Effect of supplementation with flaxseed oil and different doses of fish oil for 2 weeks on plasma phosphatidylcholine fatty acids in young women

Effect of supplementation with flaxseed oil and different doses of fish oil for 2 weeks on plasma... Background/objectives Although assumed, it remains unclear that fatty acid (FA) biomarkers of n-3 long-chain PUFA reflect wide ranges of intake. However, to be utilised as biomarkers, to predict dietary intake, dose–response curves that cover a spectrum of intakes are required. The aim of the study was to investigate whether the FA composition of plasma phosphatidylcholine (PC) is a sensitive biomarker of n-3 FAs from fish oil, across a range of supplementation doses, and alpha-linolenic acid (ALA) supplementation, in young, healthy women. Subjects/methods A total of 303 young women were randomised to intakes ranging between 0.33 and 4.50 g EPA+DHA/ day from fish oil (not all doses used in each year) or flaxseed oil (5.90–6.60 g/d) daily for 14 days in a series of trials, over 5 years. Fasting blood was collected at baseline (day 0) and day 14 and plasma PC FA composition, total and HDL-cholesterol and triglyceride concentrations measured. Results Fourteen days supplementation with fish oil significantly (P < 0.01) increased, in a dose-dependent fashion, plasma PC EPA, DPA and DHA at all doses except 1 and 3 mL/day. For the combined group of women who consumed any fish oil there was a 16% (P < 0.01) decrease in plasma triacylglycerol concentrations after 14 days supplementation. Flaxseed oil supplementation for 14 day resulted in significant (P < 0.01) increases in ALA, EPA and DPA, whilst DHA remained unchanged. Conclusion Our data demonstrate plasma PC is a sensitive biomarker of n-3 FA intake and reflects changes within 14 days across a range of intakes. Introduction 3 LCPUFA intake in a dose–response manner [1–3]. However, for FA to be utilised as biomarkers to predict Plasma, erythrocyte and platelet phospholipids are the blood dietary intake, dose–response curves that cover a spectrum lipid fractions most abundant in n-3 long-chain poly- of FA intake are required and although assumed, it remains unsaturated fatty acids (LCPUFA). Dose–response studies, unclear that FA biomarkers of n-3 LCPUFA reflect a wide typically with only three ‘distinct’ levels of n-3 fatty acid range of intakes. Another important source of n-3 LCPUFA (FA), have shown blood phospholipids reflect changes in n- could come from the precursor alpha-linolenic acid (ALA). Longer-term studies have found the abundance of ALA, EPA and docosapentaenoic acid (DPA) increased in FA biomarkers with an increase in ALA intake with the data for * Leanne Hodson DHA being less clear [4–11]. leanne.hodson@ocdem.ox.ac.uk Although evidence from randomised controlled trials has not proven n-3 LCPUFA lowers cardiovascular disease Department of Human Nutrition, University of Otago, (CVD) risk [12], intervention studies have clearly demon- Dunedin, New Zealand strated n-3 LCPUFA (as fish oil or ethyl esters of EPA and Present address: Oxford Centre for Diabetes, Endocrinology and DHA) have a triglyceride-lowering effect [13]. A large Metabolism (OCDEM), University of Oxford, Churchill Hospital, Oxford OX3 7LE, UK proportion of these studies have been undertaken in middle- aged adults [13, 14]; it remains unclear whether n-3 Present address: Institute of Applied Health Research, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK LCPUFA have a similar effect in young adults. 1234567890();,: 1234567890();,: Effect of supplementation with flaxseed oil and different doses of fish oil for 2 weeks on plasma. . . 833 We sought to investigate the effect of short-term Biochemical and lipid analysis (14 days) supplementation with EPA and DHA (given as fish oil), across a range of doses, on plasma phosphati- On days 0 and 14 venous blood samples were collected dylcholine (PC) n-3 FA levels and plasma lipid concentra- from participants after an overnight fast and plasma isolated tions, along with the effect of short-term (14 days) ALA and stored [15]. Plasma total, high-density lipoprotein supplementation on plasma PC n-3 FA status in young, (HDL) cholesterol and triglyceride concentrations were healthy women. measured and plasma low-density lipoprotein (LDL) cho- lesterol concentrations calculated [16]. The analytical coefficient of variation for the measurement of total cho- Subjects and methods lesterol was less than 3% for plasma total cholesterol, HDL- cholesterol and triglyceride. Participants Plasma lipids were extracted, after the addition of a known amount of an internal standard (diheptadecanoyl Participants were recruited from an undergraduate nutri- [17:0] PC), according to the method of Bligh and Dyer [17]. tion course at the University of Otago and were eligible if Plasma PC was separated using thin-layer chromatography they were 18 years or older and were not allergic to fish or as we have previously described for erythrocyte PC [18] and nuts. Ethical approval was given for the study from The PC FAs converted to FA methyl esters (FAMEs). Separation Human Ethics Committee, University of Otago. All par- and quantitation of the plasma PC FAMEs was achieved ticipants gave informed written consent