Probing the fibrate binding specificity of rat liver fatty acid binding protein

Liver-fatty acid binding protein (L-FABP) is found in high levels in enterocytes and is involved in cytosolic solubilization of fatty acids. In addition, L-FABP has been shown to bind endogenous and exogenous lipophilic compounds, suggesting that it may also play a role in modulating their absorption and disposition within enterocytes. Previously, we have described binding of L-FABP to a range of drugs, including a series of fibrates. In the present study, we have generated structural models of L-FABP-fibrate complexes and undertaken thermodynamic analysis of the binding of fibrates containing either a carboxylic acid or ester functionality. Analysis of the current data reveals that both the location and the energetics of binding are different for fibrates that contain a carboxylate compared to those that do not. As such, the data presented in this study suggest potential mechanisms that underpin molecular recognition and dictate specificity in the interaction between fibrates and L-FABP.

The interaction of lipophilic drugs with intestinal fatty acid-binding protein

Intestinal fatty acid-binding protein (I-FABP) is a small protein that binds long-chain dietary fatty acids in the cytosol of the columnar absorptive epithelial cells (enterocytes) of the intestine. The binding cavity of I-FABP is much larger than is necessary to bind a fatty acid molecule, which suggests that the protein may be able to bind other hydrophobic and amphipathic ligands such as lipophilic drugs. Herein we describe the binding of three structurally diverse lipophilic drugs, bezafibrate, ibuprofen (both R- and S-isomers) and nitrazepam to I-FABP. The rank order of affinity for I-FABP determined for these compounds was found to be R-ibuprofen {approx} bezafibrate > S-ibuprofen >> nitrazepam. The binding affinities were not directly related to aqueous solubility or partition coefficient of the compounds; however, the freely water-soluble drug diltiazem showed no affinity for I-FABP. Drug-I-FABP interaction interfaces were defined by analysis of chemical shift perturbations in NMR spectra, which revealed that the drugs bound within the central fatty acid binding cavity. Each drug participated in a different set of interactions within the cavity; however, a number of common contacts were observed with residues also involved in fatty acid binding. These data suggest that the binding of non-fatty acid lipophilic drugs to I-FABP may increase the cytosolic solubility of these compounds and thereby facilitate drug transport from the intestinal lumen across the enterocyte to sites of distribution and metabolism.

Evaluation of fatty acid extraction methods for Thraustochytrium sp. ONC-T18

Various extraction methods were assessed in their capacity to extract fatty acids from a dried biomass of Thraustochytrium sp. ONC-T18. Direct saponification using KOH in ethanol or in hexane:ethanol was one of the most efficient techniques to extract lipids (697 mg g^-1). The highest amount of fatty acids (714 mg g^-1) was extracted using a miniaturized Bligh and Dyer extraction technique. The use of ultrasonics to break down cell walls while extracting with solvents (methanol:chloroform) also offered high extraction yields of fatty acids (609 mg g^-1). Moreover, when the transesterification mixture used for a direct transesterification method was doubled, the extraction of fatty acids increased approximately 77% (from 392 to 696 mg g^-1). This work showed that Thraustochytrium sp. ONC-T18 has the ability to produce over 700 mg g^-1 of lipids, including more than 165 mg g-1 of docosahexaenoic acid, which makes this microorganism a potential candidate for the commercial production of polyunsaturated fatty acids. Finally, other lipids, such as myristic, palmitic, palmitoleic, and oleic acids, were also produced and recovered in significant amounts (54, 196, 123, and 81 mg g^-1), respectively.

Fatty acid composition in tissues of mice fed diets containing conjugated linolenic acid and conjugated linoleic acid

The influence of 1% alpha-eleostearic acid (α-ESA, cis9,trans11,trans 13-18:3) and 1% punicic acid (PA, cis9,trans11,cis13-18:3) on fatty acid composition in mouse tissues was compared with conjugated linoleic acid (CLA, mixture of primarily cis9,trans11- and trans10,cis12-18:2) in the present study. The content (% total fatty acids) of 18:2n-6 was significantly reduced in the heart and adipose tissues, and total polyunsaturated fatty acids (PUFAs) and n-6 PUFA were significantly reduced in adipose tissue by α-ESA, PA and CLA feeding. The content of 22:6n-3 and total n-3 PUFA were significantly increased in the liver, kidney and heart by PA feeding, but not by α-ESA. In contrast to PA, supplementation with CLA significantly decreased 22:6n-3 in the liver, kidney and heart. The content of 20:4n-6 was significantly decreased in the liver and kidney by CLA feeding, but not by α-ESA and PA. The present results indicate that α-ESA, PA and CLA have differential effects on 22:6n-3 and 20:4n-6 content in mouse tissues.

