Long-chain n-3 fatty acids and inflammation: potential application in surgical and trauma patients

Lipids used in nutritional support of surgical or critically ill patients have been based on soybean oil, which is rich in the n-6 fatty acid linoleic acid (18:2n-6). Linoleic acid is the precursor of arachidonic acid (20:4n-6). In turn, arachidonic acid in cell membrane phospholipids is the substrate for the synthesis of a range of biologically active compounds (eicosanoids) including prostaglandins, thromboxanes, and leukotrienes. These compounds can act as mediators in their own right and can also act as regulators of other processes, such as platelet aggregation, blood clotting, smooth muscle contraction, leukocyte chemotaxis, inflammatory cytokine production, and immune function. There is a view that an excess of n-6 fatty acids should be avoided since this could contribute to a state where physiological processes become dysregulated. One alternative is the use of fish oil. The rationale of this latter approach is that fish oil contains long chain n-3 fatty acids, such as eicosapentaenoic acid. When fish oil is provided, eicosapentaenoic acid is incorporated into cell membrane phospholipids, partly at the expense of arachidonic acid. Thus, there is less arachidonic acid available for eicosanoid synthesis. Hence, fish oil decreases production of prostaglandins like PGE2 and of leukotrienes like LTB4. Thus, n-3 fatty acids can potentially reduce platelet aggregation, blood clotting, smooth muscle contraction, and leukocyte chemotaxis, and can modulate inflammatory cytokine production and immune function. These effects have been demonstrated in cell culture, animal feeding and healthy volunteer studies. Fish oil decreases the host metabolic response and improves survival to endotoxin in laboratory animals. Recently clinical studies performed in various patient groups have indicated benefit from this approach.

A randomized double-blind study of the short-time treatment of obese patients with nonalcoholic fatty liver disease with ursodeoxycholic acid

In order to determine the effect of ursodeoxycholic acid on nonalcoholic fatty liver disease, 30 patients with body mass indices higher than 25, serum levels of alanine aminotransferase (ALT), aspartate aminotransferase (AST) or gamma-glutamyltransferase (gamma-GT) at least more than 1.5 times the upper limit of normality, and hepatic steatosis demonstrated by ultrasonography were randomized into two groups of 15 patients to receive placebo or 10 mg kg-1 day-1 ursodeoxycholic acid for three months. Abdominal computed tomography was performed to quantify hepatic fat content, which was significantly correlated with histological grading of steatosis (r s = -0.83, P < 0.01). Patient body mass index remained stable for both groups throughout the study, but a significant reduction in mean (± SEM) serum levels of ALT, AST and gamma-GT was observed only in the treated group (ALT = 81.2 ± 9.7, 44.8 ± 7.7, 48.1 ± 7.7 and 52.2 ± 6.3 IU/l at the beginning and after the first, second and third months, respectively, N = 14, P < 0.05). For the placebo group ALT values were 66.4 ± 9.8, 54.5 ± 7, 60 ± 7.6 and 43.7 ± 5 IU/l, respectively. No alterations in hepatic lipid content were observed in these patients by computed tomography examination (50.2 ± 4.2 Hounsfield units (HU) at the beginning versus 51.1 ± 4.1 HU at the third month). These results show that ursodeoxycholic acid is able to reduce serum levels of hepatic enzymes in patients with nonalcoholic fatty liver disease, but this effect is not related to modifications in liver fat content.

Effect of bullfrog (Rana catesbeiana) oil administered by gavage on the fatty acid composition and oxidative stress of mouse liver

