Robert F. Grimble

Association of n-3 polyunsaturated fatty acids with stability of atherosclerotic plaques: a randomised controlled trial

BACKGROUND N-3 polyunsaturated fatty acids (PUFAs) from oily fish protect against death from cardiovascular disease. We aimed to assess the hypothesis that incorporation of n-3 and n-6 PUFAs into advanced atherosclerotic plaques increases and decreases plaque stability, respectively. METHODS We did a randomised controlled trial of patients awaiting carotid endarterectomy. We randomly allocated patients control, sunflower oil (n-6), or fish-oil (n-3) capsules until surgery. Primary outcome was plaque morphology indicative of stability or instability, and outcome measures were concentrations of EPA, DHA, and linoleic acid in carotid plaques; plaque morphology; and presence of macrophages in plaques. Analysis was per protocol. FINDINGS 188 patients were enrolled and randomised; 18 withdrew and eight were excluded. Duration of oil treatment was 7-189 days (median 42) and did not differ between groups. The proportions of EPA and DHA were higher in carotid plaque fractions in patients receiving fish oil compared with those receiving control (absolute difference 0.5 [95% CI 0.3-0.7], 0.4 [0.1-0.6], and 0.2 [0.1-0.4] g/100 g total fatty acids for EPA; and 0.3 [0.0-0.8], 0.4 [0.1-0.7], and 0.3 [0.1-0.6] g/100 g total fatty acids for DHA; in plaque phospholipids, cholesteryl esters, and triacylglycerols, respectively). Sunflower oil had little effect on the fatty acid composition of lipid fractions. Fewer plaques from patients being treated with fish oil had thin fibrous caps and signs of inflammation and more plaques had thick fibrous caps and no signs of inflammation, compared with plaques in patients in the control and sunflower oil groups (odds ratio 0.52 [95% CI 0.24-0.89] and 1.19 [1.02-1.57] vs control; 0.49 [0.23-0.90] and 1.16 [1.01-1.53] vs sunflower oil). The number of macrophages in plaques from patients receiving fish oil was lower than in the other two groups. Carotid plaque morphology and infiltration by macrophages did not differ between control and sunflower oil groups. INTERPRETATION Atherosclerotic plaques readily incorporate n-3 PUFAs from fish-oil supplementation, inducing changes that can enhance stability of atherosclerotic plaques. By contrast, increased consumption of n-6 PUFAs does not affect carotid plaque fatty-acid composition or stability over the time course studied here. Stability of plaques could explain reductions in non-fatal and fatal cardiovascular events associated with increased n-3 PUFA intake.

Nutritional Modulation of Cytokine Biology

The pro-inflammatory cytokines and oxidant molecules produced during the inflammatory response, which follows infection and injury, may be beneficial, or detrimental to the patient, depending on the amounts and contexts in which they are produced. Aberrant or excessive production has been implicated in inflammatory disease, and sepsis. The upregulation of cytokine production by NF kappa B and NFIL-6 activation by oxidants increases the likelihood of cytokine-induced mortality and morbidity. Complex systems exist for the control of cytokine production and oxidant actions. The former include the hormones of the hypothalamo-pituitary-adrenal axis, acute phase proteins, and endogenous inhibitors of interleukin (IL)-1 and tumor necrosis factor (TNF). The latter include endogenously synthesized antioxidants, such as glutathione and dietary antioxidants, such as tocopherols, ascorbates and cachectins. Nutrients change cytokine production and potency by influencing tissue concentrations of many of the molecules involved in cytokine biology. Monounsaturated fatty acids and omega-3 polyunsaturated fatty acids (PUFAs) suppress TNF and IL-1 production and actions, while n-6 PUFAs exert the opposite effect. Changes in eicosanoid production are more likely to underlie this effect than alterations in membrane fluidity. Low antioxidant intake results in enhanced cytokine production and effects. The anorexia that follows infection and injury, may be purposeful to permit release of substrate from endogenous sources to support and control the inflammatory process. Therefore, prior as well as concurrent nutrient intake are of importance in determining the outcome of the inflammatory response.