after receiving using a DB-225 megabore column (25 m × 0.53 mm internal both a verbal and written explanation of the study. The diameter; film thickness 0.25 µm; J & W Scientific) installed experiment was part of an undergraduate teaching and on an HP-6890 Series Gas Chromatograph (GC) with flame learning activity that was conducted annually for five ionisation detection [15, 19]. Students performed the lipid years from 2003 to 2007. extraction and thin-layer chromatography, under super- vision, whereas a qualified research technician performed Study design and supplementation the GC analysis of samples. Plasma FA were recorded as molecular percentages (mol%), defined as the number of Participants were randomised to receive fish oil or flaxseed molecules of the individual FA as a percentage of the total oil in capsule form and consumed the allocated daily dose, number of FA molecules. The concentrations (µmol/L) of with food for 14 days. Participants were asked to maintain FAs were calculated based on the area of the internal stan- their usual diet and physical activity; if they consumed fish dard (diheptadecanoyl [17:0] PC) peak. they were instructed to continue their usual pattern of consumption. MaxEPA (Seven Seas Health Company UK) Statistical analysis fish oil capsules were used in 2003 and 2004 and Omega-3 Salmon Oil capsules (Thompson’s, Auckland, New Zeal- Data were analysed using the statistical package STATA and) from 2005 to 2007; flaxseed oil was from Waihi Bush (version 11). Statistical differences in the plasma lipids and (Geraldine, New Zealand). Manufacturer’s information FA composition of PC between day 0 and day 14 were indicated that each capsule of MaxEPA contained 190 mg determined using a paired t-test. All comparisons were two- of EPA and 110 mg of DHA, each capsule of Omega-3 sided and changes were considered statistically significant Salmon Oil contained 180 mg of EPA and 120 mg of DHA, when the P-value was less than 0.01; this value was chosen and each capsule of flaxseed oil contained 600 mg of ALA. to reduce the chances of concluding erroneously that a The doses of fish oil used in the trials were 1, 2, 3, 4, 5, 6, difference existed between days 14 and 0. 10 and 15 capsules/day, not all doses were used in each year As subjects consumed EPA and DHA (given as fish oil), (due to participant numbers), whereas the dose of flaxseed across a range of doses (Supplementary Table 1) over a 14- oil was always 10 capsules/day. Intakes (g/d) of EPA and day period, this provided the opportunity to determine if DHA and ALA were estimated on the basis of manu- there was a dose–response effect. The change from day 0 to facturers’ information about the FA content of the fish oil 14 in plasma PC n-3 FA or plasma lipid concentration was and flaxseed oil and the number of capsules assigned to be calculated for each participant and these values used in the consumed (Supplementary Table 1). Compliance was regression analysis to test for a dose–response effect. The assessed in all years by change in FA composition of dose–response relation between the increase in EPA intake plasma PC and in 2005, 2006 and 2007 (but not 2003 and and change in EPA composition of plasma PC was esti- 2004) compliance was also assessed by a daily diary of mated using regression analysis, adjusting for year of the capsule consumption. study and baseline (i.e., day 0) FA composition. The dose–response relation was expressed as the incremental 834 L. Hodson et al. Table 1 Long-chain n-3 polyunsaturated fatty acid composition (mol%) of plasma phosphatidylcholine at before and after 14 days of consuming the oil supplement Intake (g/d) n Eicosapentaenoic acid (µmol/L) Docosapentaenoic acid (µmol/L) Docosahexaenoic acid (µmol/L) a a b a a b a a b EPA DHA EPA+DHA Day 0 Day 14 Difference Day 0 Day 14 Difference Day 0 Day 14 Difference 0.19 0.14 0.33 15 1.0 (0.5) 1.3 (0.3) 0.4 (−0.1, 0.8) 0.7 (0.2) 0.8 (0.2) 0.1 (−0.1, 0.3) 3.0 (1.0) 3.3 (0.9) 0.3 (−0.6, 1.2) c c c 0.38 0.28 0.66 12 0.8 (0.2) 2.2 (0.7) 1.5 (0.7, 2.2) 0.6 (0.2) 0.9 (0.3) 0.3 (0.2, 0.5) 2.7 (0.8) 3.7 (0.8) 1.0 (0.5, 1.5) c c 0.44 0.28 0.72 19 1.0 (0.4) 1.6 (0.6) 0.8 (0.1, 1.5) 0.7 (0.2) 0.9 (0.3) 0.2 (0.0, 0.4) 3.6 (1.2) 3.6 (1.0) 0.0 (−0.8, 0.9) c c c 0.55 0.42 0.99 12 1.2 (1.0) 3.1 (1.1) 1.9 (1.1, 2.6) 0.7 (0.3) 1.0 (0.4) 0.3 (0.1, 0.5) 3.1 (1.4) 4.3 (1.3) 1.2 (0.2, 2.2) c c c 0.76 0.56 1.32 12 0.8 (0.2) 3.5 (0.7) 2.6 (2.1, 3.2) 0.7 (0.2) 1.1 (0.2) 0.4 (0.3, 0.6) 3.1 (0.8) 4.5 (0.9) 1.4 (0.8, 2.0) c c 0.88 0.56 1.44 13 1.0 (0.3) 2.5 (1.0) 1.6 (0.8, 2.4) 0.7 (0.2) 1.1 (0.3) 0.3 (0.1, 0.5) 3.4 (0.9) 4.6 (1.1) 1.2 (−0.2, 2.6) c c c 0.95 0.55 1.50 47 1.0 (0.5) 3.7 (1.3) 2.7 (2.3, 3.2) 0.8 (0.3) 1.2 (0.3) 0.4 (0.3, 0.5) 3.4 (1.1) 4.6 (1.2) 1.3 (0.9, 1.7) c c c 0.95 0.70 1.65 7 1.3 (1.0) 4.3 (0.7) 3.0 (1.2, 4.7) 0.7 (0.2) 1.1 (0.3) 0.3 (0.0, 0.7) 3.4 (0.7) 4.7 (1.1) 1.3 (0.0, 2.6) c c 1.32 0.84 2.16 10 1.0 (0.6) 3.1 (1.5) 2.2 (0.5, 3.8) 0.8 (0.2) 1.1 (0.3) 0.3 (0.1, 0.6) 3.3 (0.7) 4.4 (1.4) 1.1 (−0.7, 2.9) c c c 1.90 1.10 3.00 48 1.0 (0.5) 5.6 (2.0) 4.6 (3.8, 5.4) 0.8 (0.2) 1.4 (0.3) 0.6 (0.5, 0.7) 3.5 (1.0) 5.2 (1.3) 1.8 (1.3, 2.2) c c 1.90 1.40 3.30 8 1.3 (0.7) 5.8 (2.0) 4.5 (2.0, 7.0) 1.0 (0.2) 1.6 (0.3) 0.6 (0.1, 1.0) 4.1 (1.1) 5.6 (1.5) 1.6 (−0.2, 3.4) c