The aim of the present study was to investigate the effects of daily intragastric administration of bullfrog oil (oleic, linoleic and palmitoleic acid-rich oil), corresponding to 0.4% of body weight for four weeks, on fatty acid composition and oxidative stress (lipid peroxidation and catalase activity) in mouse liver. The activities of aspartate aminotransferase (AST), alkaline phosphatase (ALP), alanine aminotransferase (ALT), and gamma-glutamyltransferase (GGT), biomarkers of tissue injury, were determined in liver homogenates and serum. The proportions of 18:2n-6, 20:4n-6, 20:5n-3, and 22:6n-3 (polyunsaturated fatty acids, from 37 to 60%) in the total fatty acid content were increased in the liver of the bullfrog oil-treated group (P < 0.05) compared to control. At the same time, a significant decrease in the relative abundance of 14:0, 16:0, and 18:0 (saturated fatty acids, from 49 to 25%) was observed. The hepatic content of thiobarbituric acid reactive substances (TBARS) was increased from 2.3 ± 0.2 to 12.3 ± 0.3 nmol TBA-MDA/mg protein and catalase activity was increased from 840 ± 32 to 1110 ± 45 µmol reduced H2O2 min-1 mg protein-1 in the treated group. Bullfrog oil administration increased AST and ALP activities in the liver (from 234.10 ± 0.12 to 342.84 ± 0.13 and 9.38 ± 0.60 to 20.06 ± 0.27 U/g, respectively) and in serum (from 95.41 ± 6.13 to 120.32 ± 3.15 and 234.75 ± 11.5 to 254.41 ± 2.73 U/l, respectively), suggesting that this treatment induced tissue damage. ALT activity was increased from 287.28 ± 0.29 to 315.98 ± 0.34 U/g in the liver but remained unchanged in serum, whereas the GGT activity was not affected by bullfrog oil treatment. Therefore, despite the interesting modulation of fatty acids by bullfrog oil, a possible therapeutic use requires care since some adverse effects were observed in liver.

Serum laminin, type IV collagen and hyaluronan as fibrosis markers in non-alcoholic fatty liver disease

Hepatic fibrosis in patients with non-alcoholic fatty liver disease is associated with progression of the disease. In the present study, we analyzed the discriminative ability of serum laminin, type IV collagen and hyaluronan levels to predict the presence of fibrosis in these patients. In this preliminary report, we studied 30 overweight patients divided into two groups according to the absence (group I, N = 19) or presence (group II, N = 11) of fibrosis in a liver biopsy. Triglycerides, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyltranspeptidade, hyaluronan (noncompetitive fluoroassay), type IV collagen, and laminin (ELISA) were determined. Group II presented significantly higher mean laminin, hyaluronan, type IV collagen, and aspartate aminotransferase values, which were due to the correlation between these parameters and the stage of fibrosis in the biopsy (Spearman's correlation coefficient, rS = 0.65, 0.62, 0.53, and 0.49, respectively). Analysis of the ROC curve showed that laminin values >282 ng/ml were those with the best diagnostic performance, with 87% accuracy. Association of laminin with type IV collagen showed improvement in the positive predictive value (100%), but with reduction in diagnostic sensitivity (64%). When compared with the criteria of Ratziu et al. [Gastroenterology (2000) 118: 1117-1123] for the diagnosis of septal fibrosis, laminin values presented a better diagnostic accuracy (83 vs 70%). Determination of extracellular matrix components in serum, especially of laminin, may identify patients with non-alcoholic fatty liver disease and fibrosis and these components may be used as indicators for liver biopsy in these patients.

Liver mitochondrial dysfunction and oxidative stress in the pathogenesis of experimental nonalcoholic fatty liver disease

