Reto Muggli

The cardiovascular protective role of docosahexaenoic acid

Dietary fish oils rich in n-3 polyunsaturated fatty acids can modulate a diverse range of factors contributing to cardiovascular disease. This study examined the relative roles of eicosapentaenoic acid (20:5 n-3; EPA) and docosahexaenoic acid (22:6 n-3; DHA) which are the principal n-3 polyunsaturated fatty acids regarded as candidates for cardioprotective actions. At low dietary intakes (0.4-1.1% of energy (%en)), docosahexaenoic acid but not eicosapentaenoic acid inhibited ischaemia-induced cardiac arrhythmias. At intakes of 3.9-10.0%en, docosahexaenoic acid was more effective than eicosapentaenoic acid at retarding hypertension development in spontaneously hypertensive rats (SHR) and inhibiting thromboxane-like vasoconstrictor responses in aortas from SHR. In stroke-prone SHR with established hypertension, docosahexaenoic acid (3.9-10.0%en) retarded the development of salt-loading induced proteinuria but eicosapentaenoic acid alone was ineffective. The results demonstrate that purified n-3 polyunsaturated fatty acids mimic the cardiovascular actions of fish oils and imply that docosahexaenoic acid may be the principal active component conferring cardiovascular protection.

Collagen induced platelet aggregation: Requirement for tropocollagen multimers☆

Abstract Fibrillar collagen suspensions and neutral salt soluble collagen were investigated with regard to their platelet aggregating activity. The activity of fibrillar suspensions was highly dependent on the method of preparation and was observed to increase with the fineness of dispersion. Monodisperse tropocollagen was found not to aggregate platelets. However, aggregating activity developed after multimerizing the molecules by incubation at 37° C. At this stage no fibrils could be detected by measuring the opacity at 400 nm. It is proposed that the triple helix is an insufficient structure for promoting platelet aggregation; multimers of tropocollagen are required to induce this platelet reaction. Evidence for a close correlation between fibril precipitation and development of aggregating activity is presented. Treatment of soluble collagen with galactose oxidase was shown to interfere with multimerization and consequently to lead to a delayed development of aggregating activity. Enzyme treated and control collagen showed similar aggregating activities after multimerization at 37° C. Treatment of fibrillar collagen with galactose oxidase produced no loss in aggregating activity as compared to control collagen.

Collagen type III induced ex vivo thrombogenesis in humans. Role of platelets and leukocytes in deposition of fibrin.

Exposure of type III collagen coats on plastic cover slips in parallel-plate perfusion chambers to flowing nonanticoagulated human blood resulted in deposition of platelets and fibrin. Blood was drawn directly from an antecubital vein by an occlusive roller pump over the collagen coats in chambers having flow slits of different dimensions, so that wall shear rates of 100, 650, and 2600 s-1 were obtained at 10 ml/min. Coagulation was minimally activated during the passage of blood from the vein to the chamber as shown by fibrinopeptide A levels of 3.7 ng/ml after 5-minute perfusions. The surface coverage with platelets increased from 18% at 100 s-1 to 59% at 2600 s-1, and the corresponding thrombus volumes increased from 2 to 22 microns 3/microns 2, respectively. This contrasted with the coverage with fibrin on collagen, which decreased from 28% at 100 s-1 to 9% at 2600 s-1. Fibrin deposits on the thrombi covered 6% of the surface irrespective of the shear rate, indicating that some of the deposited platelets accelerated the deposition of fibrin. The type III collagen preparation did not activate factor XII and did not possess tissue factor activity, indicating that the surface itself was not procoagulant. However, a correlation between deposited leukocytes and surface coverage with fibrin was observed (r = 0.78, p less than 0.01), suggesting a role for these cells in the deposition of fibrin. The data demonstrate that thrombogenesis is triggered by pure type III collagen, although the deposition of fibrin is not initiated by the collagen itself but presumably by deposited leukocytes.(ABSTRACT TRUNCATED AT 250 WORDS)

[5] Measurements of platelet interaction with components of the vessel wall in flowing blood

Publisher Summary This chapter describes the methods that differentiate and quantify various platelet–surface interactions observed under in vitro conditions that mimic the sequence of events leading to thrombosis in various parts of the circulation in vivo . The chapter describes perfusion chambers and systems with laminar (nonturbulent) blood flow and the preparation of vascular and artificial surfaces, as well as blood perfusates for the study of platelet–surface interactions. By using perfusion systems, the importance of rheologic parameters and of various vessel-wall components for platelet–surface interactions can be understood. In the study discussed in the chapter, the results obtained from the blood of patients served as a framework for the characterization of some bleeding disorders. Quantitative techniques may improve the yield of information—for example, half of the test surface is used for radioactive counting, while the other half for morphometric evaluation. Panels of test samples are rapidly screened by means of radiolabeled platelets. Refined analysis of platelet–surface interactions is achieved only by means of cross-sectional morphometry. It is, however, important to keep in mind that washed platelets interact less extensively with the surface than nonwashed platelets. This may mask small differences in surface-mediated platelet interactions in comparative studies.