Transesterification of fish oil to produce fatty acid ethyl esters using ultrasonic energy

This study evaluated the production of fatty acid ethyl esters from fish oil using ultrasonic energy and alkaline catalysts dissolved in ethanol. The feasibility of fatty acid ethyl ester production was determined using an ultrasonic bath and probe, and between 0.5 and 1% KOH (added to the fish oil). Furthermore, factors such as ultrasonic device (bath and probe), catalyst (KOH and C2H5ONa), temperature (20 and 60 °C), and duration of exposure (10–90 min) were assessed. Sodium ethoxide was found to be a more efficient catalyst than KOH when transesterifying fish oil. Ultrasonic energy applied for greater than 30 min at 60 °C using 0.8% of C2H5ONa as a catalyst transesterified over 98% fish oil triglycerides to fatty acid ethyl esters. It is reasonable to conclude that the yield of fatty acid ethyl esters produced by applying ultrasonic energy to fish oil is related to the sonication time. Due to increases in the surface area contact between the reactants and the catalyst, ultrasonic energy has the potential to reduce the production time required by a conventional large-scale commercial transesterification method that uses agitation as a way of mixing.

Optimization of fatty acid determination in selected fish and microalgal oils

The use of ten fatty acid methyl ester reference standards coupled with a detailed quantification method was shown to significantly optimize the fatty acid determination of selected fish and microalgal oils when compared to methods that use only one reference standard (C19:0 or C23:0) as a relative response factor. When using the mixture of ten reference standards after transesterifying oils with NaOH/BF3, determination of total fatty acids, eicosapentaenoic acid and docosahexaenoic acid improved by an average of 7.3, 11.5 and 8.4%, respectively. Furthermore, improvements of 13.9, 18.9 and 6.8% of total fatty acids, EPA and DHA, respectively, were obtained when using the mixture of reference standards for fatty acid determination after directly extracting and transesterifying oil contained in microalgal cells with a mixture of methanol, HCl and chloroform. Fatty acid methyl ester standards dissolved in isooctane showed <5% variability throughout 130 days of stability testing when stored at −20 °C. The optimized method can be used for improving the quantification of fatty acids in both oils (fish and microalgal oils) and dry microalgal cells.

Dietary protein level interacts with ω-3 polyunsaturated fatty acid deficiency to induce hypertension

Background : Dietary ω-3 fatty acid deficiency can lead to hypertension in later life; however, hypertension is affected by numerous other dietary factors. We examined the effect of altering the dietary protein level on blood pressure in animals deficient or sufficient in ω-3 fatty acids.

Methods : Female rats were placed on one of four experimental diets 1 week prior to mating. Diets were either deficient (10% safflower oil; DEF) or sufficient (7% safflower oil, 3% flaxseed oil; SUF) in ω-3 fatty acids and contained 20 or 30% casein (DEF20, SUF20, DEF30, SUF30). Offspring were maintained on the maternal diet for the duration of the experiment. At 12, 18, 24, and 30 weeks, blood pressure was assessed by tail cuff plethysmography.

Results : At both 12 and 18 weeks of age, no differences in blood pressure were observed based on diet, however, by 24 weeks hypertension was evident in DEF30 animals; there were no blood pressure differences between the other groups. This hypertension in DEF30 group was increased at 30 weeks, with systolic, diastolic, and mean arterial pressure all elevated.

Conclusions : These results indicate that the hypertension previously attributed to ω-3 fatty acid deficiency is dependent on additional dietary factors, including protein content. Furthermore, this study is the first to plot the establishment of ω-3 fatty acid deficiency hypertension over time.

Fatty acid-benzendiol derivatives and methods of making and using thereof

Disclosed are compounds comprising a benzenediol derivative bound to one or more fatty acids. Also disclosed are nutritional supplements, pharmaceutical formulations, delivery devices, and foodstuffs comprising the disclosed compounds. Methods of using the disclosed compounds and compositions to improve health are also disclosed.