c c 2.20 1.40 3.60 12 1.1 (0.5) 4.5 (2.3) 3.4 (1.3, 5.5) 0.8 (0.3) 1.4 (0.3) 0.6 (0.2, 0.9) 3.3 (0.8) 5.3 (1.2) 1.9 (1.3, 2.6) c c c 2.85 1.65 4.50 10 1.0 (0.4) 7.0 (2.5) 6.1 (3.4, 8.7) 0.7 (0.3) 1.7 (0.4) 1.0 (0.5, 1.5) 3.7 (1.1) 6.4 (1.0) 2.7 (1.4, 4.1) Values are mean (SD) Values are mean (99% CI) Day 14 significantly different from day 0, paired t-test P < 0.01 change (95% CI) in mol% per g/d or µmol/L per g/d from 2005 through 2007 completed a daily diary of capsule increase in intake of EPA. A simple plot of the results consumption; self-reported compliance indicated that 97% suggested a curvilinear relation between increasing intake of the assigned capsules were consumed. of EPA and change in mol% and µmol/L EPA in plasma PC; therefore, a quadratic term was included in the model. Effect of fish oil supplementation on plasma PC n-3 The same procedure was used to explore the dose–response FA relation between DHA intake and DHA composition of plasma PC; however, a quadratic term was dropped from Daily supplementation with different amounts of fish oil the model because it was not statistically significant (P = (EPA+DHA) significantly (P < 0.01) increased plasma PC 0.150). A similar approach was used to calculate the dose EPA mol% and µmol/L at all doses of fish oil intakes except response relation between EPA+DHA intake and plasma the one mL dose (Tables 1 and 2). The mean increase, lipid concentrations (mmol/L), with the dose response unadjusted analysis, in EPA mol% and µmol/L between expressed as the incremental change (95% CI) per g/d days 0 and 14 by dose of EPA is shown in Fig. 1a, c; the increase of EPA+DHA. increase was dose-dependent and did not differ by year of study (P = 0.988, interaction between dose and year). According to this association, the dose response, over Results 14 days, in plasma PC EPA was 1.5 mol% or 76 µmol/L per g EPA (Fig. 1b, d). Notably, daily supplementation with Baseline characteristics fish oil significantly increased the abundance (as mol% and µmol/L) of DPA in plasma PC, in most, but not all doses of Three hundred and three women participated in the sup- fish oil (Tables 1 and 2). plementation trials between 2003 and 2007, of which we Daily supplementation with fish oil also significantly (P have complete data for 294. The participants’ (n = 294) < 0.01) increased plasma PC DPA and DHA mol% at all were aged 22.1 years (4.0) (mean (SD)) with a BMI of doses of fish oil intake except one and three mL (Table 1) 22.6 kg/m (2.9); mean plasma lipid concentrations were but changes occurred at fewer fish oil doses when expressed 4.45 (0.80) mmol/L for plasma total cholesterol, 2.51 (0.68) as a concentration (µmol/L) (Table 2). The mean increase, mmol/L for plasma LDL-cholesterol, 1.51 (0.35) mmol/L unadjusted analysis, in DHA mol% and µmol/L between for plasma HDL cholesterol, and 1.11 (0.40) mmol/L for day 0 and 14 by dose of fish oil intake is shown in Fig. 2a,c; plasma triglycerides. The characteristics of women assigned the increase was linearly dose-dependent and did not differ to the fish or flaxseed oil groups did not differ. The number by year of study (P = 0.524, interaction between dose and of women enrolled each year and randomised to the dif- year). The predicted mean (95%CI) increase, over 14 days, ferent fish oil groups or the flaxseed oil group is shown in in plasma PC DHA mol% and µmol/L across the range of Supplementary Table 1. Ninety-four percent of participants daily supplemental DHA intake is shown in Fig. 2b,d. Effect of supplementation with flaxseed oil and different doses of fish oil for 2 weeks on plasma. . . 835 Table 2 Long-chain n-3 polyunsaturated fatty acid composition (µmol/L) of plasma phosphatidylcholine at before and after 14 days of consuming the oil supplement Intake (g/d) n Eicosapentaenoic acid (µmol/L) Docosapentaenoic acid (µmol/L) Docosahexaenoic acid (µmol/L) a a b c a b a a b EPA DHA EPA+DHA Day 0 Day 14 Difference Day 0 Day 14 Difference Day 0 Day 14 Difference 0.19 0.14 0.33 15 43 (19) 72 (51) 29 (−15, 72) 31 (10) 43 (32) 12 (−11, 34) 144 (92) 180 (167) 37 (−89, 163) c c 0.38 0.28 0.66 12 41 (10) 122 (55) 81 (37, 125) 29 (8) 49 (24) 20 (3, 37) 151 (75) 198 (68) 47 (−2, 96) c c 0.44 0.28 0.72 19 42 (22) 78 (45) 36 (6, 65) 31 (15) 45 (24) 13 (0, 27) 157 (92) 177 (105) 20 (−61, 101) c c c 0.55 0.42 0.99 12 54 (36) 168 (79) 113 (62, 164) 35 (20) 53 (24) 18 (5, 32) 147 (80) 227 (88) 80 (28, 132) c c 0.76 0.56 1.32 1 32 (11) 147 (67) 115 (58, 171) 27 (8) 48 (20) 21 (4, 39) 121 (42) 193 (90) 72 (−3, 148) 0.88 0.56 1.44 13 53 (29) 106 (43) 52 (10, 94) 39 (19) 43 (9) 3 (−13, 19) 201 (131) 186 (30) −15 (−123, 93) c c c 0.95 0.55 1.50 47 39 (23) 136 (60) 97 (75, 119) 29 (14) 42 (19) 13 (7, 19) 126 (57) 168 (75) 42 (17, 66) c c c 0.95 0.70 1.65 7 54 (38) 174 (40) 120 (66, 173) 29 (9) 42 (9) 12 (3, 22) 141 (38) 184 (32) 43 (14, 72) 1.32 0.84 2.16 10 43 (36) 117 (37) 74 (17, 130) 32 (11) 42 (12) 10 (−3, 23) 148 (71) 176 (65) 28 (−73, 129) c c c 1.90 1.10 3.00 48 38 (18) 200 (88) 162 (128, 196) 29 (12) 49 (19) 21 (13, 28) 129 (50) 185 (70) 56 (36, 76) c c 1.90 1.40 3.30 8 56 (32) 284 (124) 228 (88, 367) 42 (8) 74 (29) 32 (2, 62) 175 (60) 274 (114) 98 (−16, 213) c c c 2.20 1.40 3.60 12 45 (23) 229 (145) 183 (61, 305) 32 (12) 72 (32) 40 (14, 66) 133 (44) 282 (135) 149 (48, 250) c c c 2.85 1.65 4.50 10 36 (15) 298 (138) 262 (121, 403) 28 (11) 74 (31) 46 (15, 77) 143 (62) 272 (106) 129 (37, 221) Values are mean (SD) Values are mean (99% CI) Day 14 significantly different from day 0, paired t-test P < 0.01 According to this association, the dose response, over 3.60 g EPA+DHA/day for 14 day; they were also sig- 14 days, in plasma PC DHA was 1.1 mol% or 61 µmol/L nificantly lower (by 15%, P < 0.001 day 14 vs day 0) when per g intake of DHA (Fig. 2b,d). results were combined for all fish oil consumers (Table 4). There was no significant dose-response relation between Effect of flaxseed oil supplementation on plasma PC total n-3 LCPUFA intake (i.e., EPA+DHA) and total n-3 FA cholesterol (P = 0.235), LDL-cholesterol (P = 0.955), or HDL-cholesterol (P = 0.440) concentrations (Table 4)or Daily supplementation with flaxseed oil for 14 days with plasma triglyceride concentrations. Consuming 10 mL increased plasma PC ALA by 0.7 mol% (95% CI, 0.6–0.9; of flaxseed oil, containing 6 g of ALA, daily for 14 days P < 0.001) or 32 µmol/L (95% CI, 14–46, P <0.001) did not significantly alter plasma lipid concentrations (Table 3). The proportions and concentrations of plasma (Table 4). PC FA as EPA and DPA were significantly (P <0.01) increased, with no change in DHA, after 14 days (Table 3). The changes in FA composition with flaxseed oil con- Discussion sumption did not differ by year in which the study was conducted (P > 0.6). As the usefulness of plasma PC as a biomarker of n-3 FA intake has not been extensively examined, we assessed Effect of fish oil and flaxseed oil supplementation changes in plasma PC n-3 FAs before and after supple- on plasma lipids mentation with fish oil (across a range of doses) or sup- plementation with ALA. Our results clearly demonstrate the Daily fish oil supplementation for 14 days did not sig- FA composition of plasma PC to be a sensitive biomarker of nificantly alter plasma total cholesterol concentrations n-3 LCPUFA intake, with small increases in EPA and DHA within any one group of fish oil intake (Table 4); however, intake (from fish oil) being reflected within 14 days, in a when the results for all participants who received fish oil dose-dependent fashion. The increase in EPA and DHA were combined (n = 234), mean plasma total and LDL abundance with increasing dose of fish oil, and the increase cholesterol concentrations were significantly (P < 0.001) in ALA abundance with flaxseed oil supplementation higher at day 14 compared with day 0. For this combined indicate a high degree of compliance over the 14-day per- group, mean intake of fish oil, weighted for the proportion iod. EPA and DPA, two FA formed by the metabolic of participants in each dose group, was 6.1 mL/d, or 1.8 g of interconversion of ALA, were also increased with flaxseed EPA+DHA (Table 4). Plasma HDL-cholesterol concentra- oil supplementation, whilst DHA content remained tions were unaltered by fish oil consumption (Table 4). unchanged. Mean plasma triglyceride concentrations were significantly Blood lipid n-3 FAs have been extensively utilised as (P < 0.01) lowered by consuming 1.50, 1.65, 3.00 and biomarkers of n-3 intakes as they are predominantly derived 836 L. Hodson et al. A C Fig. 1 Effect of fish oil supplementation on a the molecular percent (mol %) and b 300 concentration (µmol/L) of EPA in plasma phosphatidylcholine (PC). Values are unadjusted means (95% CI) of the change, from days 0 to 14, in the mol % and µmol/L of EPA in plasma 1 PC and are shown by dose of 0 0 EPA intake and by year in which 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Eicosapentaenoic acid intake (g per day) the study was conducted. The Eicosapentaenoic acid intake (g per day) point estimates occur at the particular doses used in each year. Values are predicted means (95% CI) calculated by regressing the dose of EPA on B D Dose response: Dose response: the change, from days 0 to 14, in 76 μmol/L per g intake of EPA 1.5 mol% per g intake of EPA the mol % and µmol/L of EPA in plasma PC with adjustment for year of study and baseline EPA composition of plasma PC. The 150 point estimates shown on the graph were calculated for each dose of EPA intake (g/d) on the c mol % and d µmol/L 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Eicosapentaenoic acid intake (g per day) Eicosapentaenoic acid intake (g per day) A C Fig. 2 Effect of fish oil 7.0 350 supplementation on a the 6.0 molecular percent (mol %) and b concentration (µmol/L) of DHA 5.0 250 in plasma phosphatidylcholine 4.0 (PC). Values are unadjusted 3.0 150 means (95% CI) of the change, 2.0 from days 0 to 14, in the mol % 1.0 50 and µmol/L of DHA in plasma 0.0 PC and are shown by dose of 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 DHA intake and by year in Docosahexaenoic acid intake (g per day) Docosahexaenoic acid intake (g per day) which the study was conducted. The point estimates occur at the particular doses used in each year. Values are predicted means (95%CI) calculated by B D regressing the dose of DHA on 3.5 Dose response: Dose response: the change, from days 0 to 14, in 1.1 mol% per g intake of DHA 61 μmol/L per g intake of DHA 3.0 the mol % and µmol/L of DHA 2.5 in