Eicosapentaenoic acid (EPA) from highly concentrated n-3 fatty acid ethyl esters is incorporated into advanced atherosclerotic plaques and higher plaque EPA is associated with decreased plaque inflammation and increased stability

OBJECTIVE To examine n-3 polyunsaturated fatty acid (PUFA) incorporation into atherosclerotic plaques and the association with plaque inflammation and stability. METHODS AND RESULTS Patients awaiting carotid endarterectomy (n=121) were randomised to consume control capsules or n-3 PUFA ethyl ester capsules until surgery (median 21 days). The fatty acid compositions of plasma and carotid plaque phospholipids, plaque features, and expression of inflammatory genes were determined. The proportion of eicosapentaenoic acid (EPA) was higher (P<0.0001) in carotid plaque phospholipids in patients in the n-3 PUFA group. Plaques from patients in the n-3 PUFA group had fewer foam cells (P=0.0390). There were no other differences between plaques in the two groups with regard to histological characteristics or morphology. Plaque stability was not different between the two groups. However, the EPA content of plaque phospholipids was inversely associated with plaque instability (P=0.0209), plaque inflammation (P=0.0108), the number of T cells in the plaque (P=0.0097) and a summary score considering a range of plaque features (P=0.0425). Plaques from patients who received n-3 PUFAs had significantly lower levels of mRNA for matrix metalloproteinases (MMP)-7 (P=0.0055), -9 (P=0.0048) and -12 (P=0.0044) and for interleukin-6 (P=0.0395) and intercellular adhesion molecule 1 (P=0.0142). CONCLUSIONS Atherosclerotic plaques readily incorporate EPA. A higher plaque EPA content is associated with a reduced number of foam cells and T cells, less inflammation and increased stability.

Omega-3 Fatty Acids and Inflammation: Novel Interactions Reveal a New Step in Neutrophil Recruitment

While investigating new mechanisms by which the dietary omega-3 fatty acids regulate inflammation, the authors have identified a new step in the regulation of neutrophil migration across vascular endothelial cells.

The Effects of Sulfur Amino Acid Intake on Immune Function in Humans

No direct data exist on the influence of supranormal intakes of sulfur amino acids on immune function in humans. However 3 major products of sulfur amino acids, glutathione (GSH), homocysteine (Hcy), and taurine (Tau), influence, mainly, inflammatory aspects of the immune response in vitro and in vivo. Methionine intakes above approximately 1 g/d transiently raise plasma Tau, Hcy, and GSH. Tau and GSH ameliorate inflammation. Hcy has the opposite effect. A biphasic relation, between cellular GSH and CD4+ and CD8+ numbers occurs in healthy men. How changes in sulfur amino acid intake influence this phenomenon is unknown. In animals, high Tau intakes are antiinflammatory. How immune function in humans is affected is unknown. A positive relation between plasma neopterin (a marker of a Th-1-type immune response) and Hcy indicates that Hcy may play a part in inflammatory aspects of Parkinsons disease and aging. In vitro, Hcy, at concentrations seen following consumption of approximately 6 g L-methionine/d in adults, increases the interactions among T lymphocytes, monocytes, and endothelium. Whether a similar phenomenon occurs in vivo is unknown. Polymorphisms in the methylenetetrahydrofolate reductase gene are associated with raised plasma Hcy in young but not old subjects. The relation of this observation to immune function is unknown. The relationships among Hcy, inflammatory aspects of disease, and in vitro alterations in immune cell behavior create a cautionary note about supplementation of diets with l-methionine to raise intake above approximately 1 g/d. Studies directly linking methionine intake, genetics, plasma Hcy, Tau, and GSH and immune function are needed.