Chromium-fatty acid compounds and methods of making and using thereof

Disclosed are compounds comprising one or more chromium atoms bonded to one or more fatty acids. Also disclosed are nutritional supplements, pharmaceutical formulations, delivery devices, and foodstuffs comprising the disclosed compounds. Methods of using the disclosed compounds and compositions to improve health are also disclosed

Comparative effects of tuna oil and salmon oil on liver lipid metabolism and fatty acid concentrations in rats

The comparative effect of tuna oil (TO) and salmon oil (SO) on the plasma and liver lipid and fatty acid compositions in Sprague Dawley rats was investigated. The total triacylglycerol (TG) and total cholesterol (TC) concentrations in liver was significantly decreased in the TO group; TG level in liver was also significantly decreased in the SO group. The mRNA expression of HMG-CoA reductase in liver was significantly down-regulated in the TO and SO groups relative to the control group. The plasma TG and TC were decreased in TO, but not in SO; plasma low-density lipoprotein and very low-density lipoprotein levels in TO and SO were decreased compared with the control group. The total n-3 polyunsaturated fatty acid (PUFA) in plasma and liver phospholipids was significantly elevated in the TO and SO. Docosahexaenoic acid (22:6n-3) and eicosapentaenoic acid (20:5n-3) in tissues were significantly increased in the TO and SO, respectively. In this study, TO had a more beneficial effect on liver TC and plasma TG, TC, high-density lipoprotein in rats than SO. The likely mechanism for lowering liver and plasma cholesterol by n-3 PUFA is to suppress the mRNA expression of gene encoding HMG-CoA reductase responsible for cholesterol biosynthesis.

PRACTICAL APPLICATIONS

The beneficial effects of n-3 polyunsaturated fatty acids (PUFAs) from fish and fish oil on human health is derived from their role in modulating membrane lipid composition and affecting metabolic and signal-transduction pathways. In the present study, we demonstrated that n-3 PUFA, docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) from tuna and salmon oils can be effectively incorporated into tissue membranes. Tuna oil rich in DHA has more beneficial effect on liver total cholesterol (TC) and plasma triglyceride, TC and HDL in rats than salmon oil, which is rich in EPA. The present data could provide information for the potential application of fish oils as components of functional food, and selected for fortification with different fish oils.

Factors regulating skeletal muscle fatty acid oxidative genes

Skeletal muscle is the most significant site for whole body fat utilisation. The ability to regulate fat use has a significant impact on the development of obesity and Type II diabetes. The studies conducted during this PhD provided significant insight into the complex molecular regulation of skeletal muscle fat utilisation.

Hypertension induced by ω-3 polyunsaturated fatty acid deficiency is alleviated by α-linolenic acid regardless of dietary source

Ω-3 polyunsaturated fatty acid deficiency, particularly during the prenatal period, can cause hypertension in later life. This study examined the effect of different sources of α-linolenic acid (canola oil or flaxseed oil) in the prevention of hypertension and other metabolic symptoms induced by an ω-3 fatty acid-deficient diet. Dams were provided one of three experimental diets from 1 week before mating. Diets were either deficient (10% safflower oil-DEF) or sufficient (7% safflower oil+3% flaxseed oil-SUF-F; or 10% canola oil-SUF-C) in ω-3 fatty acids. The male offspring were continued on the maternal diet from weaning for the duration of the study. Body weight, ingestive behaviors, blood pressure, body composition, metabolic rate, plasma leptin and brain fatty acids were all assessed. The DEF animals were hypertensive at 24 weeks of age compared with SUF-F or SUF-C animals; this was not evident at 12 weeks. These results suggest that different sources of ALA are effective in preventing hypertension related to ω-3 fatty acid deficiency. However, there were other marked differences between the DEF and, in particular, the SUF-C phenotype including lowered body weight, adiposity, leptin and food intake in SUF-C animals. SUF-F animals also had lower, but less marked reductions in adiposity and leptin compared with DEF animals. The differences observed between DEF, SUF-F and SUF-C phenotypes indicate that body fat and leptin may be involved in ω-3 fatty acid deficiency hypertension.

Gender-specific inhibition of platelet aggregation following omega-3 fatty acid supplementation

Background and Aims : Increased platelet aggregation is a major risk factor for heart attacks, stroke and thrombosis. Long chain omega-3 polyunsaturated fatty acids (LCn-3PUFA; eicosapentaenoic acid, EPA; docosahexaenoic acid, DHA) reduce platelet aggregation; however studies in the published literature involving EPA and/or DHA supplementation have yielded equivocal results. Recent in vitro studies have demonstrated that inhibition of platelet aggregation by LCn-3PUFA is gender specific. We examined the acute effects of dietary supplementation with EPA or DHA rich oils on platelet aggregation in healthy male and females.

