natural therapy', there is a need to scientifically evaluate its effectiveness to treat depression in the perinatal period. It is also important to investigate omega-3 as an alternative to antidepressants given the ongoing uncertainties regarding their safety in pregnancy. In this thesis a literature review presents current research relating to this field. This is followed by a description of the methodology and results for the two trials conducted. The results of the double-blind randomised placebo controlled trial of omega-3 as a treatment for depression in the perinatal period were essentially negative. However this result is limited by the small sample size in the study and therefore it may be unwise to interpret the result as conclusive. The case-control study confirmed the hypothesis that omega-3 levels were more depleted in depressed women compared to non-depressed women. A discussion of the results and trial limitations then follows in the thesis. It is concluded that further larger studies are warranted in this area.