Introduction: Non-invasive assessment of steatosis and fibrosis is of growing relevance in non-alcoholic fatty liver disease (NAFLD). 1H-Magnetic resonance spectroscopy (1H-MRS) and the ultrasound-based controlled attenuation parameter (CAP) correlate with biopsy proven steatosis, but have not been correlated with each other so far. We therefore performed a headto- head comparison between both methods. Methods: Fifty patients with biopsy-proven NAFLD and 15 healthy volunteers were evaluated with 1H-MRS and transient elastography (TE) including CAP. Steatosis was defined according to the percentage of affected hepatocytes: S1 5-33%, S2 34–66%, S3 $67%. Results: Steatosis grade in patients with NAFLD was S1 36%, S2 40% and S3 24%. CAP and 1H-MRS significantly correlated with histopathology and showed comparable accuracy for the detection of hepatic steatosis: areas under the receiveroperating characteristics curves were 0.93 vs. 0.88 for steatosis $S1 and 0.94 vs. 0.88 for $S2, respectively. Boot-strapping analysis revealed a CAP cut-off of 300 dB/m for detection of S2-3 steatosis, while retaining the lower cut-off of 215 dB/m for the definition of healthy individuals. Direct comparison between CAP and 1H-MRS revealed only modest correlation (total cohort: r = 0.63 [0.44, 0.76]; NAFLD cases: r = 0.56 [0.32, 0.74]). For detection of F2–4 fibrosis TE had sensitivity and specificity of 100% and 98.1% at a cut-off value of 8.85 kPa. Conclusion: Our data suggest a comparable diagnostic value of CAP and 1H-MRS for hepatic steatosis quantification. Combined with the simultaneous TE fibrosis assessment, CAP represents an efficient method for non-invasive characterization of NAFLD. Limited correlation between CAP and 1H-MRS may be explained by different technical aspects, anthropometry, and presence of advanced liver fibrosis.

Mice (C57BL/6J: B6) exposed to maternal dietary restriction (DR) exhibited fetal growth- restriction and as adults develop symptoms of the metabolic syndrome. We aimed to determine the impact of DR on fetal hepatic gluconeogenic pathway and insulin sensitivity in late gestation. Second, we aimed to determine whether a postweaning diet rich in omega-3 fatty acids would alter the development of glucose intolerance, insulin resistance and obesity in DR male offspring. The reduced rate of fetal glycogen synthesis by DR male offspring and altered hepatic gene expression of enzymes involved in insulin signalling and glucose metabolism suggest abnormal fetal development in response to DR that may contribute to the later development of the metabolic syndrome. The postweaning omega-3 diet improved obesity, glucose intolerance and insulin resistance in both DR and control males. These data suggest that nutrition in pregnancy and postnatal life play important roles in determining life-long metabolic health.

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.

Mice (C57BL/6J: B6) exposed to maternal dietary restriction (DR) exhibited fetal growth- restriction and as adults develop symptoms of the metabolic syndrome. We aimed to determine the impact of DR on fetal hepatic gluconeogenic pathway and insulin sensitivity in late gestation. Second, we aimed to determine whether a postweaning diet rich in omega-3 fatty acids would alter the development of glucose intolerance, insulin resistance and obesity in DR male offspring. The reduced rate of fetal glycogen synthesis by DR male offspring and altered hepatic gene expression of enzymes involved in insulin signalling and glucose metabolism suggest abnormal fetal development in response to DR that may contribute to the later development of the metabolic syndrome. The postweaning omega-3 diet improved obesity, glucose intolerance and insulin resistance in both DR and control males. These data suggest that nutrition in pregnancy and postnatal life play important roles in determining life-long metabolic health.

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 objectives were (1) to establish a profile of total fatty acids and cholesterol in amniotic fluid (AF) as well as (2) to determine possible associations between AT fatty acids (micromolar and relative proportion) with gestational age and birth weight. A total of 208 AF samples collected between 12 and 22 weeks of gestation during routine amniocentesis were analyzed using tandem column gas chromatography (GC). Smoking increased AF polyunsaturated fatty acid (PUFAs) levels while developmental stage and storage time decreased AF fatty acid quantities. AF trans fatty acids (TFAs) were negatively associated with both birth outcomes, whereas specific fatty acids including stearic acid (C18:0) and gondoic acid (C20:1n-9) were identified as negative predictors for gestational age and birth weight respectively. This study demonstrated novel relationships between fatty acids and fetal growth and gestational age in early midgestation AF, suggesting a possible role of AF fatty acids in predicting birth outcomes.

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.

