High-Dose DHA Has More Profound Effects on LDL-Related Features Than High-Dose EPA: The ComparED Study

High-Dose DHA Has More Profound Effects on LDL-Related Features Than High-Dose EPA: The ComparED... Abstract Context Supplementation with high-dose docosahexaenoic acid (DHA) increases serum low-density lipoprotein (LDL) cholesterol (LDL-C) concentrations more than high-dose eicosapentaenoic acid (EPA). The mechanisms underlying this difference are unknown. Objective To examine the phenotypic change in LDL and mechanisms responsible for the differential LDL-C response to EPA and DHA supplementation in men and women at risk of cardiovascular disease. Design, Setting, Participants, and Intervention In a double-blind, controlled, crossover study, 48 men and 106 women with abdominal obesity and subclinical inflammation were randomized to a sequence of three treatment phases: phase 1, 2.7 g/d of EPA; phase 2, 2.7 g/d of DHA; and phase 3, 3 g/d of corn oil. All supplements were provided as three 1-g capsules for a total of 3 g/d. The 10-week treatment phases were separated by a 9-week washout period. Main Outcome Measure In vivo kinetics of apolipoprotein (apo)B100-containing lipoproteins were assessed using primed-constant infusion of deuterated leucine at the end of each treatment in a subset of participants (n = 19). Results Compared with EPA, DHA increased LDL-C concentrations (+3.3%; P = 0.038) and mean LDL particle size (+0.7 Å; P < 0.001) and reduced the proportion of small LDL (−3.2%; P < 0.01). Both EPA and DHA decreased proprotein convertase subtilisin/kexin type 9 concentrations similarly (−18.2% vs −25.0%; P < 0.0001 vs control). Compared with EPA, DHA supplementation increased both the LDL apoB100 fractional catabolic rate (+11.4%; P = 0.008) and the production rate (+9.4%; P = 0.03). Conclusions The results of the present study have shown that supplementation with high-dose DHA increases LDL turnover and contributes to larger LDL particles compared with EPA. Despite having favorable effects on serum triglyceride (TG) concentrations, cardiac arrhythmia, platelet aggregation, heart rate, blood pressure, and inflammation (1), the extent to which long-chain omega-3 polyunsaturated fatty acid (LCn3-PUFA) supplementation prevents cardiovascular disease (CVD) remains controversial (2). The inconsistent effect of LCn3-PUFAs supplementation on CVD risk might be because docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are also known to increase low-density lipoprotein (LDL) cholesterol (LDL-C) concentrations (1). The extent to which different LCn3-PUFAs have distinct effects on CVD risk also remains questionable because previous studies have shown that DHA is more potent than EPA in modulating cardiovascular risk factors, including increasing LDL-C concentrations (3, 4). To the best of our knowledge, no study has yet examined how DHA and EPA influence the mechanisms underlying their differential effect on several LDL features, including LDL-C concentrations and, hence, CVD risk. It has been stressed that the cholesterol content of LDL represents only one of several features of this complex lipoprotein, which is heterogeneous in size, charge, and protein content (5, 6). Different immunochemically defined LDL subclasses are thought to have distinct metabolisms and atherogenicity (6). Specifically, small and dense LDL (sdLDL) consists largely of apolipoprotein (apo)-CIII–containing lipoproteins that originate from the remodeling in plasma of apo-CIII–rich very-low-density lipoprotein (VLDL) (7). sdLDLs have also been consistently associated with an increased risk of developing CVD, independent of the LDL-C concentration (8). Data from a limited number of trials have suggested that, unlike EPA, high-dose DHA increases LDL particle size (9). In other studies, LCn3-PUFA supplementation (4 g/d) in hypertriglyceridemic men had no substantial impact on the features of the LDL size phenotype. However, this might have been because DHA and EPA were given in combination (10, 11). Although the clinical relevance of the potential differences between DHA and EPA in modulating LDL particle size is unclear, data have suggested that both LCn3-PUFAs might differentially influence LDL metabolism. Previous studies have investigated the effect of an LCn3-PUFA–rich diet or supplementation on the kinetic of apoB100–containing lipoproteins. Ooi et al. (12) have shown that a high-fish diet providing 1.23 g/d of EPA and DHA reduced the TG-rich lipoprotein (TRL) apoB100 concentration and production rate (PR) compared with a low-fish diet in elderly men and women with moderate hyperlipidemia. These studies also showed that the high-fish diet decreased TRL apoB100–direct catabolism, rechanneling TRL toward conversion into LDL and, hence, increasing the LDL PR. These data have been reproduced in other studies that used fish oils as supplements (13–17). Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates cholesterol metabolism by degrading cellular LDL receptors, blunting the clearance of LDL from the circulation (18). LCn3-PUFAs have been shown to decrease PCSK9 concentrations in several studies (19, 20), which should, in theory, be associated with increased LDL clearance and, hence, reduced serum LDL-C. To the best of our knowledge, no study has yet compared the effect of high-dose supplementation with DHA and EPA on the kinetics of apoB100–containing lipoproteins and PCSK9 as key determinants of LDL-C concentrations. The objective of the present study was, therefore, to examine the mechanisms underlying the differential effect of DHA and EPA supplementation on LDL features, including the LDL-C concentrations in men and women at risk of CVD. Specifically, we compared the effect of high doses of DHA and of EPA on the intravascular kinetics of apoB100-containing lipoproteins, VLDL apoCIII, LDL particle size distribution, and PCSK9 levels. We hypothesized that DHA has favorable effects on LDL size features compared with EPA and that changes in the intravascular kinetics of LDL are also different between DHA and EPA, thereby partly explaining the different effect of the two LCn3-PUFAs on the serum LDL-C concentrations. Materials and Methods Study design Details of the study design have been previously reported (4). In brief, the present study used a double-blind randomized, controlled crossover design with three treatment phases: phase 1, DHA; phase 2, EPA; phase 3, corn oil as the control. Each treatment phase had a median duration of 10 weeks, separated by a 9-week washout period. The participants were randomized to one of six treatment sequences and received supplementation with three identical 1-g capsules of >90% purified LCn3-PUFA daily, providing either 2.7 g/d of DHA or 2.7 g/d of EPA. Corn oil was used as the control (0 g/d of DHA plus EPA). LCn3-PUFA supplements were formulated as re-esterified TG and provided by Douglas Laboratories (Pittsburgh, PA). The participants were instructed to maintain a constant body weight during the course of the study and were counseled on how to exclude fatty fish (including salmon, tuna, mackerel, and herring), other LCn3-PUFA supplements, flax products, walnuts, and LCn3-PUFA–enriched products during the three study phases. The primary outcome of the present study was the change in C-reactive protein (CRP) with DHA and EPA supplementation (4). All participants signed an informed consent document that had been approved by local ethics committees at the beginning of the study, and the study protocol was registered at ClinicalTrials.gov (NCT01810003) on March 4, 2013. Study population The primary eligibility criteria were abdominal obesity using the International Diabetes Federation sex-specific cutoffs (≥80 cm for women, ≥94 cm for men) (21) and a screening plasma CRP concentration >1 mg/L but <10 mg/L. The participants had to be otherwise healthy. Adult participants (aged 18 to 70 years) were recruited at the Institute of Nutrition and Functional Foods. Their body weight had to be stable for ≥3 months before randomization. The exclusion criteria were plasma CRP >10 mg/L at screening, extreme dyslipidemia such as familial hypercholesterolemia, a personal history of CVD (coronary heart disease, cerebrovascular disease, or peripheral arterial disease), use of medications or substances known to affect inflammation (e.g., steroids, binging alcohol), and the use of LCn3-PUFA supplements within 2 months of study onset. However, individuals taking lipid-lowering drugs for >1 month were eligible. Anthropometry Anthropometric measures, including waist and hip circumferences, were measured according to standardized procedures before and after each study phase (22). Body weight was measured before each kinetic protocol. Compliance Compliance to supplementation was assessed by counting the supplements that were returned to the study coordinators by the participants (4). The DHA and EPA content in red blood cells was also used as another proxy of compliance for all participants (23). Laboratory analyses Blood samples were collected after a 12-hour overnight fast on 2 consecutive days at the end of each treatment phase. The mean of the two measurements was used in the analyses of the LDL features and blood glucose. The total apoB100, apoCIII, PCSK9, and insulin concentrations were measured once after each treatment phase. The serum total apoB100, apoCIII, and PCSK9 concentrations were measured using commercial ELISA kits [catalog no. A70102 (Alerchek Inc., Springvale, ME), catalog no. EA8133-1 (Assaypro LLC, St. Charles, MO), catalog no. CY-8079 (CircuLex, Nagano, Japan)]. Serum LDL-C concentrations were calculated using the Friedewald equation. Nondenaturing 2% to 16% polyacrylamide gradient gel electrophoresis was used to characterize various features of the LDL particle size phenotype (24), including the LDL peak particle size and mean LDL particle size and the proportion of LDL in the various size categories. Fasting blood glucose levels were measured using colorimetry, and insulin concentrations were measured using electrochemiluminescence (Roche Diagnostics, Indianapolis, IN). Finally, the homeostatic model assessment of insulin resistance was measured using the formula developed by Matthews et al. (25). All personnel involved in the measurements of the study outcomes were unaware of the treatments. Experimental protocol for in vivo stable isotope kinetics Kinetic studies using primed-constant infusion of deuterated leucine were performed at the end of each treatment in a subsample of the participants. The participants in the kinetic studies were recruited as a part of the general recruitment process in the project, until 20 participants had been reached. The participants underwent a primed-constant infusion of L-[5,5,5-D3] leucine while kept in a constant fed state to determine the kinetics of apoB100. Starting at 7:00 am, the participants received one small standardized snack every 30 minutes for 15 hours, each containing 1/30th of their estimate daily food intake, according to the Harris-Benedict equation (26), with 15% of the calories from proteins, 45% from carbohydrates, and 40% from fat. The snacks were the same for each treatment. At 10:00 am, with two intravenous lines in place (one for the infusate and one for blood sampling), L-[5,5,5-D3] leucine (10 µmol/kg body weight) was injected as an intravenous bolus and then by continuous infusion (10 µmol/kg body weight per hour) for a 12-hour period. Blood samples (24 mL) were collected at 0, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 11, and 12 hours. Data on sample processing, laboratory measurements, analysis of the lipoprotein PR, and fractional catabolic rate (FCR) are provided in the Supplemental Data. Statistical analysis Differences between treatments were assessed using the MIXED procedure for repeated measures, with treatment as fixed effect and a compound symmetry matrix to account for within-subject correlations (SAS, version 9.4; SAS Institute, Cary, NC). The change vs the control treatment (post-treatment DHA minus control and EPA minus control) was used as the dependent variable in all analyses. The main treatment effect in the mixed models reflected the direct comparison of DHA and EPA and was considered the primary analysis. Adjustment for multiple comparisons was not necessary, because the main treatment effect had only two levels (DHA and EPA). In the same model and as secondary analyses, the change vs control for each treatment was tested against the null hypothesis using the LSMEANS statement. The skewness in the distribution of model residuals was considered, and the data were log-transformed when required. Wilcoxon signed-rank tests were also performed to test for the difference in the change from control after DHA and EPA supplementation, with results similar to those generated by the mixed models (data not shown). Spearman coefficient correlations among the changes in apoB100- and apoCIII-containing lipoprotein kinetic parameters, PCSK9 levels, LDL-C, LDL-apoB100, and LDL size were computed. Results Data from one participant who was ill during the first kinetic study test were excluded from the analyses. The baseline characteristics of the 19 participants who completed at least one kinetic substudy and the participants of the whole sample who completed at least one study phase are presented in Table 1. The characteristics of the subsample were similar to those of the whole group, with the exception that proportionally more women were included in the kinetic substudy. Among the participants of the kinetic substudy, one participant completed only one phase and two participants completed two study phases. The mean compliance rate based on the returned capsules was >95% for all study phases (data not shown). Among the 154 participants randomized to treatment sequences, 12 participants were taking statins. None were taking other lipid-lowering drugs. Pharmacotherapy remained unchanged in all participants throughout the study in the present crossover trial. None of the kinetic substudy participants were taking lipid-lowering drugs. Three participants had type 2 diabetes and one had type 1 diabetes; however, none of these participants were included in the substudy. Table 1. Baseline Characteristics of the Kinetic Subsample and Whole Cohort Characteristic Subsample (n = 19) Whole Cohort (n = 138) Age, y 47±16 52 ± 15 Female, n (%) 9 (47) 96 (70) Weight, kg 84.0 ± 13.8 80.6 ± 14.5 BMI, kg/m2 29.7 ± 4.8 29.4 ± 4.3 Waist circumference, cm 102.5 ± 9.9 100.7 ± 11.1 SBP, mm Hg 115.2 ± 8.5 115.6 ± 12.8 DBP, mm Hg 68.0 ± 8.1 69.7 ± 8.9 Total C, mmol/La 5.08 ± 0.64 5.18 ± 0.93 LDL-C, mmol/La 3.05 ± 0.55 3.03 ± 0.81 HDL-C, mmol/La 1.43 ± 0.43 1.52 ± 0.42 TG, mmol/La 1.30 ± 0.50 1.36 ± 0.58 CRP, mg/L 3.35 ± 2.71 3.32 ± 2.44 Glucose, mmol/La 5.26 ± 0.37 5.28 ± 0.85 Insulin, pmol/L 93.4 ± 39.9 102.54 ± 58.61 HOMA-IRa 3.11 ± 1.27 3.55 ± 2.57 Characteristic Subsample (n = 19) Whole Cohort (n = 138) Age, y 47±16 52 ± 15 Female, n (%) 9 (47) 96 (70) Weight, kg 84.0 ± 13.8 80.6 ± 14.5 BMI, kg/m2 29.7 ± 4.8 29.4 ± 4.3 Waist circumference, cm 102.5 ± 9.9 100.7 ± 11.1 SBP, mm Hg 115.2 ± 8.5 115.6 ± 12.8 DBP, mm Hg 68.0 ± 8.1 69.7 ± 8.9 Total C, mmol/La 5.08 ± 0.64 5.18 ± 0.93 LDL-C, mmol/La 3.05 ± 0.55 3.03 ± 0.81 HDL-C, mmol/La 1.43 ± 0.43 1.52 ± 0.42 TG, mmol/La 1.30 ± 0.50 1.36 ± 0.58 CRP, mg/L 3.35 ± 2.71 3.32 ± 2.44 Glucose, mmol/La 5.26 ± 0.37 5.28 ± 0.85 Insulin, pmol/L 93.4 ± 39.9 102.54 ± 58.61 HOMA-IRa 3.11 ± 1.27 3.55 ± 2.57 Data presented as mean ± SD. CRP values >10 were excluded (n = 5 for the whole cohort). For the whole cohort, the baseline characteristics of participants who completed at least one study phase are presented. Abbreviations: BMI, body mass index; C, cholesterol; DBP, diastolic blood pressure; HDL, high-density lipoprotein; HOMA-IR, homeostatic model assessment of insulin resistance; SBP, systolic blood pressure. a For the whole cohort, n = 137. View Large Table 1. Baseline Characteristics of the Kinetic Subsample and Whole Cohort Characteristic Subsample (n = 19) Whole Cohort (n = 138) Age, y 47±16 52 ± 15 Female, n (%) 9 (47) 96 (70) Weight, kg 84.0 ± 13.8 80.6 ± 14.5 BMI, kg/m2 29.7 ± 4.8 29.4 ± 4.3 Waist circumference, cm 102.5 ± 9.9 100.7 ± 11.1 SBP, mm Hg 115.2 ± 8.5 115.6 ± 12.8 DBP, mm Hg 68.0 ± 8.1 69.7 ± 8.9 Total C, mmol/La 5.08 ± 0.64 5.18 ± 0.93 LDL-C, mmol/La 3.05 ± 0.55 3.03 ± 0.81 HDL-C, mmol/La 1.43 ± 0.43 1.52 ± 0.42 TG, mmol/La 1.30 ± 0.50 1.36 ± 0.58 CRP, mg/L 3.35 ± 2.71 3.32 ± 2.44 Glucose, mmol/La 5.26 ± 0.37 5.28 ± 0.85 Insulin, pmol/L 93.4 ± 39.9 102.54 ± 58.61 HOMA-IRa 3.11 ± 1.27 3.55 ± 2.57 Characteristic Subsample (n = 19) Whole Cohort (n = 138) Age, y 47±16 52 ± 15 Female, n (%) 9 (47) 96 (70) Weight, kg 84.0 ± 13.8 80.6 ± 14.5 BMI, kg/m2 29.7 ± 4.8 29.4 ± 4.3 Waist circumference, cm 102.5 ± 9.9 100.7 ± 11.1 SBP, mm Hg 115.2 ± 8.5 115.6 ± 12.8 DBP, mm Hg 68.0 ± 8.1 69.7 ± 8.9 Total C, mmol/La 5.08 ± 0.64 5.18 ± 0.93 LDL-C, mmol/La 3.05 ± 0.55 3.03 ± 0.81 HDL-C, mmol/La 1.43 ± 0.43 1.52 ± 0.42 TG, mmol/La 1.30 ± 0.50 1.36 ± 0.58 CRP, mg/L 3.35 ± 2.71 3.32 ± 2.44 Glucose, mmol/La 5.26 ± 0.37 5.28 ± 0.85 Insulin, pmol/L 93.4 ± 39.9 