plasma PC with adjustment for year of study and baseline 2.0 DHA composition of plasma 1.5 PC. The point estimates shown 1.0 on the graph were calculated for 0.5 each dose of DHA intake (g/d) 0.0 on the c mol % and d µmol/L 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 Docosahexaenoic acid intake (g per day) Docosahexaenoic acid intake (g per day) from dietary intake [1, 20]. This relationship was reported Patterson et al. [3] undertook a dose–response study and by Lands et al. [21] who described an empirical association systematic review to investigate the relationship between between the maintenance of plasma phospholipid LCPUFA diet and blood n-3 FA. They found the abundance of whole and the dietary intakes of n-6 and n-3 FAs. More recently, blood, erythrocyte and plasma phospholipid EPA+DHA to Day 0 to day 14 difference in Docosahexaenoic acid (mol%) Day 0 to day 14 difference in Eicosapentaenoic acid (mol%) docosahexaenoic acid (mol%) eicosapentaenoic acid (mol%) Day 0 to day 14 difference in Docosahexaenoic acid (μmol/L) Day 0 to day 14 difference in Eicosapentaenoic acid (μmol/L) docosahexaenoic acid (μmol/L) eicosapentaenoic acid (μmol/L) Effect of supplementation with flaxseed oil and different doses of fish oil for 2 weeks on plasma. . . 837 Table 3 N-3 polyunsaturated Mol% µmol/L fatty acid composition of plasma a a b a a b phosphatidylcholine before and Fatty acid Day 0 Day 14 Difference Day 0 Day 14 Difference after 14 days of consuming c c flaxseed oil (10 mL/day) 18:3n-3 0.4 (0.2) 1.1 (0.5) 0.7 (0.6, 0.9) 14 (7) 46 (31) 32 (23, 42) c c 20:5n-3 1.0 (0.4) 2.0 (1.3) 1.0 (0.7, 1.4) 39 (19) 89 (82) 49 (24, 75) c d 22:5n-3 0.8 (0.2) 1.0 (0.3) 0.2 (0.1, 0.3) 30 (13) 42 (33) 12 (2, 23) 22:6n-3 3.3 (1.0) 3.4 (1.4) 0.1 (−0.4, 0.5) 132 (51) 148 (122) 16 (−24, 56) Values are mean (SD), n = 69 Values are mean (99% CI), n = 69 Day 14 significantly different from day 0, paired t-test P < 0.001 Day 14 significantly different from day 0, paired t-test P < 0.01 increase in a linear manner with dietary intakes up to 1 g/d unexpected, not least as supplementation was short-term EPA+DHA. In the present study, we found plasma PC EPA (14 days) and participants were young, healthy females with and DHA to increase in a linear manner with intakes up to relatively low baseline plasma triglyceride concentrations. 4.5 g/d EPA+DHA. As the participants in the present study In contrast, Flock et al. [28] reported no change in plasma consumed fish oil supplements for 14 days, the gen- triglyceride concentrations in healthy, young individuals eralisability of our estimates of the dose–response relation consuming up to 1.80 g of EPA plus DHA/day for between intake of n-3 LCPUFA and change in abundance 5 months. The discrepancy in findings between studies may of plasma PC EPA or DHA depend on whether this period be partly due to the higher doses of EPA plus DHA in the is sufficient for maximum changes in FA composition to present study. Supplementation with ALA did not decrease occur. Browning et al. [2] estimated the time to maximal plasma triglyceride concentrations, consistent with some [4] change in plasma PC EPA content ranged from 5 to 18 days but not all [7] previous reports. depending on the dose, with the majority of change Evidence for changes in plasma total, LDL- and HDL- occurring in the first few days of supplementation. Thus, cholesterol concentrations after n-3 LCPUFA supple- our estimate of the dose–response for EPA is unlikely to be mentation are less consistent, although it appears increases underestimated to a significant degree. In contrast, Brown- in LDL-cholesterol are more marked in individuals with ing et al. [2] found the time to peak changes in plasma PC hyper-triglyceridaemia [13, 14]. We found a small but DHA composition were longer, ranging between 12 and significant increase in plasma total cholesterol, which can 32 days, which is in-line with others [22]. Thus, it is most be explained by an increase in LDL-cholesterol after n-3 likely our estimate for dose–response for DHA under- LCPUFA supplementation, with no effect of ALA supple- estimates maximum incorporation. mentation as previously reported [14, 29]. A proposed Our findings of significant increases in plasma PC ALA, mechanism for the increase in LDL-cholesterol concentra- EPA and DPA, with no change in DHA after 14 days tions is via an increased conversion rate of VLDL to LDL supplementation with flaxseed oil are in line with previous [29]. work [4–10]. Although, Hennebelle et al. [11] reported Our study has some limitations. We used MaxEPA for ALA supplementation for 4 weeks increased total plasma two and salmon oil for three of the trials; however, as there EPA and DHA abundance in older (73y) but not younger were only small compositional differences between the oils, (25y) adults. The synthesis of ALA to EPA, DPA and DHA we combined the dose of fish oil but used actual intake of occurs primarily in liver endoplasmic reticulum where there supplemental EPA and DHA in the statistical calculations of is competition between n-6 and n-3 FA for the same elon- the dose-response relationships. Our study participants were gase and desaturase enzymes [23]. The conversion of ALA young, healthy females who were undergraduate