My thesis research has examined the fatty acid composition of serum triacylglycerol, phospholipid and cholesterol ester in 2793 participants who took part in the 1997 National Nutrition Survey - a national population-based survey of New Zealand adolescents and adults aged or [greater than or equal to]̲15 y. Differences in serum fatty acids by sex, age, ethnicity, body mass index and smoking - independent of dietary fat intake - were determined. Serum fatty acids were used as biomarkers of saturated and polyunsaturated fat intake to predict population serum total cholesterol concentrations. The association between n-3 long-chain polyunsaturated fatty acids in serum phospholipid and mental and physical wellbeing, as assessed by the short form-36, was determined. Serum fatty acids have been used as biological markers of fat intake and to predict the risk of disease. The fatty acid composition of serum triacylglycerol, phospholipid and cholesterol ester is subject to alteration by dietary fat but overall, is largely controlled by metabolic enzymes. Non-dietary variables - sex, age, body mass index or cigarette smoking - may influence the activity of these enzymes, which will subsequently alter the fatty acid composition but the extent to which these affect serum fatty acid composition in the general population is poorly understood. Our results showed that the proportion of docosahexaenoic acid in serum phospholipid and cholesterol ester was significantly greater in women by 0.15 and 0.02 mol%, respectively in comparison to men whereas, the proportion of eicosapentaenoic acid was significantly greater in men by 0.08 and 0.1 mol%, respectively, after adjusting for age, ethnicity, body mass index and smoking. A number of differences in the proportion of palmitoleic acid in serum triacylglycerol, phospholipid and cholesterol ester were detected; palmitoleic acid increased across the age categories in women (15-24, 25-44, 45-64 65+ y), was higher in women compared to men, New Zealand Europeans compared to New Zealand Maori and Pacific People, those with a body mass index or [greater than or equal to] 30 kg/m� compared to those with a body mass index < 25 kg/m� and in current smokers in comparison to non-smokers. In women, there was an inverse trend in the proportion of linoleic acid in serum phospholipid and cholesterol ester across the age categories. The proportion of linoleic acid in serum triacylglycerol, phospholipid and cholesterol ester was lower in smokers by 2.19, 1.04 and 0.75 mol%, respectively in comparison to non-smokers. None of these differences could be explained by a difference in dietary fat intake. Consequently, sex appears to affect the metabolism of n-3 long-chain polyunsaturated fatty acids independent of dietary fat intake and metabolic differences associated with age, body mass index and smoking may be at play for a number of other serum fatty acids notably, palmitoleic and linoleic acids. Evidence for a role of dietary fat as a predictor of serum cholesterol concentrations in the general population is conflicting. On one hand, results from cholesterol-lowering dietary intervention trials show unequivocally that decreasing saturated fat intake produces a meaningful reduction in serum cholesterol concentrations. On the other hand, the results of large observational studies show little association between saturated fat intake and cholesterol concentrations. The lack of association in the latter studies may result from errors in dietary assessment and therefore, using serum fatty acids as biomarkers of fat intake may overcome the limitations associated with typical dietary assessment techniques. Participants were divided into quintiles of increasing proportion of serum fatty acids. Each one SD increase in the myristic acid composition of serum cholesterol ester, triacylglycerol and phospholipid was associated with an increase in cholesterol of 0.19, 0.10 and 0.13 mmol/L, respectively after adjusting for confounding variables. The difference in cholesterol concentrations between those categorised into the highest and lowest quintiles of serum cholesterol ester myristate was 0.48 mmol/L. A one SD increase in the linoleic acid composition of serum cholesterol ester, triacylglycerol and phospholipid corresponded to a decrease in cholesterol of 0.07, 0.05 and 0.07 mmol/L, respectively. The difference in cholesterol concentrations between the 1st and 5th quintiles of serum cholesterol linoleate was 0.18 mmol/L. Intake of saturated and polyunsaturated fats, as measured using serum fatty acids, are important determinants of cholesterol concentrations in New Zealanders. It has been hypothesised that a lower intake of n-3 long-chain polyunsaturated fatty acids, largely of marine origin, is implicated in the aetiology of depressive disorder. Results from the majority of observational studies have shown that depressed participants have a lower proportion of eicosapentaenoic or docosahexaenoic acid in phospholipids compared to controls but evidence for an improvement in depressive symptoms after supplementation with n-3 long-chain polyunsaturated fatty acids is conflicting. There is little known about the role that n-3 long-chain polyunsaturated fatty acids may have as predictors of mental wellbeing in the general population. Participants were categorised into quintiles of increasing n-3 long-chain polyunsaturated fatty acids in serum phospholipid. There was no significant trend in self-reported mental wellbeing - the mental component score - across the quintiles of eicosapentaenoic, docosapentaenoic and docosahexaenoic acids or the sum of these three fatty acids after adjusting for confounding variables. There was a significant trend in the mental component score across the quintiles of the ratio of eicosapentaenoic/arachidonic acid; the difference between the highest and the lowest quintile was 6.6 points. There were significant positive trends in self-reported physical health - the physical component score - across the quintiles of eicosapentaenoic and docosapentaenoic acids as well as the ratio of eicosapentaenoic/arachidonic acid ratio; the difference between the 1st and 5th quintiles were 8.6, 6.0 and 8.9 points, respectively. Overall, there appears to be little association between the n-3 long-chain polyunsaturated fatty acid composition of serum phospholipid and self-reported mental health in a population of low fish consumers; however, the proportion of n-3 long-chain polyunsaturated fatty acids may be an important predictor of physical wellbeing in New Zealanders.