PHYSICAL FACTORS INFLUENCING PLATELET DEPOSITION ON SUBENDOTHELIUM: IMPORTANCE OF BLOOD SHEAR RATE*

Platelets circulate in the blood without attaching to each other or to the intimal lining. Removal of endothelial cells results in platelet deposition on the exposed subendothelial surface, the release of certain platelet constituents, and the formation of platelet aggregates. These platelet events play a basic role in both thrombogenesis and hemostasis. A knowledge of the factors controlling deposition is fundamental to the further understanding of these conditions. An annular perfusion chamber has been developed for the in vitro exposure of subendothelium under controlled blood flow c0nditions.l A range of physical and chemical variables has been studied with this The experimentally obtained values for platelet deposition compared satisfactorily with values predicted by available mass-transport theory. Theoretical analysis has indicated that the platelet attachment rate at the subendothelial surface is controlled predominantly by the transport of platelets through the blood7 and that this transport is substantially increased by the physical presence of red cells. The theoretical analysis will be reviewed and the implications of a diffusioncontrolled transport situation will be discussed with respect to measurement of platelet kinetics. Preliminary data using a perfusion chamber particularly designed to operate under nondiffusion-controlled conditions will be reported.

Prevention of nerve conduction deficit in diabetic rats by polyunsaturated fatty acids

The influence of diets containing gamma-linolenic acid (GLA; 18:3n-6) on sciatic nerve conduction velocity (NCV) was determined in diabetic rats. NCV was lower in diabetic rats fed diets supplemented with olive oil or sunflower seed oil than in nondiabetic rats; rats supplemented with GLA during a 5-wk diabetic period, however, did not exhibit significantly lower NCV. The mean proportion of the phospholipid fatty acid linoleic acid (18:2n-6) was higher in the sciatic nerves of diabetic rats than in the nondiabetic groups irrespective of dietary lipid treatment. Additionally, the proportion of linoleic acid was higher in the diabetic rats fed sunflower oil than in all other groups. Dietary GLA supplementation did not significantly influence the fatty acid composition of nerve membrane phospholipids and there was no obvious correlation between the fatty acid composition of nerve membrane phospholipids and NCV. The content of fructose and glucose in sciatic nerves was higher, whereas that of myo-inositol was lower, in diabetic rats than in nondiabetic rats; however, this was not significantly influenced by dietary GLA. GLA administration did not significantly influence Na(+)-K(+)-exchanging ATPase or ouabain binding activity in sciatic nerve preparations, both of which remained nonsignificantly different in the diabetic and nondiabetic groups. The results suggest that dietary GLA can prevent the deficit in NCV induced by diabetes and that this effect is independent of the nerve phospholipid fatty acid profile, sugar and polyol content, Na(+)-K(+)-exchanging ATPase activity, and ouabain binding. GLA may prevent the deficit in NCV indirectly, possibly by its role as a precursor of vasodilatory prostaglandins. These results confirm that GLA is the active component of evening primrose oil.

Determinants of plasma anti-oxidant vitamin levels in a population at high risk for stomach cancer.

Our objective was to identify the determinants of plasma levels of anti‐oxidant vitamins which have been linked with decreased risk of cancer and other chronic diseases. Correlation analyses were performed between baseline plasma levels of ascorbic acid, α‐ and β‐carotenes, cryptoxanthin, lycopene and α‐ and γ‐tocopherols and baseline information on dietary and other demographic and life‐style factors among 1,364 subjects 35–69 years of age, who are participants in a chemoprevention trial on pre‐cancerous lesions of the stomach in Venezuela. Males had lower levels of ascorbic acid, α‐ and β‐carotene and cryptoxanthin and higher levels of α‐tocopherol than females. This finding was confirmed in non‐smokers and non‐drinkers. In females, but not in males, age was positively associated with levels of ascorbic acid, cryptoxanthin, α‐ and β‐carotene and γ‐tocopherol. Male tobacco users had lower plasma levels of ascorbic acid, α‐ and β‐carotene and cryptoxanthin than non‐users, and regular alcohol drinkers had a decreased plasma levels of β‐carotene compared with non‐drinkers. Female tobacco users had lower levels of ascorbic acid and cryptoxanthin than non‐users, and regular alcohol drinkers had lower levels of ascorbic acid and lycopene than non‐drinkers. Frequencies of consumption of fresh fruits, fruit juice, raw vegetables and plantains showed weak positive associations with plasma levels of several vitamins studied in both sexes. Sex, age in females, tobacco and alcohol use and dietary consumption affected plasma anti‐oxidant vitamin levels in this population significantly. These factors may influence the effect of anti‐oxidant treatment in intervention trials.

IS A DIETARY N-3 FATTY ACID SUPPLEMENT ABLE TO INFLUENCE THE CARDIAC EFFECT OF THE PSYCHOLOGICAL STRESS?