Dietary manipulation of the inflammatory response

Inflammation involves a complex interaction of cells and soluble mediators. These components interact to produce a situation in which immune cells are attracted to the inflammatory locus and activated. Cytokines are among the potent soluble mediators produced during inflammation and include the interleukins (IL) 1-8, tumour necrosis factors (TNF), and interferons (Male et al. 1989). In addition to activating the immune system, a number of these molecules bring about metabolic changes in the subject which lead to the provision of nutrients for the activated immune system. The cytokines involved in this process are IL-1 and -6 and TNF-a and -p (Grimble, 1990). An inflammatory stimulus, such as invasion of tissue by bacteria, viruses or parasites, or tissue damage incurred by trauma, surgery or burns, will induce production of IL-1, IL-6 and TNF from a range of immune cells. These cells include phagocytic leukocytes, T and B lymphocytes, mast cells and non-immune cells such as fibroblasts and endothelial cells (Moissec & Ziff, 1986; Le et al. 1987; Dinarello et al. 1988; Gordon & Galli, 1990). Once induced, IL-1 and TNF can induce each other’s and IL-6 production, and IL-6 can induce that of IL-1. Thus, a cascade of cytokines, which are capable of producing metabolic and immune effects, occurs (Old, 1987; Heinrich et al. 1990). Inflammatory stimuli not only bring about cytokine production, free radicals are also released (Farante et al. 1988). These may enhance production of TNF and other cytokines. Clark et al. (1989) showed that experimental conditions which lead to enhanced free radical production also lead to increased TNF production, both in vitro and in vivo. The metabolic actions of cytokines provide the mechanisms for limiting the extent and duration of cytokine production, via a number of routes (Fig. 1). These mechanisms arise from the actions of IL-1 and TNF on the hypothalamo-pituitary adrenal axis. Consequently glucocorticoids are released and have the ability to suppress cytokine production (Sherry & Cerami, 1988). They also arise from the action of cytokines on peripheral tissue causing release of amino acids, and by direct action on hepatocytes (Perlmutter et al. 1986). Cytokines also act indirectly on liver by creating a hormonal milieu for enhancing production of acute-phase proteins. Some of these substances, such as orosomucoid, a-2-macroglobulin and a-l-antichymotrypsin have the ability to directly inhibit neutrophil activation and production of TNF (Costello et al. 1984; Scuderi et al. 1989). The increased production of acute-phase proteins, such as orosomucoid and caeruloplasmin (EC 1.16.3.1; CP), and of glutathione, enhance antioxidant defences and may thereby limit the stimulatory effects of free radicals released concurrently with cytokines. Acute-phase proteins are not entirely suppressive of cytokine production, since one such protein, lipopolysaccharide-binding protein, increases the sensitivity of macrophages to endotoxin and results in enhanced TNF production (Schumann et al. 1990). The essential nature of cytokines in recovery from inflammatory situations is indicated by the poor prognosis of malnourished patients who have a reduced ability for

The influence of dietary lipids on the composition and membrane fluidity of rat hepatocyte plasma membrane

Weanling male Wistar rats were fed for five weeks on standard rat chow (23 g fat/kg diet) or one of four synthetic diets with butterfat, coconut oil, corn oil, or fish oil as the main lipid source (100 g fat/kg diet). In all diets, 10% of the fat was provided as corn oil to prevent essential fatty acid deficiency. Significant differences were observed in the saturated, monounsaturated, and polyunsaturated fatty acid composition, and in the ratio of cholesterol to phospholipid, in the hepatocyte membranes. The fluidity of hepatocyte plasma membranes was assessed using the fluorescence recovery after photobleaching technique and steady-state fluorescence anisotropy of diphenylhexatriene. No significant differences were found in the fluidity of plasma membranes between animals on the different fat diets, despite diet-induced changes in their fatty acid composition. However, the proportion of lipid free to diffuse in the plasma membrane varied with diet, being significantly greater (P<0.05) in animals fed chow (63.7%), coconut oil (61.5%), and butterfat (57.6%) diets than in those fed the corn oil (47.3%) diet. Animals fed fish oil showed an intermediate (50.0%) proportion of lipid free to diffuse. The data support the hypothesis that dietary lipids can change both the chemical composition and lateral organization (lipid domain structure) of rat hepatocyte plasma membranes.

