Elsevier

Atherosclerosis

Volume 175, Issue 2, August 2004, Pages 333-343
Atherosclerosis

Effects of oils rich in eicosapentaenoic and docosahexaenoic acids on the oxidizability and thrombogenicity of low-density lipoprotein

https://doi.org/10.1016/j.atherosclerosis.2004.04.004Get rights and content

Abstract

Consumption of oily fish and fish oils is associated with protection against cardiovascular disease. Paradoxically, long-chain polyunsaturated fatty acids present in low-density lipoprotein (LDL) are suggested to be susceptible to oxidation. It is not clear whether eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have similar effects on the susceptibility of LDL to oxidation or whether they affect the thrombogenicity of oxidized LDL. This study examined the influence of highly purified preparations of EPA and DHA on LDL oxidizability and LDL-supported thrombin generation in healthy human volunteers.

Forty-two healthy volunteers were randomly assigned to receive olive oil (placebo), an EPA-rich oil or a DHA-rich oil for 4 weeks at a dose of 9 g oil/day. EPA and DHA were incorporated into LDL phospholipids and cholesteryl esters during the supplementation period, but were progressively lost during ex vivo copper-mediated oxidation. Following supplementation, the EPA treatment significantly increased the formation of conjugated dienes during LDL oxidation compared with baseline, whereas the DHA treatment had no effect. Neither treatment significantly affected the lag time for oxidation, oxidation rate during the propagation phase or maximum diene production. Neither EPA nor DHA significantly affected the thrombotic tendency of oxidized LDL compared with the placebo, although DHA tended to decrease it.

In conclusion, there are subtle differences in the effects of EPA and DHA on the oxidizability and thrombogenicity of LDL. DHA does not appear to increase the susceptibility of LDL to oxidation to the same degree as EPA and has a tendency to decrease LDL-supported thrombin generation.

Introduction

There is epidemiological evidence that consumption of fish or of long-chain n-3 polyunsaturated fatty acids (PUFA), found in oily fish and fish oils, protects against cardiovascular disease in Western populations [1], [2], [3], [4], [5]. Secondary prevention studies, providing long-chain n-3 PUFA to patients who had already suffered a myocardial infarction (MI), demonstrate significant benefit [6], [7], [8]. This effect might be due to anti-thrombotic [9] and anti-arrhythmic actions of n-3 PUFA [10] or to an increase in plaque stability [11].

Long-chain n-3 PUFA, such as EPA and DHA, are usually consumed in small quantities, and are therefore found in relatively low proportions in plasma and tissue lipids. However, increased consumption of these fatty acids is marked by an increase in their proportion in various blood and tissue lipid pools, including LDL cholesteryl esters and phospholipids [12]. Paradoxically, enrichment of LDL with n-3 PUFA is thought to enhance the susceptibility of LDL for oxidation [13], [14], [15], [16], [17], [18], [19]. The susceptibility of LDL to oxidation is influenced by its PUFA content (amount of substrate available for oxidation) and its antioxidant content (confer resistance to oxidation). Thus, at a given antioxidant content, increasing the PUFA content (or the number of double bonds) of LDL should increase its susceptibility to oxidation. A number of studies have investigated the effects of dietary or supplemented fish oil on the susceptibility of LDL to oxidation and demonstrated a marked reduction in the lag phase for oxidation of LDL by fish oil [13], [14], [15], [16], [17], [18], [19]. However, not all studies agree that n-3 PUFA increase overall susceptibility to oxidation [12], [20] and it is not clear whether the two major n-3 PUFA in fish oil, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA), are equally potent in decreasing the lag time for oxidation. Purified preparations of EPA and DHA have been demonstrated to have differential effects on blood pressure [21], heart rate [21], vascular reactivity [22], blood lipids [23], [24] and T lymphocyte activation [25]. In general, supplementation with DHA decreased all of the above, whereas EPA was not as potent and, in some cases, had no effect.

Thrombin plays a key role in haemostasis, catalyzing the formation of fibrin from fibrinogen and amplifying the coagulation cascade through the activation of factors V, VIII and XI. Thrombin also activates platelets and promotes inflammatory activities, which are associated with the initiation of thrombosis, one of the potentially fatal clinical consequences of atherosclerosis. Thrombin is formed by catalysis of prothrombin in the presence of the prothrombinase complex, which results from the assembly of activated factor V and activated factor X on a phospholipid membrane. This is usually provided in vivo by platelets [26], but may also be provided by the phospholipid surfaces of plasma lipoproteins [27]. Oxidized LDL (oxLDL) has been reported to support a significantly higher rate of thrombin generation in vitro than native LDL [28]. However, there is currently no information regarding the influence of n-3 PUFA on the ability of LDL to support thrombin generation. It was hypothesised that incorporation of these fatty acids into LDL phospholipids may alter the ability of oxLDL to support the prothrombinase complex and thereby alter its thrombotic tendency.

The aim of the current study was to investigate the influence of enrichment of LDL phospholipids with either EPA or DHA on the susceptibility of LDL to oxidation and on the ability of native and oxLDL to support thrombin generation.

Section snippets

Materials and reagents

Bovine serum albumin (BSA), CuSO4, EDTA, NaCl, Trizma (Tris base), butylated hydroxytoluene and gas chromatography standards were purchased from Sigma (Germany). Solvents, CaCl2 and KBr were obtained from Fisher Scientific (UK). Phosphate-buffered saline (PBS) was purchased from Oxoid (UK), and citric acid from BDH Laboratory Supplies (UK). Highly purified human plasma-derived prothrombin, and human coagulation factors V (FV) and activated factor X (FXa) were obtained from Diagnostica Stago

Effect of EPA versus DHA on LDL oxidation

The generation of conjugated dienes during copper-mediated oxidation of LDL is shown in Fig. 1. There was a significant effect of treatment following supplementation with the placebo and the EPA-rich oil. The placebo treatment resulted in a decrease in the generation of conjugated dienes at a few time points during the propagation phase, while supplementation with the EPA-rich oil resulted in a significant increase in the level of conjugated dienes throughout the oxidation process compared with

Discussion

This paper describes, for the first time, the separate effects of EPA and DHA on the susceptibility of LDL to oxidation. The placebo treatment resulted in a decrease in conjugated diene formation during the propagation phase of oxidation and a significant increase in the lag time, suggesting protective effects of this treatment on LDL oxidizability. Although the placebo treatment consisted of a relatively low dose of olive oil, other studies have also reported protective effects of olive oil on

Acknowledgements

This work was funded by The Nutricia Research Foundation. The capsules used in the study were a generous gift from Ocean Nutrition Ltd. (Bedford, Nova Scotia, Canada). MDM was funded by a post-doctoral scholarship from the University of Granada and the Ministry of Education, Spain. The authors would like to thank Dr. N.A. Englyst for advice on the thrombin assay.

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