Methods and Results : A blinded placebo controlled trial involving 15 male and 15 female subjects. Platelet aggregation was measured at 0, 2, 5 and 24 h post-supplementation with a single dose of either a placebo or EPA or DHA rich oil capsules. The relationship between LCn-3PUFA and platelet activity at each time point was examined according to gender vs. treatment. EPA was significantly the most effective in reducing platelet aggregation in males at 2, 5 and 24 h post-supplementation (−11%, −10.6%, −20.5% respectively) whereas DHA was not effective relative to placebo. In contrast, in females, DHA significantly reduced platelet aggregation at 24 h (−13.7%) while EPA was not effective. An inverse relationship between testosterone levels and platelet aggregation following EPA supplementation was observed.

Conclusion : Interactions between sex hormones and omega-3 fatty acids exist to differentially reduce platelet aggregation. For healthy individuals, males may benefit more from EPA supplementation while females are more responsive to DHA.

Arterial compliance in obese subjects is improved with dietary plant n-3 fatty acid from flaxseed oil despite increased LDL oxidizability

The compliance or elasticity of the arterial system, an important index of circulatory function, diminishes with increasing cardiovascular risk. Conversely, systemic arterial compliance improves through eating of fish and fish oil. We therefore tested the value of high intake of alpha-linolenic acid, the plant precursor of fish fatty acids. Fifteen obese people with markers for insulin resistance ate in turn four diets of 4 weeks each: saturated/high fat (SHF), alpha-linolenic acid/low fat (ALF), oleic/low fat (OLF), and SHF. Daily intake of alpha-linolenic acid was 20 g from margarine products based on flax oil. Systemic arterial compliance was calculated from aortic flow velocity and aortic root driving pressure. Plasma lipids, glucose tolerance, and in vitro LDL oxidizability were also measured. Systemic arterial compliance during the first and last SHF periods was 0.42 +/- 0.12 (mean +/- SD) and 0.56 +/- 0.21 units based on milliliters per millimeter of mercury. It rose significantly to 0.78 +/- 0.28 (P < .0001) with ALF; systemic arterial compliance with OLF was 0.62 +/- 0.19, lower than with ALF (P < .05). Mean arterial pressures and results of oral glucose tolerance tests were similar during ALF, OLF, and second SHF; total cholesterol levels were also not significantly different. However, insulin sensitivity and HDL cholesterol diminished and LDL oxidizability increased with ALF. The marked rise in arterial compliance at least with alpha-linolenic acid reflected rapid functional improvement in the systemic arterial circulation despite a rise in LDL oxidizability. Dietary n-3 fatty acids in flax oil thus confer a novel approach to improving arterial function.

n-3 Polyunsaturated fatty acid-rich vegetable oils and blends

α-Linseed, camelina. perilla, and echium oils are n-3 C₁₈ polyunsaturated fatty acid (PUFA)-rich vegetable oil sources viewed as favorable replacements to fish oil in aquaculture feed (aquafeed) production in consideration of their high (α-linolenic acid (ALA, 18:3n-3) and/or stearidonic acid (SDA, 18:4n-3) contents and potential for subsequent bioconversion to n-3 long-chain polyunsaturated fatty acids (LC-PUFA) in farmed aquatic species. While the total production of these oils is currently low in comparison with that of other terrestrial oil sources, their distinct fatty acid composition and high n-3 to n-6 ratio deliver a unique substitute to fish oil in aquafeeds, presently unparalleled in other alternative terrestrial oil sources. The dietary inclusion of these oil sources has therefore attracted significant research attention, resulting in a multitude of investigations across a broad range of aquatic species (finfish and crustaceans). Generally, providing that the essential fatty acid (EFA) requirements of the species under investigation were met and an adequate level of fish meal was present in the diet, it was found possible to replace 100% and 60-70% of the dietary fish oil component for freshwater and marine species, respectively, with minimal impact on growth performance indices. However, the substitution of fish oil with n-3-rich vegetable oils and/or vegetable oil blends resulted in substantially reduced concentrations of health-promoting eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) in the edible portion of the farmed species. This chapter provides an overview of the use of n-3 PUFA-rich vegetable oils and/or vegetable oil blends for use in aquafeeds. In particular, key aspects of oil production, processing, and refinement will be presented, and individual differences pertaining to the physical, chemical, and nutritional characteristics of the oil types will be highlighted. Following on from this, a summary of the key findings relevant to n-3 PUFA-rich vegetable oil inclusion in aquafeeds will be discussed, with particular emphasis placed on growth performance and nutritional modification.