Physicochemical-guided design of cathelicidin-derived peptides generates membrane active variants with therapeutic potential.

The spread of multi-drug resistance and the slow pace at which antibiotics come onto the market are undermining our ability to treat human infections, leading to high mortality rates. Aiming to overcome this global crisis, antimicrobial peptides are considered promising alternatives to counter bacterial infections with multi-drug resistant bacteria. The cathelicidins comprise a well-studied class of AMPs whose members have been used as model molecules for sequence modifications, aiming at enhanced biological activities and stability, along with reduced toxic effects on mammalian cells. Here, we describe the antimicrobial activities, modes of action and structural characterization of two novel cathelicidin-like peptides, named BotrAMP14 and CrotAMP14, which were re-designed from snake batroxicidin and crotalicidin, respectively. BotrAMP14 and CrotAMP14 showed broad-spectrum antibacterial activity against susceptible microorganisms and clinical isolates with minimal inhibitory concentrations ranging from 2-35.1 µM. Moreover, both peptides had low cytotoxicity against Caco-2 cells in vitro. In addition, in vivo toxicity against Galleria mellonella moth larvae revealed that both peptides led to>76% larval survival after 144 h. Microscopy studies suggest that BotrAMP14 and CrotAMP14 destabilize E. coli membranes. Furthermore, circular dichroism and molecular dynamics simulations indicate that, in a membrane-like environment, both peptides adopt α-helical structures that interact with bilayer phospholipids through hydrogen bonds and electrostatic interaction. Thus, we concluded that BotrAMP14 and CrotAMP14 are helical membrane active peptides, with similar antibacterial properties but lower cytotoxicity than the larger parent peptides batroxicidin and crotalicidin, having advantages for drug development strategies.

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-1beta (IL-1beta), or tumor necrosis factor alpha (TNFalpha) 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 gamma, and percentage of monocytes producing TNFalpha. 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 alter their functions.

The effect of conjugated linoleic acid on platelet function, platelet fatty acid composition, and blood coagulation in humans.

Despite extensive research on conjugated linoleic acid (CLA) showing multiple beneficial effects in animal models, little is known about the role of dietary CLA in human health. To investigate if the beneficial effects of CLA seen in animal models are relevant to humans, we conducted a study with 17 healthy female volunteers who lived in the Metabolic Research Unit of the Western Human Nutrition Research Center for 93 d. This paper reports only the results from this study that are related to the effects of CLA supplementation on blood coagulation, platelet function, and platelet fatty acid composition. Throughout the study, the subjects were fed a low-fat diet (30 en% fat, 19 en% protein, and 51 en% carbohydrate) consisting of natural foods with the recommended dietary allowances for all known nutrients. After a 30-d stabilization period, subjects were randomly assigned to either an intervention group (n = 10) whose diet was supplemented with 3.9 g/d of CLA or a control group (n = 7) who received an equivalent amount of sunflower oil consisting of 72.6% linoleic acid with no detectable CLA. Platelet aggregation was measured in platelet-rich plasma using adenosine diphosphate, collagen, and arachidonic acid agonists. No statistical difference was detected between the amount of agonist required to produce 50% aggregation of platelet-rich plasma before and after the subjects consumed the CLA, with the exception of a decrease in response to collagen. This decrease was found in both control and intervention groups with no significant difference between the groups, suggesting that both linoleic acid (sunflower oil) and CLA might have similar effects on platelet function. The prothrombin time, activated partial thromboplastin time, and the antithrombin III levels in the subjects were determined. Again, there was no statistically significant difference in these three parameters when pre- and post-CLA consumption values were compared. The in vivo bleeding times were also unaffected by CLA supplementation (10.4 + 2.8 min pre- and 10.2 + 1.6 min postconsumption). Platelet fatty acid composition was not markedly influenced by the consumption of dietary CLA, although there was a small increase in the amount of the 9 cis,11 trans-18:2 isomer normally present in platelets after feeding CLA for 63 days. In addition, small amounts of the 8 trans,10 cis-18:2 and the 10 trans,12 cis-18:2 isomers were detected in the platelets along with traces of some of the other isomers. Thus, when compared to sunflower oil, the blood-clotting parameters and in vitro platelet aggregation showed that adding 3.9 g/d of dietary CLA to a typical Western diet for 63 d produces no observable physiological change in blood coagulation and platelet function in healthy adult females. Short-term consumption of CLA does not seem to exhibit antithrombotic properties in humans.

