European Journal of Clinical Nutrition (2015) 69, 121–126 © 2015 Macmillan Publishers Limited All rights reserved 0954-3007/15 www.nature.com/ejcn
ORIGINAL ARTICLE
Effects of stearidonic acid on serum triacylglycerol concentrations in overweight and obese subjects: a randomized controlled trial DJM Pieters and RP Mensink BACKGROUND/OBJECTIVES: Eicosapentaenoic acid (EPA), which may reduce the risk for coronary heart disease (CHD), can be synthesized at low rates from α-linolenic acid (ALA). The rate-limiting step for this conversion is the Δ6-desaturation of ALA into stearidonic acid (SDA). Thus providing oils rich in SDA may increase endogenous synthesis of EPA, which may subsequently lower serum triacylglycerol concentrations, an effect frequently observed after EPA supplementation. We therefore studied the effects of Echium oil on serum triacylglycerol concentrations and the omega-3 index, which correlate negatively with the risk for CHD. SUBJECTS/METHODS: A randomized, double-blind, placebo-controlled crossover trial was conducted, in which 36 healthy overweight and slightly obese subjects daily received 10 g of Echium oil (providing 1.2 g of SDA) or a high oleic acid sunflower oil (HOSO) as control for 6 weeks, with a washout period of at least 14 days. Four subjects dropped out. Differences between periods were tested for statistical significance (P o0.05) using a paired t-test. RESULTS: Serum triacylglycerol and other lipid concentrations were not significantly affected by consumption of Echium oil compared with HOSO. Echium oil significantly increased percentage of EPA in red blood cell (RBC) membranes with 0.14 ± 0.25% (mean ± s.d.) compared with HOSO (P = 0.02). No significant effects on docosahexaenoic acid in RBC membranes or on the omega-3 index were found. CONCLUSIONS: In healthy overweight and slightly obese subjects, an increased intake of SDA from Echium oil does not lower serum triacylglycerol concentrations. Despite an increase in the percentage of EPA in RBC membranes, the omega-3 index was not changed. European Journal of Clinical Nutrition (2015) 69, 121–126; doi:10.1038/ejcn.2014.193; published online 17 September 2014
INTRODUCTION Many lines of evidence suggest that the intake of n-3 long-chain polyunsaturated fatty acids from fish such as eicosapentaenoic acid (EPA or C20:5n-3) and docosahexaenoic acid (DHA or C22:6n3) are cardioprotective.1 Therefore, specific recommendations for adequate intakes of these fatty acids have been formulated. In these guidelines, no distinction is made between EPA and DHA. In one study, however, EPA supplementation was associated with a 19% reduction in major coronary events,2 supporting dietary approaches to increase tissue EPA concentrations. Current intakes of EPA and DHA are however below recommended intakes. Therefore, alternative (plant-based) sources of n-3 polyunsaturated fatty acids may be needed to bridge the gap between guidelines and actual intakes. EPA can be synthesized from α-linolenic acid (ALA, C18:3n-3), the most common vegetableoil-based n-3 fatty acid. In humans, however, this conversion is extremely low.3 It has been suggested that the rate-limiting step for this conversion is the Δ6-desaturation of ALA into stearidonic acid (SDA, C18:4n-3).4 Thus, providing oils rich in SDA may increase the endogenous synthesis of EPA. SDA levels in vegetable oils are however low, but there are exceptions. An SDA-enriched soya bean oil increased plasma EPA concentrations as compared with a conventional soya bean oil.5 Serum triacylglycerol concentrations did not change, although such an effect can be
hypothesized, as fish-oil fatty acids have frequently been shown to lower serum triacylglycerol concentrations. In a noncontrolled study with slightly hypertriglyceridemic subjects, a daily intake of 15 g of Echium oil also increased plasma EPA concentrations.6 Although the amount of SDA in Echium oil is lower than in SDA-enriched soybean oil (12% vs 28%), serum triacylglycerol concentrations were decreased by 21%. One may speculate that this is due to the unique fatty acid composition of Echium oil or to some other minor (unknown) component in Echium oil. However, no control group was included in that study, which may have confounded the results. Therefore, we designed a randomized, placebo-controlled, double-blind study to compare the effects of Echium oil with those of high oleic sunflower oil (HOSO) on serum triacylglycerol in subjects with an increased body mass index, who are at increased risk to develop hypertriglyceridemia.7 In addition, effects on the omega-3 index were studied. This index is negatively related to cardiovascular risk, and defined as the proportion of EPA+DHA in red blood cells (RBCs).8 MATERIALS AND METHODS Study population Healthy, overweight or slightly obese subjects with a body mass index between 25 and 35 kg/m2 and aged between 18 and 70 years were
Department of Human Biology, NUTRIM School for Nutrition, Toxicology and Metabolism, Maastricht University Medical Centre, Maastricht, The Netherlands. Correspondence: Professor RP Mensink, Department of Human Biology, NUTRIM School for Nutrition, Toxicology and Metabolism, Maastricht University Medical Centre, Universiteitssingel 50, Maastricht, NL-6200 MD, The Netherlands. E-mail: [email protected] Received 4 April 2014; revised 17 July 2014; accepted 22 July 2014; published online 17 September 2014
Stearidonic acid and lipid metabolism DJM Pieters and RP Mensink
122 recruited via advertisements in local newspapers, posters in the university and hospital buildings and among subjects who participated in earlier studies at the department from July until October 2011. Forty-two subjects were invited for two screening visits. At each visit, fasting blood was sampled for analyses of serum lipids and lipoproteins. In addition, height and body weight were determined. Furthermore, subjects had to complete medical and general questionnaire. Seventeen men and nineteen women enroled into the study. Inclusion criteria were mean serum triacylglycerol o3.0 mmol/l, stable body weight (weight gain or loss o2 kg in the previous 3 months), no indications for treatment of hyperlipidemia,9 no use of medication or a diet known to affect serum lipid or glucose metabolism, no active cardiovascular disease, no drug or alcohol abuse, no use of an investigational product within the previous 30 days and willing to stop the consumption of vitamin supplements, fish oil capsules, fatty fish and products rich in plant stanol or sterol esters 3 weeks before the start of the study. This study was conducted according to the guidelines laid down in the Declaration of Helsinki and the standards for Good Clinical Practice. All procedures involving human subjects were approved by the Medical Ethical Committee of Maastricht University. Written informed consent was obtained from all subjects before the start of the study. The study was registered at clinicaltrials.gov as NCT01365078.
Study design and intervention In this randomized, double-blind, placebo-controlled crossover trial, each subject received Echium oil or HOSO for 6 weeks with a washout period of at least 2 weeks between the intervention periods. Subjects were allocated to start with either Echium oil or HOSO according to a preestablished randomization scheme. During the Echium oil period, subjects consumed daily 10 g of Echium oil (BioMega SDA Refined Echium Oil, Bioriginal Europe Asia b.v. Den Bommel, The Netherlands), providing approximately 1.2 g SDA. During the control period, subjects consumed daily 10 g of HOSO. The fatty acid composition of the oils is shown in Table 1. Both oils were provided in sachets containing 5 g of oil, which were letter-coded to blind the subjects and the investigators. Subjects consumed one sachet at lunch and one at dinner. At each visit, subjects were supplied with a number of sachets sufficient till the next visit. Sachets that were not consumed had to be returned and were used to estimate compliance. At weeks 0, 3, 5 and 6 of both intervention periods, body weight was recorded. In addition, blood pressure was measured four times (Omron M7, CEMEX Medische Techniek BV, Nieuwegein, The Netherlands) and the last three readings were averaged. At the end of both intervention periods, energy and nutrient intakes of the previous 6 weeks were assessed by a validated food frequency questionnaire,10 which was checked immediately by a registered dietician in the presence of the subjects. Subjects recorded daily signs of illness, medication used and any deviations from the protocol in diaries, which were checked at each visit. All measurements were performed at the Metabolic Research Unit Maastricht of Maastricht University.
Table 1.
Fatty acid composition of the two experimental oils HOSO
Fatty acid Saturated fatty acids C16:0 Palmitic acid C18:0 Stearic acid Monounsaturated fatty acids C18:1(n-9) Oleic acid C22:1(n-9) Erucic acid Polyunsaturated fatty acids Omega-6 fatty acids C18:2(n-6) Linoleic acid C18:3(n-6) γ-Linolenic acid Omega-3 fatty acids C18:3(n-3) α-Linolenic acid C18:4(n-3) Stearidonic acid
Echium oil
%a
g/10 g
%a
g/10 g
3.5 3.2
0.3 0.3
6.7 3.8
0.6 0.3
84.2
7.6
15.2 0.1
1.4 o0.1
7.4
0.7
15.5 10.8
1.4 1
32.3 12.8
2.9 1.2
Abbreviation: HOSO, high oleic acid sunflower oil. aValues are expressed as percentage of total fatty acids.