102.54 ± 58.61 HOMA-IRa 3.11 ± 1.27 3.55 ± 2.57 Data presented as mean ± SD. CRP values >10 were excluded (n = 5 for the whole cohort). For the whole cohort, the baseline characteristics of participants who completed at least one study phase are presented. Abbreviations: BMI, body mass index; C, cholesterol; DBP, diastolic blood pressure; HDL, high-density lipoprotein; HOMA-IR, homeostatic model assessment of insulin resistance; SBP, systolic blood pressure. a For the whole cohort, n = 137. View Large LDL particle size and PCSK9 Blood lipids and LDL particle size features before the control phase and after the three treatments are presented in Table 2 for all participants and for the substudy group only. The treatment-specific baseline values before DHA and EPA were essentially identical to the values measured before the control treatment and therefore were not presented. In all participants, DHA increased the mean LDL particle size (compared with the control: DHA, +0.32 Å; EPA, −0.41 Å; DHA vs EPA, P < 0.0001) and LDL peak particle size (compared with control: DHA, +0.58 Å; EPA, −0.32 Å; DHA vs EPA, P < 0.0001) more than did EPA. The change in the proportion of sdLDL was also significantly different statistically between EPA and DHA (compared with control: DHA, −1.10%; EPA, +2.10%; EPA vs DHA, P < 0.002). Both EPA and DHA decreased the PCSK9 concentrations similarly (compared with control: DHA, −25.0 ng/mL; EPA, −18.2 ng/mL; DHA vs EPA, P = 0.19). The changes in these cardiometabolic outcomes with DHA and EPA were generally similar in direction and magnitude among the participants of the substudy. However, only the difference between the change in the PCSK9 concentrations after DHA and EPA compared with control remained statistically significant in the subsample. Table 2. LDL Particle Size Features and PCSK9 Concentration Before and After Control, DHA, and EPA Phase in Whole and Kinetic Sample Variable Before Control Phase After Control Phase After DHA Difference (DHA vs Control), % P Value (DHA vs Controla) After EPA Difference (EPA vs Control), % P Value (EPA vs Controla) Difference (DHA vs ΔEPA) P Value (DHA vs EPAb) All participants (n = 121)c  Total cholesterol, mmol/Ld,e 5.29 ± 0.08 5.16 ± 0.08 5.33 ± 0.08 3.1 0.001f 5.15 ± 0.08 −0.4 0.62 3.6 < 0.001f  LDL-C, mmol/Ld,e 3.12 ± 0.07 2.99 ± 0.07 3.16 ± 0.08 5.4 < 0.001f 3.06 ± 0.07 2.5 0.046f 3.2 0.04f  Total apoB, g/Ld,e 1.34 ± 0.04 1.31 ± 0.04 1.34 ± 0.04 2.8 0.02f 1.32 ± 0.04 1.2 0.46 1.6 0.16  TG, mmol/Ld,e 1.34 ± 0.06 1.38 ± 0.06 1.14 ± 0.04 −21.3 < 0.001f 1.23 ± 0.05 −11.3 < 0.001f −7.1 0.005f  Mean LDL size, Å 252.10 ± 0.26 251.89 ± 0.21 252.15 ± 0.23 0.1 0.051 251.41 ± 0.20 −0.2 0.01f 0.3 < 0.001f  LDL peak, Å 251.74 ± 0.28 251.56 ± 0.24 252.08 ± 0.25 0.2 0.001f 251.19 ± 0.23 −0.1 0.059 0.4 < 0.001f  Proportion of large LDL, % 9.49 ± 0.72 9.00 ± 0.53 9.16 ± 0.62 1.8 0.85 8.42 ± 0.53 −6.4 0.20 8.8 0.19  Proportion of small LDL, % 69.29 ± 1.45 69.83 ± 1.20 68.88 ± 1.34 −1.4 0.25 72.00 ± 1.08 3.1 0.03f −4.3 0.002f  PCSK9, ng/mL NA 213.02 ± 5.65 189.12 ± 4.89 −12.6 < 0.001f 194.98 ± 5.40 −8.5 < 0.001f −3.0 0.19 Substudy (n = 19)g  Total cholesterol, mmol/L 5.18 ± 0.16 4.70 ± 0.16 5.13 ± 0.14 9.1 0.005f 5.01 ± 0.19 8.0 0.018f 2.5 0.62  LDL-C, mmol/L 3.11 ± 0.14 2.70 ± 0.13 3.10 ± 0.15 14.7 0.006f 3.05 ± 0.14 14.4 0.010f 1.6 0.88  Total apoB, g/L 1.37 ± 0.09 1.26 ± 0.08 1.40 ± 0.12 11.0 0.03f 1.35 ± 0.09 6.9 0.27 3.5 0.27  TG, mmol/L 1.29 ± 0.13 1.35 ± 0.17 1.20 ± 0.15 −11.1 0.10 1.39 ± 0.21 4.6 0.63 −13.6 0.06  Mean LDL size, Å 250.88 ± 0.75 250.95 ± 0.54 250.95 ± 0.63 0.0 0.90 250.42 ± 0.60 −0.2 0.29 0.2 0.33  LDL peak, Å 250.62 ± 0.76 250.68 ± 0.63 250.79 ± 0.71 0.0 0.89 250.20 ± 0.61 −0.2 0.12 0.2 0.17  Proportion of large LDL, % 7.52 ± 1.38 6.90 ± 1.07 7.01 ± 1.35 1.5 0.34 7.42 ± 1.12 4.2 0.12 −5.6 0.54  Proportion of small LDL, % 75.03 ± 3.16 76.13 ± 2.32 75.53 ± 2.88 −0.8 0.56 75.98 ± 2.55 0.0 0.72 −0.6 0.86  PCSK9, ng/mL NA 196.92 ± 15.23 174.43 ± 13.53 −11.4 0.11 190.08 ± 15.33 1.2 0.75 −8.2 0.04f Variable Before Control Phase After Control Phase After DHA Difference (DHA vs Control), % P Value (DHA vs Controla) After EPA Difference (EPA vs Control), % P Value (EPA vs Controla) Difference (DHA vs ΔEPA) P Value (DHA vs EPAb) All participants (n = 121)c  Total cholesterol, mmol/Ld,e 5.29 ± 0.08 5.16 ± 0.08 5.33 ± 0.08 3.1 0.001f 5.15 ± 0.08 −0.4 0.62 3.6 < 0.001f  LDL-C, mmol/Ld,e 3.12 ± 0.07 2.99 ± 0.07 3.16 ± 0.08 5.4 < 0.001f 3.06 ± 0.07 2.5 0.046f 3.2 0.04f  Total apoB, g/Ld,e 1.34 ± 0.04 1.31 ± 0.04 1.34 ± 0.04 2.8 0.02f 1.32 ± 0.04 1.2 0.46 1.6 0.16  TG, mmol/Ld,e 1.34 ± 0.06 1.38 ± 0.06 1.14 ± 0.04 −21.3 < 0.001f 1.23 ± 0.05 −11.3 < 0.001f −7.1 0.005f  Mean LDL size, Å 252.10 ± 0.26 251.89 ± 0.21 252.15 ± 0.23 0.1 0.051 251.41 ± 0.20 −0.2 0.01f 0.3 < 0.001f  LDL peak, Å 251.74 ± 0.28 251.56 ± 0.24 252.08 ± 0.25 0.2 0.001f 251.19 ± 0.23 −0.1 0.059 0.4 < 0.001f  Proportion of large LDL, % 9.49 ± 0.72 9.00 ± 0.53 9.16 ± 0.62 1.8 0.85 8.42 ± 0.53 −6.4 0.20 8.8 0.19  Proportion of small LDL, % 69.29 ± 1.45 69.83 ± 1.20 68.88 ± 1.34 −1.4 0.25 72.00 ± 1.08 3.1 0.03f −4.3 0.002f  PCSK9, ng/mL NA 213.02 ± 5.65 189.12 ± 4.89 −12.6 < 0.001f 194.98 ± 5.40 −8.5 < 0.001f −3.0 0.19 Substudy (n = 19)g  Total cholesterol, mmol/L 5.18 ± 0.16 4.70 ± 0.16 5.13 ± 0.14 9.1 0.005f 5.01 ± 0.19 8.0 0.018f 2.5 0.62  LDL-C, mmol/L 3.11 ± 0.14 2.70 ± 0.13 3.10 ± 0.15 14.7 0.006f 3.05 ± 0.14 14.4 0.010f 1.6 0.88  Total apoB, g/L 1.37 ± 0.09 1.26 ± 0.08 1.40 ± 0.12 11.0 0.03f 1.35 ± 0.09 6.9 0.27 3.5 0.27  TG, mmol/L 1.29 ± 0.13 1.35 ± 0.17 1.20 ± 0.15 −11.1 0.10 1.39 ± 0.21 4.6 0.63 −13.6 0.06  Mean LDL size, Å 250.88 ± 0.75 250.95 ± 0.54 250.95 ± 0.63 0.0 0.90 250.42 ± 0.60 −0.2 0.29 0.2 0.33  LDL peak, Å 250.62 ± 0.76 250.68 ± 0.63 250.79 ± 0.71 0.0 0.89 250.20 ± 0.61 −0.2 0.12 0.2 0.17  Proportion of large LDL, % 7.52 ± 1.38 6.90 ± 1.07 7.01 ± 1.35 1.5 0.34 7.42 ± 1.12 4.2 0.12 −5.6 0.54  Proportion of small LDL, % 75.03 ± 3.16 76.13 ± 2.32 75.53 ± 2.88 −0.8 0.56 75.98 ± 2.55 0.0 0.72 −0.6 0.86  PCSK9, ng/mL NA 196.92 ± 15.23 174.43 ± 13.53 −11.4 0.11 190.08 ± 15.33 1.2 0.75 −8.2 0.04f Data presented as unadjusted mean ± SEM. Abbreviation: NA, not available. a This analysis compared the change with DHA or EPA compared with control based on posttreatment values; P values were taken from the main treatment effect in the mixed models. b P values for the EPA and DHA changes vs control, as determined by the LSMEANS statement and tested against the null hypothesis in the mixed models. c Participants equaled 123 for DHA, 121 for EPA, 125 for control, and 138 for baseline. d Log-transformed data were used in these analyses due to skewness of the distribution of the values. e Previously reported (4). f P < 0.05; because pre-DHA and -EPA values were essentially identical to the precontrol values, only precontrol values are presented. Models were adjusted for sex, weight, age, sequence; baseline value was considered only when these covariates were found to be significant at P < 0.05 in the models. g Participants equaled 19 for DHA, 17 for EPA, and 19 for control. View Large Table 2. LDL Particle Size Features and PCSK9 Concentration Before and After Control, DHA, and EPA Phase in Whole and Kinetic Sample Variable Before Control Phase After Control Phase After DHA Difference (DHA vs Control), % P Value (DHA vs Controla) After EPA Difference (EPA vs Control), % P Value (EPA vs Controla) Difference (DHA vs ΔEPA) P Value (DHA vs EPAb) All participants (n = 121)c  Total cholesterol, mmol/Ld,e 5.29 ± 0.08 5.16 ± 0.08 5.33 ± 0.08 3.1 0.001f 5.15 ± 0.08 −0.4 0.62 3.6 < 0.001f  LDL-C, mmol/Ld,e 3.12 ± 0.07 2.99 ± 0.07 3.16 ± 0.08 5.4 < 0.001f 3.06 ± 0.07 2.5 0.046f 3.2 0.04f  Total apoB, g/Ld,e 1.34 ± 0.04 1.31 ± 0.04 1.34 ± 0.04 2.8 0.02f 1.32 ± 0.04 1.2 0.46 1.6 0.16  TG, mmol/Ld,e 1.34 ± 0.06 1.38 ± 0.06 1.14 ± 0.04 −21.3 < 0.001f 1.23 ± 0.05 −11.3 < 0.001f −7.1 0.005f  Mean LDL size, Å 252.10 ± 0.26 251.89 ± 0.21 252.15 ± 0.23 0.1 0.051 251.41 ± 0.20 −0.2 0.01f 0.3 < 0.001f  LDL peak, Å 251.74 ± 0.28 251.56 ± 0.24 252.08 ± 0.25 0.2 0.001f 251.19 ± 0.23 −0.1 0.059 0.4 < 0.001f  Proportion of large LDL, % 9.49 ± 0.72 9.00 ± 0.53 9.16 ± 0.62 1.8 0.85 8.42 ± 0.53 −6.4 0.20 8.8 0.19  Proportion of small LDL, % 69.29 ± 1.45 69.83 ± 1.20 68.88 ± 1.34 −1.4 0.25 72.00 ± 1.08 3.1 0.03f −4.3 0.002f  PCSK9, ng/mL NA 213.02 ± 5.65 189.12 ± 4.89 −12.6 < 0.001f 194.98 ± 5.40 −8.5 < 0.001f −3.0 0.19 Substudy (n = 19)g  Total cholesterol, mmol/L 5.18 ± 0.16 4.70 ± 0.16 5.13 ± 0.14 9.1 0.005f 5.01 ± 0.19 8.0 0.018f 2.5 0.62  LDL-C, mmol/L 3.11 ± 0.14 2.70 ± 0.13 3.10 ± 0.15 14.7 0.006f 3.05 ± 0.14 14.4 0.010f 1.6 0.88  Total apoB, g/L 1.37 ± 0.09 1.26 ± 0.08 1.40 ± 0.12 11.0 0.03f 1.35 ± 0.09 6.9 0.27 3.5 0.27  TG, mmol/L 1.29 ± 0.13 1.35 ± 0.17 1.20 ± 0.15 −11.1 0.10 1.39 ± 0.21 4.6 0.63 −13.6 0.06  Mean LDL size, Å 250.88 ± 0.75 250.95 ± 0.54 250.95 ± 0.63 0.0 0.90 250.42 ± 0.60 −0.2 0.29 0.2 0.33  LDL peak, Å 250.62 ± 0.76 250.68 ± 0.63 250.79 ± 0.71 0.0 0.89 250.20 ± 0.61 −0.2 0.12 0.2 0.17  Proportion of large LDL, % 7.52 ± 1.38 6.90 ± 1.07 7.01 ± 1.35 1.5 0.34 7.42 ± 1.12 4.2 0.12 −5.6 0.54  Proportion of small LDL, % 75.03 ± 3.16 76.13 ± 2.32 75.53 ± 2.88 −0.8 0.56 75.98 ± 2.55 0.0 0.72 −0.6 0.86  PCSK9, ng/mL NA 196.92 ± 15.23 174.43 ± 13.53 −11.4 0.11 190.08 ± 15.33 1.2 0.75 −8.2 0.04f Variable Before Control Phase After Control Phase After DHA Difference (DHA vs Control), % P Value (DHA vs Controla) After EPA Difference (EPA vs Control), % P Value (EPA vs Controla) Difference (DHA vs ΔEPA) P Value (DHA vs EPAb) All participants (n = 121)c  Total cholesterol, mmol/Ld,e 5.29 ± 0.08 5.16 ± 0.08 5.33 ± 0.08 3.1 0.001f 5.15 ± 0.08 −0.4 0.62 3.6 < 0.001f  LDL-C, mmol/Ld,e 3.12 ± 0.07 2.99 ± 0.07 3.16 ± 0.08 5.4 < 0.001f 3.06 ± 0.07 2.5 0.046f 3.2 0.04f  Total apoB, g/Ld,e 1.34 ± 0.04 1.31 ± 0.04 1.34 ± 0.04 2.8 0.02f 1.32 ± 0.04 1.2 0.46 1.6 0.16  TG, mmol/Ld,e 1.34 ± 0.06 1.38 ± 0.06 1.14 ± 0.04 −21.3 < 0.001f 1.23 ± 0.05 −11.3 < 0.001f −7.1 0.005f  Mean LDL size, Å 252.10 ± 0.26 251.89 ± 0.21 252.15 ± 0.23 0.1 0.051 251.41 ± 0.20 −0.2 0.01f 0.3 < 0.001f  LDL peak, Å 251.74 ± 0.28 251.56 ± 0.24 252.08 ± 0.25 0.2 0.001f 251.19 ± 0.23 −0.1 0.059 0.4 < 0.001f  Proportion of large LDL, % 9.49 ± 0.72 9.00 ± 0.53 9.16 ± 0.62 1.8 0.85 8.42 ± 0.53 −6.4 0.20 8.8 0.19  Proportion of small LDL, % 69.29 ± 1.45 69.83 ± 1.20 68.88 ± 1.34 −1.4 0.25 72.00 ± 1.08 3.1 0.03f −4.3 0.002f  PCSK9, ng/mL NA 213.02 ± 5.65 189.12 ± 4.89 −12.6 < 0.001f 194.98 ± 5.40 −8.5 < 0.001f −3.0 0.19 Substudy (n = 19)g  Total cholesterol, mmol/L 5.18 ± 0.16 4.70 ± 0.16 5.13 ± 0.14 9.1 0.005f 5.01 ± 0.19 8.0 0.018f 2.5 0.62  LDL-C, mmol/L 3.11 ± 0.14 2.70 ± 0.13 3.10 ± 0.15 14.7 0.006f 3.05 ± 0.14 14.4 0.010f 1.6 0.88  Total apoB, g/L 1.37 ± 0.09 1.26 ± 0.08 1.40 ± 0.12 11.0 0.03f 1.35 ± 0.09 6.9 0.27 3.5 0.27  TG, mmol/L 1.29 ± 0.13 1.35 ± 0.17 1.20 ± 0.15 −11.1 0.10 1.39 ± 0.21 4.6 0.63 −13.6 0.06  Mean LDL size, Å 250.88 ± 0.75 250.95 ± 0.54 250.95 ± 0.63 0.0 0.90 250.42 ± 0.60 −0.2 0.29 0.2 0.33  LDL peak, Å 250.62 ± 0.76 250.68 ± 0.63 250.79 ± 0.71 0.0 0.89 250.20 ± 0.61 −0.2 0.12 0.2 0.17  Proportion of large LDL, % 7.52 ± 1.38 6.90 ± 1.07 7.01 ± 1.35 1.5 0.34 7.42 ± 1.12 4.2 0.12 −5.6 0.54  Proportion of small LDL, % 75.03 ± 3.16 76.13 ± 2.32 75.53 ± 2.88 −0.8 0.56 75.98 ± 2.55 0.0 0.72 −0.6 0.86  PCSK9, ng/mL NA 196.92 ± 15.23 174.43 ± 13.53 −11.4 0.11 190.08 ± 15.33 1.2 0.75 −8.2 0.04f Data presented as unadjusted mean ± SEM. Abbreviation: NA, not available. a This analysis compared the change with DHA or EPA compared with control based on posttreatment values; P values were taken from the main treatment effect in the mixed models. b P values for the EPA and DHA changes vs control, as determined by the LSMEANS statement and tested against the null hypothesis in the mixed models. c Participants equaled 123 for DHA, 121 for EPA, 125 for control, and 138 for baseline. d Log-transformed data were used in these analyses due to skewness of the distribution of the values. e Previously reported (4). f P < 0.05; because pre-DHA and -EPA values were essentially identical to the precontrol values, only precontrol values are presented. Models were adjusted for sex, weight, age, sequence; baseline value was considered only when these covariates were found to be significant at P < 0.05 in the models. g Participants equaled 19 for DHA, 17 for EPA, and 19 for control. View Large Kinetic studies Both DHA and EPA tended to increase VLDL apoB100 FCR similarly compared with the control (DHA, +21%; EPA, +19%; DHA vs EPA, P = 0.73; Table 3). However, EPA tended to increase VLDL apoB100 direct catabolism more than did DHA (compared with control: DHA, −3%; EPA, +22%; DHA vs EPA, P = 0.10). Changes in the VLDL to LDL apoB100 conversion rates were similar after DHA and EPA (compared with control: DHA, +8%; EPA, +7%; DHA vs EPA, P = 0.44). LDL apoB100 FCR was significantly lower after EPA supplementation than after DHA supplementation (compared with control: DHA, 0%; EPA, −10%; DHA vs EPA, P = 0.008). In contrast, DHA increased the LDL apoB100 PR compared with EPA (compared with control: DHA, +2%; EPA, −7%; DHA vs EPA, P = 0.027). Table 3. ApoB100-Containing Lipoproteins and VLDL apoCIII Kinetics After Control, DHA, and EPA Phases Variable After Control Phase After DHA Phase % vs Control P Value ΔDHAa After EPA Control % vs Control P Value ΔEPAa DHA vs EPA (%) P Value ΔDHA vs ΔEPAb VLDL apoB100  PS, mg 336.07 ± 46.90 300.0 ± 47.4 −10.7 0.03c 336.31 ± 74.52 0.1 0.70 −10.8 0.10  FCR, pools/dd 9.38 ± 0.94 11.3 ± 1.9 20.5 0.12 11.21 ± 1.87 19.4 0.20 0.9 0.73  PR, mg/kg/dd 30.42 ± 2.21 30.5 ± 2.6 0.1 1.00 32.91 ± 3.40 8.2 0.30 −7.4 0.22  Abs. conv. VLDL to IDL apoB100, mg/kg/dd 4.14 ± 1.29 4.0 ± 1.0 −3.2 0.34 2.37 ± 0.46 −42.7 0.31 69.0 0.002c  Abs. conv. VLDL to LDL apoB100, mg/kg/d 9.40 ± 1.49 10.1 ± 1.5 7.5 0.29 10.08 ± 1.00 7.3 0.80 0.2 0.44  VLDL apoB100 direct catabolism, mg/kg/dd 16.06 ± 2.27 15.6 ± 2.6 −2.9 0.78 19.54 ± 3.22 21.7 0.25 −20.2 0.10 IDL apoB100  PS, mg 16.06 ± 2.05 20.8 ± 2.7 29.7 0.11 15.24 ± 2.28 −5.2 0.58 36.7 0.05  FCR, pools/dd 11.54 ± 1.85 14.6 ± 2.3 26.4 0.02c 13.46 ± 1.72 16.6 0.18 8.4 0.32  PR, mg/kg/dd 2.37 ± 0.64 3.4 ± 0.8 42.0 0.03c 2.38 ± 0.46 0.6 0.78 41.1 0.009c  Abs. conv. IDL to LDL apoB100, mg/kg/dd 3.77 ± 1.31 3.3 ± 0.8 −13.5 0.50 2.37 ± 0.46 −37.1 0.63 37.5 0.14 LDL apoB100  PS, mg 2700.98 ± 159.05 2811.1 ± 192.1 4.1 0.67 2785.01 ± 155.81 3.1 0.54 0.9 0.84  FCR, pools/dd 0.43 ± 0.06 0.43 ± 0.06 −0.2 0.50 0.39 ± 0.04 −10.4 0.24 11.4 0.008c  PR, mg/kg/dd 13.29 ± 1.45 13.5 ± 1.4 1.6 0.43 12.34 ± 1.04 −7.1 0.55 9.4 0.03c VLDL apoCIII  PS, mgd 109.58 ± 15.77 100.41 ± 16.50 −8.4 0.10 115.30 ± 23.62 5.2 0.96 −12.9 0.11  FCR, pools/d 0.94 ± 0.06 0.83 ± 0.05 −11.3 0.009c 0.88 ± 0.06 −6.9 0.04c −4.7 0.60  PR, mg/kg/dd 4.18 ± 0.54 3.67 ± 0.69 −12.2 0.009c 4.24 ± 0.77 1.3 0.34 −13.3 0.092 Variable After Control Phase After DHA Phase % vs Control P Value ΔDHAa After EPA Control % vs Control P Value ΔEPAa DHA vs EPA (%) P Value ΔDHA vs ΔEPAb VLDL apoB100  PS, mg 336.07 ± 46.90 300.0 ± 47.4 −10.7 0.03c 336.31 ± 74.52 0.1 0.70 −10.8 0.10  FCR, pools/dd 9.38 ± 0.94 11.3 ± 1.9 20.5 0.12 11.21 ± 1.87 19.4 0.20 0.9 0.73  PR, mg/kg/dd 30.42 ± 2.21 30.5 ± 2.6 0.1 1.00 32.91 ± 3.40 8.2 0.30 −7.4 0.22  Abs. conv. VLDL to IDL apoB100, mg/kg/dd 4.14 ± 1.29 4.0 ± 1.0 −3.2 0.34 2.37 ± 0.46 −42.7 0.31 69.0 0.002c  Abs. conv. VLDL to LDL apoB100, mg/kg/d 9.40 ± 1.49 10.1 ± 1.5 7.5 0.29 10.08 ± 1.00 7.3 0.80 0.2 0.44  VLDL apoB100 direct catabolism, mg/kg/dd 16.06 ± 2.27 15.6 ± 2.6 −2.9 0.78 19.54 ± 3.22 21.7 0.25 −20.2 0.10 IDL apoB100  PS, mg 16.06 ± 2.05 20.8 ± 2.7 29.7 0.11 15.24 ± 2.28 −5.2 0.58 36.7 0.05  FCR, pools/dd 11.54 ± 1.85 14.6 ± 2.3 26.4 0.02c 13.46 ± 1.72 16.6 0.18 8.4 0.32  PR, mg/kg/dd 2.37 ± 0.64 3.4 ± 0.8 42.0 0.03c 2.38 ± 0.46 0.6 0.78 41.1 0.009c  Abs. conv. IDL to LDL apoB100, mg/kg/dd 3.77 ± 1.31 3.3 ± 0.8 −13.5 0.50 2.37 ± 0.46 −37.1 0.63 37.5 0.14 LDL apoB100  PS, mg 2700.98 ± 159.05 2811.1 ± 192.1 4.1 0.67 2785.01 ± 155.81 3.1 0.54 0.9 0.84  FCR, pools/dd 0.43 ± 0.06 0.43 ± 0.06 −0.2 0.50 0.39 ± 0.04 −10.4 0.24 11.4 0.008c  PR, mg/kg/dd 13.29 ± 1.45 13.5 ± 1.4 1.6 0.43 12.34 ± 1.04 −7.1 0.55 9.4 0.03c VLDL apoCIII  PS, mgd 109.58 ± 15.77 100.41 ± 16.50 −8.4 0.10 115.30 ± 23.62 5.2 0.96 −12.9 0.11  FCR, pools/d 0.94 ± 0.06 0.83 ± 0.05 −11.3 0.009c 0.88 ± 0.06 −6.9 0.04c −4.7 0.60  PR, mg/kg/dd 4.18 ± 0.54 3.67 ± 0.69 −12.2 0.009c 4.24 ± 0.77 1.3 0.34 −13.3 0.092 Data presented as mean ± SEM. ApoB100: control, n = 17 (n = 18 for PS, FCR, and PR VLDL); DHA, n = 16 (18 for PS VLDL, 17 for PS IDL and LDL, FCR and PR VLDL); EPA, n = 14 (16 for PS, FCR, and PR VLDL, 15 for PS, FCR, and PR LDL); ApoCIII: control, n = 18; DHA, n = 18; EPA, n = 16. Abbreviations: abs., absolute; conv., conversion; IDL, intermediate-density lipoprotein; PS, pool size. a This analysis compared the change with DHA or EPA compared with control, using post-treatment values; P values were from the main treatment effect in the mixed models. b P values for the EPA and DHA changes vs control, as determined by the LSMEANS statement and tested against the null hypothesis in the mixed models. c P < 