Bruce E. Mackey

Docosahexaenoic acid ingestion inhibits natural killer cell activity and production of inflammatory mediators in young healthy men

The purpose of this study was to examine the effects of feeding docosahexaenoic acid (DHA) as triacylglycerol on the fatty acid composition, eicosanoid production, and select activities of human peripheral blood mononuclear cells (PBMNC). A 120-d study with 11 healthy men was conducted at the Metabolic Research Unit of Western Human Nutrition Reach Center. Four subjects (control group) were fed the stabilization diet throughout the study; the remaining seven subjects were fed the basal diet for the first 30 d, followed by 6 g DHA/d for the next 90 d. DHA replaced an equivalent amount of linoleic acid; the two diets were comparable in their total fat and all other nutrients. Both diets were supplemented with 20 mg d α-tocopherol acetate per day. PBMNC fatty acid composition and eicosanoid production were examined on day 30 and 113; immune cell functions were tested on day 22, 30, 78, 85, 106, and 113. DHA feeding increased its concentration from 2.3 to 7.4 wt% in the PBMNC total lipids, and decreased arachidonic acid concentration from 19.8 to 10.7 wt%. It also lowered prostaglandin E2 (PGE2) and leukotriene B4 (LTB4) production, in response to lipopolysaccharide, by 60–75%. Natural killer cell activity and in vitro secretion of interleukin-1β and tumor necrosis factor α were significantly reduced by DHA feeding. These parameters remained unchanged in the subjects fed the control diet. B-cell functions as reported here and T-cell functions that we reported previously were not altered by DHA feeding. Our results show that inhibitory effects of DHA on immune cell functions varied with the cell type, and that the inhibitory effects are not mediated through increased production of PGE2 and LTB4.

Arachidonic acid supplementation enhances synthesis of eicosanoids without suppressing immune functions in young healthy men

This study was conducted to determine the effects of arachidonic acid (AA) supplementation on human immune response (IR) and on the secretion of prostaglandin E2 (PGE2) and leukotriene B4 (LTB4). Ten healthy men (20–38 yr) participated in the study and lived at the Metabolic Suite of the Western Human Nutrition Research Center. They were fed a basal diet (57, 27, and 16 energy percentage from carbohydrate, fat, and protein, respectively, and AA 200 mg/d) for the first 15 d of the study. Additional AA (1.5 g/d) was added to the diet of six men from day 16 to 65, while the remaining four subjects remained on the basal diet. The diets of the two groups were crossed-over from day 66 to 115. In vitro indices of IR were examined using blood drawn on days 15, 58, 65, 108, and 115. Influenza antibody titers were determined in the sera prepared from blood drawn on days 92 and 115 (23 d postimmunization). AA supplementation caused significant increases in the in vitro secretion of LTB4, and PGE2, but it did not alter the in vitro secretion of tumor necrosis factor α; interleukins 1β, 2, 6; and the receptor for interleukin 2. Nor did it change the number of circulting lymphocytes bearing markers for specific subsets (B, T, helper, suppressor, natural killer) and the serum antibody titers against influenza vaccine. The opposing effects of PGE2 and LTB4 may have led to the lack of change in immune functions tested.

Dietary α-linolenic acid alters tissue fatty acid composition, but not blood lipids, lipoproteins or coagulation status in humans

We examined the effect of dietary α-linolenic acid (ALA) on the indices of lipid and coagulation status and on the fatty acid composition of serum and peripheral blood mononuclear cell (PBMNC) lipids in ten healthy men (age 21–37 yr) who consumed all their meals at the Western Human Nutrition Research Center for 126 d. There was a stabilization period of 14 d at the start when all 10 subjects consumed the basal diet (BD) containing 23.4 energy percent (en%) fat and two intervention periods of 56 d each. During the first intervention period, 5 subjects consumed the BD containing 23.4 en% fat, and 5 subjects consumed a diet providing 6.3% calories from α-linolenic acid [flaxseed oil (FSO) diet containing 28.8 en% fat]. Diets were crossed over between the two groups during the second intervention period. Feeding the FSO diet did not nignificantly alter serum triglycerides, cholesterol, highdensity lipoproteins, low-density lipoproteins, apoprotein A-I and apoprotein B when compared to the corresponding values in the subjects fed the BD, nor was there any effect of the FSO diet on the bleeding time, prothrombin time and partial prothrombin time for these subjects. Feeding the ALA-containing diet did cause a significant increase in ALA concentration in serum (P<0.001) and PBMNC lipids (P<0.05). It also caused a significant increase (P<0.05) in the eicosapentaenoic and docosapentaenoic acid contents of PBMNC lipids, and a decrease (P<0.01) in linoleic and eicosatrienoic acid contents of serum lipids. Thus, dietary ALA, fed for 56 d at 6.3% of calories, had no effect on plasma triglyceride or very low density lipoprotein levels or the common risk factors associated with atherosclerosis, although these parameters have been reported by others to be influenced by fatty acids, such as palmitic or linoleic acids, in the diet. Dietary ALA did significantly alter the fatty acid composition of plasma and PBMNC.