Epidemiological studies suggest that n-3 polyunsaturated fatty acids (PUFA) are involved in the prevention of cardiovascular disease. Stress is known to increase the incidence of CVD and the present study was realised to evaluate some physiological and biochemical effects of dietary docosahexaenoic acid (DHA) in male Wistar rats subjected to a psycho social stress. Rats were fed for 8 weeks a semi-purified diet containing 10% of either sunflower seed oil or the same oil supplemented with DHA. This food supply represented 50% of their daily requirement. The remaining 50% were supplied as 45 mg food pellets designed to induce stress in rats by an intermittent-feeding schedule process. The control group (n = 12) was fed the equivalent food ration as a single daily feeding. The physiological cardiovascular parameters were recorded by telemetry through a transmitter introduced in the abdomen. At the end of the experimentation, the heart and adrenals were withdrawn and the fatty acid composition and the catecholamine store were determined. Dietary DHA induced a pronounced alteration of the fatty acid profile of cardiac phospholipids (PL). The level of all the n-6 PUFAs was reduced while 22:6 n-3 was increased. The stress induced a significant increase in heart rate which was not observed in DHA-fed group. The time evolution of the systolic blood pressure was not affected by the stress and was roughly similar in the stressed rats of either dietary group. Conversely, the systolic blood pressure decreased in the unstressed rats fed DHA. Similar data were obtained for the diastolic blood pressure. The beneficial effect of DHA was also observed on cardiac contractility, since the dP/dtmax increase was prevented in the DHA-fed rats. The stress-induced modifications were associated with an increase in cardiac noradrenaline level which was not observed in DHA-fed rats. The fatty acid composition of adrenals was significantly related to the fatty acid intake particularly the neutral lipid fraction (NL) which incorporated a large amount of DHA. Conversely, n-3 PUFAs were poorly incorporated in adrenal phospholipids. Moreover the NL/PL ratio was significantly increased in the DHA fed rats. The amount of adrenal catecholamines did not differ significantly between the groups. These results show that a supplementation of the diet with DHA induced cardiovascular alterations which could be detected in conscious animals within a few weeks. These alterations were elicited by a reduced heart rate and systolic and diastolic blood pressure.

Influence of formulas with borage oil or borage oil plus fish oil on the arachidonic acid status in premature infants

Several studies have reported that feeding γ-linolenic acid (GLA) has resulted in no increase in arachidonic acid (AA) in newborns. This result was ascribed to the eicosapentaenoic acid (EPA)-rich fish oil used in these formulas. Docosahexaenoic acid (DHA) sources with only minor amounts of EPA are now available, thus the addition of GLA to infant formulas might be considered an alternative to AA supplementation. Sixty-six premature infants were randomized to feeding one of four formulas [ST: no GLA, no long-chain polyunsaturated fatty acids; BO: 0.6% GLA (borage oil); BO + FOLOW: 0.6% GLA, 0.3% DHA, 0.06% EPA; BO + FOHIGH: 0.6% GLA, 0.3% DHA, 0.2% EPA] or human milk (HM, nonrandomized) for 4 wk. Anthropometric measures and blood samples were obtained at study entry and after 14 and 28 d. There were no significant differences between groups in anthropometric measures, tocopherol, and retinol status at any of the studied time points. The AA content of plasma phospholipids was similar between groups at study start and decreased significantly until day 28 in all formulafed groups, but not in the breast-fed infants [ST: 6.6±0.2%, BO: 6.9±0.3%, BO + FOLOW: 6.9±0.4%, BO + FOHIGH: 6.7±0.2%, HM: 8.6±0.5%, where values are reported as mean ±standard error; all formulas significantly different (P≤0.05) from HM]. There was no significant influence of GLA or fish oil addition to the diet. GLA had only a very limited effect on AA status which was too small to obtain satisfactory concentrations (concentrations similar to breast-fed babies) under the circumstances tested. The effect of GLA on AA is independent of the EPA and DHA content in the diet within the dose ranges studied.

Collagen-induced platelet aggregation: Native collagen quaternary structure is not an essential structural requirement

Abstract Collagen fibrils with different quaternary structures were precipitated from a collagen solution extracted from guinea pig skin with sodium chloride and compared with respect to their activities to induce platelet aggregation. Reconstituted native type fibrils were obtained by warming to 37°C, segment long spacing collagen by precipitation with adenosine-5′-triphosphoric acid, fibrous long spacing collagen by precipitation with chondreitin sulphate and nonstriated amorphous collagen by precipitation with alcohol. Presence, symmetry and periodicity of the banding pattern reflected the quaternary structure of the fibrils, i.e. the arrangement of the tropocollagen molecules with respect to polarity and staggering. All four precipitations induced platelet aggregation. The difference in activity did not necessarily reflect differences of intrinsic potency since it was not possible to standardize the degree of dispersion. It is concluded that a molecular arrangement of regular staggering - including the native structure - is not an essential determinant for the induction of platelet aggregation.