0.05. Models were adjusted for sex, weight, age, sequence; baseline value was considered only when these covariates were found to be significant at P < 0.05 in the models. d Log-transformed data were used in these analyses owing to skewness of the value distribution. View Large Table 3. ApoB100-Containing Lipoproteins and VLDL apoCIII Kinetics After Control, DHA, and EPA Phases Variable After Control Phase After DHA Phase % vs Control P Value ΔDHAa After EPA Control % vs Control P Value ΔEPAa DHA vs EPA (%) P Value ΔDHA vs ΔEPAb VLDL apoB100  PS, mg 336.07 ± 46.90 300.0 ± 47.4 −10.7 0.03c 336.31 ± 74.52 0.1 0.70 −10.8 0.10  FCR, pools/dd 9.38 ± 0.94 11.3 ± 1.9 20.5 0.12 11.21 ± 1.87 19.4 0.20 0.9 0.73  PR, mg/kg/dd 30.42 ± 2.21 30.5 ± 2.6 0.1 1.00 32.91 ± 3.40 8.2 0.30 −7.4 0.22  Abs. conv. VLDL to IDL apoB100, mg/kg/dd 4.14 ± 1.29 4.0 ± 1.0 −3.2 0.34 2.37 ± 0.46 −42.7 0.31 69.0 0.002c  Abs. conv. VLDL to LDL apoB100, mg/kg/d 9.40 ± 1.49 10.1 ± 1.5 7.5 0.29 10.08 ± 1.00 7.3 0.80 0.2 0.44  VLDL apoB100 direct catabolism, mg/kg/dd 16.06 ± 2.27 15.6 ± 2.6 −2.9 0.78 19.54 ± 3.22 21.7 0.25 −20.2 0.10 IDL apoB100  PS, mg 16.06 ± 2.05 20.8 ± 2.7 29.7 0.11 15.24 ± 2.28 −5.2 0.58 36.7 0.05  FCR, pools/dd 11.54 ± 1.85 14.6 ± 2.3 26.4 0.02c 13.46 ± 1.72 16.6 0.18 8.4 0.32  PR, mg/kg/dd 2.37 ± 0.64 3.4 ± 0.8 42.0 0.03c 2.38 ± 0.46 0.6 0.78 41.1 0.009c  Abs. conv. IDL to LDL apoB100, mg/kg/dd 3.77 ± 1.31 3.3 ± 0.8 −13.5 0.50 2.37 ± 0.46 −37.1 0.63 37.5 0.14 LDL apoB100  PS, mg 2700.98 ± 159.05 2811.1 ± 192.1 4.1 0.67 2785.01 ± 155.81 3.1 0.54 0.9 0.84  FCR, pools/dd 0.43 ± 0.06 0.43 ± 0.06 −0.2 0.50 0.39 ± 0.04 −10.4 0.24 11.4 0.008c  PR, mg/kg/dd 13.29 ± 1.45 13.5 ± 1.4 1.6 0.43 12.34 ± 1.04 −7.1 0.55 9.4 0.03c VLDL apoCIII  PS, mgd 109.58 ± 15.77 100.41 ± 16.50 −8.4 0.10 115.30 ± 23.62 5.2 0.96 −12.9 0.11  FCR, pools/d 0.94 ± 0.06 0.83 ± 0.05 −11.3 0.009c 0.88 ± 0.06 −6.9 0.04c −4.7 0.60  PR, mg/kg/dd 4.18 ± 0.54 3.67 ± 0.69 −12.2 0.009c 4.24 ± 0.77 1.3 0.34 −13.3 0.092 Variable After Control Phase After DHA Phase % vs Control P Value ΔDHAa After EPA Control % vs Control P Value ΔEPAa DHA vs EPA (%) P Value ΔDHA vs ΔEPAb VLDL apoB100  PS, mg 336.07 ± 46.90 300.0 ± 47.4 −10.7 0.03c 336.31 ± 74.52 0.1 0.70 −10.8 0.10  FCR, pools/dd 9.38 ± 0.94 11.3 ± 1.9 20.5 0.12 11.21 ± 1.87 19.4 0.20 0.9 0.73  PR, mg/kg/dd 30.42 ± 2.21 30.5 ± 2.6 0.1 1.00 32.91 ± 3.40 8.2 0.30 −7.4 0.22  Abs. conv. VLDL to IDL apoB100, mg/kg/dd 4.14 ± 1.29 4.0 ± 1.0 −3.2 0.34 2.37 ± 0.46 −42.7 0.31 69.0 0.002c  Abs. conv. VLDL to LDL apoB100, mg/kg/d 9.40 ± 1.49 10.1 ± 1.5 7.5 0.29 10.08 ± 1.00 7.3 0.80 0.2 0.44  VLDL apoB100 direct catabolism, mg/kg/dd 16.06 ± 2.27 15.6 ± 2.6 −2.9 0.78 19.54 ± 3.22 21.7 0.25 −20.2 0.10 IDL apoB100  PS, mg 16.06 ± 2.05 20.8 ± 2.7 29.7 0.11 15.24 ± 2.28 −5.2 0.58 36.7 0.05  FCR, pools/dd 11.54 ± 1.85 14.6 ± 2.3 26.4 0.02c 13.46 ± 1.72 16.6 0.18 8.4 0.32  PR, mg/kg/dd 2.37 ± 0.64 3.4 ± 0.8 42.0 0.03c 2.38 ± 0.46 0.6 0.78 41.1 0.009c  Abs. conv. IDL to LDL apoB100, mg/kg/dd 3.77 ± 1.31 3.3 ± 0.8 −13.5 0.50 2.37 ± 0.46 −37.1 0.63 37.5 0.14 LDL apoB100  PS, mg 2700.98 ± 159.05 2811.1 ± 192.1 4.1 0.67 2785.01 ± 155.81 3.1 0.54 0.9 0.84  FCR, pools/dd 0.43 ± 0.06 0.43 ± 0.06 −0.2 0.50 0.39 ± 0.04 −10.4 0.24 11.4 0.008c  PR, mg/kg/dd 13.29 ± 1.45 13.5 ± 1.4 1.6 0.43 12.34 ± 1.04 −7.1 0.55 9.4 0.03c VLDL apoCIII  PS, mgd 109.58 ± 15.77 100.41 ± 16.50 −8.4 0.10 115.30 ± 23.62 5.2 0.96 −12.9 0.11  FCR, pools/d 0.94 ± 0.06 0.83 ± 0.05 −11.3 0.009c 0.88 ± 0.06 −6.9 0.04c −4.7 0.60  PR, mg/kg/dd 4.18 ± 0.54 3.67 ± 0.69 −12.2 0.009c 4.24 ± 0.77 1.3 0.34 −13.3 0.092 Data presented as mean ± SEM. ApoB100: control, n = 17 (n = 18 for PS, FCR, and PR VLDL); DHA, n = 16 (18 for PS VLDL, 17 for PS IDL and LDL, FCR and PR VLDL); EPA, n = 14 (16 for PS, FCR, and PR VLDL, 15 for PS, FCR, and PR LDL); ApoCIII: control, n = 18; DHA, n = 18; EPA, n = 16. Abbreviations: abs., absolute; conv., conversion; IDL, intermediate-density lipoprotein; PS, pool size. a This analysis compared the change with DHA or EPA compared with control, using post-treatment values; P values were from the main treatment effect in the mixed models. b P values for the EPA and DHA changes vs control, as determined by the LSMEANS statement and tested against the null hypothesis in the mixed models. c P < 0.05. Models were adjusted for sex, weight, age, sequence; baseline value was considered only when these covariates were found to be significant at P < 0.05 in the models. d Log-transformed data were used in these analyses owing to skewness of the value distribution. View Large The increase in LDL-C concentrations after DHA or EPA supplementation did not correlate with variations in the LDL apoB100 FCR or PR (Table 4). However, variations in the LDL apoB100 pool size correlated with change in LDL apoB100 PR after EPA (rs = 0.63; P = 0.013) and with variations in LDL apoB100 FCR after DHA (rs = −0.52; P = 0.04) and PCSK9 concentration after DHA (rs = 0.64; P < 0.01). Table 4. Spearman Correlation Coefficient Between Changes in ApoB100-Containing Lipoprotein Kinetics, PCSK9 Levels, and LDL-C After EPA and DHA vs Control Variable DHA EPA LDL-C, mmol/L LDL apoB100, mg LDL-C, mmol/L LDL apoB100, mg VLDL apoB100  FCR, pools/d −0.203 −0.424 −0.303 −0.182  PR, mg/kg/d 0.159 0.229 0.135 −0.043  VLDL to IDL apoB100, mg/kg/d 0.165 −0.291 0.420 0.305  VLDL to LDL apoB100, mg/kg/d 0.238 0.276 −0.420 0.275  VLDL apoB100 direct catabolism, mg/kg/d 0.076 0.265 −0.055 −0.196 IDL apoB100  FCR, pools/d 0.503a −0.129 0.301 0.270  PR, mg/kg/d 0.674a 0.221 0.327 0.503  IDL to LDL apoB100, mg/kg/d 0.132 −0.226 0.560a 0.380 LDL apoB100  FCR, pools/d −0.229 −0.521a −0.161 0.057  PR, mg/kg/d 0.044 −0.197 0.261 0.625a Mean LDL size, Å 0.103 −0.091 0.063 0.393 LDL peak, Å 0.148 0.324 0.062 0.254 Proportion, large LDL, % 0.059 −0.181 0.133 0.046 Proportion, small LDL, % −0.111 0.028 −0.136 −0.196 PCSK9, ng/L 0.031 0.642a 0.132 0.011 Variable DHA EPA LDL-C, mmol/L LDL apoB100, mg LDL-C, mmol/L LDL apoB100, mg VLDL apoB100  FCR, pools/d −0.203 −0.424 −0.303 −0.182  PR, mg/kg/d 0.159 0.229 0.135 −0.043  VLDL to IDL apoB100, mg/kg/d 0.165 −0.291 0.420 0.305  VLDL to LDL apoB100, mg/kg/d 0.238 0.276 −0.420 0.275  VLDL apoB100 direct catabolism, mg/kg/d 0.076 0.265 −0.055 −0.196 IDL apoB100  FCR, pools/d 0.503a −0.129 0.301 0.270  PR, mg/kg/d 0.674a 0.221 0.327 0.503  IDL to LDL apoB100, mg/kg/d 0.132 −0.226 0.560a 0.380 LDL apoB100  FCR, pools/d −0.229 −0.521a −0.161 0.057  PR, mg/kg/d 0.044 −0.197 0.261 0.625a Mean LDL size, Å 0.103 −0.091 0.063 0.393 LDL peak, Å 0.148 0.324 0.062 0.254 Proportion, large LDL, % 0.059 −0.181 0.133 0.046 Proportion, small LDL, % −0.111 0.028 −0.136 −0.196 PCSK9, ng/L 0.031 0.642a 0.132 0.011 Abbreviation: IDL, intermediate-density lipoprotein. a P < 0.05. View Large Table 4. Spearman Correlation Coefficient Between Changes in ApoB100-Containing Lipoprotein Kinetics, PCSK9 Levels, and LDL-C After EPA and DHA vs Control Variable DHA EPA LDL-C, mmol/L LDL apoB100, mg LDL-C, mmol/L LDL apoB100, mg VLDL apoB100  FCR, pools/d −0.203 −0.424 −0.303 −0.182  PR, mg/kg/d 0.159 0.229 0.135 −0.043  VLDL to IDL apoB100, mg/kg/d 0.165 −0.291 0.420 0.305  VLDL to LDL apoB100, mg/kg/d 0.238 0.276 −0.420 0.275  VLDL apoB100 direct catabolism, mg/kg/d 0.076 0.265 −0.055 −0.196 IDL apoB100  FCR, pools/d 0.503a −0.129 0.301 0.270  PR, mg/kg/d 0.674a 0.221 0.327 0.503  IDL to LDL apoB100, mg/kg/d 0.132 −0.226 0.560a 0.380 LDL apoB100  FCR, pools/d −0.229 −0.521a −0.161 0.057  PR, mg/kg/d 0.044 −0.197 0.261 0.625a Mean LDL size, Å 0.103 −0.091 0.063 0.393 LDL peak, Å 0.148 0.324 0.062 0.254 Proportion, large LDL, % 0.059 −0.181 0.133 0.046 Proportion, small LDL, % −0.111 0.028 −0.136 −0.196 PCSK9, ng/L 0.031 0.642a 0.132 0.011 Variable DHA EPA LDL-C, mmol/L LDL apoB100, mg LDL-C, mmol/L LDL apoB100, mg VLDL apoB100  FCR, pools/d −0.203 −0.424 −0.303 −0.182  PR, mg/kg/d 0.159 0.229 0.135 −0.043  VLDL to IDL apoB100, mg/kg/d 0.165 −0.291 0.420 0.305  VLDL to LDL apoB100, mg/kg/d 0.238 0.276 −0.420 0.275  VLDL apoB100 direct catabolism, mg/kg/d 0.076 0.265 −0.055 −0.196 IDL apoB100  FCR, pools/d 0.503a −0.129 0.301 0.270  PR, mg/kg/d 0.674a 0.221 0.327 0.503  IDL to LDL apoB100, mg/kg/d 0.132 −0.226 0.560a 0.380 LDL apoB100  FCR, pools/d −0.229 −0.521a −0.161 0.057  PR, mg/kg/d 0.044 −0.197 0.261 0.625a Mean LDL size, Å 0.103 −0.091 0.063 0.393 LDL peak, Å 0.148 0.324 0.062 0.254 Proportion, large LDL, % 0.059 −0.181 0.133 0.046 Proportion, small LDL, % −0.111 0.028 −0.136 −0.196 PCSK9, ng/L 0.031 0.642a 0.132 0.011 Abbreviation: IDL, intermediate-density lipoprotein. a P < 0.05. View Large Both DHA and EPA decreased VLDL apoCIII FCR similarly compared with the control (DHA, −11%; EPA, −7%; DHA vs EPA, P = 0.60). The reduction in VLDL apoCIII PR tended to be greater after DHA than after EPA (compared with control: DHA, −12%; EPA, +1%; DHA vs EPA, P = 0.09). The change in VLDL apoCIII FCR correlated inversely with the change in the PCSK9 concentration after EPA (rs = −0.58; P = 0.019; data not shown) and with the change in LDL apoB100 FCR after DHA (rs = 0.54; P = 0.030; data not shown). The change in VLDL apoCIII PR correlated inversely with LDL apoB100 FCR and PR (rs = −0.57 and rs = −0.53, respectively; P < 0.05; data not shown) and was positively associated with the change in PCSK9 concentrations after DHA (rs = 0.54; P = 0.021; data not shown). We did not find any correlation between the changes in TG and LDL-C or between the changes in VLDL apoCIII FCR and LDL apoB100 PR after DHA or EPA (data not shown). Discussion To the best of our knowledge, the present study is the first to demonstrate the mechanisms underlying the differential effects of DHA and EPA supplementation on LDL-C and other features of LDL in men and women with abdominal obesity and subclinical inflammation and at risk of CVD. The results from the present study suggest that high-dose DHA increases the LDL particle size and modifies LDL apoB100 and VLDL apoCIII kinetics compared with EPA. Although DHA and EPA reduce the PCSK9 concentration similarly, the relationships among PCSK9, LDL-C, and LDL apoB100 concentrations were different between DHA and EPA. We have previously shown that the magnitude of the reduction in TG and increase in LDL-C after DHA was greater than after EPA supplementation (4). Previous in vivo kinetic studies have documented the effects of LCn3-PUFAs, either as a dietary supplement or as part of an LCn3-PUFA–rich diet, on apoB100-containing lipoprotein metabolism (27, 28). Those studies have shown that LCn3-PUFAs reduce TG concentrations primarily by reducing the endogenous production of VLDL apoB100 and by increasing the VLDL to LDL apoB100 conversion rate (27, 28). LCn3-PUFAs have also been shown to increase the clearance of LDL apoB100 (27, 28). In contrast, Ooi et al. (12) found that a high-fish diet (containing 1.23 g EPA plus DHA daily) increases LDL apoB100 production by 32% and concomitantly decreases LDL apoB100 clearance by 44% compared with untreated baseline values. This disproportionate reduction in LDL apoB100 clearance might explain in part why the LDL-C concentrations increase after LCn3-PUFA supplementation. Because the changes in LDL-C and TG concentrations are greater with DHA supplementation than with EPA supplementation (4), we hypothesized that DHA compared with EPA induces a greater reduction in VLDL apoB100 production and a greater VLDL to LDL apoB100 conversion rate, resulting in a greater increase in LDL apoB100 production. Accordingly, DHA compared with EPA differentially influenced LDL apoB100 production and clearance rates; however, these differences were not related to the differential effects of DHA and EPA on LDL-C concentrations. DHA and EPA equally increased VLDL to LDL apoB100 conversion and VLDL apoB100 FCR. These data suggest that metabolic pathways not involving apoB100 per se might be responsible for the differential effects of DHA and EPA on LDL-C concentrations and LDL size. It is possible that DHA and EPA differentially influence apoB/C/E ratios on VLDL, which might, in turn, contribute to differences in the LDL-C concentrations and LDL size seen between DHA and EPA (6, 7). Zheng et al. (7) have shown that apoCIII-containing VLDL are the major precursor of LDL particles. Hence, the suppression of apoCIII PR with DHA might also explain to some extent its effect on LDL particle size. That total VLDL particles were converted more rapidly to intermediate-density lipoprotein after DHA than after EPA (Table 3) and that VLDL-apoCIII levels also tended to decrease with DHA compared with EPA is consistent with this hypothesis. We also hypothesized that DHA and EPA supplementation would modulate LDL particle size differently because the increase in LDL-C concentration after DHA was almost twofold greater in magnitude than the increase in total apoB concentration (4). Accordingly, DHA supplementation slightly increased mean LDL particle size and decreased the proportion of sdLDL compared with EPA supplementation. This observation is consistent with data from a few studies, which have shown that DHA, but not EPA, is associated with larger LDL (9, 29). This increase in LDL particle size after DHA can be attributed, at least in part, to the greater reduction in serum TG compared with EPA. Serum TG is an important metabolic determinant of the sdLDL phenotype through a series of metabolic transformation of the LDL particles that involve lipases and cholesteryl ester transfer protein (30). However, very few studies have compared the effect of DHA and EPA on enzyme activities. Supplementation with LCn3-PUFA has been shown to have inconsistent effects on cholesteryl ester transfer protein activity (31) and might increase lipoprotein lipase activity through upregulation of it expression (31) but might have no effect on hepatic lipase activity (31). More studies investigating these pathways in response to DHA and EPA supplementations are needed. The increase in LDL size with DHA compared with EPA might also be explained in part by a decrease in apoCIII secretion from the liver (32). DHA might reduce apoCIII production through the regulation of the forkhead box O transcription factor O1 and carbohydrate response element-binding protein (33, 34). ApoCIII inhibits the binding of apoB to hepatic apoB/E receptor and lipoprotein lipase activity (32, 35). In a small parallel study, supplementation with EPA alone tended to increase apoCIII concentrations, and DHA tended to decrease apoCIII-containing lipoprotein concentrations (36). Consistent with this, we have shown that high-dose EPA also tended to increase VLDL apoCIII mass and DHA tended to decrease the VLDL apoCIII mass compared with the control, although the differences did not reach statistical significance. DHA also tended to decrease the VLDL apoCIII PR compared with EPA; however, the difference also did not reach the statistical significance. Changes in VLDL apoCIII metabolism correlated with changes in LDL apoB100 metabolism after DHA, but not after EPA, supporting a differential effect of EPA and DHA on VLDL apoCIII and apoB100 metabolism. This apparent reduction in apoCIII production in the liver after DHA supplementation might explain the enhanced conversion of VLDL to LDL apoB100 and the formation of larger LDL particles compared with EPA. Individuals with a preponderance of sdLDL have consistently been shown to be at increased risk of myocardial infarction and CVD compared with individuals with a greater proportion of larger LDL particles (37). However, the extent to which the opposite effects of DHA on both LDL-C and LDL particle size modify CVD risk is unknown. The reduction in PCSK9 concentrations observed after DHA and EPA supplementation is consistent with data from the few available studies on this topic. A recent randomized controlled parallel study in 92 pre- and postmenopausal women has shown that supplementation with 2.2 g/d of marine oil decreased plasma PCSK9 concentrations by 11.4% in premenopausal women and 9.8% in postmenopausal women compared with baseline (19). Post hoc analyses of the Canola Oil Multicenter Intervention Trial have also shown that the PCSK9 concentration was lower after DHA-enriched canola oil than after regular canola oil supplementation (20). In the present study, DHA and EPA both reduced serum PCSK9 levels equally compared with the control. Although the PCSK9 concentrations usually correlated with LDL-C concentrations, variations in the PCSK9 levels explained <8% of the LDL-C variance (38). Furthermore, the PCSK9 concentrations might not fully reflect PCSK9 activity (38). Therefore, the increase in LDL-C after DHA and EPA despite a decrease in PCSK9 concentrations was not entirely unexpected. In contrast, changes in PCSK9 correlated positively with changes in the LDL apoB100 concentrations and negatively with changes in LDL apoB100 FCR after DHA but not after EPA, suggesting that PCSK9 might be partly involved in explaining the differential effects of DHA and EPA supplementation on the metabolic fate of the LDL particle. The present study had several strengths and limitations. A number of studies have examined the effect of an LCn3-PUFA–rich diet