1. The literature has been reviewed with respect to the dietary intake and subsequent metabolism of polyunsaturated fatty acids (PUFA), of both the n-6 and n-3 series, in teleost fish. Particular emphasis has been made to the physiological roles of PUFA with respect to cell membrane function and eicosanoid production. 2. Atlantic salmon post-smolts were fed practical-type diets, based on fish meal, in three separate dietary experiments of 10-16 weeks duration. The first trial compared dietary lipid supplied either as fish oil (FO) or as sunflower oil (SO) with the diets having an n-3/n-6 PUFA ratio of 9.4 and 0.2 respectively. The second trial used diets formulated with blends of FO, SO, grape seed oil and safflower oil to provide linoleic acid at 10, 25 and 45% of total dietary fatty acids. The third trial was similar to the first but with an additional diet in which the lipid component was supplied by linseed oil (LO). All diets satisfied the nutritional requirements of salmonid fish for n-3 PUFA. There were no statistically significant differences in final weights between dietary treatments in the third trial. However, in the second trial fish fed the intermediate level of linoleic acid (25%) attained a significantly higher final weight compared to both other treatments while fish fed the highest level of linoleic acid (45%) had significantly lower final weights compared to both other treatments. In the first trial the effect of diet on growth (weight gain) could not be ascertained as the initial weights of the fish were significantly different. 3. A number of fish fed SO developed severe cardiac lesions which caused thinning of the ventricular wall and heart muscle necrosis. In addition the fish fed diets containing SO were susceptible to a transportation-induced shock syndrome that resulted in 30% mortality. 4. Incorporation of linoleic acid (18:2n-6) into membrane phospholipids increased in response to dietary intake with fish fed SO having increased levels of 18:2n-6 (up to 15-fold), 20:2n-6 (up to 12-fold), 20:3n-6 (up to 25-fold) and arachidonic acid (AA; 20:4n-6) (up to 3-fold), and decreased levels of eicosapentaenoic acid (EPA; 20:5n-3) (up to 3-fold). The ratio of n-3/n-6 PUFA was decreased (up to 4-fold) and the20:4n-6/20:5n-3 ratio increased (up to 9-fold) in membrane phospholipids from fish fed SO compared to those fed fish oil. While the tissue phospholipids from fish fed La had increased levels of 18:2n-6, 20:2n-6 and 20:3n-6, the levels of AA, 22:4n-6 and 22:5n-6 were similar to or significantly reduced compared to fish fed FO. Membrane phospholipids from fish fed LO also had increased 18:3n-3 and 20:4n-3 compared to both other treatments while in some tissues and phospholipid classes EPA was increased compared to fish fed FO. 5. These dietary induced changes in phospholipid eicosanoid precursor ratio were reflected in altered eicosanoid production. In gill cells, stimulated with the calcium ionophore A23187, 12-hydroxy-8, 10, 14, 17-eicosapentaenoic acid (12-HEPE) was the major 12-lipoxygenase product in fish fed Fa. In stimulated gill cells from fish fed SO and LO, 12-HEPE, 12-hydroxy-5, 8, 10, 14-eicosatetraenoic acid (12-HETE), 14- hydroxy-4, 7, 10, 13, 16, 19-docosahexaenoic acid (14-HDHE) and thromboxane B2 (TXB2) were all decreased compared to fish fed FO. However, the ratio of 12- HETE/12-HEPE was significantly elevated in stimulated gill cells from SO-fed fish compared to both other treatments. In stimulated blood leucocytes leukotriene B4 (LTB4)' 12-HETE and TXB2 were significantly increased while LTB5 and 12-HEPE were significantly decreased in fish fed SO compared to those fed FO. Blood leucocytes from fish fed LO produced less TXB2 compared to fish fed SO and prostaglandin E2 was reduced compared to both other treatments. In isolated cardiac myocytes stimulated with A23187, TXB2 production was increased in SO fed fish compared to those fed FO. 6. The activity of cardiac sarcoplasmic reticulum Ca2+-Mg2+ATPase was not affected by dietary treatment. 7. An established cell line derived from chum salmon heart (CHH-1) was utilised to study PUFA metabolism. The CHH-1 cells exhibited considerable A6 desaturase activity but showed no preference towards n-3 over n-6 PUFA. CHH-1 cells did exhibit significant A5 desaturase activity which showed a preference towards n-3 PUFA. No A4 desaturation activity was observed. Elongation of C20 PUFA was especially active in CHH-1 cells with C22 PUFA being specifically incorporated into phosphatidylethanolamine (PE) and phosphatidylserine (PS). CHH-1 cells supplemented with 20:3n-6 showed reduced growth rate, cell death and unusual pycnotic appearance, compared to those supplemented with other PUFA. 8. The lipid compositions of hearts and livers from wild and farmed parr and presmolts were analysed and compared. The fatty acid compositions of triacylglycerols (TAG) and phospholipids from both farmed parr and pre-smolts contained greater amounts of monoenoic fatty acids compared to their wild counterparts. TAG, phosphatidylcholine (PC) and PE from heart and liver of wild fish contained more 18:2n-6 and AA compared to farmed fish. Linolenic acid, EPA and 22:Sn-3 were increased in hearts and livers of wild fish compared to farmed. Docosahexaenoic acid (DHA; 22:6n-3) levels were higher in heart and liver of farmed fish, particularly in heart PC, PS and TAG. The n-3/n-6 PUFA ratio was generally lower in wild compared to farmed fish, largely due to higher n-6 PUFA, in particular AA, in wild fish. 9. The results are discussed with respect to the competitive interactions between PUFA of the n-6 and n-3 series which determine the fatty acid compositions of membrane phospholipids in salmon. The ratio of n-3/n-6 PUFA in membrane phospholipids, and in particular the ratio of AAIEPA, appears important in terms of membrane physiology and biochemistry, eicosanoid production and the development of cardiac histopathological lesions.