The effect of conjugated linoleic acid on plasma lipoproteins and tissue fatty acid composition in humans.

Conjugated linoleic acid (CLA) has been suggested by some animal studies to possess antiatherogenic properties. To determine, in humans, the effect of dietary CLA on blood lipids, lipoproteins, and tissue fatty acid composition, we conducted a 93-d study with 17 healthy female volunteers at the Metabolic Research Unit of the Western Human Nutrition Research Center. Throughout the study, subjects were fed a low-fat diet [30 energy percent (en%) fat, 19 en% protein, and 51 en% carbohydrate] that consisted of natural foods with the recommended dietary allowances for all known nutrients. After a 30-d stabilization period, subjects were randomly assigned to either an intervention group (n = 10) supplemented daily with capsules containing 3.9 g of CLA or a control group (n = 7) that received an equivalent amount of sunflower oil. The CLA capsules (CLA 65%) contained four major cis/trans geometric isomers (11.4% 9 cis-,11 trans-18:2; 10.8% 8 trans-,10 cis-18:2; 15.3% 11 cis-,13 trans-18:2; and 14.7% 10 trans-,12 cis-18:2) and their corresponding cis/cis (6.74% total) and trans/trans (5.99% total) varieties in smaller amounts. Fasting blood was drawn on study days 30 (end of the stabilization period), 60 (midpoint of the intervention period), and 93 (end of the intervention period). Adipose tissue samples were taken on days 30 and 93. CLA supplementation for 63 d did not change the levels of plasma cholesterol, low density lipoprotein cholesterol, high density lipoprotein cholesterol, and triglycerides. The weight percentage of CLA in plasma increased from 0.28 +/- 0.06 to 1.09 +/- 0.31 (n = 10, P < 0.05) after the supplementation. The 9 cis-,11 trans-isomer was the most prominent variety followed by the 11 cis-,13 trans- and 10 trans-,12 cis-isomers in lesser amounts. CLA in adipose tissue was not influenced by the supplementation (0.79 +/- 0.18 to 0.83 +/- 0.19 wt%) (n = 10) and the 9 cis-,11 trans-variety was the only isomer present. Thus, contrary to findings from some animal studies, CLA does not seem to offer health benefits, in the short term, regarding the prevention of atherosclerosis in humans. CLA supplementation for 2 mon did not alter the blood cholesterol or lipoprotein levels of healthy, normolipidemic subjects. The supplementation did increase CLA in the plasma but only 4.23% of the ingested CLA was present in the plasma at any given time. No adverse effect of CLA supplementation was detected in this study.

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.

Conjugated linoleic acid supplementation in humans: effects on body composition and energy expenditure.

Recent animal studies have demonstrated that dietary conjugated linoleic acid (CLA) reduces body fat and that this decrease may be due to a change in energy expenditure. The present study examined the effect of CLA supplementation on body composition and energy expenditure in healthy, adult women. Seventeen women were fed either a CLA capsule (3 g/d) or a sunflower oil placebo for 64 d following a baseline period of 30 d. The subjects were confined to a metabolic suite for the entire 94 d study where diet and activity were controlled and held constant. Change in fat-free mass, fat mass, and percentage body fat were unaffected by CLA supplementation (0.18+/-0.43 vs. 0.09+/-0.35 kg; 0.01+/-0.64 vs. -0.19+/-0.53 kg; 0.05+/-0.62 vs. -0.67+/-0.51%, placebo vs. CLA, respectively). Likewise, body weight was not significantly different in the placebo vs. the CLA group (0.48+/-0.55 vs. -0.24+/-0.46 kg change). Energy expenditure (kcal/min), fat oxidation, and respiratory exchange ratio were measured once during the baseline period and during weeks 4 and 8 of the intervention period. At all three times, measurements were taken while resting and walking. CLA had no significant effect on energy expenditure, fat oxidation, or respiratory exchange ratio at rest or during exercise. When dietary intake was controlled, 64 d of CLA supplementation at 3 g/d had no significant effect on body composition or energy expenditure in adult women, which contrasts with previous findings in animals.