or a supplement combining EPA and DHA in various forms and proportions on apoB100-containing lipoprotein kinetics (27, 28). To the best of our knowledge, ours is the first study to compare head-to-head the effect of high-dose EPA and DHA on apoB100-containing lipoprotein kinetics. The use of a randomized crossover study design reduced the interindividual variability of the results. The baseline characteristics of the kinetic subsample were similar to the whole study cohort, and the compliance was high in all phases of the study (4). The analyses of the changes in blood lipids, LDL particle size, and PCSK9 concentrations in the substudy kinetic sample were conducted on data from fewer participants, hence influencing the statistical power. Estimates from small kinetic pool sizes have relatively high coefficients of variations and small changes in kinetic parameters can be difficult to assess. The observed effects of DHA and EPA on serum lipids, including LDL-C, might have resulted from changes in kinetics that might have been too subtle to be detected with this sample size. Corn oil was chosen as the control because of the relatively neutral effects of n6-PUFA on inflammation makers (39), which were the primary outcome of the trial (4). Supplementation with the control n6-PUFA–rich corn oil decreased total cholesterol (−0.12 mmol/L; P = 0.001), LDL-C (−0.13 mmol/L; P = 0.003), and mean LDL size (−0.22 Å; P = 0.02) compared with control-specific baseline levels. However, results were similar when the change from DHA/EPA-specific baseline values were considered. Specifically, the increase in LDL-C and the reduction in TG with DHA compared with the baseline values were significantly greater than those seen with EPA (LDL-C, +0.11 vs 0.00 mmol/L; TG, −0.26 vs −0.20 mmol/L; P < 0.01 for all). The increase from treatment-specific baseline values in LDL particle size (+0.21 Å vs −0.71 Å) and the reduction in the proportion of sdLDL (−1.36% vs +2.8%) were also greater with DHA than with EPA (P < 0.01 for all; data not shown). Because very few studies have documented the effect of n6-PUFA on apoB100- and apoCIII-containing lipoprotein metabolism, it is difficult to assess how the use of corn oil as the control treatment has affected the kinetic study data (27, 28, 40). In conclusion, the differential effects of DHA and EPA supplementation on LDL-C concentrations might not be accounted for by differences in the regulation of apoB100-containing lipoprotein metabolism and might involve other pathways that influence LDL particle size. The extent to which the greater increase in LDL-C with DHA compared with EPA, associated with larger LDL particles, influences CVD risk is unknown. Further studies are needed to better understand the changes in other metabolic factors after EPA and DHA supplementation, including the expression of the different genes involved in lipid metabolism. Abbreviations: Abbreviations: apo apolipoprotein CRP C-reactive protein CVD cardiovascular disease DHA docosahexaenoic acid EPA eicosapentaenoic acid FCR fractional catabolic rate LCn3-PUFA long-chain omega-3 polyunsaturated fatty acid LDL low-density lipoprotein LDL-C low density lipoprotein cholesterol PCSK9 proprotein convertase subtilisin/kexin type 9 PR production rate sdLDL small and dense low-density lipoprotein TG triglyceride TRL triglyceride-rich lipoprotein VLDL very-low-density lipoprotein Acknowledgments We are grateful to the subjects for their excellent collaboration and the staff of the Institute of Nutrition and Functional Foods and the CHU de Québec. Financial Support: Financial support for this randomized controlled trial was provided by a grant from the Canadian Institutes for Health Research (CIHR) (grant MOP-123494 to B.L., P.C., A.T.). Douglas Laboratories provided the EPA, DHA, and control capsules used in the present study. The CIHR and Douglas Laboratories had no role in the design of the study or analysis or interpretation of the data. J.A. is a recipient of a PhD Scholarship from the CIHR and the Fonds de recherche du Québec – Santé. C.V. is a fellow supported by the European Marie Skłodowska-Curie Actions. Clinical Trial Information: ClinicalTrials.gov. no. NCT01810003 (registered 4 March 2013). Author Contributions: B.L., P.C., and A.T. designed and obtained funding for the present study. P.C. was responsible for the medical supervision in the study. A.C. coordinated the clinical study. J.M. conducted the laboratory analyses. C.V. and A.J.T. provided significant help with modeling of the data. J.A. performed the modeling of the data and statistical analyses and wrote the manuscript, which was reviewed critically by all the authors. Disclosure Summary: B.L. is Chair of Nutrition at Laval University, which is supported by private endowments from Pfizer, La Banque Royale du Canada, and Provigo-Loblaws; has received funding in the past 5 years from the Canadian Institutes for Health Research, the Natural Sciences and Engineering Research Council of Canada, Agriculture and Agri-Food Canada (Growing Forward program supported by the Dairy Farmers of Canada, Canola Council of Canada, Flax Council of Canada, Dow Agrosciences), Dairy Research Institute, Dairy Australia, Merck & Co, Inc., Pfizer, and Atrium Innovations for which Douglas Laboratories manufacture and market omega-3 supplements; is an advisory board member of the Canadian Nutrition Society; and has received honoraria from the International Chair on Cardiometabolic risk, the Dairy Farmers of Canada, and the World Dairy Platform as an invited speaker to various conferences. P.C. has received funding in the past 5 years from the Canadian Institutes of Health Research, Agriculture and Agri-Food Canada (Growing Forward program supported by the Dairy Farmers of Canada, Canola Council of Canada, Flax Council of Canada, Dow Agrosciences), Dairy Research Institute, Dairy Australia, Merck and Co., Inc., Pfizer, Amgen, and Atrium Innovations. A.T. was funded in the past 5 years as principal investigator by the Canadian Institutes of Health Research, the Natural Sciences and Engineering Research Council of Canada, the Fonds de recherche du Québec – Santé, the Fondation de l’Institut Universitaire de Cardiologie et de Pneumologie de Québec, and investigator-initiated funding from Johnson & Johnson Medical Companies for studies unrelated to the present report. The remaining authors have nothing to disclose. References 1. Bradberry JC , Hilleman DE . Overview of omega-3 fatty acid therapies . P T . 2013 ; 38 ( 11 ): 681 – 691 . Google Scholar PubMed 2. Hooper L , Thompson RL , Harrison RA , Summerbell CD , Moore H , Worthington HV , Durrington PN , Ness AR , Capps NE , Davey Smith G , Riemersma RA , Ebrahim SB . Omega 3 fatty acids for prevention and treatment of cardiovascular disease . Cochrane Database Syst Rev . 2004 ;( 4 ): CD003177 . 3. Wei MY , Jacobson TA . Effects of eicosapentaenoic acid versus docosahexaenoic acid on serum lipids: a systematic review and meta-analysis . Curr Atheroscler Rep . 2011 ; 13 ( 6 ): 474 – 483 . Google Scholar CrossRef Search ADS PubMed 4. Allaire J , Couture P , Leclerc M , Charest A , Marin J , Lépine MC , Talbot D , Tchernof A , Lamarche B . A randomized, crossover, head-to-head comparison of eicosapentaenoic acid and docosahexaenoic acid supplementation to reduce inflammation markers in men and women: the Comparing EPA to DHA (ComparED) study . Am J Clin Nutr . 2016 ; 104 ( 2 ): 280 – 287 . Google Scholar CrossRef Search ADS PubMed 5. Berneis KK , Krauss RM . Metabolic origins and clinical significance of LDL heterogeneity . J Lipid Res . 2002 ; 43 ( 9 ): 1363 – 1379 . Google Scholar CrossRef Search ADS PubMed 6. Alaupovic P . The concept of apolipoprotein-defined lipoprotein families and its clinical significance . Curr Atheroscler Rep . 2003 ; 5 ( 6 ): 459 – 467 . Google Scholar CrossRef Search ADS PubMed 7. Zheng C , Khoo C , Ikewaki K , Sacks FM . Rapid turnover of apolipoprotein C-III-containing triglyceride-rich lipoproteins contributing to the formation of LDL subfractions . J Lipid Res . 2007 ; 48 ( 5 ): 1190 – 1203 . Google Scholar CrossRef Search ADS PubMed 8. Musunuru K , Orho-Melander M , Caulfield MP , Li S , Salameh WA , Reitz RE , Berglund G , Hedblad B , Engström G , Williams PT , Kathiresan S , Melander O , Krauss RM . Ion mobility analysis of lipoprotein subfractions identifies three independent axes of cardiovascular risk . Arterioscler Thromb Vasc Biol . 2009 ; 29 ( 11 ): 1975 – 1980 . Google Scholar CrossRef Search ADS PubMed 9. Mori TA , Burke V , Puddey IB , Watts GF , O’Neal DN , Best JD , Beilin LJ . Purified eicosapentaenoic and docosahexaenoic acids have differential effects on serum lipids and lipoproteins, LDL particle size, glucose, and insulin in mildly hyperlipidemic men . Am J Clin Nutr . 2000 ; 71 ( 5 ): 1085 – 1094 . Google Scholar CrossRef Search ADS PubMed 10. Oelrich B , Dewell A , Gardner CD . Effect of fish oil supplementation on serum triglycerides, LDL cholesterol and LDL subfractions in hypertriglyceridemic adults . Nutr Metab Cardiovasc Dis . 2013 ; 23 ( 4 ): 350 – 357 . Google Scholar CrossRef Search ADS PubMed 11. Thomas TR , Smith BK , Donahue OM , Altena TS , James-Kracke M , Sun GY . Effects of omega-3 fatty acid supplementation and exercise on low-density lipoprotein and high-density lipoprotein subfractions . Metabolism . 2004 ; 53 ( 6 ): 749 – 754 . Google Scholar CrossRef Search ADS PubMed 12. Ooi EM , Lichtenstein AH , Millar JS , Diffenderfer MR , Lamon-Fava S , Rasmussen H , Welty FK , Barrett PH , Schaefer EJ . Effects of therapeutic lifestyle change diets high and low in dietary fish-derived FAs on lipoprotein metabolism in middle-aged and elderly subjects . J Lipid Res . 2012 ; 53 ( 9 ): 1958 – 1967 . Google Scholar CrossRef Search ADS PubMed 13. Nestel PJ , Connor WE , Reardon MF , Connor S , Wong S , Boston R . Suppression by diets rich in fish oil of very low density lipoprotein production in man . J Clin Invest . 1984 ; 74 ( 1 ): 82 – 89 . Google Scholar CrossRef Search ADS PubMed 14. Bordin P , Bodamer OA , Venkatesan S , Gray RM , Bannister PA , Halliday D . Effects of fish oil supplementation on apolipoprotein B100 production and lipoprotein metabolism in normolipidaemic males . Eur J Clin Nutr . 1998 ; 52 ( 2 ): 104 – 109 . Google Scholar CrossRef Search ADS PubMed 15. Huff MW , Telford DE . Dietary fish oil increases conversion of very low density lipoprotein apoprotein B to low density lipoprotein . Arteriosclerosis . 1989 ; 9 ( 1 ): 58 – 66 . Google Scholar CrossRef Search ADS PubMed 16. Fisher WR , Zech LA , Stacpoole PW . Apolipoprotein B metabolism in hypertriglyceridemic diabetic patients administered either a fish oil- or vegetable oil-enriched diet . J Lipid Res . 1998 ; 39 ( 2 ): 388 – 401 . Google Scholar PubMed 17. Chan DC , Watts GF , Mori TA , Barrett PH , Redgrave TG , Beilin LJ . Randomized controlled trial of the effect of n-3 fatty acid supplementation on the metabolism of apolipoprotein B-100 and chylomicron remnants in men with visceral obesity . Am J Clin Nutr . 2003 ; 77 ( 2 ): 300 – 307 . Google Scholar CrossRef Search ADS PubMed 18. Tavori H , Rashid S , Fazio S . On the function and homeostasis of PCSK9: reciprocal interaction with LDLR and additional lipid effects . Atherosclerosis . 2015 ; 238 ( 2 ): 264 – 270 . Google Scholar CrossRef Search ADS PubMed 19. Graversen CB , Lundbye-Christensen S , Thomsen B , Christensen JH , Schmidt EB . Marine n-3 polyunsaturated fatty acids lower plasma proprotein convertase subtilisin kexin type 9 levels in pre- and postmenopausal women: a randomised study . Vascul Pharmacol . 2016 ; 76 : 37 – 41 . Google Scholar CrossRef Search ADS PubMed 20. Rodríguez-Pérez C , Ramprasath VR , Pu S , Sabra A , Quirantes-Piné R , Segura-Carretero A , Jones PJ . Docosahexaenoic acid attenuates cardiovascular risk factors via a decline in proprotein convertase subtilisin/kexin type 9 (PCSK9) plasma levels . Lipids . 2016 ; 51 ( 1 ): 75 – 83 . Google Scholar CrossRef Search ADS PubMed 21. Alberti KG , Zimmet P , Shaw J , Group IDFETFC ; IDF Epidemiology Task Force Consensus Group . The metabolic syndrome—a new worldwide definition . Lancet . 2005 ; 366 ( 9491 ): 1059 – 1062 . Google Scholar CrossRef Search ADS PubMed 22. Airlie LT . Roche A, Martorel R. Standardization of anthropometric measurements. The Airlie (VA) Concensus Conference . Champaign, IL : Human Kinetics ; 1988 : 39 – 80 . 23. Allaire J , Harris W , Vors C , Tchernof A , Couture P , Lamarche B. Docosahexaenoic acid is more effective than eicosapentaenoic acid in increasing the omega-3 index measured in red blood cell membranes. FASEB J. 2017 ;31(1 Supple):146.143 . 24. St-Pierre AC , Ruel IL , Cantin B , Dagenais GR , Bernard P-M , Després J-P , Lamarche B . Comparison of various electrophoretic characteristics of LDL particles and their relationship to the risk of ischemic heart disease . Circulation . 2001 ; 104 ( 19 ): 2295 – 2299 . Google Scholar CrossRef Search ADS PubMed 25. Matthews DR , Hosker JP , Rudenski AS , Naylor BA , Treacher DF , Turner RC . Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man . Diabetologia . 1985 ; 28 ( 7 ): 412 – 419 . Google Scholar CrossRef Search ADS PubMed 26. Harris JA , Benedict FG . A Biometric Study of Basal Metabolism in Man. Washington DC: Carnegie Institution of Washington ; 1919 . 27. Lamarche B , Couture P . Dietary fatty acids, dietary patterns, and lipoprotein metabolism . Curr Opin Lipidol . 2015 ; 26 ( 1 ): 42 – 47 . Google Scholar CrossRef Search ADS PubMed 28. Ooi EM , Watts GF , Ng TW , Barrett PH . Effect of dietary fatty acids on human lipoprotein metabolism: a comprehensive update . Nutrients . 2015 ; 7 ( 6 ): 4416 – 4425 . Google Scholar CrossRef Search ADS PubMed 29. Kelley DS , Siegel D , Vemuri M , Mackey BE . Docosahexaenoic acid supplementation improves fasting and postprandial lipid profiles in hypertriglyceridemic men . Am J Clin Nutr . 2007 ; 86 ( 2 ): 324 – 333 . Google Scholar CrossRef Search ADS PubMed 30. Lagrost L , Gandjini H , Athias A , Guyard-Dangremont V , Lallemant C , Gambert P . Influence of plasma cholesteryl ester transfer activity on the LDL and HDL distribution profiles in normolipidemic subjects . Arterioscler Thromb . 1993 ; 13 ( 6 ): 815 – 825 . Google Scholar CrossRef Search ADS PubMed 31. Oscarsson J , Hurt-Camejo E . Omega-3 fatty acids eicosapentaenoic acid and docosahexaenoic acid and their mechanisms of action on apolipoprotein B-containing lipoproteins in humans: a review . Lipids Health Dis . 2017 ; 16 ( 1 ): 149 . Google Scholar CrossRef Search ADS PubMed 32. Kawakami A , Yoshida M . Apolipoprotein CIII links dyslipidemia with atherosclerosis . J Atheroscler Thromb . 2009 ; 16 ( 1 ): 6 – 11 . Google Scholar CrossRef Search ADS PubMed 33. Jump DB , Botolin D , Wang Y , Xu J , Demeure O , Christian B . Docosahexaenoic acid (DHA) and hepatic gene transcription . Chem Phys Lipids . 2008 ; 153 ( 1 ): 3 – 13 . Google Scholar CrossRef Search ADS PubMed 34. Chen YJ , Chen CC , Li TK , Wang PH , Liu LR , Chang FY , Wang YC , Yu YH , Lin SP , Mersmann HJ , Ding ST . Docosahexaenoic acid suppresses the expression of FoxO and its target genes . J Nutr Biochem . 2012 ; 23 ( 12 ): 1609 – 1616 . Google Scholar CrossRef Search ADS PubMed 35. Homma Y , Ohshima K , Yamaguchi H , Nakamura H , Araki G , Goto Y . Effects of eicosapentaenoic acid on plasma lipoprotein subfractions and activities of lecithin:cholesterol acyltransferase and lipid transfer protein . Atherosclerosis . 1991 ; 91 ( 1-2 ): 145 – 153 . Google Scholar CrossRef Search ADS PubMed 36. Buckley R , Shewring B , Turner R , Yaqoob P , Minihane AM . Circulating triacylglycerol and apoE levels in response to EPA and docosahexaenoic acid supplementation in adult human subjects . Br J Nutr . 2004 ; 92 ( 3 ): 477 – 483 . Google Scholar CrossRef Search ADS PubMed 37. Hirayama S , Miida T . Small dense LDL: an emerging risk factor for cardiovascular disease . Clin Chim Acta . 2012 ; 414 : 215 – 224 . Google Scholar CrossRef Search ADS PubMed 38. Lakoski SG , Lagace TA , Cohen JC , Horton JD , Hobbs HH . Genetic and metabolic determinants of plasma PCSK9 levels . J Clin Endocrinol Metab . 2009 ; 94 ( 7 ): 2537 – 2543 . Google Scholar CrossRef Search ADS PubMed 39. Lee TC , Ivester P , Hester AG , Sergeant S , Case LD , Morgan T , Kouba EO , Chilton FH . The impact of polyunsaturated fatty acid-based dietary supplements on disease biomarkers in a metabolic syndrome/diabetes population . Lipids Health Dis . 2014 ; 13 ( 1 ): 196 . Google Scholar CrossRef Search ADS PubMed 40. Ooi EM , Ng TW , Watts GF , Barrett PHR . Dietary fatty acids and lipoprotein metabolism: new insights and updates . Curr Opin Lipidol . 2013 ; 24 ( 3 ): 192 – 197 . Google Scholar CrossRef Search ADS PubMed Copyright © 2018 Endocrine Society http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Clinical Endocrinology and Metabolism Oxford University Press

High-Dose DHA Has More Profound Effects on LDL-Related Features Than High-Dose EPA: The ComparED Study

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Endocrine Society
Copyright
Copyright © 2018 Endocrine Society
ISSN
0021-972X
eISSN
1945-7197
D.O.I.