Oxidative stress and hepatic mitochondria play a role in the pathogenesis of nonalcoholic fatty liver disease. The aim of the present study was to evaluate the role of hepatic mitochondrial dysfunction and oxidative stress in the pathogenesis of the disease. Fatty liver was induced in Wistar rats with a choline-deficient diet (CD; N = 7) or a high-fat diet enriched with PUFAs-omega-3 (H; N = 7) for 4 weeks. The control group (N = 7) was fed a standard diet. Liver mitochondrial oxidation and phosphorylation were measured polarographically and oxidative stress was estimated on the basis of malondialdehyde and glutathione concentrations. Moderate macrovacuolar liver steatosis was observed in the CD group and mild liver steatosis was observed in the periportal area in the H group. There was an increase in the oxygen consumption rate by liver mitochondria in respiratory state 4 (S4) and a decrease in respiratory control rate (RCR) in the CD group (S4: 32.70 ± 3.35; RCR: 2.55 ± 0.15 ng atoms of O2 min-1 mg protein-1) when compared to the H and control groups (S4: 23.09 ± 1.53, 17.04 ± 2.03, RCR: 3.15 ± 0.15, 3.68 ± 0.15 ng atoms of O2 min-1 mg protein-1, respectively), P < 0.05. Hepatic lipoperoxide concentrations were significantly increased and the concentration of reduced glutathione was significantly reduced in the CD group. A choline-deficient diet causes moderate steatosis with disruption of liver mitochondrial function and increased oxidative stress. These data suggest that lipid peroxidation products can impair the flow of electrons along the respiratory chain, causing overreduction of respiratory chain components and enhanced mitochondrial reactive oxygen species. These findings are important in the pathogenesis of nonalcoholic fatty liver disease.

Effect of unsaturated fatty acids on myocardial performance, metabolism and morphology

Diets rich in saturated fatty acids are one of the most important causes of atherosclerosis in men, and have been replaced with diets rich in unsaturated fatty acids (UFA) for the prevention of this disorder. However, the effect of UFA on myocardial performance, metabolism and morphology has not been completely characterized. The objective of the present investigation was to evaluate the effects of a UFA-rich diet on cardiac muscle function, oxidative stress, and morphology. Sixty-day-old male Wistar rats were fed a control (N = 8) or a UFA-rich diet (N = 8) for 60 days. Myocardial performance was studied in isolated papillary muscle by isometric and isotonic contractions under basal conditions after calcium chloride (5.2 mM) and ß-adrenergic stimulation with 1.0 µM isoproterenol. Fragments of the left ventricle free wall were used to study oxidative stress and were analyzed by light microscopy, and the myocardial ultrastructure was examined in left ventricle papillary muscle. After 60 days the UFA-rich diet did not change myocardial function. However, it caused high lipid hydroperoxide (176 ± 5 vs 158 ± 5, P < 0.0005) and low catalase (7 ± 1 vs 9 ± 1, P < 0.005) and superoxide-dismutase (18 ± 2 vs 27 ± 5, P < 0.005) levels, and discrete morphological changes in UFA-rich diet hearts such as lipid deposits and mitochondrial membrane alterations compared to control rats. These data show that a UFA-rich diet caused myocardial oxidative stress and mild structural alterations, but did not change mechanical function.

Modulation of peritoneal macrophage activity by the saturation state of the fatty acid moiety of phosphatidylcholine

To determine the effects of saturated and unsaturated fatty acids in phosphatidylcholine (PC) on macrophage activity, peritoneal lavage cells were cultured in the presence of phosphatidylcholine rich in saturated or unsaturated fatty acids (sat PC and unsat PC, respectively), both used at concentrations of 32 and 64 µM. The treatment of peritoneal macrophages with 64 µM unsat PC increased the production of hydrogen peroxide by 48.3% compared to control (148.3 ± 16.3 vs 100.0 ± 1.8%, N = 15), and both doses of unsat PC increased adhesion capacity by nearly 50%. Moreover, 64 µM unsat PC decreased neutral red uptake by lysosomes by 32.5% compared to the untreated group (67.5 ± 6.8 vs 100.0 ± 5.5%, N = 15), while both 32 and 64 µM unsat PC decreased the production of lipopolysaccharide-elicited nitric oxide by 30.4% (13.5 ± 2.6 vs 19.4 ± 2.5 µM) and 46.4% (10.4 ± 3.1 vs 19.4 ± 2.5 µM), respectively. Unsat PC did not affect anion production in non-stimulated cells or phagocytosis of unopsonized zymosan particles. A different result pattern was obtained for macrophages treated with sat PC. Phorbol 12-miristate 13-acetate-elicited superoxide production and neutral red uptake were decreased by nearly 25% by 32 and 64 µM sat PC, respectively. Sat PC did not affect nitric oxide or hydrogen peroxide production, adhesion capacity or zymosan phagocytosis. Thus, PC modifies macrophage activity, but this effect depends on cell activation state, fatty acid saturation and esterification to PC molecule and PC concentration. Taken together, these results indicate that the fatty acid moiety of PC modulates macrophage activity and, consequently, is likely to affect immune system regulation in vivo.

