Omega-3 Fatty Acids, Evolutionary Aspects and Metabolism
Artemis P. Simopoulos, M.D.
The Center for Genetics, Nutrition and Health, Washington, D.C.
Human beings evolved on a diet that was lower in total fat and saturated fat, but contained equal amounts of omega-6 and omega-3 essential fatty acids. Linoleic acid (LA) is the major omega-6 fatty acid, and alpha-linolenic acid (LNA) is the major omega-3 fatty acid. In the body, LA is metabolized to arachidonic acid (AA), and LNA is metabolized to eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). The ratio of omega-6 to 3 essential fatty acids was 1 to 2:1 with higher levels of the longer chain polyunsaturated fatty acids (PUFA), such as EPA, DHA and AA, than today’s diet. Today this ratio is about 10-20:1, indicating that Western diets are deficient in 3 fatty acids compared with the diet on which humans evolved and their genetic patterns were established. The 3 and 6 essential fatty acids are not interconvertible in the human body and are important components of practically all cell membranes. 6 and 3 fatty acids influence eicosanoid metabolism, gene expression, and intercellular cell to cell communication. The PUFA composition of cell membranes is, to a great extent, dependent on dietary intake. Therefore, appropriate amounts of dietary 6 and 3 fatty acids need to be considered in making dietary recommendations. These two classes of PUFA should be distinguished because they are metabolically and functionally distinct and have opposing physiological functions; their balance is important for homeostasis and normal development. A balanced omega-6/omega-3 ratio in the diet is essential for normal growth and development and should lead to decreases in cardiovascular disease and other chronic diseases, such as diabetes, hypertension, arthritis and other autoimmune diseases, and improve mental health. Although a recommended dietary allowance for essential fatty acids does not exist, an adequate intake (AI) has been estimated for omega-6 and omega-3 essential fatty acids by an international scientific working group. For Western societies, it will be necessary to decrease the intake of omega-6 fatty acids and increase the intake of omega-3 fatty acids. The food industry is already taking steps to return omega-3 essential fatty acids to the food supply by enriching various foods with omega-3 fatty acids. To obtain the recommended AI, it will be necessary to consider the issues involved in enriching the food supply with omega-3 PUFA in terms of dosage, safety, and sources of omega-3 fatty acids.
ω-3 LIPIDS AND VITAMIN E SUPPLEMENTS ON IMMUNE FUNCTIONS IN RA-PRONE MICE
J. T. Venkatraman, Ph.D.
Department of Physical Therapy, Exercise & Nutrition Sciences
State University of New York, Buffalo, NY 14214.
ω-3 fatty acid rich-fish oil (FO) and vitamin E (vit-E) may delay the progress of certain autoimmune (AI) diseases. The present study examined the mechanism of action of ω3 and ω6 lipids and vit-E in autoimmune-prone MRL/lpr (lpr) mice. The lpr (lymphoproliferative) gene is overexpressed in these mice and they suffer from extensive lymphoproliferation, lupus-like symptoms and accelerated aging. To determine whether the effects of ω-3 lipids in autoimmune disease (AI) is linked to vit-E levels, weanling female MRL/lpr (lpr) and congenic control MRL/+/+ (+/+) mice were fed semipurified diets containing 10% corn oil (CO), 10% fish oil (FO), at two levels of vit-E (75 IU or 500 IU/kg diet) for 4m and cellular immune functions were measured. Proliferative response to lectins was significantly higher in the groups fed the high-vit E diets. The type of oil had significant effects on IL-4, IL-10, IL-12 and pro-inflammatory cytokines (TNF-α, PGE2 and TXB2) while vit-E levels had significant effects on IL-2, IFN-γ and TNF-α levels. Strain had significant effects on IL-2, IL-10, IL-12, IFN-γ, TNF-α, PGE2 and TXB2 levels IL-1 while IL-6 levels were not affected.