10.1210/jc.2017-02745
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Abstract

Abstract Context Supplementation with high-dose docosahexaenoic acid (DHA) increases serum low-density lipoprotein (LDL) cholesterol (LDL-C) concentrations more than high-dose eicosapentaenoic acid (EPA). The mechanisms underlying this difference are unknown. Objective To examine the phenotypic change in LDL and mechanisms responsible for the differential LDL-C response to EPA and DHA supplementation in men and women at risk of cardiovascular disease. Design, Setting, Participants, and Intervention In a double-blind, controlled, crossover study, 48 men and 106 women with abdominal obesity and subclinical inflammation were randomized to a sequence of three treatment phases: phase 1, 2.7 g/d of EPA; phase 2, 2.7 g/d of DHA; and phase 3, 3 g/d of corn oil. All supplements were provided as three 1-g capsules for a total of 3 g/d. The 10-week treatment phases were separated by a 9-week washout period. Main Outcome Measure In vivo kinetics of apolipoprotein (apo)B100-containing lipoproteins were assessed using primed-constant infusion of deuterated leucine at the end of each treatment in a subset of participants (n = 19). Results Compared with EPA, DHA increased LDL-C concentrations (+3.3%; P = 0.038) and mean LDL particle size (+0.7 Å; P < 0.001) and reduced the proportion of small LDL (−3.2%; P < 0.01). Both EPA and DHA decreased proprotein convertase subtilisin/kexin type 9 concentrations similarly (−18.2% vs −25.0%; P < 0.0001 vs control). Compared with EPA, DHA supplementation increased both the LDL apoB100 fractional catabolic rate (+11.4%; P = 0.008) and the production rate (+9.4%; P = 0.03). Conclusions The results of the present study have shown that supplementation with high-dose DHA increases LDL turnover and contributes to larger LDL particles compared with EPA. Despite having favorable effects on serum triglyceride (TG) concentrations, cardiac arrhythmia, platelet aggregation, heart rate, blood pressure, and inflammation (1), the extent to which long-chain omega-3 polyunsaturated fatty acid (LCn3-PUFA) supplementation prevents cardiovascular disease (CVD) remains controversial (2). The inconsistent effect of LCn3-PUFAs supplementation on CVD risk might be because docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) are also known to increase low-density lipoprotein (LDL) cholesterol (LDL-C) concentrations (1). The extent to which different LCn3-PUFAs have distinct effects on CVD risk also remains questionable because previous studies have shown that DHA is more potent than EPA in modulating cardiovascular risk factors, including increasing LDL-C concentrations (3, 4). To the best of our knowledge, no study has yet examined how DHA and EPA influence the mechanisms underlying their differential effect on several LDL features, including LDL-C concentrations and, hence, CVD risk. It has been stressed that the cholesterol content of LDL represents only one of several features of this complex lipoprotein, which is heterogeneous in size, charge, and protein content (5, 6). Different immunochemically defined LDL subclasses are thought to have distinct metabolisms and atherogenicity (6). Specifically, small and dense LDL (sdLDL) consists largely of apolipoprotein (apo)-CIII–containing lipoproteins that originate from the remodeling in plasma of apo-CIII–rich very-low-density lipoprotein (VLDL) (7). sdLDLs have also been consistently associated with an increased risk of developing CVD, independent of the LDL-C concentration (8). Data from a limited number of trials have suggested that, unlike EPA, high-dose DHA increases LDL particle size (9). In other studies, LCn3-PUFA supplementation (4 g/d) in hypertriglyceridemic men had no substantial impact on the features of the LDL size phenotype. However, this might have been because DHA and EPA were given in combination (10, 11). Although the clinical relevance of the potential differences between DHA and EPA in modulating LDL particle size is unclear, data have suggested that both LCn3-PUFAs might differentially influence LDL metabolism. Previous studies have investigated the effect of an LCn3-PUFA–rich diet or supplementation on the kinetic of apoB100–containing lipoproteins. Ooi et al. (12) have shown that a high-fish diet providing 1.23 g/d of EPA and DHA reduced the TG-rich lipoprotein (TRL) apoB100 concentration and production rate (PR) compared with a low-fish diet in elderly men and women with moderate hyperlipidemia. These studies also showed that the high-fish diet decreased TRL apoB100–direct catabolism, rechanneling TRL toward conversion into LDL and, hence, increasing the LDL PR. These data have been reproduced in other studies that used fish oils as supplements (13–17). Proprotein convertase subtilisin/kexin type 9 (PCSK9) regulates cholesterol metabolism by degrading cellular LDL receptors, blunting the clearance of LDL from the circulation (18). LCn3-PUFAs have been shown to decrease PCSK9 concentrations in several studies (19, 20), which should, in theory, be associated with increased LDL clearance and, hence, reduced serum LDL-C. To the best of our knowledge, no study has yet compared the effect of high-dose supplementation with DHA and EPA on the kinetics of apoB100–containing lipoproteins and PCSK9 as key determinants of LDL-C concentrations. The objective of the present study was, therefore, to examine the mechanisms underlying the differential effect of DHA and EPA supplementation on LDL features, including the LDL-C concentrations in men and women at risk of CVD. Specifically, we compared the effect of high doses of DHA and of EPA on the intravascular kinetics of apoB100-containing lipoproteins, VLDL apoCIII, LDL particle size distribution, and PCSK9 levels. We hypothesized that DHA has favorable effects on LDL size features compared with EPA and that changes in the intravascular kinetics of LDL are also different between DHA and EPA, thereby partly explaining the different effect of the two LCn3-PUFAs on the serum LDL-C concentrations. Materials and Methods Study design Details of the study design have been previously reported (4). In brief, the present study used a double-blind randomized, controlled crossover design with three treatment phases: phase 1, DHA; phase 2, EPA; phase 3, corn oil as the control. Each treatment phase had a median duration of 10 weeks, separated by a 9-week washout period. The participants were randomized to one of six treatment sequences and received supplementation with three identical 1-g capsules of >90% purified LCn3-PUFA daily, providing either 2.7 g/d of DHA or 2.7 g/d of EPA. Corn oil was used as the control (0 g/d of DHA plus EPA). LCn3-PUFA supplements were formulated as re-esterified TG and provided by Douglas Laboratories (Pittsburgh, PA). The participants were instructed to maintain a constant body weight during the course of the study and were counseled on how to exclude fatty fish (including salmon, tuna, mackerel, and herring), other LCn3-PUFA supplements, flax products, walnuts, and LCn3-PUFA–enriched products during the three study phases. The primary outcome of the present study was the change in C-reactive protein (CRP) with DHA and EPA supplementation (4). All participants signed an informed consent document that had been approved by local ethics committees at the beginning of the study, and the study protocol was registered at ClinicalTrials.gov (NCT01810003) on March 4, 2013. Study population The primary eligibility criteria were abdominal obesity using the International Diabetes Federation sex-specific cutoffs (≥80 cm for women, ≥94 cm for men) (21) and a screening plasma CRP concentration >1 mg/L but <10 mg/L. The participants had to be otherwise healthy. Adult participants (aged 18 to 70 years) were recruited at the Institute of Nutrition and Functional Foods. Their body weight had to be stable for ≥3 months before randomization. The exclusion criteria were plasma CRP >10 mg/L at screening, extreme dyslipidemia such as familial hypercholesterolemia, a personal history of CVD (coronary heart disease, cerebrovascular disease, or peripheral arterial disease), use of medications or substances known to affect inflammation (e.g., steroids, binging alcohol), and the use of LCn3-PUFA supplements within 2 months of study onset. However, individuals taking lipid-lowering drugs for >1 month were eligible. Anthropometry Anthropometric measures, including waist and hip circumferences, were measured according to standardized procedures before and after each study phase (22). Body weight was measured before each kinetic protocol. Compliance Compliance to supplementation was assessed by counting the supplements that were returned to the study coordinators by the participants (4). The DHA and EPA content in red blood cells was also used as another proxy of compliance for all participants (23). Laboratory analyses Blood samples were collected after a 12-hour overnight fast on 2 consecutive days at the end of each treatment phase. The mean of the two measurements was used in the analyses of the LDL features and blood glucose. The total apoB100, apoCIII, PCSK9, and insulin concentrations were measured once after each treatment phase. The serum total apoB100, apoCIII, and PCSK9 concentrations were measured using commercial ELISA kits [catalog no. A70102 (Alerchek Inc., Springvale, ME), catalog no. EA8133-1 (Assaypro LLC, St. Charles, MO), catalog no. CY-8079 (CircuLex, Nagano, Japan)]. Serum LDL-C concentrations were calculated using the Friedewald equation. Nondenaturing 2% to 16% polyacrylamide gradient gel electrophoresis was used to characterize various features of the LDL particle size phenotype (24), including the LDL peak particle size and mean LDL particle size and the proportion of LDL in the various size categories. Fasting blood glucose levels were measured using colorimetry, and insulin concentrations were measured using electrochemiluminescence (Roche Diagnostics, Indianapolis, IN). Finally, the homeostatic model assessment of insulin resistance was measured using the formula developed by Matthews et al. (25). All personnel involved in the measurements of the study outcomes were unaware of the treatments. Experimental protocol for in vivo stable isotope kinetics Kinetic studies using primed-constant infusion of deuterated leucine were performed at the end of each treatment in a subsample of the participants. The participants in the kinetic studies were recruited as a part of the general recruitment process in the project, until 20 participants had been reached. The participants underwent a primed-constant infusion of L-[5,5,5-D3] leucine while kept in a constant fed state to determine the kinetics of apoB100. Starting at 7:00 am, the participants received one small standardized snack every 30 minutes for 15 hours, each containing 1/30th of their estimate daily food intake, according to the Harris-Benedict equation (26), with 15% of the calories from proteins, 45% from carbohydrates, and 40% from fat. The snacks were the same for each treatment. At 10:00 am, with two intravenous lines in place (one for the infusate and one for blood sampling), L-[5,5,5-D3] leucine (10 µmol/kg body weight) was injected as an intravenous bolus and then by continuous infusion (10 µmol/kg body weight per hour) for a 12-hour period. Blood samples (24 mL) were collected at 0, 0.5, 1, 1.5, 2, 3, 4, 6, 8, 10, 11, and 12 hours. Data on sample processing, laboratory measurements, analysis of the lipoprotein PR, and fractional catabolic rate (FCR) are provided in the Supplemental Data. Statistical analysis Differences between treatments were assessed using the MIXED procedure for repeated measures, with treatment as fixed effect and a compound symmetry matrix to account for within-subject correlations (SAS, version 9.4; SAS Institute, Cary, NC). The change vs the control treatment (post-treatment DHA minus control and EPA minus control) was used as the dependent variable in all analyses. The main treatment effect in the mixed models reflected the direct comparison of DHA and EPA and was considered the primary analysis. Adjustment for multiple comparisons was not necessary, because the main treatment effect had only two levels (DHA and EPA). In the same model and as secondary analyses, the change vs control for each treatment was tested against the null hypothesis using the LSMEANS statement. The skewness in the distribution of model residuals was considered, and the data were log-transformed when required. Wilcoxon signed-rank tests were also performed to test for the difference in the change from control after DHA and EPA supplementation, with results similar to those generated by the mixed models (data not shown). Spearman coefficient correlations among the changes in apoB100- and apoCIII-containing lipoprotein kinetic parameters, PCSK9 levels, LDL-C, LDL-apoB100, and LDL size were computed. Results Data from one participant who was ill during the first kinetic study test were excluded from the analyses. The baseline characteristics of the 19 participants who completed at least one kinetic substudy and the participants of the whole sample who completed at least one study phase are presented in Table 1. The characteristics of the subsample were similar to those of the whole group, with the exception that proportionally more women were included in the kinetic substudy. Among the participants of the kinetic substudy, one participant completed only one phase and two participants completed two study phases. The mean compliance rate based on the returned capsules was >95% for all study phases (data not shown). Among the 154 participants randomized to treatment sequences, 12 participants were taking statins. None were taking other lipid-lowering drugs. Pharmacotherapy remained unchanged in all participants throughout the study in the present crossover trial. None of the kinetic substudy participants were taking lipid-lowering drugs. Three participants had type 2 diabetes and one had type 1 diabetes; however, none of these participants were included in the substudy. Table 1. Baseline Characteristics of the Kinetic Subsample and Whole Cohort Characteristic Subsample (n = 19) Whole Cohort (n = 138) Age, y 47±16 52 ± 15 Female, n (%) 9 (47) 96 (70) Weight, kg 84.0 ± 13.8 80.6 ± 14.5 BMI, kg/m2 29.7 ± 4.8 29.4 ± 4.3 Waist circumference, cm 102.5 ± 9.9 100.7 ± 11.1 SBP, mm Hg 115.2 ± 8.5 115.6 ± 12.8 DBP, mm Hg 68.0 ± 8.1 69.7 ± 8.9 Total C, mmol/La 5.08 ± 0.64 5.18 ± 0.93 LDL-C, mmol/La 3.05 ± 0.55 3.03 ± 0.81 HDL-C, mmol/La 1.43 ± 0.43 1.52 ± 0.42 TG, mmol/La 1.30 ± 0.50 1.36 ± 0.58 CRP, mg/L 3.35 ± 2.71 3.32 ± 2.44 Glucose, mmol/La 5.26 ± 0.37 5.28 ± 0.85 Insulin, pmol/L 93.4 ± 39.9 102.54 ± 58.61 HOMA-IRa 3.11 ± 1.27 3.55 ± 2.57 Characteristic Subsample (n = 19) Whole Cohort (n = 138) Age, y 47±16 52 ± 15 Female, n (%) 9 (47) 96 (70) Weight, kg 84.0 ± 13.8 80.6 ± 14.5 BMI, kg/m2 29.7 ± 4.8 29.4 ± 4.3 Waist circumference, cm 102.5 ± 9.9 100.7 ± 11.1 SBP, mm Hg 115.2 ± 8.5 115.6 ± 12.8 DBP, mm Hg 68.0 ± 8.1 69.7 ± 8.9 Total C, mmol/La 5.08 ± 0.64 5.18 ± 0.93 LDL-C, mmol/La 3.05 ± 0.55 3.03 ± 0.81 HDL-C, mmol/La 1.43 ± 0.43 1.52 ± 0.42 TG, mmol/La 1.30 ± 0.50 1.36 ± 0.58 CRP, mg/L 3.35 ± 2.71 3.32 ± 2.44 Glucose, mmol/La 5.26 ± 0.37 5.28 ± 0.85 Insulin, pmol/L 93.4 ± 39.9 102.54 ± 58.61 HOMA-IRa 3.11 ± 1.27 3.55 ± 2.57 Data presented as mean ± SD. CRP values >10 were excluded (n = 5 for the whole cohort). For the whole cohort, the baseline characteristics of participants who completed at least one study phase are presented. Abbreviations: BMI, body mass index; C, cholesterol; DBP, diastolic blood pressure; HDL, high-density lipoprotein; HOMA-IR, homeostatic model assessment of insulin resistance; SBP, systolic blood pressure. a For the whole cohort, n = 137. View Large Table 1. Baseline Characteristics of the Kinetic Subsample and Whole Cohort Characteristic Subsample (n = 19) Whole Cohort (n = 138) Age, y 47±16 52 ± 15 Female, n (%) 9 (47) 96 (70) Weight, kg 84.0 ± 13.8 80.6 ± 14.5 BMI, kg/m2 29.7 ± 4.8 29.4 ± 4.3 Waist circumference, cm 102.5 ± 9.9 100.7 ± 11.1 SBP, mm Hg 115.2 ± 8.5 115.6 ± 12.8 DBP, mm Hg 68.0 ± 8.1 69.7 ± 8.9 Total C, mmol/La 5.08 ± 0.64 5.18 ± 0.93 LDL-C, mmol/La 3.05 ± 0.55 3.03 ± 0.81 HDL-C, mmol/La 1.43 ± 0.43 1.52 ± 0.42 TG, mmol/La 1.30 ± 0.50 1.36 ± 0.58 CRP, mg/L 3.35 ± 2.71 3.32 ± 2.44 Glucose, mmol/La 5.26 ± 0.37 5.28 ± 0.85 Insulin, pmol/L 93.4 ± 39.9 102.54 ± 58.61 HOMA-IRa 3.11 ± 1.27 3.55 ± 2.57 Characteristic Subsample (n = 19) Whole Cohort (n = 138) Age, y 47±16 52 ± 15 Female, n (%) 9 (47) 96 (70) Weight, kg 84.0 ± 13.8 80.6 ± 14.5 BMI, kg/m2 29.7 ± 4.8 29.4 ± 4.3 Waist circumference, cm 102.5 ± 9.9 100.7 ± 11.1 SBP, mm Hg 115.2 ± 8.5 115.6 ± 12.8 DBP, mm Hg 68.0 ± 8.1 69.7 ± 8.9 Total C, mmol/La 5.08 ± 0.64 5.18 ± 0.93 LDL-C, mmol/La 3.05 ± 0.55 3.03 ± 0.81 HDL-C, mmol/La 1.43 ± 0.43 1.52 ± 0.42 TG, mmol/La 1.30 ± 0.50 1.36 ± 0.58 CRP, mg/L 3.35 ± 2.71 3.32 ± 2.44 Glucose, mmol/La 5.26 ± 0.37 5.28 ± 0.85 Insulin, pmol/L 93.4 ± 39.9 102.54 ± 58.61 HOMA-IRa 3.11 ± 1.27 3.55 ± 2.57 Data presented as mean ± SD. CRP values >10 were excluded (n = 5 for the whole cohort). For the whole cohort, the baseline characteristics of participants who completed at least one study phase are presented. Abbreviations: BMI, body mass index; C, cholesterol; DBP, diastolic blood pressure; HDL, high-density lipoprotein; HOMA-IR, homeostatic model assessment of insulin resistance; SBP, systolic blood pressure. a For the whole cohort, n = 137. View Large LDL particle size and PCSK9 Blood lipids and LDL particle size features before the control phase and after the three treatments are presented in Table 2 for all participants and for the substudy group only. The treatment-specific baseline values before DHA and EPA were essentially identical to the values measured before the control treatment and therefore were not presented. In all participants, DHA increased the mean LDL particle size (compared with the control: DHA, +0.32 Å; EPA, −0.41 Å; DHA vs EPA, P < 0.0001) and LDL peak particle size (compared with control: DHA, +0.58 Å; EPA, −0.32 Å; DHA vs EPA, P < 0.0001) more than did EPA. The change in the proportion of sdLDL was also significantly different statistically between EPA and DHA (compared with control: DHA, −1.10%; EPA, +2.10%; EPA vs DHA, P < 0.002). Both EPA and DHA decreased the PCSK9 concentrations similarly (compared with control: DHA, −25.0 ng/mL; EPA, −18.2 ng/mL; DHA vs EPA, P = 0.19). The changes in these cardiometabolic outcomes with DHA and EPA were generally similar in direction and magnitude among the participants of the substudy. However, only the difference between the change in the PCSK9 concentrations after DHA and EPA compared with control remained statistically significant in the subsample. Table 2. LDL Particle Size Features and PCSK9 Concentration Before and After Control, DHA, and EPA Phase in Whole and Kinetic Sample Variable Before Control Phase After Control Phase After DHA Difference (DHA vs Control), % P Value (DHA vs Controla) After EPA Difference (EPA vs Control), % P Value (EPA vs Controla) Difference (DHA vs ΔEPA) P Value (DHA vs EPAb) All participants (n = 121)c  Total cholesterol, mmol/Ld,e 5.29 ± 0.08 5.16 ± 0.08 5.33 ± 0.08 3.1 0.001f 5.15 ± 0.08 −0.4 0.62 3.6 < 0.001f  LDL-C, mmol/Ld,e 3.12 ± 0.07 2.99 ± 0.07 3.16 ± 0.08 5.4 < 0.001f 3.06 ± 0.07 2.5 0.046f 3.2 0.04f  Total apoB, g/Ld,e 1.34 ± 0.04 1.31 ± 0.04 1.34 ± 0.04 2.8 0.02f 1.32 ± 0.04 1.2 0.46 1.6 0.16  TG, mmol/Ld,e 1.34 ± 0.06 1.38 ± 0.06 1.14 ± 0.04 −21.3 < 0.001f 1.23 ± 0.05 −11.3 < 0.001f −7.1 0.005f  Mean LDL size, Å 252.10 ± 0.26 251.89 ± 0.21 252.15 ± 0.23 0.1 0.051 251.41 ± 0.20 −0.2 0.01f 0.3 < 0.001f  LDL peak, Å 251.74 ± 0.28 251.56 ± 0.24 252.08 ± 0.25 0.2 0.001f 251.19 ± 0.23 −0.1 0.059 0.4 < 0.001f  Proportion of large LDL, % 9.49 ± 0.72 9.00 ± 0.53 9.16 ± 0.62 1.8 0.85 8.42 ± 0.53 −6.4 0.20 8.8 0.19  Proportion of small LDL, % 69.29 ± 1.45 69.83 ± 1.20 68.88 ± 1.34 −1.4 0.25 72.00 ± 1.08 3.1 0.03f −4.3 0.002f  PCSK9, ng/mL NA 213.02 ± 5.65 189.12 ± 4.89 −12.6 < 0.001f 194.98 ± 5.40 −8.5 < 0.001f −3.0 0.19 Substudy (n = 19)g  Total cholesterol, mmol/L 5.18 ± 0.16 4.70 ± 0.16 5.13 ± 0.14 9.1 0.005f 5.01 ± 0.19 8.0 0.018f 2.5 0.62  LDL-C, mmol/L 3.11 ± 0.14 2.70 ± 0.13 3.10 ± 0.15 14.7 0.006f 3.05 ± 0.14 14.4 0.010f 1.6 0.88  Total apoB, g/L 1.37 ± 0.09 1.26 ± 0.08 1.40 ± 0.12 11.0 0.03f 1.35 ± 0.09 6.9 0.27 3.5 0.27  TG, mmol/L 1.29 ± 0.13 1.35 ± 0.17 1.20 ± 0.15 −11.1 0.10 1.39 ± 0.21 4.6 0.63 −13.6 0.06  Mean LDL size, Å 250.88 ± 0.75 250.95 ± 0.54 250.95 ± 0.63 0.0 0.90 250.42 ± 0.60 −0.2 0.29 0.2 0.33  LDL peak, Å 250.62 ± 0.76 250.68 ± 0.63 250.79 ± 0.71 0.0 0.89 250.20 ± 0.61 −0.2 0.12 0.2 0.17  Proportion of large LDL, % 7.52 ± 1.38 6.90 ± 1.07 7.01 ± 1.35 1.5 0.34 7.42 ± 1.12 4.2 0.12 −5.6 0.54  Proportion of small LDL, % 75.03 ± 3.16 76.13 ± 2.32 75.53 ± 2.88 −0.8 0.56 75.98 ± 2.55 0.0 0.72 −0.6 0.86  PCSK9, ng/mL NA 196.92 ± 15.23 174.43 ± 13.53 −11.4 0.11 190.08 ± 15.33 1.2 0.75 −8.2 0.04f Variable Before Control Phase After Control Phase After DHA Difference (DHA vs Control), % P Value (DHA vs Controla) After EPA Difference (EPA vs Control), % P Value (EPA vs Controla) Difference (DHA vs ΔEPA) P Value (DHA vs EPAb) All participants (n = 121)c  Total cholesterol, mmol/Ld,e 5.29 ± 0.08 5.16 ± 0.08 5.33 ± 0.08 3.1 0.001f 5.15 ± 0.08 −0.4 0.62 3.6 < 0.001f  LDL-C, mmol/Ld,e 3.12 ± 0.07 2.99 ± 0.07 3.16 ± 0.08 5.4 < 0.001f 3.06 ± 0.07 2.5 0.046f 3.2 0.04f  Total apoB, g/Ld,e 1.34 ± 0.04 1.31 ± 0.04 1.34 ± 0.04 2.8 0.02f 1.32 ± 0.04 1.2 0.46 1.6 0.16  TG, mmol/Ld,e 1.34 ± 0.06 1.38 ± 0.06 1.14 ± 0.04 −21.3 < 0.001f 1.23 ± 0.05 −11.3 < 0.001f −7.1 0.005f  Mean LDL size, Å 252.10 ± 0.26 251.89 ± 0.21 252.15 ± 0.23 0.1 0.051 251.41 ± 0.20 −0.2 0.01f 0.3 < 0.001f  LDL peak, Å 251.74 ± 0.28 251.56 ± 0.24 252.08 ± 0.25 0.2 0.001f 251.19 ± 0.23 −0.1 0.059 0.4 < 0.001f  Proportion of large LDL, % 9.49 ± 0.72 9.00 ± 0.53 9.16 ± 0.62 1.8 0.85 8.42 ± 0.53 −6.4 0.20 8.8 0.19  Proportion of small LDL, % 69.29 ± 1.45 69.83 ± 1.20 68.88 ± 1.34 −1.4 0.25 72.00 ± 1.08 3.1 0.03f −4.3 0.002f  PCSK9, ng/mL NA 213.02 ± 5.65 189.12 ± 4.89 −12.6 < 0.001f 194.98 ± 5.40 −8.5 < 0.001f −3.0 0.19 Substudy (n = 19)g  Total cholesterol, mmol/L 5.18 ± 0.16 4.70 ± 0.16 5.13 ± 0.14 9.1 0.005f 5.01 ± 0.19 8.0 0.018f 2.5 0.62  LDL-C, mmol/L 3.11 ± 0.14 2.70 ± 0.13 3.10 ± 0.15 14.7 0.006f 3.05 ± 0.14 14.4 0.010f 1.6 0.88  Total apoB, g/L 1.37 ± 0.09 1.26 ± 0.08 1.40 ± 0.12 11.0 0.03f 1.35 ± 0.09 6.9 0.27 3.5 0.27  TG, mmol/L 1.29 ± 0.13 1.35 ± 0.17 1.20 ± 0.15 −11.1 0.10 1.39 ± 0.21 4.6 0.63 −13.6 0.06  Mean LDL size, Å 250.88 ± 0.75 250.95 ± 0.54 250.95 ± 0.63 0.0 0.90 250.42 ± 0.60 −0.2 0.29 0.2 0.33  LDL peak, Å 250.62 ± 0.76 250.68 ± 0.63 250.79 ± 0.71 0.0 0.89 250.20 ± 0.61 −0.2 0.12 0.2 0.17  Proportion of large LDL, % 7.52 ± 1.38 6.90 ± 1.07 7.01 ± 1.35 1.5 0.34 7.42 ± 1.12 4.2 0.12 −5.6 0.54  Proportion of small LDL, % 75.03 ± 3.16 76.13 ± 2.32 75.53 ± 2.88 −0.8 0.56 75.98 ± 2.55 0.0 0.72 −0.6 0.86  PCSK9, ng/mL NA 196.92 ± 15.23 174.43 ± 13.53 −11.4 0.11 190.08 ± 15.33 1.2 0.75 −8.2 0.04f Data presented as unadjusted mean ± SEM. Abbreviation: NA, not available. a This analysis compared the change with DHA or EPA compared with control based on posttreatment values; P values were taken from the main treatment effect in the mixed models. b P values for the EPA and DHA changes vs control, as determined by the LSMEANS statement and tested against the null hypothesis in the mixed models. c Participants equaled 123 for DHA, 121 for EPA, 125 for control, and 138 for baseline. d Log-transformed data were used in these analyses due to skewness of the distribution of the values. e Previously reported (4). f P < 0.05; because pre-DHA and -EPA values were essentially identical to the precontrol values, only precontrol values are presented. Models were adjusted for sex, weight, age, sequence; baseline value was considered only when these covariates were found to be significant at P < 0.05 in the models. g Participants equaled 19 for DHA, 17 for EPA, and 19 for control. View Large Table 2. LDL Particle Size Features and PCSK9 Concentration Before and After Control, DHA, and EPA Phase in Whole and Kinetic Sample Variable Before Control Phase After Control Phase After DHA Difference (DHA vs Control), % P Value (DHA vs Controla) After EPA Difference (EPA vs Control), % P Value (EPA vs Controla) Difference (DHA vs ΔEPA) P Value (DHA vs EPAb) All participants (n = 121)c  Total cholesterol, mmol/Ld,e 5.29 ± 0.08 5.16 ± 0.08 5.33 ± 0.08 3.1 0.001f 5.15 ± 0.08 −0.4 0.62 3.6 < 0.001f  LDL-C, mmol/Ld,e 3.12 ± 0.07 2.99 ± 0.07 3.16 ± 0.08 5.4 < 0.001f 3.06 ± 0.07 2.5 0.046f 3.2 0.04f  Total apoB, g/Ld,e 1.34 ± 0.04 1.31 ± 0.04 1.34 ± 0.04 2.8 0.02f 1.32 ± 0.04 1.2 0.46 1.6 0.16  TG, mmol/Ld,e 1.34 ± 0.06 1.38 ± 0.06 1.14 ± 0.04 −21.3 < 0.001f 1.23 ± 0.05 −11.3 < 0.001f −7.1 0.005f  Mean LDL size, Å 252.10 ± 0.26 251.89 ± 0.21 252.15 ± 0.23 0.1 0.051 251.41 ± 0.20 −0.2 0.01f 0.3 < 0.001f  LDL peak, Å 251.74 ± 0.28 251.56 ± 0.24 252.08 ± 0.25 0.2 0.001f 251.19 ± 0.23 −0.1 0.059 0.4 < 0.001f  Proportion of large LDL, % 9.49 ± 0.72 9.00 ± 0.53 9.16 ± 0.62 1.8 0.85 8.42 ± 0.53 −6.4 0.20 8.8 0.19  Proportion of small LDL, % 69.29 ± 1.45 69.83 ± 1.20 68.88 ± 1.34 −1.4 0.25 72.00 ± 1.08 3.1 0.03f −4.3 0.002f  PCSK9, ng/mL NA 213.02 ± 5.65 189.12 ± 4.89 −12.6 < 0.001f 194.98 ± 5.40 −8.5 < 0.001f −3.0 0.19 Substudy (n = 19)g  Total cholesterol, mmol/L 5.18 ± 0.16 4.70 ± 0.16 5.13 ± 0.14 9.1 0.005f 5.01 ± 0.19 8.0 0.018f 2.5 0.62  LDL-C, mmol/L 3.11 ± 0.14 2.70 ± 0.13 3.10 ± 0.15 14.7 0.006f 3.05 ± 0.14 14.4 0.010f 1.6 0.88  Total apoB, g/L 1.37 ± 0.09 1.26 ± 0.08 1.40 ± 0.12 11.0 0.03f 1.35 ± 0.09 6.9 0.27 3.5 0.27  TG, mmol/L 1.29 ± 0.13 1.35 ± 0.17 1.20 ± 0.15 −11.1 0.10 1.39 ± 0.21 4.6 0.63 −13.6 0.06  Mean LDL size, Å 250.88 ± 0.75 250.95 ± 0.54 250.95 ± 0.63 0.0 0.90 250.42 ± 0.60 −0.2 0.29 0.2 0.33  LDL peak, Å 250.62 ± 0.76 250.68 ± 0.63 250.79 ± 0.71 0.0 0.89 250.20 ± 0.61 −0.2 0.12 0.2 0.17  Proportion of large LDL, % 7.52 ± 1.38 6.90 ± 1.07 7.01 ± 1.35 1.5 0.34 7.42 ± 1.12 4.2 0.12 −5.6 0.54  Proportion of small LDL, % 75.03 ± 3.16 76.13 ± 2.32 75.53 ± 2.88 −0.8 0.56 75.98 ± 2.55 0.0 0.72 −0.6 0.86  PCSK9, ng/mL NA 196.92 ± 15.23 174.43 ± 13.53 −11.4 0.11 190.08 ± 15.33 1.2 0.75 −8.2 0.04f Variable Before Control Phase After Control Phase After DHA Difference (DHA vs Control), % P Value (DHA vs Controla) After EPA Difference (EPA vs Control), % P Value (EPA vs Controla) Difference (DHA vs ΔEPA) P Value (DHA vs EPAb) All participants (n = 121)c  Total cholesterol, mmol/Ld,e 5.29 ± 0.08 5.16 ± 0.08 5.33 ± 0.08 3.1 0.001f 5.15 ± 0.08 −0.4 0.62 3.6 < 0.001f  LDL-C, mmol/Ld,e 3.12 ± 0.07 2.99 ± 0.07 3.16 ± 0.08 5.4 < 0.001f 3.06 ± 0.07 2.5 0.046f 3.2 0.04f  Total apoB, g/Ld,e 1.34 ± 0.04 1.31 ± 0.04 1.34 ± 0.04 2.8 0.02f 1.32 ± 0.04 1.2 0.46 1.6 0.16  TG, mmol/Ld,e 1.34 ± 0.06 1.38 ± 0.06 1.14 ± 0.04 −21.3 < 0.001f 1.23 ± 0.05 −11.3 < 0.001f −7.1 0.005f  Mean LDL size, Å 252.10 ± 0.26 251.89 ± 0.21 252.15 ± 0.23 0.1 0.051 251.41 ± 0.20 −0.2 0.01f 0.3 < 0.001f  LDL peak, Å 251.74 ± 0.28 251.56 ± 0.24 252.08 ± 0.25 0.2 0.001f 251.19 ± 0.23 −0.1 0.059 0.4 < 0.001f  Proportion of large LDL, % 9.49 ± 0.72 9.00 ± 0.53 9.16 ± 0.62 1.8 0.85 8.42 ± 0.53 −6.4 0.20 8.8 0.19  Proportion of small LDL, % 69.29 ± 1.45 69.83 ± 1.20 68.88 ± 1.34 −1.4 0.25 72.00 ± 1.08 3.1 0.03f −4.3 0.002f  PCSK9, ng/mL NA 213.02 ± 5.65 189.12 ± 4.89 −12.6 < 0.001f 194.98 ± 5.40 −8.5 < 0.001f −3.0 0.19 Substudy (n = 19)g  Total cholesterol, mmol/L 5.18 ± 0.16 4.70 ± 0.16 5.13 ± 0.14 9.1 0.005f 5.01 ± 0.19 8.0 0.018f 2.5 0.62  LDL-C, mmol/L 3.11 ± 0.14 2.70 ± 0.13 3.10 ± 0.15 14.7 0.006f 3.05 ± 0.14 14.4 0.010f 1.6 0.88  Total apoB, g/L 1.37 ± 0.09 1.26 ± 0.08 1.40 ± 0.12 11.0 0.03f 1.35 ± 0.09 6.9 0.27 3.5 0.27  TG, mmol/L 1.29 ± 0.13 1.35 ± 0.17 1.20 ± 0.15 −11.1 0.10 1.39 ± 0.21 4.6 0.63 −13.6 0.06  Mean LDL size, Å 250.88 ± 0.75 250.95 ± 0.54 250.95 ± 0.63 0.0 0.90 250.42 ± 0.60 −0.2 0.29 0.2 0.33  LDL peak, Å 250.62 ± 0.76 250.68 ± 0.63 250.79 ± 0.71 0.0 0.89 250.20 ± 0.61 −0.2 0.12 0.2 0.17  Proportion of large LDL, % 7.52 ± 1.38 6.90 ± 1.07 7.01 ± 1.35 1.5 0.34 7.42 ± 1.12 4.2 0.12 −5.6 0.54  Proportion of small LDL, % 75.03 ± 3.16 76.13 ± 2.32 75.53 ± 2.88 −0.8 0.56 75.98 ± 2.55 0.0 0.72 −0.6 0.86  PCSK9, ng/mL NA 196.92 ± 15.23 174.43 ± 13.53 −11.4 0.11 190.08 ± 15.33 1.2 0.75 −8.2 0.04f Data presented as unadjusted mean ± SEM. Abbreviation: NA, not available. a This analysis compared the change with DHA or EPA compared with control based on posttreatment values; P values were taken from the main treatment effect in the mixed models. b P values for the EPA and DHA changes vs control, as determined by the LSMEANS statement and tested against the null hypothesis in the mixed models. c Participants equaled 123 for DHA, 121 for EPA, 125 for control, and 138 for baseline. d Log-transformed data were used in these analyses due to skewness of the distribution of the values. e Previously reported (4). f P < 0.05; because pre-DHA and -EPA values were essentially identical to the precontrol values, only precontrol values are presented. Models were adjusted for sex, weight, age, sequence; baseline value was considered only when these covariates were found to be significant at P < 0.05 in the models. g Participants equaled 19 for DHA, 17 for EPA, and 19 for control. View Large Kinetic studies Both DHA and EPA tended to increase VLDL apoB100 FCR similarly compared with the control (DHA, +21%; EPA, +19%; DHA vs EPA, P = 0.73; Table 3). However, EPA tended to increase VLDL apoB100 direct catabolism more than did DHA (compared with control: DHA, −3%; EPA, +22%; DHA vs EPA, P = 0.10). Changes in the VLDL to LDL apoB100 conversion rates were similar after DHA and EPA (compared with control: DHA, +8%; EPA, +7%; DHA vs EPA, P = 0.44). LDL apoB100 FCR was significantly lower after EPA supplementation than after DHA supplementation (compared with control: DHA, 0%; EPA, −10%; DHA vs EPA, P = 0.008). In contrast, DHA increased the LDL apoB100 PR compared with EPA (compared with control: DHA, +2%; EPA, −7%; DHA vs EPA, P = 0.027). Table 3. ApoB100-Containing Lipoproteins and VLDL apoCIII Kinetics After Control, DHA, and EPA Phases Variable After Control Phase After DHA Phase % vs Control P Value ΔDHAa After EPA Control % vs Control P Value ΔEPAa DHA vs EPA (%) P Value ΔDHA vs ΔEPAb VLDL apoB100  PS, mg 336.07 ± 46.90 300.0 ± 47.4 −10.7 0.03c 336.31 ± 74.52 0.1 0.70 −10.8 0.10  FCR, pools/dd 9.38 ± 0.94 11.3 ± 1.9 20.5 0.12 11.21 ± 1.87 19.4 0.20 0.9 0.73  PR, mg/kg/dd 30.42 ± 2.21 30.5 ± 2.6 0.1 1.00 32.91 ± 3.40 8.2 0.30 −7.4 0.22  Abs. conv. VLDL to IDL apoB100, mg/kg/dd 4.14 ± 1.29 4.0 ± 1.0 −3.2 0.34 2.37 ± 0.46 −42.7 0.31 69.0 0.002c  Abs. conv. VLDL to LDL apoB100, mg/kg/d 9.40 ± 1.49 10.1 ± 1.5 7.5 0.29 10.08 ± 1.00 7.3 0.80 0.2 0.44  VLDL apoB100 direct catabolism, mg/kg/dd 16.06 ± 2.27 15.6 ± 2.6 −2.9 0.78 19.54 ± 3.22 21.7 0.25 −20.2 0.10 IDL apoB100  PS, mg 16.06 ± 2.05 20.8 ± 2.7 29.7 0.11 15.24 ± 2.28 −5.2 0.58 36.7 0.05  FCR, pools/dd 11.54 ± 1.85 14.6 ± 2.3 26.4 0.02c 13.46 ± 1.72 16.6 0.18 8.4 0.32  PR, mg/kg/dd 2.37 ± 0.64 3.4 ± 0.8 42.0 0.03c 2.38 ± 0.46 0.6 0.78 41.1 0.009c  Abs. conv. IDL to LDL apoB100, mg/kg/dd 3.77 ± 1.31 3.3 ± 0.8 −13.5 0.50 2.37 ± 0.46 −37.1 0.63 37.5 0.14 LDL apoB100  PS, mg 2700.98 ± 159.05 2811.1 ± 192.1 4.1 0.67 2785.01 ± 155.81 3.1 0.54 0.9 0.84  FCR, pools/dd 0.43 ± 0.06 0.43 ± 0.06 −0.2 0.50 0.39 ± 0.04 −10.4 0.24 11.4 0.008c  PR, mg/kg/dd 13.29 ± 1.45 13.5 ± 1.4 1.6 0.43 12.34 ± 1.04 −7.1 0.55 9.4 0.03c VLDL apoCIII  PS, mgd 109.58 ± 15.77 100.41 ± 16.50 −8.4 0.10 115.30 ± 23.62 5.2 0.96 −12.9 0.11  FCR, pools/d 0.94 ± 0.06 0.83 ± 0.05 −11.3 0.009c 0.88 ± 0.06 −6.9 0.04c −4.7 0.60  PR, mg/kg/dd 4.18 ± 0.54 3.67 ± 0.69 −12.2 0.009c 4.24 ± 0.77 1.3 0.34 −13.3 0.092 Variable After Control Phase After DHA Phase % vs Control P Value ΔDHAa After EPA Control % vs Control P Value ΔEPAa DHA vs EPA (%) P Value ΔDHA vs ΔEPAb VLDL apoB100  PS, mg 336.07 ± 46.90 300.0 ± 47.4 −10.7 0.03c 336.31 ± 74.52 0.1 0.70 −10.8 0.10  FCR, pools/dd 9.38 ± 0.94 11.3 ± 1.9 20.5 0.12 11.21 ± 1.87 19.4 0.20 0.9 0.73  PR, mg/kg/dd 30.42 ± 2.21 30.5 ± 2.6 0.1 1.00 32.91 ± 3.40 8.2 0.30 −7.4 0.22  Abs. conv. VLDL to IDL apoB100, mg/kg/dd 4.14 ± 1.29 4.0 ± 1.0 −3.2 0.34 2.37 ± 0.46 −42.7 0.31 69.0 0.002c  Abs. conv. VLDL to LDL apoB100, mg/kg/d 9.40 ± 1.49 10.1 ± 1.5 7.5 0.29 10.08 ± 1.00 7.3 0.80 0.2 0.44  VLDL apoB100 direct catabolism, mg/kg/dd 16.06 ± 2.27 15.6 ± 2.6 −2.9 0.78 19.54 ± 3.22 21.7 0.25 −20.2 0.10 IDL apoB100  PS, mg 16.06 ± 2.05 20.8 ± 2.7 29.7 0.11 15.24 ± 2.28 −5.2 0.58 36.7 0.05  FCR, pools/dd 11.54 ± 1.85 14.6 ± 2.3 26.4 0.02c 13.46 ± 1.72 16.6 0.18 8.4 0.32  PR, mg/kg/dd 2.37 ± 0.64 3.4 ± 0.8 42.0 0.03c 2.38 ± 0.46 0.6 0.78 41.1 0.009c  Abs. conv. IDL to LDL apoB100, mg/kg/dd 3.77 ± 1.31 3.3 ± 0.8 −13.5 0.50 2.37 ± 0.46 −37.1 0.63 37.5 0.14 LDL apoB100  PS, mg 2700.98 ± 159.05 2811.1 ± 192.1 4.1 0.67 2785.01 ± 155.81 3.1 0.54 0.9 0.84  FCR, pools/dd 0.43 ± 0.06 0.43 ± 0.06 −0.2 0.50 0.39 ± 0.04 −10.4 0.24 11.4 0.008c  PR, mg/kg/dd 13.29 ± 1.45 13.5 ± 1.4 1.6 0.43 12.34 ± 1.04 −7.1 0.55 9.4 0.03c VLDL apoCIII  PS, mgd 109.58 ± 15.77 100.41 ± 16.50 −8.4 0.10 115.30 ± 23.62 5.2 0.96 −12.9 0.11  FCR, pools/d 0.94 ± 0.06 0.83 ± 0.05 −11.3 0.009c 0.88 ± 0.06 −6.9 0.04c −4.7 0.60  PR, mg/kg/dd 4.18 ± 0.54 3.67 ± 0.69 −12.2 0.009c 4.24 ± 0.77 1.3 0.34 −13.3 0.092 Data presented as mean ± SEM. ApoB100: control, n = 17 (n = 18 for PS, FCR, and PR VLDL); DHA, n = 16 (18 for PS VLDL, 17 for PS IDL and LDL, FCR and PR VLDL); EPA, n = 14 (16 for PS, FCR, and PR VLDL, 15 for PS, FCR, and PR LDL); ApoCIII: control, n = 18; DHA, n = 18; EPA, n = 16. Abbreviations: abs., absolute; conv., conversion; IDL, intermediate-density lipoprotein; PS, pool size. a This analysis compared the change with DHA or EPA compared with control, using post-treatment values; P values were from the main treatment effect in the mixed models. b P values for the EPA and DHA changes vs control, as determined by the LSMEANS statement and tested against the null hypothesis in the mixed models. c P < 0.05. Models were