The (n-3) fatty acid dose, independent of the (n-6) to (n-3) fatty acid ratio, affects the plasma fatty acid profile of normal dogs.

The purpose of this study was to determine whether the dose of (n-3) fatty acids (FA) administered, independent of the relative ratio of (n-6) to (n-3) FA in the food, influences plasma FA composition in dogs. Healthy female, geriatric beagles (7-10 y old) were fed foods containing (n-6) to (n-3) FA ratios of either 40.0:1 or 1.4:1 for 12 wk (study 1) or 36 wk (study 2). In study 3, beagles were fed food with the same 1:1 ratio of (n-6) to (n-3) FA, but with increasing concentrations of (n-6) and (n-3) FA. Plasma FA concentrations were measured after completing the feeding studies. In studies 1 and 2, dogs fed fish oil-enriched food with a high (n-3) FA concentration had higher plasma total (n-3) FA, eicosapentaenoic acid (EPA), and docosahexaenoic acid (DHA) concentrations and lower plasma total (n-6) FA, linoleic acid, and arachidonic acid concentrations than dogs fed corn oil-enriched food with a low (n-3) FA concentration (P < 0.001). Both inclusion of fish oil (P < 0.001) and increased food intake independent of treatment effects increased the plasma DHA (P = 0.05) concentration. Furthermore, constancy of the dose of (n-3) FA administered over long periods of time was necessary to maintain plasma levels of total (n-3) FA, EPA, and DHA. In study 3, up to certain dietary concentrations (6.3 g total (n-3) FA/kg food for DHA and 9.8 g total (n-3) FA/kg food for EPA), the dose of (n-3) FA administered, independent of the (n-6) to (n-3) FA ratio, determined the plasma (n-3) FA composition. Results from our studies indicate that approximately 175 mg DHA/(kg body weight . d) is required to attain maximum plasma levels of DHA.

Production of modified C-reactive protein in U937-derived macrophages.

Plasma C-reactive protein (CRP) has been proposed to be a strong independent predictor for cardiovascular disease. This circulating form of CRP (native CRP or nCRP) is well described. Recently, the existence of a conformationally distinct isoform of CRP (modified CRP or mCRP) has been reported. The relevance of each CRP isoform to atherosclerotic disease is unknown. The purpose of this study was to examine the natural expression of CRP in undifferentiated, differentiated, and stimulated macrophages, cells known to contribute to atherogenesis in vivo, and to determine whether transcribed CRP was expressed as nCRP or mCRP. Macrophages were generated from U937 monocytes using phorbol 12-myristate 13-acetate. Differentiated macrophages were further stimulated with lipopolysaccharides (LPS). In undifferentiated, differentiated, and stimulated cells, CRP expression was assessed by reverse transcription-polymerase chain reaction, and CRP protein production was measured by fluorescence microscopy and flow cytometry (cellular CRP) or high-sensitivity enzyme-linked immunosorbent assay (secreted CRP). CRP transcript was minimally expressed in undifferentiated cells. Expression increased markedly in macrophages during differentiation and was not affected by LPS at 24 hrs. Cellular CRP protein increased in a time-dependent manner after LPS stimulation, and this induction was mediated via interleukin (IL)-6 and IL-1beta. A small amount of secreted CRP was detected in the media of differentiated cells, but it was not significantly increased after LPS stimulation. Using specific monoclonal antibodies, our data indicate that cellular CRP is directly translated as the mCRP rather than the nCRP isomer. These results indicate that U937-derived macrophages are a good cell model to further study the production of mCRP under conditions relevant for the atherogenic process.

Reduced effect on apoptosis of 4-hydroxyhexenal and oxidized LDL enriched with n-3 fatty acids from postmenopausal women.