The level of anti-DNA antibodies in sera were significantly higher in lpr mice. FO significantly decreased IL-12, TNF-α and TXB2 levels compared to the CO fed groups. However, PGE2, IL-1, IL-2, IFN-γ, IL-4, IL-6, and IL-10 did not show differences between the two oils. The expression of IL-12 and TNF-α was significantly higher in the low vit-E groups, but not IL-1, IL-2, IFN-γ, IL-4, IL-6, IL-10 and the hormones. The levels of IL-1, IL-2, and IFN-γ were lower while IL-4, IL-6, IL-10, IL-12, and TNF-α were higher in lpr mice. The production of chemokines RANTES and MCP-1 by spleen cells was higher in MRL/lpr mice. FO decreased the production of these two chemokines by spleen cells in the MRL/lpr mice.
Autoimmune mice generally express lower activities and mRNA levels of liver antioxidant enzymes and generate higher levels of TBARS. Both FO and vit-E can significantly decrease the generation of TBARS and increase the levels of antioxidant enzymes.
In summary, data from this study suggest that adding appropriate levels of vit-E to FO diets may further enhance the beneficial effects of ω-3 lipids in delaying AI. The present study suggests that ω-3 lipids containing appropriate levels of vit-E may delay AI through modulating the levels of selected cytokines chemokines, and enzymes involved in antioxidant defense. Vit-E seems to exert selective effects on proliferative response and specific subsets and selected cytokines. (Supported by NIAMS (NIH) grant # 1R15AR/A143517).
N-3 Fatty Acid Supplements in Rheumatoid Arthritis
Joel M. Kremer M.D.
Albany Medical College, Albany, NY
Ingestion of dietary supplements of n-3 fatty acids has been consistently shown to reduce both the number of tender joints on physical examination and the amount of morning stiffness in patients with rheumatoid arthritis. In these cases, supplements were consumed daily in addition to background medications and the clinical benefits of the n-3 fatty acids were not apparent until they were consumed for 12 wks or longer. It appears that a minimum daily dose of 3g eicosapentaenoic and docosahexaenoic acids is necessary to derive the expected benefits.
These doses of n-3 fatty acids are associated with significant reductions in release of leukotriene B4 from simulated neutrophils and interleukin 1 from monocytes. Both of these mediators of inflammation are thought to contribute to the inflammatory drugs or disease modifying anti rheumatic drugs. Because the methods used to determine whether patients taking n-3 supplements can discontinue taking these agents are variable, confirmatory and definitive studies are needed to settle this issue. n-3 fatty acids have virtually no reported serious toxicity in the dose range used in rheumatoid arthritis and are generally very well tolerated.
Dietary Omega-3 Polyunsaturated Fatty Acids Reduce Interleukin-12 and Interferon-gamma Production and Can Adversely Affect Host Infectious Disease Resistance
Kevin L. Fritsche, Ph.D.
University of Missouri, Columbia, MO
The overall goal of my research program is to define the molecular and cellular mechanisms that explain how and why dietary fats both positively and negatively impact our immune system. Recently, my laboratory has focused on the effect of omega-3 fatty acids on infectious disease resistance. Specifically, we study the influence of dietary fatty acids on murine listeriosis, a well-characterized infectious disease model. During the previous five years my laboratory has demonstrated that mice fed a diet rich in n-3 fatty acids have impaired resistance to Listeria. Further, these fatty acids reduce the in vivo production of interleukin-12 (IL-12) and interferon-gamma (IFNγ) during an infectious challenge in mice. These two cytokines play a central role in regulating both innate and adaptive (i.e., cell-mediated) immune responses against many infectious pathogens. We also demonstrated that n-3 PUFA consumption leads to a reduction in the expression of IFNγ receptors on immune cells. Mice with a gene deletion for the IFNγ receptor are highly susceptible to infection from intracellular pathogens, such as Listeria and mycobacterium. The objectives of our current research efforts are to: (1) further characterize the impact of n-3 PUFA on host resistance to Listeria, with an emphasis on the adaptive arm of the immune system; (2) determine if the n-3 PUFA-mediated reduction of IFNγ receptor expression makes a significant contribution to impair host resistance against Listeria; (3) define the molecular mechanism(s) through which n-3 PUFA affect interleukin-12 production. Data from some of our most recent studies will be presented.