DHA Supplementation Decreases Serum C-Reactive Protein and Other Markers of Inflammation in Hypertriglyceridemic Men

Dietary (n-3) PUFA reduce inflammation, an independent risk factor for cardiovascular disease. The antiinflammatory effects of docosahexaenoic acid (DHA) in hypertriglyceridemic men have not been previously reported, to our knowledge, and were the focus of this study. Hypertriglyceridemic men (n = 17 per group) aged 39-66 y, participated in a double-blind, randomized, placebo-controlled parallel study. They received no supplements for the first 8 d and then received either 7.5 g/d DHA oil (3 g DHA/d) or olive oil (placebo) for the last 90 d. Blood samples were collected from fasting men on study days -7, 0, 45, 84, and 91. DHA supplementation for 45 and 91 d decreased the number of circulating neutrophils by 11.7 and 10.5%, respectively (P < 0.05). It did not alter the circulating concentrations of other inflammatory markers tested within 45 d, but at 91 d it reduced (P < 0.05) concentrations of C-reactive protein (CRP) by 15%, interleukin-6 by 23%, and granulocyte monocyte-colony stimulating factor by 21% and DHA increased the concentration of antiinflammatory matrix metalloproteinase-2 by 7%. The number of circulating neutrophils was positively associated with the weight percent (wt %) of 20:4(n-6) in RBC lipids, and negatively to the wt % of 20:5(n-3) and 22:6(n-3). Concentrations of CRP and serum amyloid A were positively associated with the sum of SFA and negatively with the wt % of 18:1(n-9) and 17:0 in RBC lipids; CRP was also positively associated with the wt % of 20:2(n-6). The mean size of VLDL particles was positively associated with plasma concentrations of neutrophils and CRP. In conclusion, DHA may lessen the inflammatory response by altering blood lipids and their fatty acid composition.

Dietary Docosahexaenoic Acid and Immunocompetence in Young Healthy Men

The purpose of this study was to examine the effect of dietary docosahexaenoic acid (DHA), in the absence of eicosapentaenoic acid, on human immune response (IR). A 120-d study with 11 healthy men was conducted at the Metabolic Research Unit of the Western Human Nutrition Research Center. Four subjects (control group) were fed the stabilization or basal diet (15, 30, and 55% energy from protein, fat, and carbohydrate, respectively) throughout the study; the remaining seven subjects (DHA group) were fed the basal diet for the first 30 d, followed by 6 g DHA/d for the next 90 d. DHA replaced an equivalent amount of linoleic acid; the two diets were comparable in their total fat and all other nutrients. Both diets were supplemented with 20 mg d-α-tocopherol acetate per day. Indices of IR were examined on study day 22, 30, 78, 85, 106, and 113. Addition of DHA at moderately high levels did not alter the proliferation of peripheral blood mononuclear cells cultured with phytohemag-glutinin or concanavalin A, or the delayed hypersensitivity skin response. Also, additional DHA did not alter the number of T cells producing interleukin 2 (IL2), the ratio between the helper/suppressor T cells in circulation, or the serum concentrations of immunoglobulin G, C3, and interleukin 2 receptor (IL2R). DHA supplementation, however, caused a significant (P=0.0001) decrease in the number of circulating white blood cells which was mainly due to a decrease in the number of circulating granulocytes. The number of lymphocytes in peripheral circulation was not affected by Dietary DHA enrichment, but the percentage of lymphocytes in white blood cells increased because of a reduction in granulocyte numbers. None of these indices was changed in the control group. Our results show that when total fat intake is low and held constant, DHA consumption does not inhibit many of the lymphocyte functions which have been reported to be inhibited by fish oil consumption.