Conjugated linoleic acid supplementation in humans: effects on circulating leptin concentrations and appetite.

Conjugated linoleic acid (CLA) has been demonstrated to reduce body fat in animals. However, the mechanism by which this reduction occurs is unknown. Leptin may mediate the effect of CLA to decrease body fat. We assessed the effects of 64 d of CLA supplementation (3 g/d) on circulating leptin, insulin, glucose, and lactate concentrations in healthy women. Appetite was assessed as a physiological correlate of changes in circulating leptin levels. Analysis of plasma leptin concentrations adjusted for adiposity by using fat mass as a covariate showed that CLA supplementation significantly decreased circulating leptin concentrations in the absence of any changes of fat mass. Mean leptin levels decreased over the first 7 wk and then returned to baseline levels over the last 2 wk of the study in the CLA-treated group. Appetite parameters measured at around the time when the greatest decreases in leptin levels were observed showed no significant differences between supplementation and baseline determinations in the CLA-supplemented group or between the CLA and placebo-supplemented groups. There was a nonsignificant trend for mean insulin levels to increase toward the end of the supplementation period in CLA-treated subjects. CLA did not affect plasma glucose and lactate over the treatment period. Thus, 64 d of CLA supplementation in women produced a transient decrease in leptin levels but did not alter appetite. CLA did not affect these parameters in a manner that promoted decreases of adiposity.

Effect of dietary docosahexaenoic acid on desaturation and uptake in vivo of isotope-labeled oleic, linoleic, and linolenic acids by male subjects.

The effect of dietary docosahexaenoic acid (22:6n-3, DHA) on the metabolism of oleic, linoleic, and linolenic acids was investigated in male subjects (n = 6) confined to a metabolic unit and fed diets containing 6.5 or <0.1 g/d of DHA for 90 d. At the end of the diet period, the subjects were fed a mixture of deuterated triglycerides containing 18:1n-9[d6], 18:2n-6[d2], and 18:3n-3[d4]. Blood samples were drawn at 0, 2, 4, 6, 8, 12, 24, 48, and 72 h. Methyl esters of plasma total lipids, triglycerides, phospholipids, and cholesterol esters were analyzed by gas chromatography-mass spectrometry. Chylomicron triglyceride results show that the deuterated fatty acids were equally well absorbed and diet did not influence absorption. Compared to the low-DHA diet (LO-DHA), clearance of the labeled fatty acids from chylomicron triglycerides was modestly higher for subjects fed the high DHA diet (HI-DHA). DHA supplementation significantly reduced the concentrations of most n-6[d2] and n-3[d4] long-chain fatty acid (LCFA) metabolites in plasma lipids. Accumulation of 20:5n-3[d4] and 22:6n-3[d4] was depressed by 76 and 88%, respectively. Accumulations of 20:3n-6[d2] and 20:4n-6[d2] were both decreased by 72%. No effect of diet was observed on acyltransferase selectivity or on uptake and clearance of 18:1n-9[d6], 18:2n-6[d2], and 18:3n-3[d4]. The results indicate that accumulation of n-3 LCFA metabolites synthesized from 18:3n-3 in typical U.S. diets would be reduced from about 120 to 30 mg/d by supplementation with 6.5 g/d of DHA. Accumulation of n-6 LCFA metabolites synthesized from 18:2n-6 in U.S. diets is estimated to be reduced from about 800 to 180 mg/d. This decrease is two to three times the amount of n-6 LCFA in a typical U.S. diet. These results support the hypothesis that health benefits associated with DHA supplementation are the combined result of reduced accretion of n-6 LCFA metabolites and an increase in n-3 LCFA levels in tissue lipids.