Development of a Safe and Effective Dietary Fatty Acid Supplement that Reduces Leukotriene Generation in Humans.
Floyd H.Chilton, Ph.D.
Wake Forest University, Winston-Salem, NC
Studies carried out over the last two decades have revealed that controlling dietary fatty acid intake in a number of animal models has great potential in reducing arachidonic acid metabolism and ameliorating inflammation in models which mimic human arthritis, asthma or glomerulonephritis. However, dietary modifications in humans on Western diets have shown only modest efficacy. Over the past decade, our laboratory has performed dietary fatty acid reduction and supplementation studies in humans fed in a General Clinical Research Center in an attempt to influence arachidonic acid metabolism and clinical outcomes. Initial studies revealed that n-3 fatty acids provided with Western diets were rapidly incorporated into membrane phospholipids of inflammatory cells. However, dietary n-3 fatty acids at supplement concentrations as high as 5.6g eicosapentaenoic (EPA) acid/day did not reduce cellular arachidonic acid content nor significantly reduce the biosynthesis of lipid mediators of inflammation. In contrast, supplementation of human diets with γ-linolenic acid (GLA), an 18-carbon n-6 fatty acid, dramatically reduced leukotriene production in neutrophils and whole blood. However, this same fatty acid caused a marked increase in serum arachidonate (AA) levels, a potentially harmful side effect. This paradox was resolved by experiments that demonstrated that inflammatory cells such as human neutrophils contain an elongase but not a Δ-5 desaturase activity, and thus dietary GLA leads to the accumulation of dihomogammalinolenic acid (an inhibitor of AA metabolism) and not AA in neutrophil lipids. In contrast, the liver converts GLA to AA utilizing both elongase and Δ-5 desaturase activities, and this causes an increase in serum AA levels.
Thus, dietary supplement strategies were designed to maintain the capacity of GLA to reduce leukotrienes without causing elevations in serum AA levels. Initial in vitro experiments revealed that addition of the n-3 fatty acid, EPA, to liver cells blocked Δ-5 desaturase, the terminal enzyme step in AA synthesis. Addition of 3.0g/day of EPA and GLA to human diets decreased leukotrienes, increased serum levels of EPA and concomitantly prevented the increase in serum AA levels. Clinical trials were then performed to optimize GLA and EPA concentrations required to reduce whole blood leukotriene generation and prevent serum AA accumulation, respectively. In collaboration with Pilot Therapeutics, oils containing these fatty acids have been formulated into a good-tasting concentrated liquid emulsion that can be easily mixed with water or juice and taken as a single daily dose. Clinical trials are currently ongoing to test the capacity of this formulation to reduce leukotriene generation in asthmatics.
Diet Modulation of Apoptosis.
Robert S. Chapkin1,3, David N. McMurray 1,2,3 and Joanne R. Lupton 1,3,
Faculty of Nutrition, 2
Department of Medical Microbiology and Immunology, and the 3
Center for Environmental and Rural Health,
Texas A&M University, College Station, TX.
We have demonstrated that the balance between cell proliferation and apoptosis can be favorably modulated by feeding fish oil, containing n-3 polyunsaturated fatty acids (PUFA). For example, dietary fish oil reduces O6-methylguanine DNA adduct levels in the rat colon in part by increasing targeted apoptosis during tumor initiation. This is significant because dietary upregulation of apoptosis can prevent malignant transformation of genetically damaged cells, lymphoproliferative disorders and autoimmunity. We have subsequently utilized the colonic epithelium and spleen-derived T-lymphocytes as model systems to determine the mechanisms by which n-3 PUFA promote apoptosis.