Nonalcoholic fatty liver disease (NAFLD) is categorized into nonalcoholic fatty liver (NAFL) and nonalcoholic steatohepatitis (NASH) and has emerged as a risk factor for more critical clinical conditions. However, the underlying mechanisms of NAFLD pathogenesis are not fully understood. In this study, expression of proteins associated with endoplasmic reticulum (ER) stress, apoptosis and autophagy were analyzed in normal, NAFL and NASH human livers by western blotting. Levels of some ER stress-transducing transcription factors, including cleaved activating transcription factor 6, were higher in NASH than in the normal tissues. However, the expression of a majority of the ER chaperones and foldases analyzed, including glucose-regulated protein 78 and ER protein 44, was lower in NASH than in the normal tissues. Levels of apoptosis markers, such as cleaved poly (ADP-ribose) polymerase, were also lower in NASH tissues, in which expression of some B-cell lymphoma-2 family proteins was up-or down-regulated compared to the normal tissues. The level of the autophagy substrate p62 was not different in NASH and normal tissues, although some autophagy regulators were up-or down-regulated in the NASH tissues compared to the normal tissues. Levels of most of the proteins analyzed in NAFL tissues were either similar to those in one of the other two types, NASH and normal, or were somewhere in between. Together, these findings suggest that regulation of certain important tissues processes involved in protein quality control and cell survival were broadly compromised in the NAFLD tissues.

Abstract In eukaryotic cells, fatty acids are mainly synthesized in the cytoplasm, but recently, in yeast and in humans, the ability to synthesize fatty acids has been characterized in mitochondria. This mitochondrial pathway is similar to bacterial type II fatty acid synthesis (FAS). The main feature of mitochondrial FAS in yeast is the respiratory deficient phenotype and loss of cytochromes when any of genes encoding enzymes for mitochondrial FAS is deleted. Mitochondrial FAS has been demonstrated to have an important role in lipoic acid production, namely it synthesizes octanoyl-ACP, which is used as a precursor for lipoic acid. However, the role and function of mitochondrial FAS is not yet fully understood. Many components of the mitochondrial FAS pathway in yeast have been identified according to their similarity to bacterial counterparts, but 3-hydroxyacyl-ACP dehydratase does not show any easily recognizable similarity to bacterial dehydratases and thus remained unidentified. In this study 3-hydroxyacyl-ACP dehydratases of mitochondrial FAS were characterized from the yeast Saccharomyces cerevisiae, humans, and the human pathogen Trypanosoma brucei. The yeast 3-hydroxyacyl-ACP dehydratase (Htd2p) was identified by using a genetic screen, and this protein was shown to be encoded by open reading frame (ORF) YHR067w. The product of this gene shows mitochondrial localization and exhibits hydratase 2 activity. The deletion of HTD2 leads to a respiratory deficient phenotype, loss of cytochromes, reduced lipoic acids levels and changes in mitochondrial morphology. The ORF encoding human 3-hydroxyacyl-ACP hydratase (HsHTD2) was identified by functional complementation of the respiratory deficient phenotype of the yeast htd2 mutant with a human cDNA library. The complementing cDNA was previously identified as the RPP14 transcript encoding the 14 kDa subunit of the human RNase P complex. It was found that this transcript contains another 3' ORF, which encodes a protein that displays hydratase 2 activity and has mitochondrial localization. The bicistronic nature of the transcript is conserved in vertebrates and indicates a genetic link between mitochondrial FAS and RNA processing. The mitochondrial 3-hydroxyacyl-ACP hydratase in T. brucei is homologous to human HTD2, can complement the yeast respiratory deficient phenotype, exhibits hydratase 2 activity and localizes to the T. brucei mitochondrion.