BACKGROUND: Oxidized low-density lipoprotein (oxLDL) promotes apoptosis in atherosclerotic plaques in the vascular wall, a process mediated through its oxidized lipids. 4-Hydroxynonenal (HNE) and 4-hydroxyhexenal (HHE), derived from oxidation of n-6 and n-3 fatty acids, respectively, are among the major oxidized products in oxLDL. HYPOTHESIS: This study hypothesized that eicosapentaenoic acid/docosahexaenoic acid (EPA/DHA)-rich versus linoleic acid-rich oxLDL obtained from postmenopausal women and HNE versus HHE differentially influence apoptosis in U937 cells. EXPERIMENTAL DESIGN: Thirty healthy postmenopausal women were supplemented with 14 g/day safflower oil (SO), 7 g/day of both fish oil and SO (low dose LFO) or 14 g/day fish oil (high dose HFO) for 5 weeks. Low-density lipoprotein, obtained after supplementation, was oxidized with 5 microM CuSO(4) at 37 degrees C for 6 h. The concentration of cholesteryl ester hydroperoxides (CEOOH) and conjugated dienes was measured in the oxidized LDL (oxLDL). U937 cells were incubated with the oxLDL, 10 microM of HHE, 7 muM of HHE plus 3 microM of HNE, 5 microM of both HHE and HNE or 10 microM of HNE and the extent of apoptosis measured three ways. RESULTS: The concentration of CEOOH and conjugated dienes in oxLDL did not differ among the three treatment groups. The percent of apoptotic cells was approximately 40% lower when incubated with oxLDL obtained from the HFO-supplemented group than the SO-supplemented group measured by both the Annexin V and the DNA fragmentation assays (P = .04 and .004, respectively). Apoptosis of U937 cells was significantly lower in cells incubated with 10 microM of HHE, and mixtures of HHE and HNE than the 10 microM HNE when measured by the Annexin V, DNA fragmentation and 4,6-diamidino-2-phenylindole (DAPI) staining. CONCLUSIONS: These data suggest that the cardioprotective properties of n-3 fatty acids may derive in part from their less reactive oxidized lipid metabolites.

Dietary (n-3) fatty acids from menhaden fish oil alter plasma fatty acids and leukotriene B synthesis in healthy horses.

The study objective was to determine the effect of feeding corn oil or fish oil to horses on plasma fatty acid profiles and leukotriene B (LTB) synthesis by stimulated peripheral blood neutrophils. Two groups of horses (n = 5) were randomly assigned to diets supplemented with either 3.0% (by weight) corn oil or fish oil for a period of 14 weeks. The ratio of (n-6) to (n-3) fatty acids in oil supplements was 68.1:1 for corn oil and 0.12:1 for fish oil. Production of LTB4 and LTB, by peripheral blood neutrophils stimulated with calcium ionophore A23187 and plasma cholesterol, triacylglycerol, and alpha-tocopherol concentrations were measured. At 12 weeks, horses fed fish oil had increased plasma concentrations of eicosapentaenoic acid (27-fold; 8.5 versus 0.3 g/100 g fatty acids; P < .0001), docosahexaenoic acid (34-fold; 5.1 versus 0.1 g/100 g fatty acids; P < .0001), and arachidonic acid (8.3-fold; 4.1 versus 0.5 g/100 g fatty acids; P < .0001) compared with horses fed corn oil. Neutrophils from horses fed fish oil produced 78-fold (P = .01) more LTB5 and 9.5-fold (P = .003) more LTB4 compared with predietary levels, and 17.6-fold (P = .01) and 3.3-fold (P = .02), respectively, more than horses fed corn oil, and the ratio of LTB5 to LTB4 concentrations was 4.0-fold (P = .002) higher in horses fed fish oil. This study suggests that dietary polyunsaturated fatty acids modulate the leukotriene inflammatory response of horses. If the ratio of LTB5 to LTB4 concentrations is important in determining how inflammatory processes are mediated, then fish oil supplementation may have value in treatment of equine inflammatory diseases.

Effect of type of dietary polyunsaturated fatty acid supplement (corn oil or fish oil) on immune responses in healthy horses.