Dietary fish oil blunts signaling programs which attenuate apoptosis. Specifically, fish oil blocks carcinogen-induced suppressors of apoptosis in the colon, i.e., oncogenic ras activation, COX II and bcl-2 expression, diacylglycerol formation and protein kinase C (PKC) beta II and lambda/iota activation in the colon. This may partly explain why dietary fish oil protects against colon cancer development. In parallel studies, mice fed highly purified ethyl esters of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) had suppressed levels of several mitogenic second messengers, i.e., diacylglycerol, ceramide, IL-2 and IL-2 receptor alpha in stimulated T-lymphocytes, resulting in a proapoptotic, antiinflammatory phenotype. Collectively, these data demonstrate that the cellular homeostatic balance between cell proliferation and apoptosis can be favorably modified in diverse cell types solely by ingesting n-3 PUFA. This research was supported by grants from the American Institute for Cancer Research 00A055, NIH CA59034, CA61750, DK53055, and NIEHS ES09106.
N-3 Fatty Acids, Calories, and SLE
Gabriel J. Fernandes, PhD
University of Texas Health Science Center, San Antonio, Texas
Systemic Lupus Erythematosus (SLE) is an autoimmune disease that affects 1/2 to 2 million people in the USA, mostly women. SLE primarily affects the joints, skin, kidneys, lungs, heart, and GI tract, etc. Treatment includes immunosuppressive drugs but side effects are often noted. In limited clinical trials omega-3 fatty acids were used. The results were somewhat inconclusive. One of the earlier negative results may have been due to large doses of fish oil without adequate antioxidant supplements, which may have caused side effects. Well designed studies with concentrated odorless fish oil capsules may provide beneficial effects including likely reduction in the dosage of drugs used to treat the disease. Fish oil studies carried out in autoimmune prone mice by several investigators consistently revealed beneficial effects such as delayed onset of renal disease, decreased anti-DNA antibodies, and decreased proinflammatory cytokines. Our earlier studies were designed to investigate the role of corn oil (n-6) vs n-3 fatty acid supplementation. The results showed that n-3 fatty acid supplementation causes elevated antioxidant enzymes, decreased pro-inflammatory cytokine mRNA, reduced renal disease and prolonged life span in (NZBxNZW)F1 (B/W) female mice.
It is also well known that calorie restriction prevents obesity and delays age-related immune dysfunction and prolongs life span in multiple animal models. The amount and type of dietary fatty acids can also profoundly affect life span. We examined the influence of calorie restriction (40% reduction in energy intake) combined with substitution of fish oil for corn oil (5%). Autoimmune-prone B/W female mice, which develop fatal autoimmune renal disease, were used. The calorie-restricted/fish oil diet maximally extended median life span to 645 days (vs. 494 days for the calorie-restricted corn oil diet). Similarly, fish oil prolonged life span in the ad libitum fed mice to 345 days (vs. 242 for the ad libitum/corn oil diet, p<0.001). Increased life span was partially associated with decreased body weight, blunting renal proinflammatory cytokine (interferon-γ, interleukins-10 and -12 and tumor necrosis factor-α) levels and lower nuclear factor kappa B (NF-κB). Reductions in NF-κB were preceded by enhanced superoxide dismutase, catalase, and glutathione peroxidase activities in young mice before the onset of autoimmune disease. Our current findings demonstrate the profound additive effects of caloric restriction and n-3 fatty acids in prolonging life span in B/W mice (submitted). In summary, it appears that either a modest reduction in food or calorie intake and/or following a moderate regimen of exercise along with drug therapy and/or n-3 fatty acid supplements may have several beneficial effects, including decreased free radicals and increased T cell immunity, in the management of obesity, diabetes, cancer and/or the aging process including various autoimmune disorders. New experimental and clinical studies are needed to confirm or refute these hypotheses.