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 alpha-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-1beta and tumor necrosis factor alpha 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.

Dietary fat, trans fatty acids, and risk of coronary heart disease.

Results from human feeding studies and recent large-scale epidemiologic surveys suggest that dietary trans fatty acids enhance the risk of developing coronary heart diseases. Despite a lack of accurate data regarding dietary intake of trans fatty acids, existing epidemiologic data and evidence from experimental feeding studies support the idea that lowering current intakes of trans fatty acids may lower the risk of coronary heart disease.

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-alpha-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 phytohemagglutinin 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.

Effect of dietary arachidonic acid on metabolism of deuterated linoleic acid by adult male subjects.

The influence of dietary supplementation with 20:4n-6 on uptake and turnover of deuterium-labeled linoleic acid (18:2n-6[d2]) in human plasma lipids and the synthesis of desaturated and elongated n-6 fatty acids from 18:2n-6[d21 were investigated in six adult male subjects. The subjects were fed either a high-arachidonic acid (HIAA) diet containing 1.7 g/d or a low-AA (LOAA) diet containing 0.21 g/d of AA for 50 d. Each subject was then dosed with about 3.5 g of 18:2n-6[d2] as the triglyceride (TG) at 8:00 A.M., 12:00, and 5:00 P.M. The total 18:2n-6[d21] fed to each subject was about 10.4 g and is approximately equal to one-half of the daily intake of 18:2n-6 in a typical U.S. male diet. Nine blood samples were drawn over a 96-h period. Methyl esters of plasma total lipid (TL), TG, phospholipid, and cholesterol ester were analyzed by gas chromatography-mass spectroscopy. Dietary 20:4n-6 supplementation did not affect uptake of 18:2n-6[d2] in plasma lipid classes over the 4-d study period nor the estimated half-life of 24-36 h for 18:2n-6[d2]. The percentages of major deuterium-labeled desaturation and elongation products in plasma TL, as a percentage of total deuterated fatty acids, were 1.35 and 1.34% 18:3n-6[d2]; 0.53 and 0.50% 20:2n-6[d2]; 1.80 and 0.92% 20:3n-6[d2] and 3.13 and 1.51% 20:4n-6[d2] for the LOAA and HIAA diet groups, respectively. Trace amounts (<0.1%) of the 22:4n-6[d2] and 22:5n-6[d2] metabolites were present. Plasma TL concentration data for both 20:3n-6[d2] and 20:4n-6[d2] were 48% lower (P < 0.05) in samples from the HIAA diet group than in samples from the LOAA diet group. For a normal adult male consuming a typical U.S. diet, the estimated accumulation in plasma TL of 20:4n-6 synthesized from 20 g/d (68 mmole) of 18:2n-6 is 677 mg/d (2.13 mmole). Dietary supplementation with 1.5 g/d of 20:4n-6 reduced accumulation of 20:4n-6 synthesized from 20 g/d of 18:2n-6 to about 326 mg/d (1.03 mmole).

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 alpha; interleukins 1 beta, 2, 6; and the receptor for interleukin 2. Nor did it change the number of circulating 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.

The effect of dietary docosahexaenoic acid on platelet function, platelet fatty acid composition, and blood coagulation in humans.