Uncoupling protein, UCP, present in the inner mitochondrial membrane of brown adipose tissue (BAT) contributes to adaptive thermogenesis. UCP functions as a proton pore and can dissipate the proton electrochemical gradient established by the respiratory chain during fuel oxidation, and thus generates heat without producing ATP. However, the brown adipose tissue thermogenesis is not likely to be a major mechanism in controlling energy expenditure for humans because adults have only residual amounts of the tissue. Two new members of the UCP family have been identified based on their high sequence homology to UCP in BAT and named UCP2 and UCP3. The original UCP was renamed UCP1. At the amino acid level, human UCP2 and UCP3 are 59% and 57% identical to UCP1, respectively. In contrast to UCP1, UCP2 is expressed in many tissues such as brown adipose tissue, white adipose tissue, muscle, spleen and macrophages. UCP3 is expressed preferentially in skeletal muscle in humans, and brown adipose tissue and skeletal muscle in rodents. Since their identification many functional studies, including transgenic animals and ectopic expression of UCP2 or UCP3 in yeast, showed uncoupling activity of UCP2 and UCP3. A number of studies have been done that show increased expression of UCP2 and UCP3 by fasting, high-fat diets and suckling of newborn mice. A common characteristic of these circumstances is an associated increase in plasma free fatty acid levels. This study aimed to investigate effects of fatty acids, peroxisome proliferator-activated receptors (PPARs) and other transcription factors on UCP2 and UCP3 gene expression and to explore the molecular mechanism of their regulation through analysis of the promoter of the UCP2 and UCP3 genes. The 3.1 kb and 3.2 kb 5�-flanking regions of the mouse UCP2 and UCP3 genes, respectively, were cloned and used to construct promoter reporter gene (firefly luciferase) plasmids. The cloned region of the UCP2 and UCP3 genes contained putative binding motifs for several transcription factors, including PPAR, myogenin, and MyoD. Luciferase assays of both constructs showed basal promoter activity with 20~190-fold induction for the UCP2 promoter and 1.3~23-fold induction for the UCP3 promoter in several transfected cell lines, including 3T3-L1, C2C12, L6, COS7 and HepG2. Oleic acid (0.3 mM) up-regulated endogenous UCP2 mRNA by 2.3-fold in 3T3-L1 preadipocytes but not in C2C12 myotubes, and UCP3 mRNA by 2.5-fold in C2C12 myotubes. Responsiveness of the cloned promoter to oleic acid reflected the tissue-specific responsiveness of their endogenous genes but with less fold induction, 1.4-fold for UCP2 promoter in 3T3-L1 preadipocytes and 1.5-fold for UCP3 promoter in C2C12 myotubes. Forced expression of PPAR isotypes (PPARα, PPAR[delta] and PPARγ) showed tissue and isotype-specific activation of the UCP2 promoter. UCP2 promoter activity was induced by 2-fold by PPARγ in 3T3-L1 and by 2.8-fold by PPAR[delta] in C2C12. Treatment of oleic acid (0.3 mM) brought about further induction of the UCP2 promoter activity only in 3T3-L1. In contrast, all three isotypes induced activation of the UCP3 promoter in 3T3-L1, C2C12 and HepG2 cells. Treatment with oleic acid (0.3 mM) or isotype-specific agonist (10 [mu]M) resulted in further increased activity of the UCP3 promoter in 3T3-L1 and HepG2 cells. In particular, rosiglitazone (10 [mu]M) induced a 41-fold increase in UCP3 promoter activity in PPARγ transfected HepG2 cells, and this induction returned to basal level by treatment with bisphenol A diglycidyl ether (BADGE) (50 [mu]M), an antagonist for PPARγ. In addition, UCP3 promoter activity increased up to 20-fold 4 days after induction of C2C12 myoblasts differentiation, whereas UCP2 promoter activity increased only up to 2-fold. Forced expression of myogenin and MyoD in C2C12 myoblasts to mimic differentiation, induced UCP3 promoter activity in an additive manner, consistent with UCP3 being regulated by muscle differentiation. In the present study, it has been shown that UCP2 and UCP3 genes are regulated differently by fatty acids. The tissue-type dependence in regulation of endogenous UCP2 and UCP3 paralleled the cell type-specific effect of oleic acid on the promoter-reporter constructs, suggesting that fatty acid effects are at the transcriptional level. UCP2 and UCP3 promoters showed differences in their response to PPARs. Mediation of the fatty acid effect through PPARs has been also demonstrated, but direct binding of PPARs and particular regulatory motifs on the cloned promoter region have not yet been investigated.