Beneficial effects of exercise training (treadmill) on insulin resistance and nonalcoholic fatty liver disease in high-fat fed C57BL/6 mice

C57BL/6 mice develop signs and symptoms comparable, in part, to the human metabolic syndrome. The objective of the present study was to evaluate the effects of exercise training on carbohydrate metabolism, lipid profile, visceral adiposity, pancreatic islet alterations, and nonalcoholic fatty liver disease in C57BL/6 mice. Animals were fed one of two diets during an 8-week period: standard (SC, N = 12) or very high-fat (HF, N = 24) chow. An exercise training protocol (treadmill) was then established and mice were divided into SC and HF sedentary (SC-Sed, HF-Sed), exercised groups (SC-Ex, HF-Ex), or switched from HF to SC (HF/SC-Sed and HF/SC-Ex). HF/HF-Sed mice had the greatest body mass (65% more than SC/SC-Sed; P < 0.0001), and exercise reduced it by 23% (P < 0.0001). Hepatic enzymes ALP (+80%), ALT (+100%) and AST (+70%) were higher in HF/HF mice than in matched SC/SC. Plasma insulin was higher in both the HF/HF-Sed and HF/SC-Sed groups than in the matched exercised groups (+85%; P < 0.001). Pancreatic islets, adipocytes and liver structure were greatly affected by HF, ultimately resulting in islet β-cell hypertrophy and severe liver steatosis. The HF group had larger islets than the SC/SC group (+220%; P < 0.0001), and exercise significantly reduced liver steatosis and islet size in HF. Exercise attenuated all the changes due to HF, and the effects were more pronounced in exercised mice switched from an HF to an SC diet. Exercise improved the lipid profile by reducing body weight gain, visceral adiposity, insulin resistance, islet alterations, and fatty liver, contributing to obesity and steatohepatitis control.

Reversible flow of cholesteryl ester between high-density lipoproteins and triacylglycerol-rich particles is modulated by the fatty acid composition and concentration of triacylglycerols

We determined the influence of fasting (FAST) and feeding (FED) on cholesteryl ester (CE) flow between high-density lipoproteins (HDL) and plasma apoB-lipoprotein and triacylglycerol (TG)-rich emulsions (EM) prepared with TG-fatty acids (FAs). TG-FAs of varying chain lengths and degrees of unsaturation were tested in the presence of a plasma fraction at d > 1.21 g/mL as the source of CE transfer protein. The transfer of CE from HDL to FED was greater than to FAST TG-rich acceptor lipoproteins, 18% and 14%, respectively. However, percent CE transfer from HDL to apoB-containing lipoproteins was similar for FED and FAST HDL. The CE transfer from HDL to EM depended on the EM TG-FA chain length. Furthermore, the chain length of the monounsaturated TG-containing EM showed a significant positive correlation of the CE transfer from HDL to EM (r = 0.81, P < 0.0001) and a negative correlation from EM to HDL (r = -041, P = 0.0088). Regarding the degree of EM TG-FAs unsaturation, among EMs containing C18, the CE transfer was lower from HDL to C18:2 compared to C18:1 and C18:3, 17.7%, 20.7%, and 20%, respectively. However, the CE transfer from EMs to HDL was higher to C18:2 than to C18:1 and C18:3, 83.7%, 51.2%, and 46.3%, respectively. Thus, the EM FA composition was found to be the rate-limiting factor regulating the transfer of CE from HDL. Consequently, the net transfer of CE between HDL and TG-rich particles depends on the specific arrangement of the TG acyl chains in the lipoprotein particle core.