The effect of dietary docosahexaenoic acid (DHA) in the absence of eicosapentaenoic acid (EPA) has been studied infrequently in humans under controlled conditions. This 120-d study followed healthy, adult male volunteers who lived in the metabolic research unit (MRU) of the Western Human Nutrition Research Center for the entire study. The basal (low-DHA) diet consisted of natural foods (30 en% fat, 15 en% protein, and 55 en% carbohydrate), containing < 50 mg/d of DHA, and met the recommended daily intake for all essential nutrients. The high-DHA (intervention) diet was similar except that 6 g/d of DHA in the form of a triglyceride containing 40% DHA replaced an equal amount of safflower oil in the basal diet. The subjects (ages 20 to 39) were within -10 to +20% of ideal body weight, nonsmoking, and not allowed alcohol in the MRU. Their exercise level was constant, and their body weights were maintained within 2% of entry level. They were initially fed the low-DHA diet for 30 d. On day 31, six subjects (intervention, group A) were placed on the high-DHA diet; the other four subjects (controls, group B) remained on the low-DHA diet. Platelet aggregation in platelet-rich plasma was determined using ADP, collagen, and arachidonic acid. No statistical differences could be detected between the amount of agonist required to produce 50% aggregation of platelet-rich plasma before and after the subjects consumed the high-DHA diet. The prothrombin time, activated partial thromboplastin time, and the antithrombin-III levels in the subjects were determined, and, again, there were no statistically significant differences in these three parameters when their values were compared before and after the subjects consumed the high-DHA diet. In addition, the in vivo bleeding times did not show any significant difference before and after the subjects consumed the high-DHA diet (9.4 +/- 3.1 min before and 8.0 +/- 3.4 min after). Platelets from the volunteers exhibited more than a threefold increase in their DHA content from 1.54 +/- 0.16 to 5.48 +/- 1.21 (wt%) during the DHA feeding period. The EPA content of the subjects' platelets increased from 0.34 +/- 0.12 to 2.67 +/- 0.91 (wt%) during the high-DHA diet despite the absence of EPA in the subjects' diets. The results from this study on blood clotting parameters and in vitro platelet aggregation suggest that adding 6 g/d of dietary DHA for 90 d to a typical Western diet containing less than 50 mg/d of DHA produces no observable physiological changes in blood coagulation, platelet function, or thrombotic tendencies in healthy, adult males.

The effect of dietary docosahexaenoic acid on plasma lipoproteins and tissue fatty acid composition in humans.

Normal, healthy male volunteers (n = 6) were fed diets [high docosahexaenoic acid-DHA] containing 6 g/d of DHA for 90 d. The stabilization (low-DHA) diet contained less than 50 mg/d of DHA. A control group (n = 4) remained on the low-DHA diet for the duration of the study (120 d). Blood samples were drawn on study days 30 (end of the stabilization period), 75 (midpoint of the intervention period), and 120 (end of the intervention period). Adipose tissue (AT) samples were taken on days 30 and 120. The plasma cholesterol (C), low density lipoprotein (LDL)-C and apolipoproteins (apo) [Al, B, and lipoprotein (a)] were unchanged after 90 d, but the triglycerides (TAG) were reduced from a mean value of 76.67 +/- 24.32 to 63.83 +/- 16.99 mg/dL (n = 6, P < 0.007 using a paired t-test) and the high density lipoprotein (HDL)-C increased from 34.83 +/- 4.38 mg/dL to 37.83 +/- 3.32 mg/dL (n = 6, P < 0.017 using a paired t-test). The control group showed no significant reduction in plasma TAG levels. Apo-E, however, showed a marked increase in the volunteers' plasma after 90 d on the high-DHA diet, from 7.06 +/- 4.47 mg/dL on study day 30 to 12.01 +/- 4.96 mg/dL on study day 120 (P < 0.002 using a paired t-test). The control subjects showed no significant change in the apo-E in their plasma (8.46 +/- 2.90 on day 30 vs. 8.59 +/- 2.97 on day 120). The weight percentage of plasma DHA rose from 1.83 +/- 0.22 to 8.12 +/- 0.76 after 90 d on the high-DHA diet. Although these volunteers were eating a diet free of eicosapentaenoic acid (EPA), plasma EPA levels rose from 0.38 +/- 0.05 to 3.39 +/- 0.52 (wt%) after consuming the high-DHA diet. The fatty acid composition of plasma lipid fractions--cholesterol esters, TAG, and phospholipid--showed marked similarity in the enrichment of DHA, about 10%, after the subjects consumed the high-DHA diet. The DHA content of these plasma lipid fractions varied from less than 1% (TAG) to 3.5% (phospholipids) at baseline, study day 30. EPA also increased in all plasma lipid fractions after the subjects consumed the high-DHA diet. There were no changes in the plasma DHA or EPA levels in the control group. Consumption of DHA also caused an increase in AT levels of DHA, from 0.10 +/- 0.02 to 0.31 +/- 0.07 (wt%) (n = 6, P < 0.001 using a paired t-test), but the amount of EPA in their AT did not change. Thus, dietary DHA will lower plasma TAG without EPA, and DHA is retroconverted to EPA in significant amounts. Dietary DHA appears to enhance apo-E synthesis in the liver. It appears that DHA can be a safe and perhaps beneficial supplement to human diets.