students may be downregulated by increased availability of con- studying nutrition. We did not assess participants’ back- version products; consumption of a fish—compared to a ground diet or lifestyle habits at baseline or over the course beef-based diet decreased conversion of DPA to DHA [24]. of the study. Although they were instructed to make no Additional factors that may influence the synthesis of DHA changes other than taking the oil supplements, it is possible from ALA include smoking tobacco [25], alcohol con- that alterations to diet and lifestyle occurred during the sumption [26] and hormonal status [27]. course of the study. Nor did we control for hormone status/ Numerous studies have demonstrated the hypo- phase of menstrual cycle or use of oral contraceptives. As triglyceridaemic effect of n-3 LCPUFA (from fish or sup- participants undertook a component of the analytical work, plements) [13, 14]. The significant decrease in plasma tri- on their own samples, and although clear instructions were glyceride concentrations in the present study was provided and they were supervised, the variation between 838 L. Hodson et al. Table 4 Plasma lipid concentrations before and after 14 days of consuming the oil supplement Intake n Total cholesterol (mmol/L) LDL cholesterol (mmol/L) HDL cholesterol (mmol/L) Triacylglycerol (mmol/L) (g/d) a b a b a b a b EPA DHA EPA Day 0 Day Difference Day 0 Day Difference Day 0 Day Difference Day 0 Day Difference a a a a +DHA 14 14 14 14 0.19 0.14 0.33 15 4.29 (0.52) 4.40 0.11 (−0.11, 2.50 (0.51) 2.59 0.10 (−0.13, 1.40 (0.20) 1.37 −0.02 0.86 (0.17) 0.95 0.08 (−0.06, (0.61) 0.33) (0.59) 0.32) (0.24) (−0.14, 0.10) (0.29) 0.23) 0.38 0.28 0.66 12 5.15 (0.90) 5.35 0.20 (−0.41, 2.74 (0.74) 3.01 0.27 (−0.37, 1.74 (0.36) 1.77 0.03 (−0.13, 1.48 (0.43) 1.25 −0.23 (−0.80, (1.09) 0.80) (1.01) 0.91) (0.33) 0.19) (0.51) 0.35) 0.44 0.28 0.72 19 4.20 (0.66) 4.31 0.11 (−0.12, 2.56 (0.54) 2.72 0.16 (−0.09, 1.47 (0.33) 1.42 −0.05 1.07 (0.34) 1.05 −0.01 (−0.17, (0.75) 0.34) (0.59) 0.41) (0.32) (−0.19, 0.10) (0.32) 0.14) 0.55 0.42 0.99 12 4.15 (0.77) 4.41 0.26 (−0.11, 2.27 (0.67) 2.58 0.31 (−0.06, 1.42 (0.26) 1.47 0.04 (−0.16, 1.02 (0.30) 0.80 −0.22 (−0.56, (0.93) 0.63) (0.81) 0.69) (0.27) 0.25) (0.20) 0.13) 0.76 0.56 1.32 12 4.25 (0.39) 4.51 0.26 (−0.26, 2.34 (0.39) 2.64 0.30 (−0.15, 1.48 (0.35) 1.46 −0.02 0.96 (0.20) 0.91 −0.05 (−0.42, (0.67) 0.77) (0.63) 0.76) (0.35) (−0.25, 0.21) (0.36) 0.32) 0.88 0.56 1.44 13 4.64 (0.85) 4.55 −0.09 2.74 (0.91) 2.67 −0.07 1.73 (0.38) 1.74 0.00 (−0.14, 1.06 (0.28) 0.92 −0.14 (−0.32, (0.92) (−0.51, 0.33) (0.86) (−0.48, 0.35) (0.44) 0.15) (0.27) 0.03) 0.95 0.55 1.50 47 4.50 (0.92) 4.61 0.11 (−0.08, 2.36 (0.61) 2.59 0.23 (0.04, 1.51 (0.35) 1.58 0.06 (−0.03, 1.20 (0.51) 1.00 −0.20 (−0.33, (0.84) 0.30) (0.63) 0.42) (0.35) 0.16) (0.44) −0.07) 0.95 0.70 1.65 7 3.81 (0.73) 4.09 0.28 (−0.20, 2.14 (0.58) 2.44 0.30 (−0.16, 1.19 (0.37) 1.31 0.12 (−0.06, 1.04 (0.40) 0.75 −0.29 (−0.43, (0.90) 0.77) (0.82) 0.75) (0.32) 0.29) (0.41) −0.16) 1.32 0.84 2.16 10 4.64 (0.80) 4.39 −0.25 3.08 (0.68) 2.85 −0.23 1.35 (0.43) 1.39 0.03 (−0.14, 1.27 (0.39) 0.93 −0.34 (−0.71, (0.80) (−0.74, 0.24) (0.68) (−0.73, 0.27) (0.39) 0.21) (0.30) 0.03) 1.90 1.10 3.00 48 4.67 (0.90) 4.73 0.06 (−0.12, 2.62 (0.81) 2.81 0.19 (−0.03, 1.51 (0.34) 1.54 0.03 (−0.07, 1.15 (0.33) 0.91 −0.24 (−0.38, (0.87) 0.24) (0.83) 0.41) (0.38) 0.13) (0.37) −0.10) 1.90 1.40 3.30 8 4.07 (0.48) 4.45 0.38 (−0.29, 2.12 (0.29) 2.50 0.37 (−0.07, 1.49 (0.42) 1.60 0.12 (−0.12, 0.99 (0.28) 0.75 −0.25 (−0.49, (0.57) 1.04) (0.37) 0.82) (0.48) 0.35) (0.24) −0.01) 2.20 1.40 3.60 12 4.29 (0.39) 4.34 0.05 (−0.32, 2.46 (0.47) 2.59 0.13 (−0.23, 1.65 (0.39) 1.61 −0.04 1.07 (0.25) 0.83 −0.23 (−0.37, (0.52) 0.42) (0.50) 0.48) (0.35) (−0.24, 0.17) (0.23) −0.10) 2.85 1.65 4.50 10 4.39 (0.78) 4.82 0.43 (−0.04, 1.38 (0.50) 1.27 −0.11 (−0.62, (0.92) 0.91) (0.53) 0.40) All doses of fish oil 225 4.46 (0.81) 4.58 0.12 (0.04, 2.51 (0.67) 2.68 0.17 (0.09, 1.51 (0.36) 1.53 0.02 (−0.02, 1.13 (0.39) 0.96 −0.17 (−0.24, c c c (0.84) 0.20) (0.71) 0.26) (0.36) 0.06) (0.38) −0.11) Flaxseed oil 10 69 4.44 4.49 0.05 (−0.06, 2.55 2.63 (0.73) 0.08 (−0.02, 1.50 1.50 (0.32) 0.00 (−0.04, 1.03 0.96 (0.35) −0.07 (−0.14, (0.73) (0.88) 0.16) (0.68) 0.17) (0.30) 0.04) (0.36) −0.01) Mean difference (99% CI) between days 14 and 0 for all doses of fish oil Paired t-test day 14 compared with day 0 Day 14 significantly different from day 0, paired t-test P < 0.01 Results for combined group of all participants consuming fish oil Effect of supplementation with flaxseed oil and different doses of fish oil for 2 weeks on plasma. . . 