Effect of silybin on high-fat-induced fatty liver in rats

Silybin, a natural antioxidant, has been traditionally used against a variety of liver ailments. To investigate its effect and the underlying mechanisms of action on non-alcoholic fatty liver in rats, we used 60 4-6-week-old male Sprague-Dawley rats to establish fatty liver models by feeding a high-fat diet for 6 weeks. Hepatic enzyme, serum lipid levels, oxidative production, mitochondrial membrane fluidity, homeostasis model assessment-insulin resistance index (HOMA-IR), gene and protein expression of adiponectin, and resistin were evaluated by biochemical, reverse transcription polymerase chain reaction (RT-PCR) and Western blot analysis. Compared with the model group, silybin treatment (26.25 mg·kg-1·day-1, started at the beginning of the protocol) significantly protected against high-fat-induced fatty liver by stabilizing mitochondrial membrane fluidity, reducing serum content of alanine aminotransferase (ALT) from 450 to 304 U/L, decreasing hepatic malondialdehyde (MDA) from 1.24 to 0.93 nmol/mg protein, but increasing superoxide dismutase (SOD) and glutathione (GSH) levels from 8.03 to 9.31 U/mg protein and from 3.65 to 4.52 nmol/mg protein, respectively. Moreover, silybin enhanced the gene and protein expression of adiponectin from 215.95 to 552.40, but inhibited that of resistin from 0.118 to 0.018. Compared to rosiglitazone (0.5 mg·kg-1·day-1, started at the beginning of the protocol), silybin was effective in stabilizing mitochondrial membrane fluidity, reducing SOD as well as ALT, and regulating gene and protein expression of adiponectin (P < 0.05). These results suggest that mitochondrial membrane stabilization, oxidative stress inhibition, as well as improved insulin resistance, may be the essential mechanisms for the hepatoprotective effect of silybin on non-alcoholic fatty liver disease in rats. Silybin was more effective than rosiglitazone in terms of maintaining mitochondrial membrane fluidity and reducing oxidative stress.

TRP channels, omega-3 fatty acids, and oxidative stress in neurodegeneration: from the cell membrane to intracellular cross-links

The transient receptor potential channels family (TRP channels) is a relatively new group of cation channels that modulate a large range of physiological mechanisms. In the nervous system, the functions of TRP channels have been associated with thermosensation, pain transduction, neurotransmitter release, and redox signaling, among others. However, they have also been extensively correlated with the pathogenesis of several innate and acquired diseases. On the other hand, the omega-3 polyunsaturated fatty acids (n-3 fatty acids) have also been associated with several processes that seem to counterbalance or to contribute to the function of several TRPs. In this short review, we discuss some of the remarkable new findings in this field. We also review the possible roles played by n-3 fatty acids in cell signaling that can both control or be controlled by TRP channels in neurodegenerative processes, as well as both the direct and indirect actions of n-3 fatty acids on TRP channels.

Trans fatty acid intake is associated with insulin sensitivity but independently of inflammation

High saturated and trans fatty acid intake, the typical dietary pattern of Western populations, favors a proinflammatory status that contributes to generating insulin resistance (IR). We examined whether the consumption of these fatty acids was associated with IR and inflammatory markers. In this cross-sectional study, 127 non-diabetic individuals were allocated to a group without IR and 56 to another with IR, defined as homeostasis model assessment-IR (HOMA-IR) >2.71. Diet was assessed using 24-h food recalls. Multiple linear regression was employed to test independent associations with HOMA-IR. The IR group presented worse anthropometric, biochemical and inflammatory profiles. Energy intake was correlated with abdominal circumference and inversely with adiponectin concentrations (r = -0.227, P = 0.002), while saturated fat intake correlated with inflammatory markers and trans fat with HOMA-IR (r = 0.160, P = 0.030). Abdominal circumference was associated with HOMA-IR (r = 0.430, P < 0.001). In multiple analysis, HOMA-IR remained associated with trans fat intake (β = 1.416, P = 0.039) and body mass index (β = 0.390, P < 0.001), and was also inversely associated with adiponectin (β = -1.637, P = 0.004). Inclusion of other nutrients (saturated fat and added sugar) or other inflammatory markers (IL-6 and CRP) into the models did not modify these associations. Our study supports that trans fat intake impairs insulin sensitivity. The hypothesis that its effect could depend on transcription factors, resulting in expression of proinflammatory genes, was not corroborated. We speculate that trans fat interferes predominantly with insulin signaling via intracellular kinases, which alter insulin receptor substrates.