A human dietary arachidonic acid supplementation study conducted in a metabolic research unit: rationale and design.

While there are many reports of studies that fed arachidonic acid (AA) to animals, there are very few reports of AA feeding to humans under controlled conditions. This 130-d study was conceived as a controlled, symmetrical crossover design with healthy, adult male volunteers. They lived in the metabolic research unit (MRU) of the Western Human Nutrition Research (WHNRC) for the entire study. All food was prepared by the WHNRC kitchen. The basal (low-AA) diet consisted of natural foods (30 en% fat, 15 en% protein, and 55 en% carbohydrate), containing 210 mg/d of AA, and met the recommended daily allowance for all nutrients. The high-AA (intervention) diet was similar except that 1.5 g/d of AA in the form of a triglyceride containing 50% AA replaced an equal amount of high-oleic safflower oil in the basal diet. The subjects (ages 20 to 39) were within -10 to +20% of ideal body weight, nonsmoking, and not allowed alcohol in the MRU. Their exercise level was constant, and their body weights were maintained within 2% of entry level. Subjects were initially fed the low-AA diet for 15 d. On day 16, half of the subjects (group A) wee placed on the high-AA diet, and the other group (B) remained on the low-AA diets. On day 65, the two groups switched diets. On day 115, group B returned to the low-AA diet. This design, assuming no carryover effect, allowed us to merge the data from the two groups, with the data comparison days being 65 (low-AA) and 115 (high-AA) for group B and 130 (low-AA) and 65 (high-AA) for group A. The main indices studied were the fatty acid composition of the plasma, red blood cells, platelets, and adipose tissue; in vitro platelet aggregation, bleeding times, clotting factors; immune response as measured by delayed hypersensitivity skin tests, cellular proliferation of peripheral blood mononuclear cells in response to various mitogens and antigens, natural killer cell activity, and response to measles/mumps/rubella and influenza vaccines; the metabolic conversion of deuterated linoleic acid to AA and the metabolic fate of deuterated AA in the subjects on and off the high-AA diet; and the production of eicosanoids as measured by excretion of 11-DTXB2 and PGI2-M in urine. The results of these studies will be presented in the next five papers from this symposium.

The effect of dietary arachidonic acid on plasma lipoprotein distributions, apoproteins, blood lipid levels, and tissue fatty acid composition in humans.