Abstract Fatty acid degradation in living organisms occurs mainly via the β-oxidation pathway. When this work was started, it was known that the hydration and dehydrogenation reactions in mammalian peroxisomal β-oxidation were catalyzed by only multifunctional enzyme type 1 (MFE-1; Δ2-Δ3-enoyl-CoA isomerase/2-enoyl-CoA hydratase 1/(3S)-hydroxyacyl-CoA dehydrogenase) via the S-specific pathway, whereas in the yeast peroxisomes via the R-specific pathway by multifunctional enzyme type 2 (MFE-2; 2-enoyl-CoA hydratase 2/(3R)-hydroxyacyl-CoA dehydrogenase). The work started with the molecular cloning of the rat 2-enoy-CoA hydratase 2 (hydratase 2). The isolated cDNA (2205 bp) encodes a polypeptide with a predicted molecular mass of 79.3 kDa, which contains a potential peroxisomal targeting signal (AKL) in the carboxyl terminus. The hydratase 2 is an integral part of the cloned polypeptide, which is assigned to be a novel mammalian peroxisomal MFE-2. The physiological role of the mammalian hydratase 2 was investigated with the recombinant hydratase 2 domain derived from rat MFE-2. The protein hydrates a physiological intermediate (24E)-3α, 7α, 12α-trihydroxy-5β-cholest-24-enoyl-CoA to (24R, 25R)-3α, 7α, 12α, 24-tetrahydroxy-5β-cholestanoyl-CoA in bile acid synthesis. The sequence alignment of human MFE-2 with MFE-2(s) of different species reveals 12 conserved protic amino acid residues, which are potential candidates for catalysis of the hydratase 2. Each of these residues was replaced by alanine. Complementation of Saccharomyces cerevisiae fox-2 (devoid of endogenous MFE-2) with human MFE-2 provided a model system for examing the in vivo function of the variants. Two protic residues, Glu366 and Asp510, of the hydratase 2 domain of human MFE-2 have been identified and are proposed to act as a base and an acid in catalysis. Mammalian MFE-2 has a (3R)-hydroxyacyl-CoA dehydrogenase domain, whereas the yeast MFE-2 has two dehydrogenase domains, A and B. The present work, applying site-directed mutagenesis to dissect the two domains, shows that the growth rates of fox-2 cells expressing a single functional domain are lower than those of cells expressing S. cerevisiae MFE-2. Kinetic experiments with the purified proteins demonstrate that domain A is more active than domain B in catalysis of medium- and long-chain (3R)-hydroxyacyl-CoA, whereas domain B is solely responsible for metabolism of short-chain substrates. Both domains are required when yeast cells utilize fatty acids as the carbon source.

This study investigated the effect of nutritional energy levels on meat quality characteristics, through effects on the fatty acid profiles of the subcutaneous fat in sheep. Two rations containing 11. 76MJ ME/kg DM and 10 .18MJ ME/kg DM were fed to Dorper and SA Mutton Merino wethers from respectively 20.51 ± 2.51kg and 22.30 ± 3.99kg to 25, 31, 37 and 43kg live mass. M.longissimus lumborum samples were removed and a trained taste panel evaluated sensory parameters on a lOcm unstructured scale. Subcutaneous fat samples and plasma samples were collected, and various carcass measurements were taken. It was found that high energy nutrition significantly increased the concentration of unsaturated fatty acids in the subcutaneous adipose tissue, with subsequent effects on the fat quality and sensory properties of lamb. Both the aroma and incipient juiciness of taste samples from wethers on the high energy treatment were noticeably improved, while the increased amount of fat, coupled with its poor consistency significantly impaired the overall acceptability of taste samples. / Dissertation (MSc Agric)--University of Pretoria, 1992. / gm2014 / Animal and Wildlife Sciences / Unrestricted

Magister Scientiae - MSc / 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 (0.05) in the PC/PE and cholesterol/phospholipids (chol/PL) ratios, and increase (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. / South Africa