Dietary conjugated linoleic acid did not alter immune status in young healthy women

The purpose of this study was to examine whether conjugated linoleic acid (CLA) supplementation in human diets would enhance indices of immune status as reported by others for animal models. Seventeen women, 20–41 yr, participated in a 93-d study conducted in two cohorts of 9 and 8 women at the Metabolic Research Unit of Western Human Nutrition Research Center. Seven subjects were fed the basal diet (19, 30 and 51% energy from protein, fat, and carbohydrate, respectively) throughout the study. The remaining 10 subjects were fed the basal diet for the first 30 d, followed by 3.9 g CLA (Tonalin)/d for the next 63 d. CLA made up 65% of the fatty acids in the Tonalin capsules, with the following isomeric composition: t10, c12, 22.6%; c11, t13, 23.6%; c9, t11, 17.6%; t8, c10, 16.6%; and other isomers 19.6%. Most indices of immune response were tested at weekly intervals, three times at the end of each period (stabilization/intervention); delayed-type hypersensitivity (DTH) to a panel of six recall antigens was tested on study day 30 and 90; all subjects were immunized on study day 65 with an influenza vaccine, and antibody titers were examined in the sera collected on day 65 and 92. None of the indices of immune status tested (number of circulating white blood cells, granulocytes, monocytes, lymphocytes, and their subsets, lymphocytes proliferation in response to phytohemagglutinin, and influenza vaccine, serum influenza antibody titers, and DTH response) were altered during the study in either dietary group. Thus, in contrast to the reports with animal models, CLA feeding to young healthy women did not alter any of the indices of immune status tested. These data suggest that short-term CLA supplementation in healthy volunteers is safe, but it does not have any added benefit to their immune status.

Effects of dietary arachidonic acid on human immune response

Arachidonic acid (AA) is a precursor of eicosanoids, which influence human health and the in vitro activity of immune cells. We therefore examined the effects of dietary AA on the immune response (IR) of 10 healthy men living at our metabolic suite for 130 d. All subjects were fed a basal diet containing 27 energy percentage (en%) fat, 57 en% carbohydrate, and 16 en% protein (AA, 200 mg/d) for the first and last 15 d of the study. Additional AA (1.5 g/d) was incorporated into the diet of six men from day 16 to 65 while the remaining four subjects continued to eat the basal diet. The diets of the two groups were crossed-over from day 66 to 115. In vitro indexes of IR were examined using the blood samples drawn on days 15, 58, 65, 108, 115, and 127. The subjects were immunized with the measles/mumps/rubella vaccine on day 35 and with the influenza vaccine on day 92. Dietary AA did not influence many indexes of IR (peripheral blood mononuclear cell proliferation in response to phytohemagglutinin, Concanavalin A, pokeweed, measles/mumps/rubella, and influenza vaccines prior to immunization, and natural killer cell activity). The postimmunization proliferation in response to influenza vaccine was about fourfold higher in the group receiving high-AA diet compared to the group receiving low-AA diet (P=0.02). Analysis of variance of the data pooled from both groups showed that the number of circulating granulocytes was significantly (P=0.03) more when the subjects were fed the high-AA diet than when they were fed the low-AA diet. The small increases in granulocyte count and the in vitro proliferation in response to influenza vaccine caused by dietary AA may not be of clinical significance. However, the lack of any adverse effects on IR indicates that supplementation with AA may be done safely when needed for other health reasons.

Trans-10,cis-12 CLA Increases Liver and Decreases Adipose Tissue Lipids in Mice: Possible Roles of Specific Lipid Metabolism Genes

Although consumption of CLA mixtures has been associated with several health effects, less is known about the actions of specific CLA isomers. There is evidence that the t10,c12-CLA isomer is associated with alterations in body and organ weights in animals fed CLA, but the mechanisms leading to these changes are unclear. The purpose of this study was to determine the effects of two commonly occurring isomers of CLA on body composition and the transcription of genes associated with lipid metabolism. Eight-week-old female mice (n=11 or 12/group) were fed either a control diet or diets supplemented with 0.5% c9,t11-CLA or t10,c12-CLA isomers or 0.2% of the peroxisome proliferator-activated receptor α (PPARα) agonist fenofibrate for 8 wk. Body and retroperitoneal adipose tissue weights were significantly lower (6–10 and 50%, respectively), and liver weights were significantly greater (100%) in the t10,c12-CLA and the fenofibrate groups compared with those in the control group; body and tissue weights in the c9,t11-CLA group did not differ from those in the control group. Livers from animals in the t10,c12-CLA group contained five times more lipids than in the control group, whereas the lipid content of the fenofibrate group did not differ from that in the control group. Although fenofibrate increased the mRNA for PPARα, t10,c12-CLA decreased it. These results suggest that PPARα did not mediate the effects of t10,c12-CLA on body composition. The CLA isomers and fenofibrate altered mRNA levels for several proteins involved in lipid metabolism, but the most striking difference was the reduction of mRNA for leptin and adiponectin in the t10,c12-CLA group. These initial results suggest that changes associated with energy homeostasis and insulin action may mediate the effects of t10,c12-CLA on lipid metabolism.