Normal healthy male volunteers (n = 10) were fed diets (high-AA) containing 1.7 g/d of arachidonic acid (AA) for 50 d. The control (low-AA) diet contained 210 mg/d of AA. Dietary AA had no statistically significant effect on the blood cholesterol levels, lipoprotein distribution, or apoprotein levels. Adipose tissue fatty acid composition was not influenced by AA feeding. The plasma total fatty acid composition was markedly enriched in AA after 50 d (P < 0.005). The fatty acid composition of plasma lipid fractions, cholesterol esters, triglycerides, free fatty acids, and phospholipid (PL) showed marked differences in the degree of enrichment in AA. The PL plasma fraction from the subjects consuming the low-AA diet contained 10.3% AA while the subjects who consumed the high-AA diet had plasma PL fractions containing 19.0% AA. The level of 22:4n-6 also was different (0.67 to 1.06%) in the plasma PL fraction after 50 d of AA feeding. After consuming the high-AA diet, the total red blood cell fatty acid composition was significantly enriched in AA which mainly replaced linoleic acid. These results indicate that dietary AA is incorporated into tissue lipids, but selectively into different tissues and lipid classes. Perhaps more importantly, the results demonstrate that dietary AA does not alter blood lipids or lipoprotein levels or have obvious adverse health effects at this level and duration of feeding.

The effect of dietary arachidonic acid on platelet function, platelet fatty acid composition, and blood coagulation in humans.

Arachidonic acid (AA) is the precursor of thromboxane and prostacyclin, two of the most active compounds related to platelet function. The effect of dietary AA on platelet function in humans is not understood although a previous study suggested dietary AA might have adverse physiological consequences on platelet function. Here normal healthy male volunteers (n = 10) were fed diets containing 1.7 g/d of AA for 50 d. The control diet contained 210 mg/d of AA. Platelet aggregation in the platelet-rich plasma was determined using ADP, collagen, and AA. No statistical differences could be detected between the aggregation before and after consuming the high-AA diet. The prothrombin time, partial thromboplastin time, and the antithrombin III levels in the subjects were determined also. There were no statistically significant differences in these three parameters when the values were compared before and after they consumed the high-AA diet. The in vivo bleeding times also did not show a significant difference before and after the subjects consumed the high-AA diet. Platelets exhibited only small changes in their AA content during the AA feeding period. The results from this study on blood clotting parameters and in vitro platelet aggregation suggest that adding 1.5 g/d of dietary AA for 50 d to a typical Western diet containing about 200 mg of AA produces no observable physiological changes in blood coagulation and thrombotic tendencies in healthy, adult males compared to the unsupplemented diet. Thus, moderate intakes of foods high in AA have few effects on blood coagulation, platelet function, or platelet fatty acid composition.

Increased dietary arachidonic acid enhances the synthesis of vasoactive eicosanoids in humans.

Data on the effect of dietary arachidonic acid (AA) (20:4n-6) on the synthesis of thromboxane and prostacyclin (PGI2) in humans are lacking. We measured the effect of 1.5 g/d (ca. 0.5 en%) of 20:4n-6 added isocalorically to a stabilization (low-AA) diet on the excretion of 11-dehydrothromboxane B2 (11-DTXB2) and 2,3-dinor-6-oxo-PGF1 alpha (PGI2-M). In a crossover design, 10 healthy men, living in a metabolic unit, were fed a diet (low-AA) containing 210 mg/d of 20:4n-6 for 65 d and an identical diet (high-AA) that contained 1.5 g/d of additional 20:4n-6 for 50 d. Three-day urine pools were collected at the end of each dietary period and analyzed for eicosanoids by gas chromatography-electron capture negative ion-tandem mass spectrometry. Mean excretion of 11-dehydrothromboxane B2 was 515 +/- 76, 493 +/- 154, and 696 +/- 144 ng/d (SD; n = 10) during the acclimation (15 d) low-AA diet and high-AA diet periods, respectively (41% increase from low-AA to high-AA diet, P = 0.0037); mean excretion of PGI2-M was 125 +/- 40, 151 +/- 36, and 192 +/- 55 ng/d (SD; n = 10) during acclimation (15 d) low-AA and high-AA diets; P = 0.0143). Thus both the metabolites of thromboxane and PGI2 increase on the high-AA diet. Furthermore, both indicated changes in metabolite excretion may be associated with measurable effects on several physiologically significant cellular functions, such as platelet aggregation in vivo and inflammation in response to immune challenges.

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 post-immunization 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.