European Journal of Clinical Nutrition (2015) 121 – 126
Blood sampling Subjects were asked to fast overnight (from 20.00 hours) and not to perform any strenuous physical exercise or to consume alcohol 1 day before blood sampling. Venous blood was sampled at weeks 0, 3, 5 and 6 of both intervention periods in BD Vacutainer tubes (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Serum was obtained after coagulation for 30 min at 21 °C and centrifugation at 1300 g at 21 °C for 15 min. EDTA and NaF tubes were placed on ice directly after sampling, plasma was obtained by centrifugation at 1300 g at 4 °C for 15 min. Serum and plasma aliquots were snap frozen and stored at − 80 °C until analysis. RBCs were obtained from EDTA blood samples, after removal of the plasma and buffy coat, at weeks 0, 5 and 6 of both intervention periods. After the RBCs were washed twice with a physiological salt solution, 2 μl butylated hydroxytoluene (0.5 mg/ml methanol) was added per 1 ml RBC as an antioxidant, where after cups were capped under a nitrogen flow and stored at − 80 °C until analysis.
Serum lipids, glucose and insulin All serum samples were analysed for total cholesterol, high-density lipoprotein cholesterol, and triacylglycerol (corrected for free glycerol concentrations) concentrations at the department of Clinical Chemistry, University Hospital Maastricht (Beckman Coulter Synchron LX20 PRO Clinical Systems, Beckman Coulter Inc., Fullerton, CA, USA). Serum low-density lipoprotein cholesterol was calculated with the Friedewald formula.11 All NaF plasma samples were analysed for free fatty acids (Wako Nefa C test kit; Wako Chemicals, Neuss, Germany) and glucose (hexokinase method; Roche Diagnostic Systems, Hoffmann-La Roche, Basel, Switzerland). Serum insulin concentrations from weeks 0 and 6 were analysed with a human insulinspecific RIA kit (Linco Research, St Charles, MO, USA). All samples from one subject were analysed within the same run.
Fatty acid analyses in RBCs Total lipids from RBCs were extracted according to the method of Sonja Garcia, using 1,2-dinonadecanoyl phosphatidylcholine (C19:0) as internal standard. Phospholipids were isolated by thin-layer chromatography (precoated glass plates, Silica gel 60, 0.5 mm; 20 × 20 cm), and subsequently hydrolysed and methylated into their corresponding fatty acid methyl esters. For separation and quantification of a GC2010 gas chromatography was used (Shimadzu, Duisburg, Germany) equipped with a CP-Sil 88 capillary column (50 m × 0.25 mm, 0.20 μm film thickness; Agilent Technologies, Santa Clara, CA, USA), using helium as the carrier gas (injector inlet pressure of 130 kPa). The injection and the detector temperature were set at 300 °C. The oven temperature started at 160 °C and increased for 10 min to 190 °C in steps of 5 °C/min. Temperature was kept constant at 190 °C for 15 min and then increased at 5 °C/min to 230 °C, where it was kept steady for 7 min. Fatty acid composition of RBC membranes was reported as weight percentage of the following 22 selected fatty acids: saturated (14:0, 16:0, 18:0 and 24:0); cis monounsaturated (16:1, 18:1, 20:1 and 24:1); trans (16:1, 18:1 and 18:2); cis n-6 polyunsaturated (18:2, 18:3, 20:2, 20:3, 20:4, 22:4 and 22:5); cis n-3 polyunsaturated (18:3, 20:5, 22:5 and 22:6).12 All samples from one subject were analysed within the same run. The omega-3 index was calculated as the sum of EPA and DHA expressed as a percentage of the selected fatty acids.
General health parameters From weeks 0 and 6 of both intervention periods, serum concentrations of markers of liver function (total bilirubin, aspartate aminotransferase, alanine-aminotransferase, alkaline phosphatase, γ-glutamyl transpeptidase) and kidney function (creatinine) were determined at the Department of Clinical Chemistry, University Hospital Maastricht (Beckman Coulter Synchron LX20 PRO Clinical Systems, Beckman Coulter Inc.). Haematological parameters in EDTA blood (number of white blood cells, percentage of lymphocytes, percentage of monocytes, percentage of granulocytes, number of RBCs, haemoglobin, haematocrit, mean corpuscular volume and the number of platelets) were determined at the Department of Haematology, University Hospital Maastricht on a Coulter AcT diff (Coulter Corporation, Miami, FL, USA). High-sensitive C-reactive protein was measured in all serum samples with a highly sensitive immunoturbidimetric assay (Kamiya Biomedical, Seattle, WA, USA). All samples from one subject were analysed within the same run. © 2015 Macmillan Publishers Limited
Stearidonic acid and lipid metabolism DJM Pieters and RP Mensink
123 Statistics Before the start of the study, it was calculated that with 32 subjects the statistical power to detect a true difference of 0.20 mmol/l in serum triacylglycerol concentrations between the treatments was 80%. The effect size of 0.20 mmol/l was derived from the study of Surette et al.6 For the calculations, a within-subject variability of 0.40 mmol/l in serum triacylglycerol concentrations was used. First, values of weeks 5 and 6 were averaged when available. Period and carryover effects, which were absent indicating that 2 weeks washout was sufficient, were examined as described.13 Differences between the periods were tested for significance using a paired samples t-test. A Po 0.05 was considered to be statistically significant. Results are presented as means ± s.d. Statistical analyses were performed using SPSS version 19.0 for Mac (SPSS Inc., Chicago, IL, USA).
RESULTS Baseline characteristics of the subjects who completed the study are shown in Table 2. A total of 36 subjects were included. One man and three women dropped out due to various personal reasons, unrelated to the intake of the test products. Analyses were performed for the 32 subjects who completed the study. Food intake is shown in Table 3. The proportion of energy from
Table 2.
protein was slightly increased during the Echium oil period (P = 0.04). Differences in the intake of oleic, linoleic and α-linoleic acids—due to the fatty acid composition of the experimental oils—were as expected (Table 3). Mean body weights were 85.6 ± 12.2 kg at the end of the HOSO period and 85.4 ± 12.0 kg at the end of the Echium oil period. Compliance, as estimated from the returned number of sachets, was 96.2% during the HOSO period and 94.4% during the Echium oil period. Serum triacylglycerol concentrations, the primary end point of the study, were 1.26 ± 0.73 mmol/l after HOSO consumption and 1.36 ± 0.59 mmol/l after the intake of Echium oil (Table 4). The difference of 0.11 ± 0.47 mmol/l did not reach statistical significance (P = 0.21). Also, serum total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol concentrations and the total to high-density lipoprotein cholesterol ratio were comparable between the two intervention periods. Plasma free fatty acids, glucose, insulin and high-sensitive C-reactive protein concentrations did not also change during the study. Five subjects had, unrelated to the dietary treatments, on one or more occasion high-sensitive C-reactive protein concentrations 410 mg/l. When these subjects were excluded from the analysis, conclusions did not change.
Study population characteristics at screening Men Mean
Number of subjects Age (years) BMI (kg/m2) Total cholesterol (mmol/l) LDL-cholesterol (mmol/l) HDL-cholesterol (mmol/l) Triacylglycerol (mmol/l) Total to HDL-cholesterol ratio Mean SBP (mm Hg) Mean DBP (mm Hg)
Women s.d.
Mean
14 2.4 0.86 0.71 0.24 0.7 0.78 18 12
50 29.3 5.97 3.72 1.75 1.08 3.53 119 80
16 56 28.5 5.92 3.65 1.57 1.51 3.85 142 90
All subjects s.d.
Mean
14 3.5 1.02 0.78 0.45 0.43 0.72 14 9
51 28.9 5.94 3.69 1.66 1.3 3.69 130 85
16
s.d. 32 15 3 0.93 0.73 0.37 0.61 0.75 20 12
Abbreviations: BMI, body mass index; DBP, diastolic blood pressure; HDL, high-density lipoprotein; LDL, low-density lipoprotein; SBP, systolic blood pressure.
Table 3.
Energy and nutrient intake during the two intervention periods according to the food frequency questionnaires HOSO
Energy MJ/day Kcal/day Protein (En%) Carbohydrates (En%) Total fat (En%) Saturated fatty acids (En%) Monounsaturated fatty acids (En%) Oleic acid (En%) Polyunsaturated fatty acids (En%) Linoleic acid (En%) α-Linoleic acid (En%) α-Linoleic acid (g/day) Trans fatty acids (En%) Alcohol (En%) Cholesterol (mg/day) Fibre (g/day)
Echium oil
P-value
Change
Mean
s.d.