839 participants and across years is likely to be higher than if the References analytical work had been undertaken by more experienced 1. Hodson L, Skeaff CM, Fielding BA. Fatty acid composition of technicians. Finally, we only studied young, healthy adipose tissue and blood in humans and its use as a biomarker of females so we can only speculate that the dose–response dietary intake. Prog Lipid Res. 2008;47:348–80. incorporation of EPA and DHA into plasma PC would be 2. Browning LM, Walker CG, Mander AP, West AL, Madden J, similar in young, healthy men. This seems likely, given the Gambell JM, et al. Incorporation of eicosapentaenoic and doc- osahexaenoic acids into lipid pools when given as supplements results of a population survey reporting men and women providing doses equivalent to typical intakes of oily fish. Am J (25–44 years) had similar-3 LCPUFA status [30]. Clin Nutr. 2012;96:748–58. Taken together, our data describe, the dose-response 3. Patterson AC, Chalil A, Aristizabal Henao JJ, Streit IT, Stark KD. relationship between EPA and DHA intake and plasma PC Omega-3 polyunsaturated fatty acid blood biomarkers increase linearly in men and women after tightly controlled intakes of 0.25, EPA and DHA mol% and µmol/L after 14 days of sup- 0.5, and 1 g/d of EPA+DHA. Nutr Res. 2015;35:1040–51. plementation, in young healthy women. We found that per g 4. Barcelo-Coblijn G, Murphy EJ, Othman R, Moghadasian MH, intake per day increase in EPA results in a 1.5 mol% or 76 Kashour T, Friel JK. Flaxseed oil and fish-oil capsule consump- µmol/L increase in plasma PC EPA abundance whilst per g tion alters human red blood cell n-3 fatty acid composition: a multiple-dosing trial comparing 2 sources of n-3 fatty acid. Am J intake per day increase in DHA results in an increase of Clin Nutr. 2008;88:801–9. 1.1 mol% or 61 µmol/L in plasma PC DHA abundance. 5. Goyens PL, Spilker ME, Zock PL, Katan MB, Mensink RP. These data highlight that even modest changes in dietary fat Conversion of alpha-linolenic acid in humans is influenced by the intake are reflected rapidly by plasma PC n-3 FA. In long- 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. term studies where n-3 FA biomarkers are only measured at 6. Mantzioris E, James MJ, Gibson RA, Cleland LG. Dietary sub- the study beginning and end to determine compliance, it stitution with an alpha-linolenic acid-rich vegetable oil increases may prove difficult to distinguish true compliers from non- eicosapentaenoic acid concentrations in tissues. Am J Clin Nutr. compliers if measuring plasma lipid pools [15, 31, 32]; 1994;59:1304–9. 7. Schwab US, Callaway JC, Erkkila AT, Gynther J, Uusitupa MI, however, the proportion of erythrocyte DHA has been Jarvinen T. Effects of hempseed and flaxseed oils on the profile of reported to characterise adherence to EPA and DHA intakes serum lipids, serum total and lipoprotein lipid concentrations and in long-term interventions [31]. Finally, our data highlight haemostatic factors. Eur J Nutr. 2006;45:470–7. that short-term supplementation with ALA is reflected 8. Wallace FA, Miles EA, Calder PC. Comparison of the effects of linseed oil and different doses of fish oil on mononuclear cell rapidly by plasma PC and has only a modest, if any effect function in healthy human subjects. Br J Nutr. 2003;89:679–89. on n-3 LCPUFA status in this cohort. 9. Wilkinson P, Leach C, Ah-Sing EE, Hussain N, Miller GJ, Millward DJ, et al. Influence of alpha-linolenic acid and fish-oil on Acknowledgements We thank Margaret Waldron and Maggie Oakley, markers of cardiovascular risk in subjects with an atherogenic Research Nurses who assisted with the blood collection; Ashley lipoprotein phenotype. Atherosclerosis. 2005;181:115–24. Duncan, and Michelle Harper, Laboratory Technicians, who advised 10. Zhao G, Etherton TD, Martin KR, West SG, Gillies PJ, Kris- and assisted with the laboratory analyses; and the participants, without Etherton PM. Dietary alpha-linolenic acid reduces inflammatory whom this study would not have been possible. and lipid cardiovascular risk factors in hypercholesterolemic men and women. J Nutr. 2004;134:2991–7. Funding The University of Otago funded the study. LH is British 11. Hennebelle M, Courchesne-Loyer A, St-Pierre V, Vandenberghe Heart Foundation Senior Research Fellow in Basic Science. C, Castellano CA, Fortier M, et al. Preliminary evaluation of a differential effect of an alpha-linolenate-rich supplement on ketogenesis and plasma omega-3 fatty acids in young and older Compliance with ethical standards adults. Nutrition. 2016;32:1211–6. 12. Rizos EC, Ntzani EE, Bika E, Kostapanos MS, Elisaf MS. Conflict of interest The authors declare that they have no conflict of Association between omega-3 fatty acid supplementation and risk interest. of major cardiovascular disease events: a systematic review and meta-analysis. JAMA. 2012;308:1024–33. Open Access This article is licensed under a Creative Commons 13. Harris WS. n-3 fatty’ acids and serum lipoproteins: human studies. Attribution 4.0 International License, which permits use, sharing, Am J Clin Nutr. 1997;65:1645S–1654S. adaptation, distribution and reproduction in any medium or format, as 14. Balk EM, Lichtenstein AH, Chung M, Kupelnick B, Chew P, Lau long as you give appropriate credit to the original author(s) and the J. Effects of omega-3 fatty acids on serum markers of cardio- source, provide a link to the Creative Commons license, and indicate if vascular disease risk: a systematic review. Atherosclerosis. changes were made. The images or other third party material in this 2006;189:19–30. article are included in the article’s Creative Commons license, unless 15. Hodson L, Eyles HC, McLachlan KJ, Bell ML, Green TJ, Skeaff indicated otherwise in a credit line to the material. If material is not CM. Plasma and erythrocyte fatty acids reflect intakes of saturated included in the article’s Creative Commons license and your intended and n-6 PUFA within a similar time frame. J Nutr. use is not permitted by statutory regulation or exceeds the permitted 2014;144:33–41. use, you will need to obtain permission directly from the copyright 16. Hodson L, Skeaff CM, Chisholm WA. The effect of replacing holder. To view a copy of this license, visit http://creativecommons. dietary saturated fat with polyunsaturated or monounsaturated fat org/licenses/by/4.0/. on plasma lipids in free-living young adults. Eur J Clin Nutr. 2001;55:908–15. 