N-3 fatty acid supplementation mediates lipid profile, including small dense LDL, when combined with statins: a randomized double blind placebo controlled trial

In this randomized, parallel-group, double-blind, placebo-controlled trial of men and women with hyperlipidemia on statins, 4 g/d EPA + DHA supplementation produced significant reductions in the primary endpoints of non-HDL-C and LDL-III particle concentration (− 9.47 ± 4.58, p < 0.001 and − 67.5 (− 100, − 31.25), P < 0.01, respectively) compared with OO.

There are relatively few studies investigating the role of marine n-3 FA in altering LDL-C particle phenotype. In a recent randomized controlled trial (n = 53), 4 g/day prescription n-3 FA supplementation for 8 weeks resulted in significant increases in LDL particle sizes and significant decreases in blood lipid, lipoprotein and apolipoprotein concentrations in the intervention arm [29]. An earlier single-blind placebo controlled study that randomized hyperlipidemic subjects (n = 33) into three groups (pravastatin, 6 g/day EPA and DHA, placebo) initially for 6 weeks and then provided combined pravastatin and 6 g/day EPA and DHA therapy to all subjects for 12 weeks, reported a significant increase in LDL stokes’ diameter from 25.0 to 25.9 nm (P < 0.05) in the n-3 FA group, although LDL III particle concentration was not measured in this study [30]. In another randomized controlled trial (n = 42), in which subjects were randomized to atorvastatin alone or atorvastatin and 1.68 g/day EPA and DHA for 5 weeks after dietary run in, a significant reduction in LDL III particle concentration (− 1.23 mmol/L, P < 0.05) in the treatment arm was reported [21]. However, this change was not significantly different when compared to the change in the placebo group. Maki et al. found a significant increase in LDL-C particle size from 19.9 (19.2, 22.0) nm to 20.4 (19.3, 21.7) nm (P = 0.024) in their cross-over study (n = 39) when 4 g/day EPA and DHA supplementation was combined with simvastatin [22]. Different from a previous study [30], Maki et al. also measured LDL III particle concentration and reported no significant change between control and intervention arms. However, in more recent studies, n-3 FA together with statins partially improved both lipoprotein particle size and concentration [25, 26, 29]. It is of particular importance that this present study showed a difference in LDL III particle concentration, given the fact that LDL III and IV particle concentration is suggested to be a stronger predictor of CVD than LDL-C particle size [31, 32].

Previous studies have shown similar results regarding the effect of n-3 FA supplementation on serum non-HDL-C concentrations. The COMBOS trial reported a 9.1% reduction (P < 0.001) in non-HDL-C with dietary supplementation of 4 g/day EPA and DHA during 40 mg/day simvastatin therapy compared to 2.2% (P < .001) with corn oil [17]. The same dose of n-3 FA and statin caused 6.9% reduction (P < 0.01) in ESPIRIT trial, compared to 0.9% (P > 0.05) with olive oil [18]. Similarly, in respective studies of combination therapy, approximately 10% significant reduction in non-HDL-C was reported [16, 21, 24, 33]. Higher reductions in have also reported in the past. Maki et al. for instance, showed 40% reduction (P < 0.001) in their study of a similar design with 39 patients, however, the control group also showed a large significant reduction in serum non-HDL-C concentrations (34%, P < 0.001) [22]. Furthermore, in the ANCHOR trial, a 15% reduction (P < 0.0001) in non-HDL-C was also reported [20]. This could be due to the use of EPA ethyl esters alone in this study, given that EPA might lead to greater average reductions in non-HDL-C than DHA [34]. However, little is known about the individual effects of these fatty acids on distinct lipids, and studies suggest they have complementary roles to each other, therefore their combined use is more widely preferred, as in the present study.

In the present study, the decrease in non-HDL-C cholesterol in the n-3 FA group was likely achieved mainly due to lowering of VLDL concentrations (− 36.88 ± 11.75%, P < 0.0001), as well as other triglyceride-rich lipoproteins such as chylomicron remnants. The significant reduction in VLDL-C concentration is consistent with the previous evidence [17,18,19,20, 22]. In human physiology, increased concentrations of VLDL-C particles positively correlate with increased TG concentrations. TG-reducing effects of n-3 FAs are mediated by transcription of several nuclear receptors that play a key role in lipid metabolism, including PPAR-α, which increases fatty acid oxidation in the liver, adipose, heart and skeletal muscle, as well as sterol regulatory element binding proteins (SREBP), especially SREBP-1c, the major activator of hepatic lipogenesis [11]. PUFA metabolites, such as eicosanoids and oxylipins, are potent activators of PPARs [35]. Through these mechanisms, marine n-3 FA may downregulate VLDL metabolism through decreasing TG synthesis and increasing triglyceride clearance.