Gene transfer of Chlorella vulgaris n-3 fatty acid desaturase optimizes the fatty acid composition of human breast cancer cells

Chlorella vulgaris has the gene of n-3 fatty acid desaturase (CvFad3), which can synthesize the precursor of n-3 polyunsaturated fatty acids (PUFAs) or convert n-6 to n-3 PUFAs. The objective of the present study was to examine whether the CvFad3 gene from C. vulgaris can be functionally and efficiently expressed in human breast cancer cells and whether its expression can exert a significant effect on cell fatty acid composition. We inserted the CvFad3 gene into the plasmid pEGFP-C3 to construct the eukaryotic expression vector pEGFP-C3-n-3 and to express the n-3 Fad gene in human breast cancer cells (MCF-7 cells). Transfection of MCF-7 cells with the recombinant vector resulted in a high expression of n-3 fatty acid desaturase. Lipid analysis indicated that the ratio of n-6/n-3 PUFAs was decreased from 6:1 in the control cells to about 1:1 in the cells expressing the n-3 fatty acid desaturase. Accordingly, the CvFad3 gene significantly decreased the ratio of n-6/n-3 PUFAs of the MCF-7 cell membrane. The expression of the CvFad3 gene can decrease cell proliferation and promote cell apoptosis. This study demonstrates that the CvFad3 gene can dramatically balance the ratio of n-6/n-3 PUFAs and may provide an effective approach to the modification of the fatty acid composition of mammalian cells, also providing a basis for potential applications of its transfer in experimental and clinical settings.

Effect and the probable mechanisms of silibinin in regulating insulin resistance in the liver of rats with non-alcoholic fatty liver

Our previous study has shown that reduced insulin resistance (IR) was one of the possible mechanisms for the therapeutic effect of silibinin on non-alcoholic fatty liver disease (NAFLD) in rats. In the present study, we investigated the pathways of silibinin in regulating hepatic glucose production and IR amelioration. Forty-five 4- to 6-week-old male Sprague Dawley rats were divided into a control group, an HFD group (high-fat diet for 6 weeks) and an HFD + silibinin group (high-fat diet + 0.5 mg kg-1·day-1 silibinin, starting at the beginning of the protocol). Both subcutaneous and visceral fat was measured. Homeostasis model assessment-IR index (HOMA-IR), intraperitoneal glucose tolerance test and insulin tolerance test (ITT) were performed. The expression of adipose triglyceride lipase (ATGL) and of genes associated with hepatic gluconeogenesis was evaluated. Silibinin intervention significantly protected liver function, down-regulated serum fat, and improved IR, as shown by decreased HOMA-IR and increased ITT slope. Silibinin markedly prevented visceral obesity by reducing visceral fat, enhanced lipolysis by up-regulating ATGL expression and inhibited gluconeogenesis by down-regulating associated genes such as Forkhead box O1, phosphoenolpyruvate carboxykinase and glucose-6-phosphatase. Silibinin was effective in ameliorating IR in NAFLD rats. Reduction of visceral obesity, enhancement of lipolysis and inhibition of gluconeogenesis might be the underlying mechanisms.

Nitro-fatty acids: novel anti-inflammatory lipid mediators

Nitro-fatty acids are formed and detected in human plasma, cell membranes, and tissue, modulating metabolic as well as inflammatory signaling pathways. Here we discuss the mechanisms of nitro-fatty acid formation as well as their key chemical and biochemical properties. The electrophilic properties of nitro-fatty acids to activate anti-inflammatory signaling pathways are discussed in detail. A critical issue is the influence of nitroarachidonic acid on prostaglandin endoperoxide H synthases, redirecting arachidonic acid metabolism and signaling. We also analyze in vivo data supporting nitro-fatty acids as promising pharmacological tools to prevent inflammatory diseases.