adjusted for sex, weight, age, sequence; baseline value was considered only when these covariates were found to be significant at P < 0.05 in the models. d Log-transformed data were used in these analyses owing to skewness of the value distribution. View Large Table 3. ApoB100-Containing Lipoproteins and VLDL apoCIII Kinetics After Control, DHA, and EPA Phases Variable After Control Phase After DHA Phase % vs Control P Value ΔDHAa After EPA Control % vs Control P Value ΔEPAa DHA vs EPA (%) P Value ΔDHA vs ΔEPAb VLDL apoB100  PS, mg 336.07 ± 46.90 300.0 ± 47.4 −10.7 0.03c 336.31 ± 74.52 0.1 0.70 −10.8 0.10  FCR, pools/dd 9.38 ± 0.94 11.3 ± 1.9 20.5 0.12 11.21 ± 1.87 19.4 0.20 0.9 0.73  PR, mg/kg/dd 30.42 ± 2.21 30.5 ± 2.6 0.1 1.00 32.91 ± 3.40 8.2 0.30 −7.4 0.22  Abs. conv. VLDL to IDL apoB100, mg/kg/dd 4.14 ± 1.29 4.0 ± 1.0 −3.2 0.34 2.37 ± 0.46 −42.7 0.31 69.0 0.002c  Abs. conv. VLDL to LDL apoB100, mg/kg/d 9.40 ± 1.49 10.1 ± 1.5 7.5 0.29 10.08 ± 1.00 7.3 0.80 0.2 0.44  VLDL apoB100 direct catabolism, mg/kg/dd 16.06 ± 2.27 15.6 ± 2.6 −2.9 0.78 19.54 ± 3.22 21.7 0.25 −20.2 0.10 IDL apoB100  PS, mg 16.06 ± 2.05 20.8 ± 2.7 29.7 0.11 15.24 ± 2.28 −5.2 0.58 36.7 0.05  FCR, pools/dd 11.54 ± 1.85 14.6 ± 2.3 26.4 0.02c 13.46 ± 1.72 16.6 0.18 8.4 0.32  PR, mg/kg/dd 2.37 ± 0.64 3.4 ± 0.8 42.0 0.03c 2.38 ± 0.46 0.6 0.78 41.1 0.009c  Abs. conv. IDL to LDL apoB100, mg/kg/dd 3.77 ± 1.31 3.3 ± 0.8 −13.5 0.50 2.37 ± 0.46 −37.1 0.63 37.5 0.14 LDL apoB100  PS, mg 2700.98 ± 159.05 2811.1 ± 192.1 4.1 0.67 2785.01 ± 155.81 3.1 0.54 0.9 0.84  FCR, pools/dd 0.43 ± 0.06 0.43 ± 0.06 −0.2 0.50 0.39 ± 0.04 −10.4 0.24 11.4 0.008c  PR, mg/kg/dd 13.29 ± 1.45 13.5 ± 1.4 1.6 0.43 12.34 ± 1.04 −7.1 0.55 9.4 0.03c VLDL apoCIII  PS, mgd 109.58 ± 15.77 100.41 ± 16.50 −8.4 0.10 115.30 ± 23.62 5.2 0.96 −12.9 0.11  FCR, pools/d 0.94 ± 0.06 0.83 ± 0.05 −11.3 0.009c 0.88 ± 0.06 −6.9 0.04c −4.7 0.60  PR, mg/kg/dd 4.18 ± 0.54 3.67 ± 0.69 −12.2 0.009c 4.24 ± 0.77 1.3 0.34 −13.3 0.092 Variable After Control Phase After DHA Phase % vs Control P Value ΔDHAa After EPA Control % vs Control P Value ΔEPAa DHA vs EPA (%) P Value ΔDHA vs ΔEPAb VLDL apoB100  PS, mg 336.07 ± 46.90 300.0 ± 47.4 −10.7 0.03c 336.31 ± 74.52 0.1 0.70 −10.8 0.10  FCR, pools/dd 9.38 ± 0.94 11.3 ± 1.9 20.5 0.12 11.21 ± 1.87 19.4 0.20 0.9 0.73  PR, mg/kg/dd 30.42 ± 2.21 30.5 ± 2.6 0.1 1.00 32.91 ± 3.40 8.2 0.30 −7.4 0.22  Abs. conv. VLDL to IDL apoB100, mg/kg/dd 4.14 ± 1.29 4.0 ± 1.0 −3.2 0.34 2.37 ± 0.46 −42.7 0.31 69.0 0.002c  Abs. conv. VLDL to LDL apoB100, mg/kg/d 9.40 ± 1.49 10.1 ± 1.5 7.5 0.29 10.08 ± 1.00 7.3 0.80 0.2 0.44  VLDL apoB100 direct catabolism, mg/kg/dd 16.06 ± 2.27 15.6 ± 2.6 −2.9 0.78 19.54 ± 3.22 21.7 0.25 −20.2 0.10 IDL apoB100  PS, mg 16.06 ± 2.05 20.8 ± 2.7 29.7 0.11 15.24 ± 2.28 −5.2 0.58 36.7 0.05  FCR, pools/dd 11.54 ± 1.85 14.6 ± 2.3 26.4 0.02c 13.46 ± 1.72 16.6 0.18 8.4 0.32  PR, mg/kg/dd 2.37 ± 0.64 3.4 ± 0.8 42.0 0.03c 2.38 ± 0.46 0.6 0.78 41.1 0.009c  Abs. conv. IDL to LDL apoB100, mg/kg/dd 3.77 ± 1.31 3.3 ± 0.8 −13.5 0.50 2.37 ± 0.46 −37.1 0.63 37.5 0.14 LDL apoB100  PS, mg 2700.98 ± 159.05 2811.1 ± 192.1 4.1 0.67 2785.01 ± 155.81 3.1 0.54 0.9 0.84  FCR, pools/dd 0.43 ± 0.06 0.43 ± 0.06 −0.2 0.50 0.39 ± 0.04 −10.4 0.24 11.4 0.008c  PR, mg/kg/dd 13.29 ± 1.45 13.5 ± 1.4 1.6 0.43 12.34 ± 1.04 −7.1 0.55 9.4 0.03c VLDL apoCIII  PS, mgd 109.58 ± 15.77 100.41 ± 16.50 −8.4 0.10 115.30 ± 23.62 5.2 0.96 −12.9 0.11  FCR, pools/d 0.94 ± 0.06 0.83 ± 0.05 −11.3 0.009c 0.88 ± 0.06 −6.9 0.04c −4.7 0.60  PR, mg/kg/dd 4.18 ± 0.54 3.67 ± 0.69 −12.2 0.009c 4.24 ± 0.77 1.3 0.34 −13.3 0.092 Data presented as mean ± SEM. ApoB100: control, n = 17 (n = 18 for PS, FCR, and PR VLDL); DHA, n = 16 (18 for PS VLDL, 17 for PS IDL and LDL, FCR and PR VLDL); EPA, n = 14 (16 for PS, FCR, and PR VLDL, 15 for PS, FCR, and PR LDL); ApoCIII: control, n = 18; DHA, n = 18; EPA, n = 16. Abbreviations: abs., absolute; conv., conversion; IDL, intermediate-density lipoprotein; PS, pool size. a This analysis compared the change with DHA or EPA compared with control, using post-treatment values; P values were from the main treatment effect in the mixed models. b P values for the EPA and DHA changes vs control, as determined by the LSMEANS statement and tested against the null hypothesis in the mixed models. c P < 0.05. Models were adjusted for sex, weight, age, sequence; baseline value was considered only when these covariates were found to be significant at P < 0.05 in the models. d Log-transformed data were used in these analyses owing to skewness of the value distribution. View Large The increase in LDL-C concentrations after DHA or EPA supplementation did not correlate with variations in the LDL apoB100 FCR or PR (Table 4). However, variations in the LDL apoB100 pool size correlated with change in LDL apoB100 PR after EPA (rs = 0.63; P = 0.013) and with variations in LDL apoB100 FCR after DHA (rs = −0.52; P = 0.04) and PCSK9 concentration after DHA (rs = 0.64; P < 0.01). Table 4. Spearman Correlation Coefficient Between Changes in ApoB100-Containing Lipoprotein Kinetics, PCSK9 Levels, and LDL-C After EPA and DHA vs Control Variable DHA EPA LDL-C, mmol/L LDL apoB100, mg LDL-C, mmol/L LDL apoB100, mg VLDL apoB100  FCR, pools/d −0.203 −0.424 −0.303 −0.182  PR, mg/kg/d 0.159 0.229 0.135 −0.043  VLDL to IDL apoB100, mg/kg/d 0.165 −0.291 0.420 0.305  VLDL to LDL apoB100, mg/kg/d 0.238 0.276 −0.420 0.275  VLDL apoB100 direct catabolism, mg/kg/d 0.076 0.265 −0.055 −0.196 IDL apoB100  FCR, pools/d 0.503a −0.129 0.301 0.270  PR, mg/kg/d 0.674a 0.221 0.327 0.503  IDL to LDL apoB100, mg/kg/d 0.132 −0.226 0.560a 0.380 LDL apoB100  FCR, pools/d −0.229 −0.521a −0.161 0.057  PR, mg/kg/d 0.044 −0.197 0.261 0.625a Mean LDL size, Å 0.103 −0.091 0.063 0.393 LDL peak, Å 0.148 0.324 0.062 0.254 Proportion, large LDL, % 0.059 −0.181 0.133 0.046 Proportion, small LDL, % −0.111 0.028 −0.136 −0.196 PCSK9, ng/L 0.031 0.642a 0.132 0.011 Variable DHA EPA LDL-C, mmol/L LDL apoB100, mg LDL-C, mmol/L LDL apoB100, mg VLDL apoB100  FCR, pools/d −0.203 −0.424 −0.303 −0.182  PR, mg/kg/d 0.159 0.229 0.135 −0.043  VLDL to IDL apoB100, mg/kg/d 0.165 −0.291 0.420 0.305  VLDL to LDL apoB100, mg/kg/d 0.238 0.276 −0.420 0.275  VLDL apoB100 direct catabolism, mg/kg/d 0.076 0.265 −0.055 −0.196 IDL apoB100  FCR, pools/d 0.503a −0.129 0.301 0.270  PR, mg/kg/d 0.674a 0.221 0.327 0.503  IDL to LDL apoB100, mg/kg/d 0.132 −0.226 0.560a 0.380 LDL apoB100  FCR, pools/d −0.229 −0.521a −0.161 0.057  PR, mg/kg/d 0.044 −0.197 0.261 0.625a Mean LDL size, Å 0.103 −0.091 0.063 0.393 LDL peak, Å 0.148 0.324 0.062 0.254 Proportion, large LDL, % 0.059 −0.181 0.133 0.046 Proportion, small LDL, % −0.111 0.028 −0.136 −0.196 PCSK9, ng/L 0.031 0.642a 0.132 0.011 Abbreviation: IDL, intermediate-density lipoprotein. a P < 0.05. View Large Table 4. Spearman Correlation Coefficient Between Changes in ApoB100-Containing Lipoprotein Kinetics, PCSK9 Levels, and LDL-C After EPA and DHA vs Control Variable DHA EPA LDL-C, mmol/L LDL apoB100, mg LDL-C, mmol/L LDL apoB100, mg VLDL apoB100  FCR, pools/d −0.203 −0.424 −0.303 −0.182  PR, mg/kg/d 0.159 0.229 0.135 −0.043  VLDL to IDL apoB100, mg/kg/d 0.165 −0.291 0.420 0.305  VLDL to LDL apoB100, mg/kg/d 0.238 0.276 −0.420 0.275  VLDL apoB100 direct catabolism, mg/kg/d 0.076 0.265 −0.055 −0.196 IDL apoB100  FCR, pools/d 0.503a −0.129 0.301 0.270  PR, mg/kg/d 0.674a 0.221 0.327 0.503  IDL to LDL apoB100, mg/kg/d 0.132 −0.226 0.560a 0.380 LDL apoB100  FCR, pools/d −0.229 −0.521a −0.161 0.057  PR, mg/kg/d 0.044 −0.197 0.261 0.625a Mean LDL size, Å 0.103 −0.091 0.063 0.393 LDL peak, Å 0.148 0.324 0.062 0.254 Proportion, large LDL, % 0.059 −0.181 0.133 0.046 Proportion, small LDL, % −0.111 0.028 −0.136 −0.196 PCSK9, ng/L 0.031 0.642a 0.132 0.011 Variable DHA EPA LDL-C, mmol/L LDL apoB100, mg LDL-C, mmol/L LDL apoB100, mg VLDL apoB100  FCR, pools/d −0.203 −0.424 −0.303 −0.182  PR, mg/kg/d 0.159 0.229 0.135 −0.043  VLDL to IDL apoB100, mg/kg/d 0.165 −0.291 0.420 0.305  VLDL to LDL apoB100, mg/kg/d 0.238 0.276 −0.420 0.275  VLDL apoB100 direct catabolism, mg/kg/d 0.076 0.265 −0.055 −0.196 IDL apoB100  FCR, pools/d 0.503a −0.129 0.301 0.270  PR, mg/kg/d 0.674a 0.221 0.327 0.503  IDL to LDL apoB100, mg/kg/d 0.132 −0.226 0.560a 0.380 LDL apoB100  FCR, pools/d −0.229 −0.521a −0.161 0.057  PR, mg/kg/d 0.044 −0.197 0.261 0.625a Mean LDL size, Å 0.103 −0.091 0.063 0.393 LDL peak, Å 0.148 0.324 0.062 0.254 Proportion, large LDL, % 0.059 −0.181 0.133 0.046 Proportion, small LDL, % −0.111 0.028 −0.136 −0.196 PCSK9, ng/L 0.031 0.642a 0.132 0.011 Abbreviation: IDL, intermediate-density lipoprotein. a P < 0.05. View Large Both DHA and EPA decreased VLDL apoCIII FCR similarly compared with the control (DHA, −11%; EPA, −7%; DHA vs EPA, P = 0.60). The reduction in VLDL apoCIII PR tended to be greater after DHA than after EPA (compared with control: DHA, −12%; EPA, +1%; DHA vs EPA, P = 0.09). The change in VLDL apoCIII FCR correlated inversely with the change in the PCSK9 concentration after EPA (rs = −0.58; P = 0.019; data not shown) and with the change in LDL apoB100 FCR after DHA (rs = 0.54; P = 0.030; data not shown). The change in VLDL apoCIII PR correlated inversely with LDL apoB100 FCR and PR (rs = −0.57 and rs = −0.53, respectively; P < 0.05; data not shown) and was positively associated with the change in PCSK9 concentrations after DHA (rs = 0.54; P = 0.021; data not shown). We did not find any correlation between the changes in TG and LDL-C or between the changes in VLDL apoCIII FCR and LDL apoB100 PR after DHA or EPA (data not shown). Discussion To the best of our knowledge, the present study is the first to demonstrate the mechanisms underlying the differential effects of DHA and EPA supplementation on LDL-C and other features of LDL in men and women with abdominal obesity and subclinical inflammation and at risk of CVD. The results from the present study suggest that high-dose DHA increases the LDL particle size and modifies LDL apoB100 and VLDL apoCIII kinetics compared with EPA. Although DHA and EPA reduce the PCSK9 concentration similarly, the relationships among PCSK9, LDL-C, and LDL apoB100 concentrations were different between DHA and EPA. We have previously shown that the magnitude of the reduction in TG and increase in LDL-C after DHA was greater than after EPA supplementation (4). Previous in vivo kinetic studies have documented the effects of LCn3-PUFAs, either as a dietary supplement or as part of an LCn3-PUFA–rich diet, on apoB100-containing lipoprotein metabolism (27, 28). Those studies have shown that LCn3-PUFAs reduce TG concentrations primarily by reducing the endogenous production of VLDL apoB100 and by increasing the VLDL to LDL apoB100 conversion rate (27, 28). LCn3-PUFAs have also been shown to increase the clearance of LDL apoB100 (27, 28). In contrast, Ooi et al. (12) found that a high-fish diet (containing 1.23 g EPA plus DHA daily) increases LDL apoB100 production by 32% and concomitantly decreases LDL apoB100 clearance by 44% compared with untreated baseline values. This disproportionate reduction in LDL apoB100 clearance might explain in part why the LDL-C concentrations increase after LCn3-PUFA supplementation. Because the changes in LDL-C and TG concentrations are greater with DHA supplementation than with EPA supplementation (4), we hypothesized that DHA compared with EPA induces a greater reduction in VLDL apoB100 production and a greater VLDL to LDL apoB100 conversion rate, resulting in a greater increase in LDL apoB100 production. Accordingly, DHA compared with EPA differentially influenced LDL apoB100 production and clearance rates; however, these differences were not related to the differential effects of DHA and EPA on LDL-C concentrations. DHA and EPA equally increased VLDL to LDL apoB100 conversion and VLDL apoB100 FCR. These data suggest that metabolic pathways not involving apoB100 per se might be responsible for the differential effects of DHA and EPA on LDL-C concentrations and LDL size. It is possible that DHA and EPA differentially influence apoB/C/E ratios on VLDL, which might, in turn, contribute to differences in the LDL-C concentrations and LDL size seen between DHA and EPA (6, 7). Zheng et al. (7) have shown that apoCIII-containing VLDL are the major precursor of LDL particles. Hence, the suppression of apoCIII PR with DHA might also explain to some extent its effect on LDL particle size. That total VLDL particles were converted more rapidly to intermediate-density lipoprotein after DHA than after EPA (Table 3) and that VLDL-apoCIII levels also tended to decrease with DHA compared with EPA is consistent with this hypothesis. We also hypothesized that DHA and EPA supplementation would modulate LDL particle size differently because the increase in LDL-C concentration after DHA was almost twofold greater in magnitude than the increase in total apoB concentration (4). Accordingly, DHA supplementation slightly increased mean LDL particle size and decreased the proportion of sdLDL compared with EPA supplementation. This observation is consistent with data from a few studies, which have shown that DHA, but not EPA, is associated with larger LDL (9, 29). This increase in LDL particle size after DHA can be attributed, at least in part, to the greater reduction in serum TG compared with EPA. Serum TG is an important metabolic determinant of the sdLDL phenotype through a series of metabolic transformation of the LDL particles that involve lipases and cholesteryl ester transfer protein (30). However, very few studies have compared the effect of DHA and EPA on enzyme activities. Supplementation with LCn3-PUFA has been shown to have inconsistent effects on cholesteryl ester transfer protein activity (31) and might increase lipoprotein lipase activity through upregulation of it expression (31) but might have no effect on hepatic lipase activity (31). More studies investigating these pathways in response to DHA and EPA supplementations are needed. The increase in LDL size with DHA compared with EPA might also be explained in part by a decrease in apoCIII secretion from the liver (32). DHA might reduce apoCIII production through the regulation of the forkhead box O transcription factor O1 and carbohydrate response element-binding protein (33, 34). ApoCIII inhibits the binding of apoB to hepatic apoB/E receptor and lipoprotein lipase activity (32, 35). In a small parallel study, supplementation with EPA alone tended to increase apoCIII concentrations, and DHA tended to decrease apoCIII-containing lipoprotein concentrations (36). Consistent with this, we have shown that high-dose EPA also tended to increase VLDL apoCIII mass and DHA tended to decrease the VLDL apoCIII mass compared with the control, although the differences did not reach statistical significance. DHA also tended to decrease the VLDL apoCIII PR compared with EPA; however, the difference also did not reach the statistical significance. Changes in VLDL apoCIII metabolism correlated with changes in LDL apoB100 metabolism after DHA, but not after EPA, supporting a differential effect of EPA and DHA on VLDL apoCIII and apoB100 metabolism. This apparent reduction in apoCIII production in the liver after DHA supplementation might explain the enhanced conversion of VLDL to LDL apoB100 and the formation of larger LDL particles compared with EPA. Individuals with a preponderance of sdLDL have consistently been shown to be at increased risk of myocardial infarction and CVD compared with individuals with a greater proportion of larger LDL particles (37). However, the extent to which the opposite effects of DHA on both LDL-C and LDL particle size modify CVD risk is unknown. The reduction in PCSK9 concentrations observed after DHA and EPA supplementation is consistent with data from the few available studies on this topic. A recent randomized controlled parallel study in 92 pre- and postmenopausal women has shown that supplementation with 2.2 g/d of marine oil decreased plasma PCSK9 concentrations by 11.4% in premenopausal women and 9.8% in postmenopausal women compared with baseline (19). Post hoc analyses of the Canola Oil Multicenter Intervention Trial have also shown that the PCSK9 concentration was lower after DHA-enriched canola oil than after regular canola oil supplementation (20). In the present study, DHA and EPA both reduced serum PCSK9 levels equally compared with the control. Although the PCSK9 concentrations usually correlated with LDL-C concentrations, variations in the PCSK9 levels explained <8% of the LDL-C variance (38). Furthermore, the PCSK9 concentrations might not fully reflect PCSK9 activity (38). Therefore, the increase in LDL-C after DHA and EPA despite a decrease in PCSK9 concentrations was not entirely unexpected. In contrast, changes in PCSK9 correlated positively with changes in the LDL apoB100 concentrations and negatively with changes in LDL apoB100 FCR after DHA but not after EPA, suggesting that PCSK9 might be partly involved in explaining the differential effects of DHA and EPA supplementation on the metabolic fate of the LDL particle. The present study had several strengths and limitations. A number of studies have examined