Aquaculture feeds, traditionally composed mainly of fishmeal and fish oil, currently represent the largest cost to fish farmers. With aquaculture growing at an average of 8.8% per year and limited supply of fishmeal and fish oil, suitable alternatives must be found. In addition to increasing sustainability and lowering production costs, the use of plant and/or animal ingredients has the potential to lower flesh levels of persistent organic pollutants (POPs) such as polychlorinated biphenyls. Fish oil and to a lesser extent fishmeal, are considered to be the largest source POPs in farmed fish. Using alternative feed ingredients however, can compromise fish growth and the flesh quality of the final product. Lipid sources including flaxseed oil, canola oil, poultry fat and the protein sources canola protein concentrate, soy protein concentrate and poultry by-product meal were examined as alternatives to fish oil and fishmeal in one on-farm field study and one laboratory feeding trial with Atlantic salmon (Salmon salar) and two laboratory feeding trials conducted on sablefish (Anoplopoma fimbria), a relatively new marine aquaculture species. The nutritive value of the alternative ingredients was assessed on the basis of fish growth performance, proximate composition, fatty acid composition and apparent digestibility coefficients. Sensory attributes were evaluated in the sablefish studies while flesh POP levels were determined in both species. The use of alternative dietary lipids showed no negative effects on fish performance. However replacement of fishmeal with plant proteins in some cases, negatively affected fish growth. Flesh levels of persistent organic pollutants were significantly decreased (p<0.05) with the use of alternative dietary lipids, and flesh levels of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were also depressed. Activated carbon treated anchovy oil and finishing diets were examined in the Atlantic salmon laboratory feeding trial and were effective at lowering flesh POP levels while providing high levels of EPA and DHA. The use of alternative feed ingredients will soon be inevitable in aquaculture feeds. The current research shows alternative lipids and proteins can be incorporated successfully in sablefish and Atlantic salmon feeds with minimal effects on fish growth and quality. / Land and Food Systems, Faculty of / Graduate

The objective of the study was to determine growth performance, physico-chemical attributes, oxidative stability and sensory quality of pork from pigs fed one of three dietary treatments, eac containing 0%, 2.5% or 5% (T1, T2 and T3, respectively) Moringa oleifera leaf meal (MOLM). Dietary treatments were formulated to be isonitrogenous and isoenergetic for weaner (6 – 8 weeks) and grower (9 – 13 weeks) phases. Twelve Large White (LW) and 12 Kolbroek (KB) male pigs at 6 weeks of age, initially weighing an average 10 kg, were randomly allocated to one of the dietary treatments, each with four replicates, in individual pens. Feed and water were offered ad libitum. The Longissimus thoracis et lumborum (LTL) muscle of each carcass (n = 24) was sampled for meat quality and fatty acid analyses. Breed differences in measured parameters were observed. In pigs receiving 5% MOLM, the LW had significantly the highest ADFI (P<0.05) than KB pigs. In pigs receiving 2.5% and 5% MOLM, backfat thickness was significantly highest (P<0.05) compared to those receiving 0% MOML. Dietary inclusion of MOLM had no significant effects (P>0.05) on the physico-chemical quality of pork from LW pigs; although it significantly (P<0.05) increased a* and reduced WBSF values in pork from KB pigs. Pork from LW pigs receiving 5% MOLM had significantly increased (P<0.05) n-3 content of the subcutaneous tissue of LW pigs compared to KB, and also when compread to other treatment groups. In both breeds, the composition of PUFA: SFA and n-6: n-3 in the subcutaneous adipose tissue was significantly (P<0.05) lower in T2 and T3 than in T1. The n-3 levels for pork muscle from LW pigs receiving 2.5% and 5% MOLM, and from KB pigs receiving 5% MOLM, were significantly (P<0.05) lower. Inclusions of MOLM significantly (P<0.05) reduced the n-6: n-3 fatty acids in pork from both LW pigs and KB pigs. Consumer scores on sensory attributes (aroma intensity, initial impression of juiciness, first bite, sustained impression of juiciness, muscle fibre and overall tenderness and overall flavor intensity) significantly (P<0.05) increased as the level of MOLM inclusion in the diet increased; and higher scores were observed in fried meat than in boiled for most sensory attributes. It may be concluded that inclusion of MOLM in pig diets up to 5% improved the ADFI, a*, tenderness of pork without adversely affecting the FCR and other physico-chemical quality attributes; and resulted in desired increase in levels of n-3 and reduced the n-6: n-3 fatty acid ratio.