1. Avula, C. P., and Fernandes, G., Modulation of antioxidant enzymes and apoptosis in mice by dietary lipids and treadmill exercise, J Clin Immunol, 19, 35-44 1999.
2. Jolly, C. A., and Fernandes, G., Dietary N-3 fatty acids and calorie restriction in autoimmune disease: Influence in different immune compartments, Current Organic Chemistry, 4, 945-957 2000.
3. Lim, B. O., Jolly, C. A., Zaman, K., and Fernandes, G., Dietary (n-6) and (n-3) fatty acids and energy restriction modulate mesenteric lymph node lymphocyte function in autoimmune-prone (NZB x NZW)F1 mice, J Nutr, 130, 1657-64 2000.
4. Muthukumar, A. R., Jolly, C. A., Zaman, K., and Fernandes, G., Calorie restriction and n-3 fatty acid supplementation modulate pro-inflammatory cytokines and polymeric Ig receptor expression in the submandibular glands of autoimmune prone (NZB x NZW) F1 mice, J Clin Immunol, 20, 354-361 2000.
Omega-3 Fatty Acids in the Treatment of Patients with IgA Nephropathy: The Rationale and the Studies
James V. Donadio, M.D.
Mayo Clinic and Foundation, Rochester, MN
Idiopathic immunoglobulin A nephropathy (IgAN) is an immune-complex mediated glomerulonephritis characterized by the predominant or codominant deposition of IgA within the mesangial regions of the glomeruli associated with a variety of histopathologic lesions. IgAN is the most common glomerular disease worldwide. IgAN occurs at all ages with the usual age of clinical onset in the second and third decades of life. Clinical presentation includes episodic, macroscopic hematuria often coinicident with upper respiratory infection or asymptomatic proteinuria and microscopic hematuria. Renal biopsy is essential for making an accurate diagnosis. The disease has a poorly understood pathogenesis. Progressive renal failure develops up to 25 yr after diagnosis in 20%-40% of patients, although there is considerable variability in the clinical course of different groups of patients. There is no currently approved treatment for patients with IgAN. Three therapeutic agents have emerged as the most promising -- corticosteroids, angiotensin-converting-enzyme inhibitors, and omega-3 fatty acids -- and all have been tested, or are currently undergoing testing, in randomized clinical trials. Of the 4 trials testing efficacy of omega-3 fatty acids, 2 showed that such treatment stabilized renal function while 2 reported a decline in renal function. The findings of the largest study, which comprised 106 patients and was conducted by my collaborative group, provided strong evidence that in high-risk patients, treatment for 2-yr with a daily dose of 1.8g EPA and 1.2g DHA retarded the loss of renal function. From our 2-yr double-blind study and our extended observational study we conclude that early and prolonged treatment with omega-3 fatty acids slows renal disease progression for high-risk patients with IgAN.
Omega-3 fatty acids inhibit mesangial cell proliferation in vitro and in vivo.
Joseph P Grande, M.D.