The objective of this study was to compare effects of dietary polyunsaturated fatty acid supplementation (corn oil or fish oil) on selected immune responses in normal horses. Two groups of horses (n = 5) were randomly assigned a dietary supplement with either 3.0% corn oil or fish oil for a period of 14 weeks. Plasma fatty acid profiles were monitored to ensure uptake of dietary fatty acids. Cell-mediated immunity was assessed by a delayed-type hypersensitivity (DTH) skin test to keyhole limpet hemocyanin (KLH), and humoral immunity was assessed by measuring antibody titers to KLH. Production of prostaglandin E2 (PGE2), expression of tumor necrosis factor-alpha (TNF-alpha), and phagocytosis of latex beads by bronchoalveolar lavage fluid (BALF) cells were also assessed. Lipopolysaccharide (LPS)-stimulated BALF cells from horses fed corn oil showed a higher production of PGE2 compared with those from horses fed fish oil at 6 and 12 weeks. Production of TNF-alpha by LPS-stimulated BALF cells was higher in both groups of horses at 6, 8, and 12 weeks compared with pretrial values, and phagocytic activity of BALF cells was higher at 8 and 12 weeks, however, there were no differences between the 2 groups of horses. The DTH skin test and antibody titers to KLH revealed no differences between horses fed corn or fish oil. Based on these studies, dietary polyunsaturated fatty acids modulate the inflammatory response of horses. Both fatty acid supplements increased production of the proinflammatory cytokine TNF-alpha, whereas only corn oil increased production of the proinflammatory eicosanoid PGE2 by LPS-stimulated BALF cells. It is possible that fish oil, because it did not increase production of PGE2, could have value in the treatment of equine recurrent airway obstruction or other equine inflammatory diseases.

Dietary fish oil decreases C-reactive protein, interleukin-6, and triacylglycerol to HDL-cholesterol ratio in postmenopausal women on HRT.

BACKGROUND: Atherogenesis is a complex process involving both a low-grade inflammation and a disturbed lipid profile. Although dietary fish and fish oil improve the latter of these two risk factors, their impact on the former is less clear. OBJECTIVE: This study addressed the effect of supplementation with fish oil in doses achievable with diet on serum C-reactive protein (CRP), interleukin-6 (IL-6), and the lipid profile. METHODS AND RESULTS: Thirty healthy subjects taking HRT were randomly divided into three groups and supplemented for five weeks with 14 g/day safflower oil (SO), 7 g/day of both safflower oil and fish oil (LFO), or 14 g/day fish oil (HFO). Measurements included serum high-sensitivity CRP, IL-6 in plasma and in cell culture supernatant collected from 24-hr lipopolysaccharide (LPS)-stimulated whole blood, and lipid profile markers. CRP and IL-6 were adjusted for body mass index (BMI). Fish oil supplementation significantly decreased CRP and IL-6 compared to SO, with a greater effect in the LFO than HFO groups. Plasma triacylglycerol (TG) and the TG/HDL-C ratio were significantly lower in the HFO compared to the SO group. CONCLUSIONS: These results suggest that dietary fish oil may decrease the risk for cardiovascular disease through the modulation of both plasma lipids and inflammatory markers in healthy postmenopausal women.

Biologic and quality-of-life outcomes from the Mediterranean Lifestyle Program: a randomized clinical trial.

OBJECTIVE: Few multiple lifestyle behavior change programs have been designed to reduce the risk of coronary heart disease in postmenopausal women with type 2 diabetes. This study tested the effectiveness of the Mediterranean Lifestyle Program (MLP), a comprehensive lifestyle self-management program (Mediterranean low-saturated fat diet, stress management training, exercise, group support, and smoking cessation), in reducing cardiovascular risk factors in postmenopausal women with type 2 diabetes. RESEARCH DESIGN AND METHODS: Postmenopausal women with type 2 diabetes (n = 279) were randomized to either usual care (control) or treatment (MLP) conditions. MLP participants took part in an initial 3-day retreat, followed by 6 months of weekly meetings, to learn and practice program components. Biological end points were changes in HbA(1c), lipid profiles, BMI, blood pressure, plasma fatty acids, and flexibility. Impact on quality of life was assessed. RESULTS: Multivariate ANCOVAs revealed significantly greater improvements in the MLP condition compared with the usual care group on HbA(1c), BMI, plasma fatty acids, and quality of life at the 6-month follow-up. Patterns favoring intervention were seen in lipids, blood pressure, and flexibility but did not reach statistical significance. CONCLUSIONS: These results demonstrate that postmenopausal women with type 2 diabetes can make comprehensive lifestyle changes that may lead to clinically significant improvements in glycemic control, some coronary heart disease risk factors, and quality of life.