Mean
s.d.
Mean
s.d.
9.7 2328 15.1 43.9 38.5 10.8 15.2 13.8 9.1 8 0.4 0.9 1 2.6 166.3 26
3.1 752 4 8.7 6.7 2.5 3.8 3.7 3.4 3 0.1 0.4 0.6 3.9 73.5 7.1
9.4 2247 16.3 41.2 39.9 11.3 13.3 11.8 9 10.3 1.7 4 1.1 2.6 181.2 25
2.5 602 3.6 7.7 6.8 2.1 4.5 4.4 3.3 3.3 0.5 0.5 0.8 4 92.4 9.2
− 0.3 − 81 1.3 − 2.7 1.4 0.5 − 1.9 −2 − 0.1 2.3 1.3 3 0.1 0 14.9 − 0.8
2.8 667 3.3 8.9 8.2 1.8 4.4 4.1 3.7 3.6 0.4 0.5 0.6 1.2 71.9 6.4
0.5 0.5 0.04 0.1 0.35 0.1 0.02 0.01 0.84 o 0.01 o 0.01 o 0.01 0.26 0.99 0.25 0.51
Abbreviations: En%, energy percentage; HOSO, high oleic sunflower oil. P-values for diet effects were calculated by a paired t-test, P o0.05 was considered to be statistically significant.
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European Journal of Clinical Nutrition (2015) 121 – 126
Stearidonic acid and lipid metabolism DJM Pieters and RP Mensink
124
Proportion of the various fatty acids at the start of the first and second intervention periods did not differ between the groups, who started at these time points with, respectively, the HOSO and Echium oil periods (Supplementary Table S1 and S2). Echium oil decreased the proportions of myristic (C14:0) and oleic acid (C18:1n-9) and increased those of γ-linoleic (C18:3n-6), dihomogamma-linoleic (C20:3n-6) and α-linoleic acid (C18:3n-3). More interestingly, EPA (C20:5n-3) in RBC membranes increased with 0.14% to 0.60% after the Echium oil period. Also the proportion of docosapentaenoic acid (22:5n-3) was significantly greater after the Echium oil period (P = 0.02), whereas there was no difference in the proportion of DHA (C22:6n-3) after consumption of HOSO and Echium oil (P = 0.96). Despite the change in the proportion of EPA, the omega-3 index was not significantly affected (P = 0.68; Table 5). Systolic blood pressure was 127 ± 17 mm Hg and 129 ± 16 mm Hg (P = 0.24), and diastolic blood pressure was 81 ± 9 mm Hg and 82 ± 2 mm Hg (P = 0.39) after the HOSO and Echium oil periods, respectively. Finally, no significant effects of Echium oil Table 4.
consumption on markers of liver and kidney function compared with HOSO consumption were observed (data not shown). DISCUSSION In the present study, a daily intake of 1.2 g SDA from Echium oil for 6 weeks did not change serum triacylglycerol concentrations or the omega-3 index in overweight and slightly obese subjects. Our results contrast the findings of Surette et al.,6 who reported a reduction in serum triacylglycerol concentrations of 21% after consumption of 15 g of Echium oil providing 1.88 g SDA a day for 4 weeks. In that study, subjects were hypertriglyceridemic, which may explain the different findings, as effects of fish oils depend on initial triacylglycerol concentrations.1,14 Although we deliberately chose for overweight and slightly obese subjects, who have increased serum triacylglycerol concentrations compared with lean subjects,15 levels were not as high as in the study of Surette et al.6 A potential flaw of that study; however, was that no control group was included. Therefore, another explanation is that
Mean concentrations of lipids and lipoproteins, high-sensitive CRP (hsCRP), glucose and insulin at the end of the two intervention periods HOSO
Total cholesterol (mmol/l) LDL-cholesterol (mmol/l) HDL-cholesterol (mmol/l) Total to HDL-cholesterol ratio Triacylglycerol (mmol/l) Free fatty acids (μmol/l) hsCRP (mg/l) Glucose (mmol/l) Insulin (mU/l)
Echium oil
P-value
Change
Mean
s.d.
Mean
s.d.
Mean
s.d.
5.6 3.71 1.33 4.37 1.26 426 2.05 5.7 17.5
1 0.75 0.33 0.97 0.73 154 1.72 0.69 8.2
5.7 3.66 1.36 4.32 1.36 442 3.55 5.75 17
1 0.76 0.37 1.01 0.59 145 5.77 0.73 7.1
0.03 − 0.04 0.03 − 0.06 0.11 16 1.5 0.05 − 0.4
0.39 0.33 0.12 0.44 0.47 153 5.25 0.36 4.8
95%CI − 0.18 − 0.08 − 0.07 − 0.1 − 0.27 − 71 − 3.39 − 0.18 − 1.3
to to to to to to to to to
0.11 0.16 0.01 0.21 0.06 39 0.39 0.08 2.1
0.62 0.46 0.18 0.48 0.21 0.57 0.12 0.42 0.62
Abbreviations: HDL, high-density lipoprotein; HOSO, high oleic sunflower oil; hsCRP, high-sensitive C-reactive protein; LDL, low-density lipoprotein. P-values for oil effects were calculated by a paired t-test.
Table 5.
Mean fatty acid composition of red blood cell membranes at the end of the two intervention periods HOSO
Echium oil
P-value
Change
Mean
s.d.
Mean
s.d.
Mean
s.d.
Total saturated fatty acids Palmitic acid Stearic acid
C16:0 C18:0
49.6 24.3 17.8
4.5 2.3 1.5
48.7 23.9 17.9
2.6 1.6 1
− 0.88 − 0.43 0.06
4.48 2.39 1.17
0.35 0.39 0.79
Total monounsaturated fatty acids Oleic acid
C18:1n-9
19.5 12.5
1.7 1.1
18.8 12
1.1 1.1
− 0.73 − 0.56
1.48 0.91
0.03 0.01
Total trans fatty acids Trans palmitoleic acid Trans oleic acid Trans linoleic acid
C16:1n-7tr C18:1tr C18:2n-6tr
0.69 0.1 0.55 0.05
0.3 0.04 0.27 0.04
0.69 0.09 0.55 0.05
0.43 0.03 0.41 0.04
0 − 0.01 0 0
0.29 0.02 0.3 0.03
0.97 0.17 1 0.72
30.2 24.5 9 11.3 5.7 0.1 0.5 2.1 3.1 3.5
4.9 3.7 1.2 2.3 1.8 0 0.2 0.4 1.3 1.5
31.8 25.7 9 12.1 6.1 0.2 0.6 2.3 3 3.7
2.7 2.1 0.9 1.6 1.3 0.1 0.3 0.3 1 1.2
1.6 1.2 − 0.01 0.76 0.4 0.06 0.14 0.22 −0.02 0.12
5.3 3.87 0.87 2.38 1.71 0.08 0.25 0.44 1.29 1.4
0.15 0.14 0.94 0.13 0.26 0 0.02 0.02 0.96 0.68
Total polyunsaturated fatty acids Total omega-6 fatty acids Linoleic acid Arachidonic acid Total omega-3 fatty acids α-Linolenic acid (ALA) Eicosapentaenoic acid (EPA) Docosapentaenoic acid Docosahexaenoic acid (DHA) Omega-3 index
C18:2n-6 C20:4n-6 C18:3n-3 C20:5n-3 C22:5n-3 C22:6n-3
Abbreviation: HOSO, high oleic sunflower oil. Values are means ± s.d. and are expressed as weight percentage of 22 fatty acids identified.12 P-values for oil effects were calculated by an independent samples t-test.