Mayo Clinic and Foundation, Rochester, MN
Mesangial cell proliferation is a characteristic feature of IgA nephropathy. Recent clinical studies have shown that dietary ω
-3 fatty acid supplementation retards renal disease progression in patients with IgA nephropathy. Potential mechanisms underlying this protective effect of ω
-3 fatty acids have not been defined. We sought to test the hypothesis that ω
-3 fatty acids reduce mesangial cell proliferation following acute renal injury, using the anti-thy-1.1 (ATS) model of mesangial proliferative glomerulonephritis. Subcultured rat mesangial cells were used to determine the in vitro effects of eicosapentaenoic acid (EPA) and docosahexanoic acid (DHA), the primary components of fish oil, on proliferation. ω
-3 fatty acids were extracted from OmacorTM capsules, and administered twice daily by gavage; the daily dose was approximately 4.7 g/kg EPA and 3.7 g/kg DHA. Following ATS administration, proteinuria was significantly decreased in animals treated with ω
-3 fatty acids, compared with sesame oil-treated controls. In ATS rats given ω
-3 fatty acids, there was less mesangial cell and matrix expansion, mesangiolysis, or basement membrane disruption (Δ
% = -40%). ATS rats receiving fish oil had less glomerular cell proliferation (PCNA (Δ
% = -50%) and a reduction of α smooth muscle actin expression (Δ
% = -27%) by glomerular mesangial cells. In subcultured rat mesangial cells, DHA, but not EPA, significantly inhibited proliferation. In the ATS glomerulonephritis model, ω
-3 fatty acids inhibit mesangial cell activation and proliferation, reduces proteinuria, and decreases histologic evidence of glomerular damage. In vitro, the antiproliferative effects are more likely related to the action of DHA. We suggest that orally administered fish oil, or purified DHA, may have a suppressive effect in the acute phase or relapses of glomerulopathies by inhibiting activation and proliferation of mesangial cells.
Dietary Supplementation with L-arginine and Canola Oil Reduces Rejection Episodes, Renal Biopsies, Rehospitalization and Blood Pressure Following Renal Transplantation
J. Wesley Alexander 1, Trisha Ofstedal 1, Libby Stanley 1, Ann Erickson 2, Norman Greenberg 2, Laura James 1, M. Roy First 1, James F. Whiting 1 and E. Steve Woodle 1. 1, University of Cincinnati College of Medicine, Cincinnati, Ohio; 2, and Novartis Nutrition, St Louis Park , Minnesota.
Studies in laboratory animals receiving minimal immunosuppression have shown that supplementation of the diet with immunonutrients can prolong allograft survival. The present study was performed to determine whether similar results could be achieved in adult renal allograft transplant patients receiving standard Neoral®, CellCept® and steroids as their initial immunosuppression. Patients were stratified for major risk factors and prospectively randomized to control (C, 54) of supplement (S, 49). All patients received a low fat diet, but S patients also received arginine (4.5g BID) and canola oil (15g BID) daily. Demographics for C and S patients were similar with 55% Cad donor in the C group and 52% Cad donor in the S group. Patients were censored at different times for noncompliance, withdrawal, or loss of kidney. Follow-up was available for 40, 35 and 23 S patients and 49, 46 and 26 C patients at 3, 6 and 12 months respectively. Excluding 2 C and 2 S patients with a rejection episode in the first week, there were 13 rejection episodes in 12 C patients compared to 3 in 2 S patients (p=0.029). Furthermore, there were 24 renal biopsies for dysfunction in 19 C patients compared to 11 in 8 S patients (p=0.025). Serum creatinines at 3, 6 and 12 months for C patients were 1.52, 1.52 and 1.58 compared to 1.45, 1.42 and 1.61 for S patients (p=NS). Systolic blood pressure fell by 10.5%, 9.6% and 19.26% in C patients vs. 12.8%, 16.5% and 26.6% in S patients at 3,6 and 12 months (p=.0001) despite 71% and 52% C patients taking antihypertensive drugs at 6 and 12 months compared to 52% and 47% S patients, respectively (p=NS). C patients gained 8.8 kg or 12.4% body weight at 12 months compared to 5.1 kg or 6.9% for S patients (p=NS). In patients with one year follow-up, 54% C and 39% S patients developed one or more infections; 4% in C group vs. 2% in S group developed CMV infections. Two patients lost a kidney from rejection in the C group whereas no kidneys were lost from rejection in the S group. The length of rehospitalization per patient over 1 year for C patients was 6.5 + 1.4 days compared to 3.0 + 0.9 days for S (p=0.039). We conclude that dietary supplementation with arginine and canola oil results in significant reduction in rejection episodes, improved blood pressure and shorter rehospitalization stay in renal transplant patients receiving initial CsA base therapy without having any adverse effects. Transplant 2000 Joint Meeting.