The significant 21.51 ± 12.15% (P < 0.001) reduction in TG in the present study is slightly lower in magnitude than the findings of some o previous studies. Kastelein et al. reported a 31% reduction (P < 0.001) and Bays et al. reported 33% (P < 0.0001) reduction in serum TG concentrations with 4 g/day EPA and DHA monotherapy [12, 13]. When the same n-3 FA dose was combined with a statin, a 28% reduction was reported [17]. Similarly, in a randomized controlled trial of hyperlipidemic patients (n = 56), co-administration of 4 g/day n-3 FA with statin treatment for 16 weeks reduced serum TG concentrations more effectively than statin monotherapy (− 34.8% vs. -15.2%, P = 0.0176) [36]. The relatively smaller reduction in TG achieved in the present study may be due to the difference in the baseline characteristics of the patients when compared to the aforementioned studies. Mean (±SD) baseline serum TG concentrations of the intervention group in the present study (145.05 ± 52.67 mg/dL, 1.62 ± 0.60 mmol/l) were much lower than the EVOLVE and MARINE trials (655 and 679 mg/dL, 7.40 and 7.67 mmol/l, median values, respectively) and the percent reduction in TG concentrations highly depends on the baseline values [37, 38]. This is supported by the mean (±SD) baseline TG concentration of the n-3 FA groups in ESPIRIT trial which was 287 ± 82.8 mg/dL (3.25 ± 0/94 mmol/l) and the study achieved a reduction in serum TG concentration of − 20%, (P < 0.01), similar to the present study [18].

Reducing serum TG concentration is important for two reasons. Firstly, TG, as a substrate of LDL-C synthesis, is deemed a target in clinical management of dyslipidemia and each 1 mmol/l reduction in TG concentrations is believed to reduce CVD risk by 14% in men and 37% in women [39]. More importantly, it has been long known that clinically significant reductions in TG concentrations are typically accompanied by a shift in LDL particle size from smaller and denser particles (LDL III, IV) to larger and more buoyant particles (LDL-C I, II) [40]. Serum TG concentration are inversely related to the LDL-C particle size and it can been hypothesized that reducing TG would result in an increase in LDL-C particle size [41]. In the present study, the significant decrease in serum concentration of LDL III (− 67%, P < 0.01) from 3.5 mg/dL to 1 mg/dL in the n-3 FA group is likely to be linked to the significant decrease in serum TG.

In the placebo group, serum TC, TG and HDL-C concentrations at week 8 had shown minor significant changes from baseline. One possible explanation for changes in the placebo group in the present study is that late-onset effects of the dietary run-in and continued adherence to the healthy and balanced diet might have improved these variables.

Strengths and limitations

The main strength of the present study is its prospective, randomized, double-blinded and placebo-controlled design. Furthermore, the study population was carefully determined and there were no baseline differences between control and intervention groups. Additionally, small and dense LDL-C was measured with gel electrophoresis that is a strong method for detecting types of lipoprotein particles.

The present study also has certain limitations. First, the small sample size (although powered) and short intervention duration, limits the conclusions that can be drawn. Although the lipid alterations achieved in this study are consistent with the findings of larger studies with longer duration, findings should still be confirmed in a larger population. Furthermore, the capsule load was high (4 capsules per day), and compliance was measured only by capsule count. Ideally, compliance needs to be assessed by plasma and erythrocytes EPA and DHA content. Indeed, the plasma and erythrocyte fatty acid composition should be measured and compared in any further studies.

Like many other studies, this study tested a supplemental form of n-3 FA containing EPA and DHA in ethyl ester (EE) form. However, n-3 FA supplements are also available in TG form and there is ongoing debate about whether different chemical forms of EPA and DHA are absorbed in an identical way by the human body. Some previous findings suggest a comparable bioavailability, whereas others reported a higher bioavailability from TG. An earlier RCT (n = 150) that tested long-term (6 months) moderate consumption (1.68 g/day) of both chemical forms concluded that TG n-FA led to a faster and higher increase in the erythrocyte’s membrane EPA and DHA content [42]. In a more recent trial (n = 22), short term bioavailability of the EE and TG, measured by plasma concentrations of EPA, and DHA, did not differ after 24 hours a single oral dose of ∼1.2 g [43]. Taken together, there is limited evidence to compare bioavailability of EE and TG n-3 FA. However, recent studies point out that EPA and DHA in free fatty acids (FFA) form may have 4-fold greater bioavailability than n-3 FA ethyl esters given that their absorption does not involve pancreatic lipase. Particularly when taken on an empty stomach, the FFA formulation may have provided great flexibility in the dosing schedule. Unfortunately, this prescription n-3 FA was not available to the researchers.

The use of OO as a placebo may have had non-neutral effects on the outcome variables due to its high oleic acid content and the role of oleic acid in CVD prevention [44]. However, 4 g/day was deemed too low a dose to bias the result, especially when compared to the amount used in studies such as PREDIMED, which showed that 50 g/day use of OO reduced CVD risk [45], several studies have used OO as placebo given the lack of a true placebo.

The present study included participants with a normal body weight – which was preserved throughout the intervention. In obese subjects, similar results in lipid profile might be observed [46], also accompanied by a reduction in inflammatory markers [47].

Additionally, physical activity was not monitored throughout the trial. Physical activity is an important factor that effects lipid metabolism and ideally should be evaluated in studies looking into blood lipids.

Lastly, although the present studies and others have demonstrated beneficial effects of n-3 FAs in relation risk, it should be noted that dietary pattern, and more generally, lifestyle factors are stronger determinants of CVD risk compared to the effect of single nutrients alone.

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