European Journal of Clinical Nutrition (2015) 121 – 126
© 2015 Macmillan Publishers Limited
Stearidonic acid and lipid metabolism DJM Pieters and RP Mensink
125 aspecific drifts with time may have biased results. In fact, our results do agree with other placebo-controlled studies looking at the effect of SDA intake.5,16–19 In these studies, subjects received SDA-enhanced soybean oil for 12 and 14 weeks, providing daily 1.5, 4.2 and 3.7 g SDA,5,16,19 respectively, or SDA provided as ethyl esters in doses ranging from 0.43 to 5.2 g a day for 12 weeks17 or 0.75 g and then 1.5 g for a period of 3 weeks each.18 In none of these studies, significant changes in serum triacylglycerol concentrations were observed. It should be noted, however, that none of these studies were specifically designed to examine effects on serum triacylglycerol, while subjects were also not hypertriglyceridemic. In the present study, serum total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol concentrations and the total to high-density lipoprotein cholesterol ratio were also comparable between the two intervention periods. These results are in line with other studies.5,6,16,18,19 Krul et al.,17 however did find a significant increase in the total to high-density lipoprotein cholesterol ratio at the end of treatment relative to control in several treatment groups receiving different doses of SDA, but this may have been a chance finding. Mean percentage changes from baseline ranged from − 12% (1.5 g SDA per day) to 7% (3.0 g SDA/day), compared with − 8% in the control group, with no indication of a pattern related to dosage.17 The intake of Echium oil significantly increased the proportion of EPA and docosapentaenoic acid in RBC membranes, indicating that dietary SDA is desaturated and elongated in humans. The proportion of DHA in RBC membranes remained unaffected. These findings agree with those of previous studies5,6,16–19 and do indicate that the conversion of docosapentaenoic acid into DHA by Δ6-desaturase is rate limiting in the conversion of SDA into DHA. Despite the increase in EPA, there was no significant increase in the omega-3 index, as shown in other studies.5,16,19 The increases in the omega-3 indexes in the previous studies were the result of greater increases in EPA content of the RBC membranes, which may have been due to different intakes of SDA, background diets or study duration. During the Echium oil period, the intake of linoleic acid was significantly higher (10.3 ± 3.3 energy percentage) compared with the HOSO period (8.0 ± 3.0 energy percentage), due to the relatively high linoleic acid content in Echium oil. Previous studies have indicated that linoleic acid lowers the conversion of ALA into EPA,3,20 because these two essential fatty acids compete for the Δ6-desaturase.21 The Echium oil also contained a relatively high amount of ALA, resulting in a significantly higher ALA intake during the Echium oil period (1.7 ± 0.5 energy percentage) compared with the HOSO period (0.4 ± 0.1 energy percentage). This was also reflected by changes in the proportion of ALA in RBC membranes. Although ALA can be converted to EPA and DHA, the efficiency of this conversion will be lower than that of SDA. A meta-regression of six ALA supplementation studies that ranged from 2 to 14 g/day however, showed a dose–response increase in EPA levels without detectable increases in DHA.22 Therefore, it can be speculated that the increase in the proportion of EPA and docosapentaenoic acid in RBC membranes in the Echium oil period was also partly due to the higher ALA intake during this period. Noteworthy, the Japan EPA lipid intervention study suggested that EPA supplementation alone reduced the risks of cardiac events.2 Although it is not known whether results can be extrapolated to other populations, it does support the notion that increasing tissue EPA concentrations can have beneficial effects on heart health. In summary, our findings suggest that low daily intake of SDA in the form of Echium oil, easily achievable through dietary means, does not lower serum triacylglycerol concentrations or improve the omega-3 index, but does raise the proportion of EPA in RBC phospholipids in non-hypertriglyceridemic overweight and slightly obese subjects. Whether this latter effect is translated into © 2015 Macmillan Publishers Limited
beneficial health effects on the longer term, remains to be investigated. CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS We would like to thank M Hulsbosch, H Aydeniz and W Sluijsmans for their technical support, and N Wystyrk for her dietary assistance. Bioriginal Europe Asia b.v., Den Bommel, The Netherlands, funded the study and provided the test products. Bioriginal Europe Asia b.v. had no role in the design, analysis or writing of this article.
AUTHOR CONTRIBUTION RPM designed the research; DJMP conducted the research; DJMP and RPM analysed the data and wrote the paper; RPM had primary responsibility for final content. Both authors read and approved the final manuscript.
REFERENCES 1 Roth EM, Harris WS. Fish oil for primary and secondary prevention of coronary heart disease. Curr Atheroscler Rep 2010; 12: 66–72. 2 Yokoyama M, Origas H, Matsuzaki M, Matsuzawa Y, Saito Y, Ishikawa Y et al. Effects of eicosapentaenoic acid on major coronary events in hypercholesterolaemic patients (JELIS): a randomised open-label, blinded endpoint analysis. Lancet 2007; 369: 1090–1098. 3 Goyens PL, Spilker ME, Zock PL, Katan MB, Mensink RP. Conversion of alphalinolenic acid in humans is influenced by the absolute amounts of alpha-linolenic acid and linoleic acid in the diet and not by their ratio. Am J Clin Nutr 2006; 84: 44–53. 4 Yamazaki K, Fujikawa M, Hamazaki T, Yano S, Shono T. Comparison of the conversion rates of alpha-linolenic acid (18:3(n - 3)) and stearidonic acid (18:4(n - 3)) to longer polyunsaturated fatty acids in rats. Biochim Biophys Acta 1992; 1123: 18–26. 5 Lemke SL, Vicini JL, Su H, Goldstein DA, Nemeth MA, Krul ES et al. Dietary intake of stearidonic acid-enriched soybean oil increases the omega-3 index: randomized, double-blind clinical study of efficacy and safety. Am J Clin Nutr 2010; 92: 766–775. 6 Surette ME, Edens M, Chilton FH, Tramposch KM. Dietary echium oil increases plasma and neutrophil long-chain (n-3) fatty acids and lowers serum triacylglycerols in hypertriglyceridemic humans. J Nutr 2004; 134: 1406–1411. 7 Tirosh A, Shai I, Bitzur R, Kochba I, Tekes-Manova D, Israeli E et al. Changes in triglyceride levels over time and risk of type 2 diabetes in young men. Diabetes Care 2008; 31: 2032–2037. 8 Harris WS, Von Schacky C. The Omega-3 Index: a new risk factor for death from coronary heart disease? Prev Med 2004; 39: 212–220. 9 Netherlands Centraal begeleidingsorgaan voor de intercollegiale toetsing. Behandeling en preventie van coronaire hartziekten door verlaging van de plasma cholesterolconcentratie. Utrecht, 1998. 10 Plat J, Mensink RP. Vegetable oil based versus wood based stanol ester mixtures: effects on serum lipids and hemostatic factors in non-hypercholesterolemic subjects. Atherosclerosis 2000; 148: 101–112. 11 Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem 1972; 18: 499–502. 12 Harris WS, Pottala JV, Vasan RS, Larson MG, Robins SJ. Changes in erythrocyte membrane trans and marine fatty acids between 1999 and 2006 in older Americans. J Nutr 2012; 147: 1297–1303. 13 Pocock SJ. Clinical Trials. A practical approach. John Wiley & Sons: Hoboken, 1983. 14 Balk EM, Lichtenstein AH, Chung M, Kupelnick B, Chew P, Lau J. Effects of omega3 fatty acids on serum markers of cardiovascular disease risk: a systematic review. Atherosclerosis 2006; 189: 19–30. 15 Sanders TAB, Filippou A, Berry SE, Baumgartner S, Mensink RP. Palmitic acid in the sn-2 position of triacylglycerols acutely influences postprandial lipid metabolism. Am J Clin Nutr 2011; 94: 1433–1441. 16 Harris WS, Lemke SL, Hansen SN, Goldstein DA, DiRienzo MA, Su H et al. Stearidonic Acid-Enriched Soybean Oil Increased the Omega-3 Index, an Emerging Cardiovascular Risk Marker. Lipids 2008; 43: 805–811. 17 Krul ES, Lemke SL, Mukherjea R, Taylor ML, Goldstein DA, Su H et al. Effects of duration of treatment and dosage of eicosapentaenoic acid and stearidonic acid
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126 on red blood cell eicosapentaenoic acid content. Prostaglandins Leukot Essent Fatty Acids 2012; 86: 51–59. 18 James MJ, Ursin VM, Cleland LG. Metabolism of stearidonic acid in human subjects: comparison with the metabolism of other n-3 fatty acids. Am J Clin Nutr 2003; 77: 1140–1145. 19 Lemke SL, Maki KC, Hughes G, Taylor ML, Krul AS, Goldstein DA et al. Consumption of stearidonic acid-rich oil in foods increases red blood cell eicosapentaenoic acid. J Acad Nutr Diet 2013; 113: 1044–1056.
20 Pawlosky RJ, Hibbeln JR, Novotny JA, Salem N Jr. Physiological compartmental analysis of a-linolenic acid metabolism in adult humans. J Lipid Res 2001; 42: 1257–1265. 21 Plourde M, Cunnane SC. Extremely limited synthesis of long chain polyunsaturates in adults: implications for their dietary essentiality and use as supplements. Appl Physiol Nutr Metab 2007; 32: 619–634. 22 Arterburn LM, Hall EB, Oken H. Distribution, interconversion, and dose response of n-3 fatty acids in humans. Am J Clin Nutr 2006; 83: 1467S–1476S.