Dietary supplementation with conjugated linoleic acid increased its concentration in human peripheral blood mononuclear cells, but did not alter their function

The purpose of this study was to examine if conjugated linoleic acid (CLA) supplementation of diets would alter fatty acid (FA) composition and function of peripheral blood mononuclear cells (PBMC). Seventeen women, 20–41 yr, participated in a 93-d study conducted at the Metabolic Research Unit. The same diet (19, 30, and 51% energy from protein, fat, and carbohydrate, respectively) was fed to all subjects throughout the study. Seven subjects (control group) supplemented their diet with six daily capsules (1 g each) of placebo oil (sunflower) for 93 d. For the other 10 subjects (CLA group), the supplement was changed to an equivalent amount of Tonalin capsules for the last 63 d of the study. Tonalin provided 3.9 g/d of a mixture of CLA isomers (trans-10,cis-12, 22.6%; cis-11,trans-13, 23.6%; cis-9,trans-11, 17.6%; trans-8,cis-10, 16.6%; other isomers 19.6%), and 2.1 g/d of other FA. PBMC isolated on study days 30 and 90 were used to assess intracellular cytokines by flow cytometry, secreted cytokines, and eicosanoid by enzyme-linked immonosorbent assay, and FA composition by gas-liquid chromatography. After supplementation, total CLA concentration increased from 0.012 to 0.97% (P<0.0001) in PBMC lipids, but it did not significantly alter the concentration of other FA. CLA supplementation did not alter the in vitro secretion of prostaglandin E2, leukotriene B4, interleukin-1β (IL-1β), or tumor necrosis factor α (TNFα) by PBMC simulated with lipopolysaccharide, and the secretion of IL-2 by PBMC stimulated with phytohemagglutinin. Nor did it alter the percentage T cells producing IL-2, interferon γ, and percentage of monocytes producing TNFα. The intracellular concentration of these cytokines was also not altered. None of the variables tested changed in the control group. Our results show that CLA supplementation increased its concentration in PBMC lipids, but did not alte their functions.

Contrasting Effects of t10,c12- and c9,t11-Conjugated Linoleic Acid Isomers on the Fatty Acid Profiles of Mouse Liver Lipids

The purpose of this study was to examine the effects of two purified isomers of CLA (c9,t11-CLA and t10,c12-CLA) on the weights and FA compositions of hepatic TG, phospholipids, cholesterol esters, and FFA. Eight-week-old female mice (n=6/group) were fed either a control diet or diets supplemented with 0.5% c9,t11-CLA or t10, c12-CLA isomers for 8 wk. Weights of liver total lipids and those of individual lipid fractions. did not differ between the control and the c9,t11-CLA groups. Livers from animals fed the t10,c12-CLA diet contained four times more lipids than those of the control group; this was mainly due to an increase in the TG fractions (fivefold), but cholesterol (threefold), cholesterol esters (threefold), and FFA (twofold) were also significantly increased. Although c9,t11-CLA did not significantly alter the weights of liver lipids when compared with the control group, its intake was associated with significant reductions in the weight percentage (wt% of total FAME) of 18∶1n−9 and 18∶1n−7 in the TG fraction and with significant increases in the weight percentage of 18∶2n−6 in the TG, cholesterol ester, and phospholipid fractions. on the other hand, t10,c12-CLA intake was linked with a significant increase in the weight percentage of 18∶1n−9 and a decrease in that of 18∶2n−6 in all lipid fractions. These changes may be the result of alterations in the activity of Δ9-desaturase (stearoyl CoA desaturase) and the enzymes involved in the metabolism of 18∶2n−6. Thus, the two isomers differed not only in their effects on the weights of total liver lipids and lipid fractions but also on the FA profile of the lipid fractions.