Fatty acid distributions in tissues from salamanders of the family Plethodontidae were compared to determine the feasibility of using such differences as a taxonomic tool. Intraspecific, generic, and interspecific variations in the fatty acid composition of one species of genus Desmognathus, two species of genus Eurycea, four species of genus Plethodon, and one species each of genus Gyrinophilus and genus Pseudotriton were compared.Intraspecific variation in fatty acid percentages were found when sex, size, season of collection, geographic locality, and altitude were compared on a variety of tissue extracts. Sex and geographic differences seemed to be of the least importance, while differences in the other three comparisons were distinct. Fatty acid compositions of salamanders of various sizes were compared and it was found that tissues of larger salamanders had smaller percentages of short chain fatty acids.Seasonal variation was apparent in that there was an increase in short chain fatty acid percentages of tissues of salamanders collected toward the fall, compared to those collected in the spring. Tissues of specimens from higher elevations were found to have more short chain fatty acids than those from lower elevations.A trend toward larger proportions of short chain fatty acids was found among salamanders of genus Eurycea, which has the greatest diversity in habitat. This trend graded toward lower percentages as the genera became more aquatic, as in genus Gyrinophilus and genus Pseudotriton, or more terrestrial, as in genus Desmognathus and genus Plethodon.Few interspecific variations were found which would allow consistent differentiation between species. One notable exception was the presence of fatty acid 17:2 in Eurycea multiplicata multiplicata, and not in the other species of Eurycea.This study suggests that the biochemical taxonomic differentiation of salamanders of family Plethodontidae using fatty acid distribution, may be possible in a more comprehensive investigation using larger sample sizes.Ball State UniversityMuncie, IN 47306

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Docosahexaenoic acid (DHA) is an omega-3 polyunsaturated fatty acid (n-3 PUFA) required for fetal neurodevelopment. Increased DHA levels are associated with 17β-estradiol levels, as DHA is higher in women relative to men and in pregnant relative to non-pregnant women, suggesting a maternal adaptation to supply DHA to the fetus. DHA can be synthesized in the body from shorter n-3 PUFA through sequential elongation-desaturation, with Δ6-desaturase being the rate-limiting enzyme. The goal of the present thesis was to characterize the mechanism underlying higher DHA in situations of altered 17β-estradiol status by examining the expression of DHA synthesis enzymes in rodent models. Fatty acid composition of several lipid classes was measured by gas chromatography and enzyme expression was measured by RT-qPCR and immunoblotting. Hepatic Δ6-desaturase and phospholipid DHA was higher in female relative to male, and in pregnant relative to non-pregnant rats. Similarly, 17β-estradiol supplementation of ovariectomized rats resulted in increased hepatic Δ6-desaturase expression and DHA content, while ovariectomy itself had no effects on DHA levels despite controlling for hyperphagia. Mice deficient in the DNA binding activity of estrogen receptor α (ERα) had no differences in hepatic Δ6-desaturase or DHA levels. These results suggest that 17β-estradiol mediates the higher DHA levels in females and during pregnancy through increasing hepatic Δ6-desaturase expression, although the effects of removing 17β-estradiol signalling through ovariectomy or ERα disruption are less clear. This work helps to explain findings of altered DHA status in response to changes in 17β-estradiol concentrations, possibly resulting in more appropriately tailored dietary DHA recommendations. Also, increased understanding of the regulation of DHA synthesis may improve DHA yields in agri/aquaculture and enable increased content of DHA in the food supply.

Analytical instrumentation for quantitative in vivo stable isotope metabolic studies has included gas chromatography-mass spectrometry (GC-MS) and gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Limitations of existing MS techniques include excessive parent ion fragmentation, time-consuming sample preparation, and complex instrument operating parameters. In this thesis, procedures for set up and implementation of four GC-MS techniques are described. The first three replicate existing GC-MS single quadrupole methods on an ion trap, and are electron ionization (EI), external methane positive chemical ionization (PCI), and methane negative chemical ionization (NCI). The fourth method is a novel GC-MS mass-selected ion trap internal isobutane positive chemical ionization technique. Four groups of rats were administered isotopic linoleic acid, and liver tissue was collected for labelled linoleic and n-6 polyunsaturated fatty acids (PUFA) metabolites analyses. Qualitative utility of EI was confirmed, and its quantitative limitations exposed. Labelled 18:2n-6 and n-6 PUFA metabolites were detected using external methane PCI, though limited due to significant fragmentation. Methane NCI also detected labelled 18:2n-6, as well as minimizing fragmentation. However, time-consuming sample preparation and non-linear responses were major limitations. Internal isobutane PCI was useful in detecting isotopic 18:2n-6 and n-6 PUFA metabolites. Fragmentation was reduced compared to EI and external methane PCI. Limitations include fragmentation of HUFAs such as EPA and DHA. The novel internal isobutane PCI is more sensitive than external methane PCI and NCI, produces highly linear responses, is simpler and less expensive to operate than C-IRMS, offers reliable instrument operation, and sample preparation time is minimal. Regular quantitative analyses of HUFAs such as EPA and DHA may require further refinements such as using lower energy reagents than isobutane, including acetonitrile and ammonia.

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.

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.

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.