the effect of an LCn3-PUFA–rich diet or a supplement combining EPA and DHA in various forms and proportions on apoB100-containing lipoprotein kinetics (27, 28). To the best of our knowledge, ours is the first study to compare head-to-head the effect of high-dose EPA and DHA on apoB100-containing lipoprotein kinetics. The use of a randomized crossover study design reduced the interindividual variability of the results. The baseline characteristics of the kinetic subsample were similar to the whole study cohort, and the compliance was high in all phases of the study (4). The analyses of the changes in blood lipids, LDL particle size, and PCSK9 concentrations in the substudy kinetic sample were conducted on data from fewer participants, hence influencing the statistical power. Estimates from small kinetic pool sizes have relatively high coefficients of variations and small changes in kinetic parameters can be difficult to assess. The observed effects of DHA and EPA on serum lipids, including LDL-C, might have resulted from changes in kinetics that might have been too subtle to be detected with this sample size. Corn oil was chosen as the control because of the relatively neutral effects of n6-PUFA on inflammation makers (39), which were the primary outcome of the trial (4). Supplementation with the control n6-PUFA–rich corn oil decreased total cholesterol (−0.12 mmol/L; P = 0.001), LDL-C (−0.13 mmol/L; P = 0.003), and mean LDL size (−0.22 Å; P = 0.02) compared with control-specific baseline levels. However, results were similar when the change from DHA/EPA-specific baseline values were considered. Specifically, the increase in LDL-C and the reduction in TG with DHA compared with the baseline values were significantly greater than those seen with EPA (LDL-C, +0.11 vs 0.00 mmol/L; TG, −0.26 vs −0.20 mmol/L; P < 0.01 for all). The increase from treatment-specific baseline values in LDL particle size (+0.21 Å vs −0.71 Å) and the reduction in the proportion of sdLDL (−1.36% vs +2.8%) were also greater with DHA than with EPA (P < 0.01 for all; data not shown). Because very few studies have documented the effect of n6-PUFA on apoB100- and apoCIII-containing lipoprotein metabolism, it is difficult to assess how the use of corn oil as the control treatment has affected the kinetic study data (27, 28, 40). In conclusion, the differential effects of DHA and EPA supplementation on LDL-C concentrations might not be accounted for by differences in the regulation of apoB100-containing lipoprotein metabolism and might involve other pathways that influence LDL particle size. The extent to which the greater increase in LDL-C with DHA compared with EPA, associated with larger LDL particles, influences CVD risk is unknown. Further studies are needed to better understand the changes in other metabolic factors after EPA and DHA supplementation, including the expression of the different genes involved in lipid metabolism. Abbreviations: Abbreviations: apo apolipoprotein CRP C-reactive protein CVD cardiovascular disease DHA docosahexaenoic acid EPA eicosapentaenoic acid FCR fractional catabolic rate LCn3-PUFA long-chain omega-3 polyunsaturated fatty acid LDL low-density lipoprotein LDL-C low density lipoprotein cholesterol PCSK9 proprotein convertase subtilisin/kexin type 9 PR production rate sdLDL small and dense low-density lipoprotein TG triglyceride TRL triglyceride-rich lipoprotein VLDL very-low-density lipoprotein Acknowledgments We are grateful to the subjects for their excellent collaboration and the staff of the Institute of Nutrition and Functional Foods and the CHU de Québec. Financial Support: Financial support for this randomized controlled trial was provided by a grant from the Canadian Institutes for Health Research (CIHR) (grant MOP-123494 to B.L., P.C., A.T.). Douglas Laboratories provided the EPA, DHA, and control capsules used in the present study. The CIHR and Douglas Laboratories had no role in the design of the study or analysis or interpretation of the data. J.A. is a recipient of a PhD Scholarship from the CIHR and the Fonds de recherche du Québec – Santé. C.V. is a fellow supported by the European Marie Skłodowska-Curie Actions. Clinical Trial Information: ClinicalTrials.gov. no. NCT01810003 (registered 4 March 2013). Author Contributions: B.L., P.C., and A.T. designed and obtained funding for the present study. P.C. was responsible for the medical supervision in the study. A.C. coordinated the clinical study. J.M. conducted the laboratory analyses. C.V. and A.J.T. provided significant help with modeling of the data. J.A. performed the modeling of the data and statistical analyses and wrote the manuscript, which was reviewed critically by all the authors. Disclosure Summary: B.L. is Chair of Nutrition at Laval University, which is supported by private endowments from Pfizer, La Banque Royale du Canada, and Provigo-Loblaws; has received funding in the past 5 years from the Canadian Institutes for Health Research, the Natural Sciences and Engineering Research Council of Canada, Agriculture and Agri-Food Canada (Growing Forward program supported by the Dairy Farmers of Canada, Canola Council of Canada, Flax Council of Canada, Dow Agrosciences), Dairy Research Institute, Dairy Australia, Merck & Co, Inc., Pfizer, and Atrium Innovations for which Douglas Laboratories manufacture and market omega-3 supplements; is an advisory board member of the Canadian Nutrition Society; and has received honoraria from the International Chair on Cardiometabolic risk, the Dairy Farmers of Canada, and the World Dairy Platform as an invited speaker to various conferences. P.C. has received funding in the past 5 years from the Canadian Institutes of Health Research, Agriculture and Agri-Food Canada (Growing Forward program supported by the Dairy Farmers of Canada, Canola Council of Canada, Flax Council of Canada, Dow Agrosciences), Dairy Research Institute, Dairy Australia, Merck and Co., Inc., Pfizer, Amgen, and Atrium Innovations. A.T. was funded in the past 5 years as principal investigator by the Canadian Institutes of Health Research, the Natural Sciences and Engineering Research Council of Canada, the Fonds de recherche du Québec – Santé, the Fondation de l’Institut Universitaire de Cardiologie et de Pneumologie de Québec, and investigator-initiated funding from Johnson & Johnson Medical Companies for studies unrelated to the present report. The remaining authors have nothing to disclose. References 1. Bradberry JC , Hilleman DE . Overview of omega-3 fatty acid therapies . P T . 2013 ; 38 ( 11 ): 681 – 691 . Google Scholar PubMed 2. Hooper L , Thompson RL , Harrison RA , Summerbell CD , Moore H , Worthington HV , Durrington PN , Ness AR , Capps NE , Davey Smith G , Riemersma RA , Ebrahim SB . Omega 3 fatty acids for prevention and treatment of cardiovascular disease . Cochrane Database Syst Rev . 2004 ;( 4 ): CD003177 . 3. Wei MY , Jacobson TA . Effects of eicosapentaenoic acid versus docosahexaenoic acid on serum lipids: a systematic review and meta-analysis . Curr Atheroscler Rep . 2011 ; 13 ( 6 ): 474 – 483 . Google Scholar CrossRef Search ADS PubMed 4. Allaire J , Couture P , Leclerc M , Charest A , Marin J , Lépine MC , Talbot D , Tchernof A , Lamarche B . A randomized, crossover, head-to-head comparison of eicosapentaenoic acid and docosahexaenoic acid supplementation to reduce inflammation markers in men and women: the Comparing EPA to DHA (ComparED) study . Am J Clin Nutr . 2016 ; 104 ( 2 ): 280 – 287 . Google Scholar CrossRef Search ADS PubMed 5. Berneis KK , Krauss RM . Metabolic origins and clinical significance of LDL heterogeneity . J Lipid Res . 2002 ; 43 ( 9 ): 1363 – 1379 . Google Scholar CrossRef Search ADS PubMed 6. Alaupovic P . The concept of apolipoprotein-defined lipoprotein families and its clinical significance . Curr Atheroscler Rep . 2003 ; 5 ( 6 ): 459 – 467 . Google Scholar CrossRef Search ADS PubMed 7. Zheng C , Khoo C , Ikewaki K , Sacks FM . Rapid turnover of apolipoprotein C-III-containing triglyceride-rich lipoproteins contributing to the formation of LDL subfractions . J Lipid Res . 2007 ; 48 ( 5 ): 1190 – 1203 . Google Scholar CrossRef Search ADS PubMed 8. Musunuru K , Orho-Melander M , Caulfield MP , Li S , Salameh WA , Reitz RE , Berglund G , Hedblad B , Engström G , Williams PT , Kathiresan S , Melander O , Krauss RM . Ion mobility analysis of lipoprotein subfractions identifies three independent axes of cardiovascular risk . Arterioscler Thromb Vasc Biol . 2009 ; 29 ( 11 ): 1975 – 1980 . Google Scholar CrossRef Search ADS PubMed 9. Mori TA , Burke V , Puddey IB , Watts GF , O’Neal DN , Best JD , Beilin LJ . Purified eicosapentaenoic and docosahexaenoic acids have differential effects on serum lipids and lipoproteins, LDL particle size, glucose, and insulin in mildly hyperlipidemic men . Am J Clin Nutr . 2000 ; 71 ( 5 ): 1085 – 1094 . Google Scholar CrossRef Search ADS PubMed 10. Oelrich B , Dewell A , Gardner CD . Effect of fish oil supplementation on serum triglycerides, LDL cholesterol and LDL subfractions in hypertriglyceridemic adults . Nutr Metab Cardiovasc Dis . 2013 ; 23 ( 4 ): 350 – 357 . Google Scholar CrossRef Search ADS PubMed 11. Thomas TR , Smith BK , Donahue OM , Altena TS , James-Kracke M , Sun GY . Effects of omega-3 fatty acid supplementation and exercise on low-density lipoprotein and high-density lipoprotein subfractions . Metabolism . 2004 ; 53 ( 6 ): 749 – 754 . Google Scholar CrossRef Search ADS PubMed 12. Ooi EM , Lichtenstein AH , Millar JS , Diffenderfer MR , Lamon-Fava S , Rasmussen H , Welty FK , Barrett PH , Schaefer EJ . Effects of therapeutic lifestyle change diets high and low in dietary fish-derived FAs on lipoprotein metabolism in middle-aged and elderly subjects . J Lipid Res . 2012 ; 53 ( 9 ): 1958 – 1967 . Google Scholar CrossRef Search ADS PubMed 13. Nestel PJ , Connor WE , Reardon MF , Connor S , Wong S , Boston R . Suppression by diets rich in fish oil of very low density lipoprotein production in man . J Clin Invest . 1984 ; 74 ( 1 ): 82 – 89 . Google Scholar CrossRef Search ADS PubMed 14. Bordin P , Bodamer OA , Venkatesan S , Gray RM , Bannister PA , Halliday D . Effects of fish oil supplementation on apolipoprotein B100 production and lipoprotein metabolism in normolipidaemic males . Eur J Clin Nutr . 1998 ; 52 ( 2 ): 104 – 109 . Google Scholar CrossRef Search ADS PubMed 15. Huff MW , Telford DE . Dietary fish oil increases conversion of very low density lipoprotein apoprotein B to low density lipoprotein . Arteriosclerosis . 1989 ; 9 ( 1 ): 58 – 66 . Google Scholar CrossRef Search ADS PubMed 16. Fisher WR , Zech LA , Stacpoole PW . Apolipoprotein B metabolism in hypertriglyceridemic diabetic patients administered either a fish oil- or vegetable oil-enriched diet . J Lipid Res . 1998 ; 39 ( 2 ): 388 – 401 . Google Scholar PubMed 17. Chan DC , Watts GF , Mori TA , Barrett PH , Redgrave TG , Beilin LJ . Randomized controlled trial of the effect of n-3 fatty acid supplementation on the metabolism of apolipoprotein B-100 and chylomicron remnants in men with visceral obesity . Am J Clin Nutr . 2003 ; 77 ( 2 ): 300 – 307 . Google Scholar CrossRef Search ADS PubMed 18. Tavori H , Rashid S , Fazio S . On the function and homeostasis of PCSK9: reciprocal interaction with LDLR and additional lipid effects . Atherosclerosis . 2015 ; 238 ( 2 ): 264 – 270 . Google Scholar CrossRef Search ADS PubMed 19. Graversen CB , Lundbye-Christensen S , Thomsen B , Christensen JH , Schmidt EB . Marine n-3 polyunsaturated fatty acids lower plasma proprotein convertase subtilisin kexin type 9 levels in pre- and postmenopausal women: a randomised study . Vascul Pharmacol . 2016 ; 76 : 37 – 41 . Google Scholar CrossRef Search ADS PubMed 20. Rodríguez-Pérez C , Ramprasath VR , Pu S , Sabra A , Quirantes-Piné R , Segura-Carretero A , Jones PJ . Docosahexaenoic acid attenuates cardiovascular risk factors via a decline in proprotein convertase subtilisin/kexin type 9 (PCSK9) plasma levels . Lipids . 2016 ; 51 ( 1 ): 75 – 83 . Google Scholar CrossRef Search ADS PubMed 21. Alberti KG , Zimmet P , Shaw J , Group IDFETFC ; IDF Epidemiology Task Force Consensus Group . The metabolic syndrome—a new worldwide definition . Lancet . 2005 ; 366 ( 9491 ): 1059 – 1062 . Google Scholar CrossRef Search ADS PubMed 22. Airlie LT . Roche A, Martorel R. Standardization of anthropometric measurements. The Airlie (VA) Concensus Conference . Champaign, IL : Human Kinetics ; 1988 : 39 – 80 . 23. Allaire J , Harris W , Vors C , Tchernof A , Couture P , Lamarche B. Docosahexaenoic acid is more effective than eicosapentaenoic acid in increasing the omega-3 index measured in red blood cell membranes. FASEB J. 2017 ;31(1 Supple):146.143 . 24. St-Pierre AC , Ruel IL , Cantin B , Dagenais GR , Bernard P-M , Després J-P , Lamarche B . Comparison of various electrophoretic characteristics of LDL particles and their relationship to the risk of ischemic heart disease . Circulation . 2001 ; 104 ( 19 ): 2295 – 2299 . Google Scholar CrossRef Search ADS PubMed 25. Matthews DR , Hosker JP , Rudenski AS , Naylor BA , Treacher DF , Turner RC . Homeostasis model assessment: insulin resistance and beta-cell function from fasting plasma glucose and insulin concentrations in man . Diabetologia . 1985 ; 28 ( 7 ): 412 – 419 . Google Scholar CrossRef Search ADS PubMed 26. Harris JA , Benedict FG . A Biometric Study of Basal Metabolism in Man. Washington DC: Carnegie Institution of Washington ; 1919 . 27. Lamarche B , Couture P . Dietary fatty acids, dietary patterns, and lipoprotein metabolism . Curr Opin Lipidol . 2015 ; 26 ( 1 ): 42 – 47 . Google Scholar CrossRef Search ADS PubMed 28. Ooi EM , Watts GF , Ng TW , Barrett PH . Effect of dietary fatty acids on human lipoprotein metabolism: a comprehensive update . Nutrients . 2015 ; 7 ( 6 ): 4416 – 4425 . Google Scholar CrossRef Search ADS PubMed 29. Kelley DS , Siegel D , Vemuri M , Mackey BE . Docosahexaenoic acid supplementation improves fasting and postprandial lipid profiles in hypertriglyceridemic men . Am J Clin Nutr . 2007 ; 86 ( 2 ): 324 – 333 . Google Scholar CrossRef Search ADS PubMed 30. Lagrost L , Gandjini H , Athias A , Guyard-Dangremont V , Lallemant C , Gambert P . Influence of plasma cholesteryl ester transfer activity on the LDL and HDL distribution profiles in normolipidemic subjects . Arterioscler Thromb . 1993 ; 13 ( 6 ): 815 – 825 . Google Scholar CrossRef Search ADS PubMed 31. Oscarsson J , Hurt-Camejo E . Omega-3 fatty acids eicosapentaenoic acid and docosahexaenoic acid and their mechanisms of action on apolipoprotein B-containing lipoproteins in humans: a review . Lipids Health Dis . 2017 ; 16 ( 1 ): 149 . Google Scholar CrossRef Search ADS PubMed 32. Kawakami A , Yoshida M . Apolipoprotein CIII links dyslipidemia with atherosclerosis . J Atheroscler Thromb . 2009 ; 16 ( 1 ): 6 – 11 . Google Scholar CrossRef Search ADS PubMed 33. Jump DB , Botolin D , Wang Y , Xu J , Demeure O , Christian B . Docosahexaenoic acid (DHA) and hepatic gene transcription . Chem Phys Lipids . 2008 ; 153 ( 1 ): 3 – 13 . Google Scholar CrossRef Search ADS PubMed 34. Chen YJ , Chen CC , Li TK , Wang PH , Liu LR , Chang FY , Wang YC , Yu YH , Lin SP , Mersmann HJ , Ding ST . Docosahexaenoic acid suppresses the expression of FoxO and its target genes . J Nutr Biochem . 2012 ; 23 ( 12 ): 1609 – 1616 . Google Scholar CrossRef Search ADS PubMed 35. Homma Y , Ohshima K , Yamaguchi H , Nakamura H , Araki G , Goto Y . Effects of eicosapentaenoic acid on plasma lipoprotein subfractions and activities of lecithin:cholesterol acyltransferase and lipid transfer protein . Atherosclerosis . 1991 ; 91 ( 1-2 ): 145 – 153 . Google Scholar CrossRef Search ADS PubMed 36. Buckley R , Shewring B , Turner R , Yaqoob P , Minihane AM . Circulating triacylglycerol and apoE levels in response to EPA and docosahexaenoic acid supplementation in adult human subjects . Br J Nutr . 2004 ; 92 ( 3 ): 477 – 483 . Google Scholar CrossRef Search ADS PubMed 37. Hirayama S , Miida T . Small dense LDL: an emerging risk factor for cardiovascular disease . Clin Chim Acta . 2012 ; 414 : 215 – 224 . Google Scholar CrossRef Search ADS PubMed 38. Lakoski SG , Lagace TA , Cohen JC , Horton JD , Hobbs HH . Genetic and metabolic determinants of plasma PCSK9 levels . J Clin Endocrinol Metab . 2009 ; 94 ( 7 ): 2537 – 2543 . Google Scholar CrossRef Search ADS PubMed 39. Lee TC , Ivester P , Hester AG , Sergeant S , Case LD , Morgan T , Kouba EO , Chilton FH . The impact of polyunsaturated fatty acid-based dietary supplements on disease biomarkers in a metabolic syndrome/diabetes population . Lipids Health Dis . 2014 ; 13 ( 1 ): 196 . Google Scholar CrossRef Search ADS PubMed 40. Ooi EM , Ng TW , Watts GF , Barrett PHR . Dietary fatty acids and lipoprotein metabolism: new insights and updates . Curr Opin Lipidol . 2013 ; 24 ( 3 ): 192 – 197 . Google Scholar CrossRef Search ADS PubMed Copyright © 2018 Endocrine Society

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Journal of Clinical Endocrinology and MetabolismOxford University Press

Published: Aug 1, 2018

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