Aldo Ferretti

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.

A human dietary arachidonic acid supplementation study conducted in a metabolic research unit: Radionale 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) were placed on the high-AA diet, and the other group (B) remained on the low-AA diet. 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 mitogene 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 with be presented in the next five papers from this symposium.

Antithromboxane activity of dietary alpha-linolenic acid: a pilot study.

Two decades of research conclusively demonstrated the antithrombotic properties of the long-chain n-3 polyunsaturated fatty acids (PUFA) present in lipids from marine fishes. Most American consumers, however, given their preference for meat, will not realize the benefits of a fish-rich diet. Could alpha-linolenic acid (18:3, n-3) be similarly effective via modulation of the synthesis of vasoactive eicosanoids, i.e., thromboxane and prostacyclin? The present pilot study is a contribution toward answering this question. We determined that the urinary excretion of 11-dehydrothromboxane B2 declined by 34% from baseline level 7 weeks after the n-6/n-3 ratio of dietary PUFA was reduced from 28:1 to 1:1. The excretion of 2,3-dinor-6-oxo-prostaglandin F1 alpha was similarly affected. The dietary adjustment was brought about by substituting measured amounts of canola and flaxseed oils (3:1) for measured amounts of olive and corn oils (3:1) in an otherwise fat-free basal diet. This study demonstrates that dietary alpha-linolenic acid is an effective modulator of thromboxane and prostacyclin biosynthesis. Therefore, we can expect that the eicosanoid-mediated effects of alpha-linolenic acid are similar to those elicited by marine lipids.

Effect of fish oil supplementation on the excretion of the major metabolite of prostaglandin E in healthy male subjects

We investigated the effect of fish oil supplementation on the synthesis of prostaglandin E (PGE)in vivo by measuring the excretion of its catabolite, PGE-M, in 24-hr urine by gas chromatography/mass spectrometry. Forty healthy male volunteers (24–57 years of age) consumed a controlled basal diet providing 40% of energy from fat (P/S ratio about 0.8∶1), 130 mg/1000 kcal cholesterol, and a minimum of 22 mg/day of α-tocopherol (α-T), for three experimental periods lasting a total of 28 weeks. During period 1 (10 weeks) the diet was supplemented with placebo (PO) capsules (15×1 g/day) consisting of a blend of fats approaching the fatty acid profile of the basal diet. This was followed by a second 10-week period during which the subjects received 15×1 g/day capsules of fish oil concentrate (FOC). During period 3 (8 weeks) they continued the 15 g/day intake of FOC but received an additional 200 mg/day of α-T. PO and FOC capsules contained 1 mg α-T/g fat as antioxidant. A 14% reduction of PGE-M excretion was observed after 10 weeks of FOC supplementation (period 2), compared to an identical period of placebo supplementation (period 1), P=0.009. PGE-M excretion during the last week of period 3 was not significantly different from that at the end of period 2. The reduction in PGE synthesis in response to the relatively high marine oil supplementation was large in many subjects participating in this study.

Isolation and measurement of urinary 8-iso-prostaglandin F2α by high-performance liquid chromatography and gas chromatography–mass spectrometry

8-iso-Prostaglandin F2alpha (8-iso-PGF2alpha) is a product of free radical-catalyzed peroxidation of arachidonic acid. Measurement of its urinary excretion has been proposed as an index of oxidative status in vivo. A stable isotope dilution method for its quantification by gas chromatography-electron capture chemical ionization mass spectrometry is described. Sample cleanup required the combined use of high-performance liquid chromatography and thin-layer chromatography. The inter-assay R.S.D. in two separate determinations was 1.6 (n=4) and 2.3% (n=4). The accuracy of the assay was evaluated through recovery experiments. The equation of the regression plot correlating the amounts added and recovered was y=0.91x-0.31, r=0.9916 (n=12). The pair of fragment ions ([M-181]-) at m/z 569 and m/z 573 was monitored for quantification. The mean 8-iso-PGF2alpha excretion rate was 528 +/- 127 (S.D.) ng per day in five male volunteers and 730 +/- 305 ng per day in six females. Intake of 80 mg of lycopene per day by eleven volunteers for four weeks resulted in a non-significant reduction of 8-iso-PGF2alpha excretion.

Dietary docosahexaenoic acid reduces the thromboxane/prostacyclin synthetic ratio in humans

Abstract The effect of fish oils, rich in docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA), on eicosanoid production in vivo has been extensively studied, but data on the effects of dietary DHA alone on the synthesis of thromboxane (TXA2) and prostacyclin (PGI2) in humans are lacking. We quantified the effect of an isocaloric shift in DHA intake from trace (low-DHA diet) to about 6% of total fatty acids (high-DHA diet), ca. 2 en%, on the excretion of 11-dehydrothromboxane B2 (11-DTXB2) and 2, 3-dinor-6-oxo-prostaglandin F1α (PGI2-M). In a longitudinal study, seven healthy men, living in a metabolic unit, were fed a 30% fat low-DHA diet for 30 days, then a high-DHA diet containing 6g/day of DHA for 90 days. A control group of four subjects remained on the low-DHA diet for the duration of the study (120 days). Three-day urine pools were collected at the end of each dietary period (around day 30 and day 120) and analyzed for eicosanoids by gas chromatography-electron capture negative ion-tandem mass spectrometry. Mean excretion of 11-DTXB2 was 590 ± 256 ng/d (SD; n = 7) with the low-DHA diet, and 385 ± 148 ng/d (n = 7) with the high-DHA diet, a 35% reduction (p = 0.013, n = 11 including the control group, when log transformed data were used). Production of 11-DTXB2 in the control group was unchanged. Mean excretion of PGI2-M was 229 ± 73 ng/day (SD; n = 7) and 210 ± 102 ng/day with the low-DHA and the high DHA diet, respectively (a nonsignificant reduction). This study confirms that the synthesis of TXA2 is more open to diet-induced modulation than the synthesis of PGI2. The observed reduction of 11-DTXB2 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.