Analytical instrumentation for quantitative in vivo stable isotope metabolic studies has included gas chromatography-mass spectrometry (GC-MS) and gas chromatography-combustion-isotope ratio mass spectrometry (GC-C-IRMS). Limitations of existing MS techniques include excessive parent ion fragmentation, time-consuming sample preparation, and complex instrument operating parameters. In this thesis, procedures for set up and implementation of four GC-MS techniques are described. The first three replicate existing GC-MS single quadrupole methods on an ion trap, and are electron ionization (EI), external methane positive chemical ionization (PCI), and methane negative chemical ionization (NCI). The fourth method is a novel GC-MS mass-selected ion trap internal isobutane positive chemical ionization technique. Four groups of rats were administered isotopic linoleic acid, and liver tissue was collected for labelled linoleic and n-6 polyunsaturated fatty acids (PUFA) metabolites analyses. Qualitative utility of EI was confirmed, and its quantitative limitations exposed. Labelled 18:2n-6 and n-6 PUFA metabolites were detected using external methane PCI, though limited due to significant fragmentation. Methane NCI also detected labelled 18:2n-6, as well as minimizing fragmentation. However, time-consuming sample preparation and non-linear responses were major limitations. Internal isobutane PCI was useful in detecting isotopic 18:2n-6 and n-6 PUFA metabolites. Fragmentation was reduced compared to EI and external methane PCI. Limitations include fragmentation of HUFAs such as EPA and DHA. The novel internal isobutane PCI is more sensitive than external methane PCI and NCI, produces highly linear responses, is simpler and less expensive to operate than C-IRMS, offers reliable instrument operation, and sample preparation time is minimal. Regular quantitative analyses of HUFAs such as EPA and DHA may require further refinements such as using lower energy reagents than isobutane, including acetonitrile and ammonia.

Fatty acid profiling provides information on dietary intakes and an understanding of lipid metabolism. High throughput techniques such as fingertip prick (FTP) sampling has gained popularity in recent years as a simplified method for basic research, and could be further used to assess disease risk in the population, and other similar high-throughput techniques have the potential to assist in the monitoring and labeling of fatty acids in the food supply. With the advancement of high-throughput sample analysis techniques, a more complete understanding of storage stability is required as a larger volume of samples are produced with equal amounts of time to analyze them. Energy-assisted analysis techniques have the potential to help ameliorate some of these issues. Presently, FTP blood, whole blood and salmon storage stability is assessed under various storage conditions, and both microwave-assisted direct transesterification and indirect ultrasound-assisted extraction techniques are assessed. It is determined that storage of FTP blood and whole blood samples at -20°C results in significant and nearly complete highly unsaturated fatty acid (HUFA) degradation compared to all other temperatures examined. This degradation is determined to be the result of hemolysis and subsequent iron release from erythrocytes initiating fatty acid peroxidation reactions. Direct transesterification of FTP blood is reduced from as long as three hours to one minute with microwave-assisted energy and fatty acid extraction from ground flaxseed is reduced to 40 minutes from as long as 24 hours without compromising fatty acid profiles. Results of the current study provides insight into the storage stability of food sample and blood samples collected via high-throughput techniques, and provides support for the utilization of further high-throughput energy-assisted analytical methods that can help to minimize the potentially detrimental effects that long-term storage can have on fatty acid profiles.

The objective of this study was to determine the effects of method of barley grain processing (dry-rolled vs. pelleted barley) and source of oilseed (ground canola vs. ground flaxseed), arranged as a 2 x 2 factorial, on feed intake, ruminal fermentation, nutrient flow to the duodenum, and milk production and composition in dairy cows. Eight Holstein cows (655 ± 69 kg; 83 ± 16 DIM) were used in a replicated 4 x 4 Latin square with 28-d periods. Cows in one square were fitted with ruminal and duodenal cannulae. Cows fed dry-rolled barley consumed 1.8 to 3.5 kg/d more (P = 0.02) DM than those fed pelleted barley; however, source of supplemental dietary fat had no effect on DM intake. Ruminal pH was lower (P = 0.045) in cows fed pelleted barley compared to those fed dry-rolled barley. Ruminal concentration of acetate was greater (P = 0.001), whereas ruminal concentration of propionate tended to be lower (P = 0.11), in cows fed dry-rolled barley compared to those fed pelleted barley; consequently, the acetate:propionate ratio was higher (P = 0.01) in cows fed dry-rolled barley compared to those fed pelleted barley. Ruminal concentration of total VFA was unaffected (P > 0.05) by diet. Source of dietary fat had no effect on ruminal digestion of OM, NDF, ADF or starch; however, ruminal starch digestion was slightly higher in cows fed pelleted barley compared to those fed dry-rolled barley (90.8 vs. 89.5%). Total dietary fatty acid intake was higher (P < 0.05) in cows consuming dry-rolled barley compared to those fed pelleted barley. Duodenal flow of C18:0 was lower, whereas that of C18:2n6c was higher (P < 0.05) in cows fed pelleted barley compared to those fed dry-rolled barley. Feeding flaxseed increased duodenal flows of C18:3n3, cis-9, trans-11 and trans-10, cis-12 conjugated linoleic acid compared to feeding canola. Milk yield was unaffected (P > 0.05) by diet; however, milk fat content was higher (P = 0.004) in cows fed dry-rolled barley compared to those fed pelleted barley. Milk fat content of C18:3 was higher (P = 0.005) in cows fed canola compared to those fed flax. Milk fat content of C18:3 and cis-9, trans-11 C18:2 were higher in cows fed pelleted barley compared to those fed dry-rolled barley with flax as the source of oilseed, but not with canola (interaction, P < 0.01). Milk fat content of saturated fatty acids decreased (P < 0.001) and that of polyunsaturated fatty acids increased (P = 0.003) in cows fed pelleted barley compared to those fed dry-rolled barley. In summary, milk fatty acid profiles were altered by method of grain processing and source of oilseed.