This study (Wassell & Young 2007; Wassell et al., 2010a) shows that behenic (C22:0) fatty acid rich Monoacylglycerol (MAG), or its significant inclusion, has a pronounced effect on crystallisation (Wassell et al., 2010b; 2012; Young et al., 2008) and interfacial kinetics (3.0; 4.0). New interfacial measurements demonstrate an unusual surface-interactive relationship of long chain MAG compositions, with and without Polyglycerol Polyricinoleate (PGPR). A novel MAG synthesised from Moringa oleifera Triacylglycerol (TAG) influenced textural behaviour of water-in-oil (W/O) emulsions and anhydrous TAG systems (4.0: 5.0; 6.0). Emulsifier mixtures of PGPR and MAG rich in C18:1 / 18:2 and C16:0 / C18:0 do not decrease interfacial tension compared with PGPR alone. Only those containing MAG with significant proportion of C22:0 impacted interfacial behaviour. A mixture of C22:0 based MAG and PGPR results with decreasing tension from ~20°C and is initially dominated by PGPR, then through rearrangement, the surface is rapidly dominated by C22:0 fatty acids. A Moringa oleifera based MAG showed unusual decreased interfacial behaviour not dissimilar to PGPR. All other tested MAG (excluding a C22:0 based MAG), irrespective of fatty acid composition resulted with high interfacial tension values across the measured temperature spectrum (50°C to 5°C). A relative decrease of interfacial tension, with decreased temperature, was greater, the longer the chain length (Krog & Larsson 1992). Moreover, results from bulk and interfacial rheology showed that the presence of C22:0 based MAG has a pronounced effect on both elastic modulus (G’) and viscous modulus (G’’). Through a multidisciplinary approach, results were verified in relevant product applications. By means of ultrasonic velocity profiling with pressure difference (UVP-PD) technique, it was possible to examine the effect of a C22:0 based MAG in an anhydrous TAG system whilst in a dynamic non-isothermal condition (3.0). The non-invasive UVP-PD technique conclusively validated structural events. The application of a Moringa oleifera based MAG in low TAG (35% - 41%), W/O emulsions, results in high emulsion stability without a co-surfactant (PGPR). The bi-functional behaviour of Moringa oleifera based MAG is probably attributed to miscibility (Ueno et al., 1994) of its fatty acids, ranging ~30% of saturated fatty acids (SAFA), with ~70% of C18:1 (5.0). It is concluded that the surface-interactive behaviour of Moringa oleifera based MAG, is attributed to approximately 10% of its SAFA commencing from C20:0. When examined separately and compared, results showed that physical effect of a Moringa oleifera based MAG was not dissimilar to PGPR, influencing the crystallisation kinetics of the particular anhydrous TAG system. When either was combined with a C22:0 rich MAG, enhanced gelation onset and strong propensity to form dendrite structure occurred (5.0). Macrobeam and synchrotron radiation microbeam small angle x-ray diffraction (SR-μ-SAXD) was utilized (6.0) to assess behavior of C22:0 rich MAG, with and without PGPR (Wassell et al., 2012). The C22:0 based MAG combined with PGPR promoted TAG crystallisation as observed by differential scanning calorimetry (DSC). Polarised optical microscopy (POM) observations indicated that C22:0 based MAG eliminates formation of large crystal aggregates, resulting in the likely formation of tiny Pickering TAG / MAG crystals (6.0). It is concluded that the presence and interactive behaviour of Pickering surface-active MAG, is strongly linked to increased fatty acid chain length, which induce increased textural resilience owing to viscoelasticity (4.0; 5.0). A multidisciplinary approach was able to verify structuring behaviour (4.0; 5.0), using multiple analyses (Wassell et al., 2010b; 2012; Young et al., 2008). Novel structuring solutions in reduced TAG based systems have been provided (4.0; 5.0). This study both enhances current understanding of structuring in low TAG W/O emulsions and has led to novel MAG compositions, which address emulsification, structuring and texture in TAG based food systems (Wassell et al., 2010a; 2012a; 2012b; 2012c; 2012d; 2012e; Bech et al., 2013).

Heat exposure causes several physiological and metabolic alterations. Although lipids are vital in sustaining energy production, heat-induced alterations in lipid metabolism have not been clearly established. CHAPTER 1 reviews the known metabolic alterations resulting from heat stress, with a specific focus on changes in whole-body lipid utilization and plasma lipids. CHAPTER 1also outlines the physiological changes caused by heat stress, and their role in reducing exercise performance. The study presented in CHAPTER 2 has shown that, compared to thermoneutral conditions, NEFA concentrations were 37% higher following passive heating and 34% higher following exercise in the heat, without significant changes in whole-body lipid utilization. In addition, the level of hyperthermia attained during passive pre-heating and exercise in the heat resulted in a 13% decrease in total external work and a significantly higher rate of perceived exertion. CHAPTER 3 summarizes the study results and presents the limitations and applications of the study.