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Abnormalities in lipoprotein profiles (size, distribution and concentration) play an important role in the pathobiology of atherosclerosis and coronary artery disease. Dietary fat, among other factors, has been demonstrated to modulate lipoprotein profiles. We aimed to investigate if background dietary fat (saturated, SFA versus omega-6 polyunsaturated fatty acids, n-6PUFA) was a determinant of the effects of LCn-3PUFA supplementation on lipoprotein profiles.A randomized controlled clinical intervention trial in a parallel design was conducted. Healthy subjects (n = 26) were supplemented with 400 mg eicosapentaenoic acid plus 2000 mg docosahexaenoic acid daily and randomized to consume diets rich in either SFA or n-6PUFA for a period of 6 weeks. Blood samples, collected at baseline and after 6 weeks of intervention, were assessed for plasma lipoprotein profiles (lipoprotein size, concentration and distribution in subclasses) determined using nuclear magnetic resonance spectroscopy.Study participants receiving the SFA or the n-6PUFA enriched diets consumed similar percentage energy from fat (41 and 42% respectively, P = 0.681). However, subjects on the SFA diet consumed 50% more energy as saturated fat and 77% less as linoleic acid than those consuming the n-6PUFA diet (P < 0.001). The diets rich in SFA and n-6PUFA reduced the concentration of total very-low-density lipoprotein (VLDL) particles (P < 0.001, both), and their subclasses and increased VLDL (P = 0.042 and P = 0.007, respectively) and LDL (P = 0.030 and 0.027, respectively) particle size. In addition, plasma triglyceride concentration was significantly reduced by LCn-3PUFA supplementation irrespective of the dietary fat.LCn-3PUFA modulated lipoprotein profiles in a similar fashion when supplemented in diets rich in either SFA or n-6PUFA.