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European Journal of Clinical Nutrition (2015) 121 – 126
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ORIGINAL ARTICLE
Effects of stearidonic acid on serum triacylglycerol concentrations in overweight and obese subjects: a randomized controlled trial DJM Pieters and RP Mensink BACKGROUND/OBJECTIVES: Eicosapentaenoic acid (EPA), which may reduce the risk for coronary heart disease (CHD), can be synthesized at low rates from α-linolenic acid (ALA). The rate-limiting step for this conversion is the Δ6-desaturation of ALA into stearidonic acid (SDA). Thus providing oils rich in SDA may increase endogenous synthesis of EPA, which may subsequently lower serum triacylglycerol concentrations, an effect frequently observed after EPA supplementation. We therefore studied the effects of Echium oil on serum triacylglycerol concentrations and the omega-3 index, which correlate negatively with the risk for CHD. SUBJECTS/METHODS: A randomized, double-blind, placebo-controlled crossover trial was conducted, in which 36 healthy overweight and slightly obese subjects daily received 10 g of Echium oil (providing 1.2 g of SDA) or a high oleic acid sunflower oil (HOSO) as control for 6 weeks, with a washout period of at least 14 days. Four subjects dropped out. Differences between periods were tested for statistical significance (P o0.05) using a paired t-test. RESULTS: Serum triacylglycerol and other lipid concentrations were not significantly affected by consumption of Echium oil compared with HOSO. Echium oil significantly increased percentage of EPA in red blood cell (RBC) membranes with 0.14 ± 0.25% (mean ± s.d.) compared with HOSO (P = 0.02). No significant effects on docosahexaenoic acid in RBC membranes or on the omega-3 index were found. CONCLUSIONS: In healthy overweight and slightly obese subjects, an increased intake of SDA from Echium oil does not lower serum triacylglycerol concentrations. Despite an increase in the percentage of EPA in RBC membranes, the omega-3 index was not changed. European Journal of Clinical Nutrition (2015) 69, 121–126; doi:10.1038/ejcn.2014.193; published online 17 September 2014
INTRODUCTION Many lines of evidence suggest that the intake of n-3 long-chain polyunsaturated fatty acids from fish such as eicosapentaenoic acid (EPA or C20:5n-3) and docosahexaenoic acid (DHA or C22:6n3) are cardioprotective.1 Therefore, specific recommendations for adequate intakes of these fatty acids have been formulated. In these guidelines, no distinction is made between EPA and DHA. In one study, however, EPA supplementation was associated with a 19% reduction in major coronary events,2 supporting dietary approaches to increase tissue EPA concentrations. Current intakes of EPA and DHA are however below recommended intakes. Therefore, alternative (plant-based) sources of n-3 polyunsaturated fatty acids may be needed to bridge the gap between guidelines and actual intakes. EPA can be synthesized from α-linolenic acid (ALA, C18:3n-3), the most common vegetableoil-based n-3 fatty acid. In humans, however, this conversion is extremely low.3 It has been suggested that the rate-limiting step for this conversion is the Δ6-desaturation of ALA into stearidonic acid (SDA, C18:4n-3).4 Thus, providing oils rich in SDA may increase the endogenous synthesis of EPA. SDA levels in vegetable oils are however low, but there are exceptions. An SDA-enriched soya bean oil increased plasma EPA concentrations as compared with a conventional soya bean oil.5 Serum triacylglycerol concentrations did not change, although such an effect can be
hypothesized, as fish-oil fatty acids have frequently been shown to lower serum triacylglycerol concentrations. In a noncontrolled study with slightly hypertriglyceridemic subjects, a daily intake of 15 g of Echium oil also increased plasma EPA concentrations.6 Although the amount of SDA in Echium oil is lower than in SDA-enriched soybean oil (12% vs 28%), serum triacylglycerol concentrations were decreased by 21%. One may speculate that this is due to the unique fatty acid composition of Echium oil or to some other minor (unknown) component in Echium oil. However, no control group was included in that study, which may have confounded the results. Therefore, we designed a randomized, placebo-controlled, double-blind study to compare the effects of Echium oil with those of high oleic sunflower oil (HOSO) on serum triacylglycerol in subjects with an increased body mass index, who are at increased risk to develop hypertriglyceridemia.7 In addition, effects on the omega-3 index were studied. This index is negatively related to cardiovascular risk, and defined as the proportion of EPA+DHA in red blood cells (RBCs).8 MATERIALS AND METHODS Study population Healthy, overweight or slightly obese subjects with a body mass index between 25 and 35 kg/m2 and aged between 18 and 70 years were
Department of Human Biology, NUTRIM School for Nutrition, Toxicology and Metabolism, Maastricht University Medical Centre, Maastricht, The Netherlands. Correspondence: Professor RP Mensink, Department of Human Biology, NUTRIM School for Nutrition, Toxicology and Metabolism, Maastricht University Medical Centre, Universiteitssingel 50, Maastricht, NL-6200 MD, The Netherlands. E-mail: [email protected] Received 4 April 2014; revised 17 July 2014; accepted 22 July 2014; published online 17 September 2014
Stearidonic acid and lipid metabolism DJM Pieters and RP Mensink
122 recruited via advertisements in local newspapers, posters in the university and hospital buildings and among subjects who participated in earlier studies at the department from July until October 2011. Forty-two subjects were invited for two screening visits. At each visit, fasting blood was sampled for analyses of serum lipids and lipoproteins. In addition, height and body weight were determined. Furthermore, subjects had to complete medical and general questionnaire. Seventeen men and nineteen women enroled into the study. Inclusion criteria were mean serum triacylglycerol o3.0 mmol/l, stable body weight (weight gain or loss o2 kg in the previous 3 months), no indications for treatment of hyperlipidemia,9 no use of medication or a diet known to affect serum lipid or glucose metabolism, no active cardiovascular disease, no drug or alcohol abuse, no use of an investigational product within the previous 30 days and willing to stop the consumption of vitamin supplements, fish oil capsules, fatty fish and products rich in plant stanol or sterol esters 3 weeks before the start of the study. This study was conducted according to the guidelines laid down in the Declaration of Helsinki and the standards for Good Clinical Practice. All procedures involving human subjects were approved by the Medical Ethical Committee of Maastricht University. Written informed consent was obtained from all subjects before the start of the study. The study was registered at clinicaltrials.gov as NCT01365078.
Study design and intervention In this randomized, double-blind, placebo-controlled crossover trial, each subject received Echium oil or HOSO for 6 weeks with a washout period of at least 2 weeks between the intervention periods. Subjects were allocated to start with either Echium oil or HOSO according to a preestablished randomization scheme. During the Echium oil period, subjects consumed daily 10 g of Echium oil (BioMega SDA Refined Echium Oil, Bioriginal Europe Asia b.v. Den Bommel, The Netherlands), providing approximately 1.2 g SDA. During the control period, subjects consumed daily 10 g of HOSO. The fatty acid composition of the oils is shown in Table 1. Both oils were provided in sachets containing 5 g of oil, which were letter-coded to blind the subjects and the investigators. Subjects consumed one sachet at lunch and one at dinner. At each visit, subjects were supplied with a number of sachets sufficient till the next visit. Sachets that were not consumed had to be returned and were used to estimate compliance. At weeks 0, 3, 5 and 6 of both intervention periods, body weight was recorded. In addition, blood pressure was measured four times (Omron M7, CEMEX Medische Techniek BV, Nieuwegein, The Netherlands) and the last three readings were averaged. At the end of both intervention periods, energy and nutrient intakes of the previous 6 weeks were assessed by a validated food frequency questionnaire,10 which was checked immediately by a registered dietician in the presence of the subjects. Subjects recorded daily signs of illness, medication used and any deviations from the protocol in diaries, which were checked at each visit. All measurements were performed at the Metabolic Research Unit Maastricht of Maastricht University.
Table 1.
Fatty acid composition of the two experimental oils HOSO
Fatty acid Saturated fatty acids C16:0 Palmitic acid C18:0 Stearic acid Monounsaturated fatty acids C18:1(n-9) Oleic acid C22:1(n-9) Erucic acid Polyunsaturated fatty acids Omega-6 fatty acids C18:2(n-6) Linoleic acid C18:3(n-6) γ-Linolenic acid Omega-3 fatty acids C18:3(n-3) α-Linolenic acid C18:4(n-3) Stearidonic acid
Echium oil
%a
g/10 g
%a
g/10 g
3.5 3.2
0.3 0.3
6.7 3.8
0.6 0.3
84.2
7.6
15.2 0.1
1.4 o0.1
7.4
0.7
15.5 10.8
1.4 1
32.3 12.8
2.9 1.2
Abbreviation: HOSO, high oleic acid sunflower oil. aValues are expressed as percentage of total fatty acids.