Moderate changes in linoleate intake do not influence the systemic production of E prostaglandins

A pilot study was undertaken to determine if moderate changes in linoleate (18∶2ω6) intake would modulate the prostaglandin E turnover concurrently with, or independently of, changes in the plasma prostaglandin (PG) precursor levels. Four adult male volunteers in good health were fed two controlled diets containing 35% of energy from fat, with either 10 (diet L) or 30 g (diet H) linoleate/day, 30 to 50 g saturated fatty acids/day, and the balance mainly monounsaturated fatty acids. All four subjects were consuming sufficient amounts of polyunsaturates before the study. Protein (13–14%) and carbohydrate (51–53%) contribution to total caloric intake was kept constant. The menu cycle was 7 days, and all diets were calculated to provide adequate amounts of nutrients known to be required by man when data were available. Plasma fatty acids were determined by gas-liquid chromatography, and the turnover of E prostaglandins was assessed by measuring the urinary output of the major metabolite of PGE1+PGE2 (PGE-M). Whereas we found a clear correlation between 18∶2ω6 intake and 18∶2ω6 concentrations in the neutral lipid (P=0.007) and phosphoglyceride (P=0.012) fractions of plasma, arachidonate (20∶4ω6) concentrations in those same plasma fractions did not respond significantly to changes in linoleate intake. Moreover, we could not detect an influence of moderate changes in dietary levels of 18∶2ω6 on the systemic production of PGE as measured by the daily urinary output of PGE-M.

Occurrence of prostaglandin E3 in human urine as a result of marine oil ingestion: gas chromatographic-mass spectrometric evidence

This pilot study is an effort to elucidate the metabolic fate of dietary eicosapentaenoate in vivo and its influence on arachidonate cyclooxygenation at the renal level. The ultimate objective is to shed light on the biochemical mechanisms responsible for the physiologic effects of marine oil on humans. We found prostaglandin E3 (PGE3) in urine of a female volunteer who ingested 10-50 g/day of MaxEPA fish oil concentrate for 4 years. PGE3, a cyclooxygenase metabolite of eicosapentaenoate, could not be detected in 24-h urine pools from the same subject 16 weeks after fish oil supplementation ended. The appearance of PGE3 was concurrent with a reduction of urinary PGE2. Identification of the trienoic prostaglandin was based on comparison of chromatographic behavior of three distinct derivatives of endogenous PGE3 with that of authentic material, and on selected ion monitoring mass spectrometry.

Modulating influence of dietary lipid intake on the prostaglandin system in adult men

We evaluated the effect of moderate dietary changes on the prostaglandin system by measuring the urinary excretion of 7α-hydroxy-5,11-dioxo-tetranorprostane-1,16-dioic acid (PGE-M). In a crossover design, twenty-four free-living male subjects in good health (24 to 54 years of age) were fed two diets: (i) Regular (R) diet, 41% energy (en%) from fat, P/S 0.59, M/S 0.96; (ii) Experimental (E) diet, 19 en% from fat, P/S 1.31, M/S 1.48. Diet R contained 13.9 g/day of neutral detergent fiber (NDF) and about 600 mg/day of cholesterol per 3200 kcal; Diet E contained 35.5 g/day NDF and about 280 mg/day cholesterol. Each controlled-diet period lasted ten weeks. The menu cycle was 7 days, and all diets were calculated to provide adequate amounts of essential nutrients. The PGE-M excretion rates were determined in 24-hr urine by stable-isotope dilution gas chromatography-mass spectrometry in the selected ion-monitoring mode. Low-fat Diet E, with an intake of 6.6 en% from polyunsaturates, was associated with an average 14.2% reduction in PGE-M daily output, compared to high-fat Diet R with a 9.3 en% from polyunsaturates (P=0.046). These results support the view that dietary lipid changes can significantly alter thein vivo production of E-series prostaglandins. We cannot conclude, however, if this apparent diet effect was brought about by the change in linoleate intake alone or was the result of complex biochemical interactions among individual fatty acids, both saturated and unsaturated.

Modification of prostaglandin metabolism in vivo by long-chain omega-3 polyunsaturates

In a pilot study conducted with a healthy male volunteer we determined that short-term dietary supplementation with fish oil markedly suppresses the systemic production of prostaglandin E (P = 0.04). This biochemical effect is observable after an incubation period of several days. The potential consequences of a reduced PGE synthetic rate on renal function, immune system and vascular dynamics, must be considered in the overall evaluation of the safety of fish oil supplements.