Effects of dietary n-6 and n-3 fatty acids and vitamin E on the immune response of healthy geriatric dogs.

OBJECTIVE: To determine the effect of dietary n-6 to n-3 fatty acid ratios and alpha-tocopheryl acetate concentration on immune functions andT cell subpopulations in healthy dogs. ANIMALS: Thirty-two 7- to 10-year old female Beagles. PROCEDURE: For 17 weeks, dogs were fed food that contained low (1.4:1) or high (40:1) ratios of n-6 to n-3 fatty acids in combination with 3 concentrations of all rac-alpha-tocopheryl acetate (low, 17 mg/kg of food; medium, 101 mg/kg; high, 447 mg/kg). Dogs were inoculated twice with a keyhole limpet hemocyanin suspension at 13 and 15 weeks. RESULTS: After 12 weeks, dogs consuming low concentrations of alpha-tocopheryl acetate had lower percentages of CD8+ T cells, compared with dogs consuming medium or high alpha-tocopheryl acetate concentrations. Also, dogs consuming low alpha-tocopheryl acetate concentrations had higher CD4+ to CD8+ T cell ratios. On day 4 of week 15, the percentage of CD8+ T cells was highest in dogs fed medium concentrations of alpha-tocopheryl acetate, compared with other dogs; however, the CD4+ to CD8+ T cell ratio was higher only in dogs fed low concentrations of alpha-tocopheryl acetate with high concentrations of n-3 fatty acids. Dogs consuming low concentrations of n-3 fatty acids with medium concentrations of alpha-tocopheryl acetate had the largest delayed-type hypersensitivity (DTH) skin test response. CONCLUSIONS AND CLINICAL RELEVANCE: An optimum amount of dietary alpha-tocopheryl acetate concentration, regardless of the dietary n-6 to n-3 fatty acid ratio, stimulates the CD8+ T cell population. Effects of an optimum amount of dietary alpha-tocopheryl acetate concentration on the DTH response are blunted by dietary n-3 fatty acids.

Physical activity, body fat, and serum C-reactive protein in postmenopausal women with and without hormone replacement.

The objective was to determine whether higher physical activity is associated with lower serum C-reactive protein (CRP), independent of oral hormone replacement therapy (HRT) status and body fatness, in 133 postmenopausal women using a cross-sectional exploratory design at a university research laboratory. The subjects were 133 postmenopausal women, age 50-73 years, with no evidence of coronary artery disease or diabetes. The main outcome measures were: serum CRP, physical activity as measured by Stanford 7-day activity recall, body fat (both total and regional) as measured by dual energy X-ray absorptiometry (DXA), and anthropometry (waist and hip circumference). Secondary outcome measures included fasting plasma glucose and insulin as well as fasting serum triglycerides, total cholesterol, high-density lipoprotein cholesterol, and low-density lipoprotein cholesterol. Higher physical activity energy expenditures were significantly associated with lower serum CRP levels (r = -0.18, P = 0.041), independent of oral HRT use, age, smoking behavior, alcohol consumption, aspirin use, and statin use. However, in the complete multivariate model, which included body fat, older ages (P = 0.047), greater trunk fat masses (P < 0.001), any oral HRT use (P < 0.001), and unopposed oral estrogen use (P = 0.012) were the sole independent predictors of higher serum CRP levels. The complete multivariate model accounted for 58% of the variance in serum CRP. We conclude that the association between higher physical activity and lower serum CRP levels is dependent on the lower body fat of the more active women, yet independent of oral HRT use. Future intervention trials should determine whether diet- and exercise-related reductions in body fat may be effective ways to diminish the proinflammatory effects of oral HRT in postmenopausal women.