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Blood sampling Subjects were asked to fast overnight (from 20.00 hours) and not to perform any strenuous physical exercise or to consume alcohol 1 day before blood sampling. Venous blood was sampled at weeks 0, 3, 5 and 6 of both intervention periods in BD Vacutainer tubes (Becton, Dickinson and Company, Franklin Lakes, NJ, USA). Serum was obtained after coagulation for 30 min at 21 °C and centrifugation at 1300 g at 21 °C for 15 min. EDTA and NaF tubes were placed on ice directly after sampling, plasma was obtained by centrifugation at 1300 g at 4 °C for 15 min. Serum and plasma aliquots were snap frozen and stored at − 80 °C until analysis. RBCs were obtained from EDTA blood samples, after removal of the plasma and buffy coat, at weeks 0, 5 and 6 of both intervention periods. After the RBCs were washed twice with a physiological salt solution, 2 μl butylated hydroxytoluene (0.5 mg/ml methanol) was added per 1 ml RBC as an antioxidant, where after cups were capped under a nitrogen flow and stored at − 80 °C until analysis.
Serum lipids, glucose and insulin All serum samples were analysed for total cholesterol, high-density lipoprotein cholesterol, and triacylglycerol (corrected for free glycerol concentrations) concentrations at the department of Clinical Chemistry, University Hospital Maastricht (Beckman Coulter Synchron LX20 PRO Clinical Systems, Beckman Coulter Inc., Fullerton, CA, USA). Serum low-density lipoprotein cholesterol was calculated with the Friedewald formula.11 All NaF plasma samples were analysed for free fatty acids (Wako Nefa C test kit; Wako Chemicals, Neuss, Germany) and glucose (hexokinase method; Roche Diagnostic Systems, Hoffmann-La Roche, Basel, Switzerland). Serum insulin concentrations from weeks 0 and 6 were analysed with a human insulinspecific RIA kit (Linco Research, St Charles, MO, USA). All samples from one subject were analysed within the same run.
Fatty acid analyses in RBCs Total lipids from RBCs were extracted according to the method of Sonja Garcia, using 1,2-dinonadecanoyl phosphatidylcholine (C19:0) as internal standard. Phospholipids were isolated by thin-layer chromatography (precoated glass plates, Silica gel 60, 0.5 mm; 20 × 20 cm), and subsequently hydrolysed and methylated into their corresponding fatty acid methyl esters. For separation and quantification of a GC2010 gas chromatography was used (Shimadzu, Duisburg, Germany) equipped with a CP-Sil 88 capillary column (50 m × 0.25 mm, 0.20 μm film thickness; Agilent Technologies, Santa Clara, CA, USA), using helium as the carrier gas (injector inlet pressure of 130 kPa). The injection and the detector temperature were set at 300 °C. The oven temperature started at 160 °C and increased for 10 min to 190 °C in steps of 5 °C/min. Temperature was kept constant at 190 °C for 15 min and then increased at 5 °C/min to 230 °C, where it was kept steady for 7 min. Fatty acid composition of RBC membranes was reported as weight percentage of the following 22 selected fatty acids: saturated (14:0, 16:0, 18:0 and 24:0); cis monounsaturated (16:1, 18:1, 20:1 and 24:1); trans (16:1, 18:1 and 18:2); cis n-6 polyunsaturated (18:2, 18:3, 20:2, 20:3, 20:4, 22:4 and 22:5); cis n-3 polyunsaturated (18:3, 20:5, 22:5 and 22:6).12 All samples from one subject were analysed within the same run. The omega-3 index was calculated as the sum of EPA and DHA expressed as a percentage of the selected fatty acids.
General health parameters From weeks 0 and 6 of both intervention periods, serum concentrations of markers of liver function (total bilirubin, aspartate aminotransferase, alanine-aminotransferase, alkaline phosphatase, γ-glutamyl transpeptidase) and kidney function (creatinine) were determined at the Department of Clinical Chemistry, University Hospital Maastricht (Beckman Coulter Synchron LX20 PRO Clinical Systems, Beckman Coulter Inc.). Haematological parameters in EDTA blood (number of white blood cells, percentage of lymphocytes, percentage of monocytes, percentage of granulocytes, number of RBCs, haemoglobin, haematocrit, mean corpuscular volume and the number of platelets) were determined at the Department of Haematology, University Hospital Maastricht on a Coulter AcT diff (Coulter Corporation, Miami, FL, USA). High-sensitive C-reactive protein was measured in all serum samples with a highly sensitive immunoturbidimetric assay (Kamiya Biomedical, Seattle, WA, USA). All samples from one subject were analysed within the same run. © 2015 Macmillan Publishers Limited
Stearidonic acid and lipid metabolism DJM Pieters and RP Mensink
123 Statistics Before the start of the study, it was calculated that with 32 subjects the statistical power to detect a true difference of 0.20 mmol/l in serum triacylglycerol concentrations between the treatments was 80%. The effect size of 0.20 mmol/l was derived from the study of Surette et al.6 For the calculations, a within-subject variability of 0.40 mmol/l in serum triacylglycerol concentrations was used. First, values of weeks 5 and 6 were averaged when available. Period and carryover effects, which were absent indicating that 2 weeks washout was sufficient, were examined as described.13 Differences between the periods were tested for significance using a paired samples t-test. A Po 0.05 was considered to be statistically significant. Results are presented as means ± s.d. Statistical analyses were performed using SPSS version 19.0 for Mac (SPSS Inc., Chicago, IL, USA).
RESULTS Baseline characteristics of the subjects who completed the study are shown in Table 2. A total of 36 subjects were included. One man and three women dropped out due to various personal reasons, unrelated to the intake of the test products. Analyses were performed for the 32 subjects who completed the study. Food intake is shown in Table 3. The proportion of energy from
Table 2.
protein was slightly increased during the Echium oil period (P = 0.04). Differences in the intake of oleic, linoleic and α-linoleic acids—due to the fatty acid composition of the experimental oils—were as expected (Table 3). Mean body weights were 85.6 ± 12.2 kg at the end of the HOSO period and 85.4 ± 12.0 kg at the end of the Echium oil period. Compliance, as estimated from the returned number of sachets, was 96.2% during the HOSO period and 94.4% during the Echium oil period. Serum triacylglycerol concentrations, the primary end point of the study, were 1.26 ± 0.73 mmol/l after HOSO consumption and 1.36 ± 0.59 mmol/l after the intake of Echium oil (Table 4). The difference of 0.11 ± 0.47 mmol/l did not reach statistical significance (P = 0.21). Also, serum total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol concentrations and the total to high-density lipoprotein cholesterol ratio were comparable between the two intervention periods. Plasma free fatty acids, glucose, insulin and high-sensitive C-reactive protein concentrations did not also change during the study. Five subjects had, unrelated to the dietary treatments, on one or more occasion high-sensitive C-reactive protein concentrations 410 mg/l. When these subjects were excluded from the analysis, conclusions did not change.
Study population characteristics at screening Men Mean
Number of subjects Age (years) BMI (kg/m2) Total cholesterol (mmol/l) LDL-cholesterol (mmol/l) HDL-cholesterol (mmol/l) Triacylglycerol (mmol/l) Total to HDL-cholesterol ratio Mean SBP (mm Hg) Mean DBP (mm Hg)
Women s.d.
Mean
14 2.4 0.86 0.71 0.24 0.7 0.78 18 12
50 29.3 5.97 3.72 1.75 1.08 3.53 119 80
16 56 28.5 5.92 3.65 1.57 1.51 3.85 142 90
All subjects s.d.
Mean
14 3.5 1.02 0.78 0.45 0.43 0.72 14 9
51 28.9 5.94 3.69 1.66 1.3 3.69 130 85
16
s.d. 32 15 3 0.93 0.73 0.37 0.61 0.75 20 12
Abbreviations: BMI, body mass index; DBP, diastolic blood pressure; HDL, high-density lipoprotein; LDL, low-density lipoprotein; SBP, systolic blood pressure.
Table 3.
Energy and nutrient intake during the two intervention periods according to the food frequency questionnaires HOSO
Energy MJ/day Kcal/day Protein (En%) Carbohydrates (En%) Total fat (En%) Saturated fatty acids (En%) Monounsaturated fatty acids (En%) Oleic acid (En%) Polyunsaturated fatty acids (En%) Linoleic acid (En%) α-Linoleic acid (En%) α-Linoleic acid (g/day) Trans fatty acids (En%) Alcohol (En%) Cholesterol (mg/day) Fibre (g/day)
Echium oil
P-value
Change
Mean
s.d.
Mean
s.d.
Mean
s.d.