Differences in Matrix Metalloproteinase-1 and Matrix Metalloproteinase-12 Transcript Levels Among Carotid Atherosclerotic Plaques with Different Histopathological Characteristics

Background and Purpose— Previous studies have shown that atherosclerotic lesions express a number of matrix metalloproteinases (MMPs). Here we investigated whether transcript levels of MMP-1, -3, -7, -9, and -12 in carotid atherosclerotic plaques were correlated with histological features and clinical manifestations. Methods— Atherosclerotic plaques (n=50) removed from patients undergoing carotid endarterectomy were classified histologically using a system proposed by Virmani et al, and MMP-1, -3, -7, -9, and -12 transcript levels in these tissues were quantified by real-time reverse-transcriptase polymerase chain reaction. Results— Compared to plaques with a thick fibrous cap, those with a thin cap had a 7.8-fold higher MMP-1 transcript level (P=0.006). MMP-3, -7, and -12 were 1.5-fold, 1.8-fold, and 2.1-fold, respectively, higher in thin cap plaques, but the differences did not reach statistical significance. MMP-12 transcript levels were significantly increased in ruptured plaques compared with lesions without cap disruption (P=0.001). MMP-9 transcript levels were similar among the different types of lesion. MMP-1 and -12 transcript levels were significantly higher in plaques from patients with amaurosis fugax, than in those from asymptomatic patients (P=0.029 and P=0.008 for MMP-1 and MMP-12, respectively), than in those from patients with stroke (P=0.027 and P=0.001, respectively), and than in those from patients with transient ischemic attack (P=0.046 and P=0.008, respectively). Conclusions— These data support a role of MMP-1 and -12 in determining atherosclerotic plaque stability.

2. Impact of nutrients on cytokine biology in infection

Interleukins 1 and 6 and tumour necrosis factor orchestrate a co-ordinated series of metabolic changes following invasions by pathogens. The changes are designed to destroy the pathogen. The response is characterized by fever, proteolysis in peripheral tissues, acute phase protein and antioxidant synthesis, and enhancement of the activity of the immune system. Cytokine production is enhanced by free radicals. Damage to the host may occur as a consequence. The deterious actions of these molecules are held in check by sophisticated antioxidant defences and systems which exert feedback control on cytokine biology. Nutrients have a profound effect upon the production and actions of cytokines. Protein energy malnutrition, dietary n-3 polyunsaturated fatty acids and vitamin E suppress cytokine production and actions. An opposite influence is exerted by n-6 polyunsaturated fatty acids, poor antioxidant defence, and supplementation of the diet with protein and branched chain amino acids. The synthesis of acute phase proteins and glutathione is dependent upon the adequacy of dietary sulphur amino acid intake. The consequences of the modulatory effects of previous and concurrent nutrient intake on cytokine biology are depletion of resources and damage to the host, which ranges from mild and temporary to severe, chronic or lethal.

Gene polymorphisms, inflammatory diseases and cancer.

Genes whose products play a critical role in regulation of the immune response include the human leucocyte antigen (HLA) and cytokine families of genes. The HLA genes are the most polymorphic found in the human genome, and the bulk of this polymorphism results in functional differences in expressed HLA molecules, resulting in inter-individual differences in presentation of peptide antigens to T-cells. In addition, a considerable number of cytokine-associated gene polymorphisms have been identified, the bulk of which occur in the upstream promoter sequences of these genes, which in many cases results in differential in vitro expression of the respective pro- or anti-inflammatory gene product. Particular HLA polymorphisms result in well-defined associations with a large number of immunologically-mediated diseases, including some diseases with known dietary risk factors. For example, individuals of HLA-DQA1*0501, DQB1*0201 genotype have a greater than 200-fold increased risk of developing intolerance to dietary wheat gluten (coeliac disease), and additional HLA-related factors may influence the development of malignant lymphoma within pre-existing coeliac disease. Similarly, HLA-DRB1 alleles sharing a common sequence motif constitute the primary known genetic risk factor for rheumatoid arthritis. The influence of polymorphisms associated with differential cytokine expression on disease susceptibility is currently of much interest. Most attention has been focused on associations with susceptibility to benign immunologically-mediated diseases, including a number of gut diseases. However, recent work from our laboratory indicates that cytokine polymorphisms may influence susceptibility to and prognosis in a number of different cancers, including malignant melanoma skin cancer and solid tumours which may be influenced by diet, such as prostate cancer (collaboration with the CRC/BPG UK Familial Prostate Cancer study). In addition, preliminary work suggests that dietary modulation of expression levels of certain cytokines in healthy human subjects may be genotype dependent.