Enrichment of LDL with EPA and DHA decreased oxidized LDL-induced apoptosis in U937 cells.

Oxidized LDL (oxLDL) may contribute to the accumulation of apoptotic cells in atherosclerotic plaques. Although it is well established in monophasic chemical systems that the highly unsaturated EPA and DHA will oxidize more readily than FA that contain fewer double bonds, our previous studies showed that enrichment of LDL, which has discrete polar and nonpolar phases, with these FA did not increase oxidation. The objective of this study was to compare the extent of apoptosis induced by EPA/DHA-rich oxLDL to that induced by EPA/DHA-non-rich oxLDL in U937 cells. LDL was obtained from one healthy subject three times before and after supplementation for 5 wk with 15 g/d of fish oil (FO), an amount easily obtainable from a diet that contains fatty fish. After supplementation, an EPA/DHA-rich LDL was obtained. Oxidative susceptibility of LDL, as determined by measuring the formation of conjugated dienes and the accumulation of cholesteryl ester hydroperoxides, was not higher in EPA/DHA-rich LDL. The oxLDL-induced cell apoptosis was detected by the activation of caspase-3, the translocation of PS to the outer surface of the plasma membrane using the Annexin V-fluorescein isothiocyanate binding assay, and the presence of chromatin condensation and nuclear fragmentation using the 4,6-diamidino-2-phenylindole staining assay. All three measures showed that after FO supplementation, EPA/DHA-rich oxLDL-induced cell apoptosis decreased. The decrease was not related to the concentration of lipid hydroperoxides. This study suggests that a possible protective effect of EPA/DHA-rich diets on atherosclerosis may be through lessening cell apoptosis in the arterial wall.

Influence of dietary long-chain n-3 fatty acids from menhaden fish oil on plasma concentrations of alpha-tocopherol in geriatric beagles.

OBJECTIVE: To determine effects of dietary n-3 fatty acids from Menhaden fish oil on plasma alpha-tocopherol concentrations in Beagles. ANIMALS: 32 female Beagles. PROCEDURE: For 82 days, dogs were fed diets that contained 1 of 2 ratios of n-6:n-3 fatty acids (40:1 [low n-3] and 1.4:1 [high n-3]) and 1 of 3 concentrations of all-rac-alpha-tocopheryl acetate (low, 17 mg/kg of diet; medium, 101 mg/kg; and high, 447 mg/kg) in a 2 X 3 factorial study. RESULTS: Diets high in n-3 fatty acids significantly increased total content of n-3 fatty acids in plasma (17.0 g/100 g of fatty acids), compared with low n-3 diets (2.02 g/100 g of fatty acids). Mean +/- SEM plasma concentration of cholesterol was significantly lower in dogs consuming high n-3 diets (4.59 +/- 0.48 mmol/L), compared with dogs consuming low n-3 diets (5.71 +/- 0.48 mmol/L). A significant interaction existed between the ratio for n-6 and n-3 fatty acids and amount of alpha-tocopheryl acetate in the diet (plasma alpha-tocopherol concentration expressed on a molar basis), because the plasma concentration of alpha-toco-pherol was higher in dogs consuming low n-3 diets, compared with those consuming high n-3 diets, at the 2 higher amounts of dietary alpha-tocopheryl acetate. Plasma alpha-tocopherol concentration expressed relative to total lipid content did not reveal effects of dietary n-3 fatty acids on concentration of alpha-tocopherol. CONCLUSIONS AND CLINICAL RELEVANCE: Plasma alpha-tocopherol concentration is not dependent on dietary ratio of n-6 and n-3 fatty acids when alpha-tocopherol concentration is expressed relative to the total lipid content of plasma.