9.7 2328 15.1 43.9 38.5 10.8 15.2 13.8 9.1 8 0.4 0.9 1 2.6 166.3 26
3.1 752 4 8.7 6.7 2.5 3.8 3.7 3.4 3 0.1 0.4 0.6 3.9 73.5 7.1
9.4 2247 16.3 41.2 39.9 11.3 13.3 11.8 9 10.3 1.7 4 1.1 2.6 181.2 25
2.5 602 3.6 7.7 6.8 2.1 4.5 4.4 3.3 3.3 0.5 0.5 0.8 4 92.4 9.2
− 0.3 − 81 1.3 − 2.7 1.4 0.5 − 1.9 −2 − 0.1 2.3 1.3 3 0.1 0 14.9 − 0.8
2.8 667 3.3 8.9 8.2 1.8 4.4 4.1 3.7 3.6 0.4 0.5 0.6 1.2 71.9 6.4
0.5 0.5 0.04 0.1 0.35 0.1 0.02 0.01 0.84 o 0.01 o 0.01 o 0.01 0.26 0.99 0.25 0.51
Abbreviations: En%, energy percentage; HOSO, high oleic sunflower oil. P-values for diet effects were calculated by a paired t-test, P o0.05 was considered to be statistically significant.
© 2015 Macmillan Publishers Limited
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Stearidonic acid and lipid metabolism DJM Pieters and RP Mensink
124
Proportion of the various fatty acids at the start of the first and second intervention periods did not differ between the groups, who started at these time points with, respectively, the HOSO and Echium oil periods (Supplementary Table S1 and S2). Echium oil decreased the proportions of myristic (C14:0) and oleic acid (C18:1n-9) and increased those of γ-linoleic (C18:3n-6), dihomogamma-linoleic (C20:3n-6) and α-linoleic acid (C18:3n-3). More interestingly, EPA (C20:5n-3) in RBC membranes increased with 0.14% to 0.60% after the Echium oil period. Also the proportion of docosapentaenoic acid (22:5n-3) was significantly greater after the Echium oil period (P = 0.02), whereas there was no difference in the proportion of DHA (C22:6n-3) after consumption of HOSO and Echium oil (P = 0.96). Despite the change in the proportion of EPA, the omega-3 index was not significantly affected (P = 0.68; Table 5). Systolic blood pressure was 127 ± 17 mm Hg and 129 ± 16 mm Hg (P = 0.24), and diastolic blood pressure was 81 ± 9 mm Hg and 82 ± 2 mm Hg (P = 0.39) after the HOSO and Echium oil periods, respectively. Finally, no significant effects of Echium oil Table 4.
consumption on markers of liver and kidney function compared with HOSO consumption were observed (data not shown). DISCUSSION In the present study, a daily intake of 1.2 g SDA from Echium oil for 6 weeks did not change serum triacylglycerol concentrations or the omega-3 index in overweight and slightly obese subjects. Our results contrast the findings of Surette et al.,6 who reported a reduction in serum triacylglycerol concentrations of 21% after consumption of 15 g of Echium oil providing 1.88 g SDA a day for 4 weeks. In that study, subjects were hypertriglyceridemic, which may explain the different findings, as effects of fish oils depend on initial triacylglycerol concentrations.1,14 Although we deliberately chose for overweight and slightly obese subjects, who have increased serum triacylglycerol concentrations compared with lean subjects,15 levels were not as high as in the study of Surette et al.6 A potential flaw of that study; however, was that no control group was included. Therefore, another explanation is that
Mean concentrations of lipids and lipoproteins, high-sensitive CRP (hsCRP), glucose and insulin at the end of the two intervention periods HOSO
Total cholesterol (mmol/l) LDL-cholesterol (mmol/l) HDL-cholesterol (mmol/l) Total to HDL-cholesterol ratio Triacylglycerol (mmol/l) Free fatty acids (μmol/l) hsCRP (mg/l) Glucose (mmol/l) Insulin (mU/l)
Echium oil
P-value
Change
Mean
s.d.
Mean
s.d.
Mean
s.d.
5.6 3.71 1.33 4.37 1.26 426 2.05 5.7 17.5
1 0.75 0.33 0.97 0.73 154 1.72 0.69 8.2
5.7 3.66 1.36 4.32 1.36 442 3.55 5.75 17
1 0.76 0.37 1.01 0.59 145 5.77 0.73 7.1
0.03 − 0.04 0.03 − 0.06 0.11 16 1.5 0.05 − 0.4
0.39 0.33 0.12 0.44 0.47 153 5.25 0.36 4.8
95%CI − 0.18 − 0.08 − 0.07 − 0.1 − 0.27 − 71 − 3.39 − 0.18 − 1.3
to to to to to to to to to
0.11 0.16 0.01 0.21 0.06 39 0.39 0.08 2.1
0.62 0.46 0.18 0.48 0.21 0.57 0.12 0.42 0.62
Abbreviations: HDL, high-density lipoprotein; HOSO, high oleic sunflower oil; hsCRP, high-sensitive C-reactive protein; LDL, low-density lipoprotein. P-values for oil effects were calculated by a paired t-test.
Table 5.
Mean fatty acid composition of red blood cell membranes at the end of the two intervention periods HOSO
Echium oil
P-value
Change
Mean
s.d.
Mean
s.d.
Mean
s.d.
Total saturated fatty acids Palmitic acid Stearic acid
C16:0 C18:0
49.6 24.3 17.8
4.5 2.3 1.5
48.7 23.9 17.9
2.6 1.6 1
− 0.88 − 0.43 0.06
4.48 2.39 1.17
0.35 0.39 0.79
Total monounsaturated fatty acids Oleic acid
C18:1n-9
19.5 12.5
1.7 1.1
18.8 12
1.1 1.1
− 0.73 − 0.56
1.48 0.91
0.03 0.01
Total trans fatty acids Trans palmitoleic acid Trans oleic acid Trans linoleic acid
C16:1n-7tr C18:1tr C18:2n-6tr
0.69 0.1 0.55 0.05
0.3 0.04 0.27 0.04
0.69 0.09 0.55 0.05
0.43 0.03 0.41 0.04
0 − 0.01 0 0
0.29 0.02 0.3 0.03
0.97 0.17 1 0.72
30.2 24.5 9 11.3 5.7 0.1 0.5 2.1 3.1 3.5
4.9 3.7 1.2 2.3 1.8 0 0.2 0.4 1.3 1.5
31.8 25.7 9 12.1 6.1 0.2 0.6 2.3 3 3.7
2.7 2.1 0.9 1.6 1.3 0.1 0.3 0.3 1 1.2
1.6 1.2 − 0.01 0.76 0.4 0.06 0.14 0.22 −0.02 0.12
5.3 3.87 0.87 2.38 1.71 0.08 0.25 0.44 1.29 1.4
0.15 0.14 0.94 0.13 0.26 0 0.02 0.02 0.96 0.68
Total polyunsaturated fatty acids Total omega-6 fatty acids Linoleic acid Arachidonic acid Total omega-3 fatty acids α-Linolenic acid (ALA) Eicosapentaenoic acid (EPA) Docosapentaenoic acid Docosahexaenoic acid (DHA) Omega-3 index
C18:2n-6 C20:4n-6 C18:3n-3 C20:5n-3 C22:5n-3 C22:6n-3
Abbreviation: HOSO, high oleic sunflower oil. Values are means ± s.d. and are expressed as weight percentage of 22 fatty acids identified.12 P-values for oil effects were calculated by an independent samples t-test.