Dietary omega-3 long chain polyunsaturated fatty acids (n-3 LCPUFAs), eicosapentaenoic acid (EPA, 20:5n-3), docosapentaenoic acid (DPA, 22:5n-3) and docosahexaenoic acid (DHA, 22:6n-3), have beneficial health effects and hence increasing the consumption of these fatty acids is recommended by health authorities. The most common dietary source of EPA, DPA and DHA is seafood, but few Australians habitually consume fish and on average eat less than one meal of fish per week. Thus if Australians are to meet the dietary guidelines for n-3 fatty acid intake, there is a need to develop a source of n-3 rich foods that fit into a typical Australian diet. Feeding fish oils rich in n-3 LCPUFA to chickens has proven problematic due to alteration in organoleptic properties. The incorporation of vegetable oils rich in n-3 PUFA, alpha-linolenic acid (ALA, 18:3n-3) into the diet of chickens is potentially an alternative way to provide meat rich in n-3 LCPUFAs as ALA is the precursor of EPA and DHA. However, most vegetable oils also contain the n-6 (n-6) PUFA, linoleic acid (LA, 18:2n-6) which competes with ALA for the same enzymes in their metabolism to LCPUFA. This thesis addressed two crucial issues relating to the conversion of ALA into EPA, DPA and DHA of chicken tissues. The objectives of the first experiment were to examine the effects of increasing the ALA content of diets on the conversion of ALA into EPA, DPA and DHA by measuring their accumulation in chicken meat (breast and thigh) and to determine if there was an optimum level of ALA (at a fixed level of LA) in this process. The ratio of LA to ALA of the diets ranged from 10.5:1 to 0.6:1. The findings in this study demonstrated that there was no optimum level of dietary ALA and as indicated by the observation that EPA, DPA and DHA continued to increase in breast and thigh as the ratio of LA to ALA decreased in the diet. In general, DPA achieved higher levels than DHA. The experimental diets with the lowest LA to ALA ratio elevated the incorporation of EPA and DHA into breast and thigh meat to levels 5 and 4-fold, respectively relative to birds fed the highest LA to ALA ratio. In contrast, arachidonic acid (AA, 20:4n-6) in all groups reduced with decreasing LA to ALA ratio in the diets. The results indicated that the dietary treatments did not significantly change the growth performance of chickens. The objective of the second experiment was to assess the regulatory effect of dietary LA on the conversion of ALA into EPA, DPA and DHA. While in the first experiment the diets varied in the level of ALA but had a constant LA level, in this experiment the level of ALA in the diets was held constant and the level of LA was varied. The LA to ALA ratio of experimental diets ranged from 1.4:1 to 2.1:1. The results of this study indicated that the highest LA to ALA ratio (2.1:1) resulted in the lowest n-3 LCPUFAs, EPA, DPA and DHA in meat samples. For example, the total n-3 LCPUFA levels in the breast meat of birds fed with the lowest LA to ALA ratio was 16% higher than the n-3 LCPUFA in the breast of birds fed the highest LA to ALA ratio. This study indicated that the strongest influence on EPA, DPA and DHA accumulation in chicken tissues was the level of ALA in the diet. The experimental diets did not appear to affect the growth performance of chickens. In conclusion, increasing the ALA content of chicken diets may result in a meat source high in n-3 LCPUFAs that may reduce pressure on diminishing marine stocks as well as offering health benefits to Australians. / http://proxy.library.adelaide.edu.au/login?url= http://library.adelaide.edu.au/cgi-bin/Pwebrecon.cgi?BBID=1369065 / Thesis (M.Ag.Sc.) - University of Adelaide, School of Agriculture, Food and Wine, 2009