European Journal of Clinical Nutrition (2015) 121 – 126
© 2015 Macmillan Publishers Limited
Stearidonic acid and lipid metabolism DJM Pieters and RP Mensink
125 aspecific drifts with time may have biased results. In fact, our results do agree with other placebo-controlled studies looking at the effect of SDA intake.5,16–19 In these studies, subjects received SDA-enhanced soybean oil for 12 and 14 weeks, providing daily 1.5, 4.2 and 3.7 g SDA,5,16,19 respectively, or SDA provided as ethyl esters in doses ranging from 0.43 to 5.2 g a day for 12 weeks17 or 0.75 g and then 1.5 g for a period of 3 weeks each.18 In none of these studies, significant changes in serum triacylglycerol concentrations were observed. It should be noted, however, that none of these studies were specifically designed to examine effects on serum triacylglycerol, while subjects were also not hypertriglyceridemic. In the present study, serum total cholesterol, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol concentrations and the total to high-density lipoprotein cholesterol ratio were also comparable between the two intervention periods. These results are in line with other studies.5,6,16,18,19 Krul et al.,17 however did find a significant increase in the total to high-density lipoprotein cholesterol ratio at the end of treatment relative to control in several treatment groups receiving different doses of SDA, but this may have been a chance finding. Mean percentage changes from baseline ranged from − 12% (1.5 g SDA per day) to 7% (3.0 g SDA/day), compared with − 8% in the control group, with no indication of a pattern related to dosage.17 The intake of Echium oil significantly increased the proportion of EPA and docosapentaenoic acid in RBC membranes, indicating that dietary SDA is desaturated and elongated in humans. The proportion of DHA in RBC membranes remained unaffected. These findings agree with those of previous studies5,6,16–19 and do indicate that the conversion of docosapentaenoic acid into DHA by Δ6-desaturase is rate limiting in the conversion of SDA into DHA. Despite the increase in EPA, there was no significant increase in the omega-3 index, as shown in other studies.5,16,19 The increases in the omega-3 indexes in the previous studies were the result of greater increases in EPA content of the RBC membranes, which may have been due to different intakes of SDA, background diets or study duration. During the Echium oil period, the intake of linoleic acid was significantly higher (10.3 ± 3.3 energy percentage) compared with the HOSO period (8.0 ± 3.0 energy percentage), due to the relatively high linoleic acid content in Echium oil. Previous studies have indicated that linoleic acid lowers the conversion of ALA into EPA,3,20 because these two essential fatty acids compete for the Δ6-desaturase.21 The Echium oil also contained a relatively high amount of ALA, resulting in a significantly higher ALA intake during the Echium oil period (1.7 ± 0.5 energy percentage) compared with the HOSO period (0.4 ± 0.1 energy percentage). This was also reflected by changes in the proportion of ALA in RBC membranes. Although ALA can be converted to EPA and DHA, the efficiency of this conversion will be lower than that of SDA. A meta-regression of six ALA supplementation studies that ranged from 2 to 14 g/day however, showed a dose–response increase in EPA levels without detectable increases in DHA.22 Therefore, it can be speculated that the increase in the proportion of EPA and docosapentaenoic acid in RBC membranes in the Echium oil period was also partly due to the higher ALA intake during this period. Noteworthy, the Japan EPA lipid intervention study suggested that EPA supplementation alone reduced the risks of cardiac events.2 Although it is not known whether results can be extrapolated to other populations, it does support the notion that increasing tissue EPA concentrations can have beneficial effects on heart health. In summary, our findings suggest that low daily intake of SDA in the form of Echium oil, easily achievable through dietary means, does not lower serum triacylglycerol concentrations or improve the omega-3 index, but does raise the proportion of EPA in RBC phospholipids in non-hypertriglyceridemic overweight and slightly obese subjects. Whether this latter effect is translated into © 2015 Macmillan Publishers Limited
beneficial health effects on the longer term, remains to be investigated. CONFLICT OF INTEREST The authors declare no conflict of interest.
ACKNOWLEDGEMENTS We would like to thank M Hulsbosch, H Aydeniz and W Sluijsmans for their technical support, and N Wystyrk for her dietary assistance. Bioriginal Europe Asia b.v., Den Bommel, The Netherlands, funded the study and provided the test products. Bioriginal Europe Asia b.v. had no role in the design, analysis or writing of this article.
AUTHOR CONTRIBUTION RPM designed the research; DJMP conducted the research; DJMP and RPM analysed the data and wrote the paper; RPM had primary responsibility for final content. Both authors read and approved the final manuscript.
REFERENCES 1 Roth EM, Harris WS. Fish oil for primary and secondary prevention of coronary heart disease. Curr Atheroscler Rep 2010; 12: 66–72. 2 Yokoyama M, Origas H, Matsuzaki M, Matsuzawa Y, Saito Y, Ishikawa Y et al. Effects of eicosapentaenoic acid on major coronary events in hypercholesterolaemic patients (JELIS): a randomised open-label, blinded endpoint analysis. Lancet 2007; 369: 1090–1098. 3 Goyens PL, Spilker ME, Zock PL, Katan MB, Mensink RP. Conversion of alphalinolenic acid in humans is influenced by the absolute amounts of alpha-linolenic acid and linoleic acid in the diet and not by their ratio. Am J Clin Nutr 2006; 84: 44–53. 4 Yamazaki K, Fujikawa M, Hamazaki T, Yano S, Shono T. Comparison of the conversion rates of alpha-linolenic acid (18:3(n - 3)) and stearidonic acid (18:4(n - 3)) to longer polyunsaturated fatty acids in rats. Biochim Biophys Acta 1992; 1123: 18–26. 5 Lemke SL, Vicini JL, Su H, Goldstein DA, Nemeth MA, Krul ES et al. Dietary intake of stearidonic acid-enriched soybean oil increases the omega-3 index: randomized, double-blind clinical study of efficacy and safety. Am J Clin Nutr 2010; 92: 766–775. 6 Surette ME, Edens M, Chilton FH, Tramposch KM. Dietary echium oil increases plasma and neutrophil long-chain (n-3) fatty acids and lowers serum triacylglycerols in hypertriglyceridemic humans. J Nutr 2004; 134: 1406–1411. 7 Tirosh A, Shai I, Bitzur R, Kochba I, Tekes-Manova D, Israeli E et al. Changes in triglyceride levels over time and risk of type 2 diabetes in young men. Diabetes Care 2008; 31: 2032–2037. 8 Harris WS, Von Schacky C. The Omega-3 Index: a new risk factor for death from coronary heart disease? Prev Med 2004; 39: 212–220. 9 Netherlands Centraal begeleidingsorgaan voor de intercollegiale toetsing. Behandeling en preventie van coronaire hartziekten door verlaging van de plasma cholesterolconcentratie. Utrecht, 1998. 10 Plat J, Mensink RP. Vegetable oil based versus wood based stanol ester mixtures: effects on serum lipids and hemostatic factors in non-hypercholesterolemic subjects. Atherosclerosis 2000; 148: 101–112. 11 Friedewald WT, Levy RI, Fredrickson DS. Estimation of the concentration of low-density lipoprotein cholesterol in plasma, without use of the preparative ultracentrifuge. Clin Chem 1972; 18: 499–502. 12 Harris WS, Pottala JV, Vasan RS, Larson MG, Robins SJ. Changes in erythrocyte membrane trans and marine fatty acids between 1999 and 2006 in older Americans. J Nutr 2012; 147: 1297–1303. 13 Pocock SJ. Clinical Trials. A practical approach. John Wiley & Sons: Hoboken, 1983. 14 Balk EM, Lichtenstein AH, Chung M, Kupelnick B, Chew P, Lau J. Effects of omega3 fatty acids on serum markers of cardiovascular disease risk: a systematic review. Atherosclerosis 2006; 189: 19–30. 15 Sanders TAB, Filippou A, Berry SE, Baumgartner S, Mensink RP. Palmitic acid in the sn-2 position of triacylglycerols acutely influences postprandial lipid metabolism. Am J Clin Nutr 2011; 94: 1433–1441. 16 Harris WS, Lemke SL, Hansen SN, Goldstein DA, DiRienzo MA, Su H et al. Stearidonic Acid-Enriched Soybean Oil Increased the Omega-3 Index, an Emerging Cardiovascular Risk Marker. Lipids 2008; 43: 805–811. 17 Krul ES, Lemke SL, Mukherjea R, Taylor ML, Goldstein DA, Su H et al. Effects of duration of treatment and dosage of eicosapentaenoic acid and stearidonic acid
European Journal of Clinical Nutrition (2015) 121 – 126
Stearidonic acid and lipid metabolism DJM Pieters and RP Mensink
126 on red blood cell eicosapentaenoic acid content. Prostaglandins Leukot Essent Fatty Acids 2012; 86: 51–59. 18 James MJ, Ursin VM, Cleland LG. Metabolism of stearidonic acid in human subjects: comparison with the metabolism of other n-3 fatty acids. Am J Clin Nutr 2003; 77: 1140–1145. 19 Lemke SL, Maki KC, Hughes G, Taylor ML, Krul AS, Goldstein DA et al. Consumption of stearidonic acid-rich oil in foods increases red blood cell eicosapentaenoic acid. J Acad Nutr Diet 2013; 113: 1044–1056.
20 Pawlosky RJ, Hibbeln JR, Novotny JA, Salem N Jr. Physiological compartmental analysis of a-linolenic acid metabolism in adult humans. J Lipid Res 2001; 42: 1257–1265. 21 Plourde M, Cunnane SC. Extremely limited synthesis of long chain polyunsaturates in adults: implications for their dietary essentiality and use as supplements. Appl Physiol Nutr Metab 2007; 32: 619–634. 22 Arterburn LM, Hall EB, Oken H. Distribution, interconversion, and dose response of n-3 fatty acids in humans. Am J Clin Nutr 2006; 83: 1467S–1476S.
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European Journal of Clinical Nutrition (2015) 121 – 126
© 2015 Macmillan Publishers Limited
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