840 L. Hodson et al. 17. Bligh DG, Dyer WJ. A rapid method of total lipid extraction and 26. Pawlosky RJ, Hibbeln JR, Herion D, Kleiner DE, Salem N Jr. purification. Can J Biochem Physiol. 1959;37:911–7. Compartmental analysis of plasma and liver n-3 essential fatty 18. Hodson L, Skeaff CM, Wallace AJ, Arribas GL. Stability of acids in alcohol-dependent men during withdrawal. J Lipid Res. plasma and erythrocyte fatty acid composition during cold sto- 2009;50:154–61. rage. Clin Chim Acta. 2002;321:63–7. 27. Giltay EJ, Gooren LJ, Toorians AW, Katan MB, Zock PL. Doc- 19. Holub BJ, Skeaff CM. Nutritional regulation of cellular phos- osahexaenoic acid concentrations are higher in women than in phatidylinositol. Methods Enzymol. 1987;141:234–44. men because of estrogenic effects. Am J Clin Nutr. 20. Serra-Majem L, Nissensohn M, Overby NC, Fekete K. Dietary 2004;80:1167–74. methods and biomarkers of omega 3 fatty acids: a systematic 28. Flock MR, Skulas-Ray AC, Harris WS, Etherton TD, Fleming JA, review. Br J Nutr. 2012;107(Suppl 2):S64–76. Kris-Etherton PM. Determinants of erythrocyte omega-3 fatty acid 21. Lands WE, Libelt B, Morris A, Kramer NC, Prewitt TE, Bowen P, content in response to fish oil supplementation: a dose-response et al. Maintenance of lower proportions of (n - 6) eicosanoid randomized controlled trial. J Am Heart Assoc. 2013;2:e000513. precursors in phospholipids of human plasma in response to added 29. Barcelo-Coblijn G, Murphy EJ. Alpha-linolenic acid and its dietary (n - 3) fatty acids. Biochim Biophys Acta. conversion to longer chain n-3 fatty acids: benefits for human 1992;1180:147–62. health and a role in maintaining tissue n-3 fatty acid levels. Prog 22. Katan MB, Deslypere JP, van Birgelen AP, Penders M, Zegwaard Lipid Res. 2009;48:355–74. M. Kinetics of the incorporation of dietary fatty acids into serum 30. Crowe FL, Skeaff CM, Green TJ, Gray AR. Serum n-3 long-chain cholesteryl esters, erythrocyte membranes, and adipose tissue: an PUFA differ by sex and age in a population-based survey of New 18-month controlled study. J Lipid Res. 1997;38:2012–22. Zealand adolescents and adults. Br J Nutr. 2008;99:168–74. 23. Arterburn LM, Hall EB, Oken H. Distribution, interconversion, 31. Patterson AC, Metherel AH, Hanning RM, Stark KD. The per- and dose response of n-3 fatty acids in humans. Am J Clin Nutr. centage of DHA in erythrocytes can detect non-adherence to 2006;83:1467S–1476S. advice to increase EPA and DHA intakes. Br J Nutr. 24. Pawlosky R, Hibbeln J, Lin Y, Salem N Jr. n-3 fatty acid meta- 2014;111:270–8. bolism in women. Br J Nutr. 2003;90:993–4. discussion 994-5 32. Skeaff CM, Hodson L, McKenzie JE. Dietary-induced changes in 25. Pawlosky RJ, Hibbeln JR, Salem N Jr. Compartmental analyses of fatty acid composition of human plasma, platelet, and erythrocyte plasma n-3 essential fatty acids among male and female smokers lipids follow a similar time course. J Nutr. 2006;136:565–9. and nonsmokers. J Lipid Res. 2007;48:935–43. http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png European Journal of Clinical Nutrition Springer Journals

Effect of supplementation with flaxseed oil and different doses of fish oil for 2 weeks on plasma phosphatidylcholine fatty acids in young women

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Nature Publishing Group UK
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Copyright © 2018 by Macmillan Publishers Limited, part of Springer Nature
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Medicine & Public Health; Medicine/Public Health, general; Public Health; Epidemiology; Internal Medicine; Clinical Nutrition; Metabolic Diseases
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0954-3007
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1476-5640
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10.1038/s41430-018-0174-2
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Abstract

Background/objectives Although assumed, it remains unclear that fatty acid (FA) biomarkers of n-3 long-chain PUFA reflect wide ranges of intake. However, to be utilised as biomarkers, to predict dietary intake, dose–response curves that cover a spectrum of intakes are required. The aim of the study was to investigate whether the FA composition of plasma phosphatidylcholine (PC) is a sensitive biomarker of n-3 FAs from fish oil, across a range of supplementation doses, and alpha-linolenic acid (ALA) supplementation, in young, healthy women. Subjects/methods A total of 303 young women were randomised to intakes ranging between 0.33 and 4.50 g EPA+DHA/ day from fish oil (not all doses used in each year) or flaxseed oil (5.90–6.60 g/d) daily for 14 days in a series of trials, over 5 years. Fasting blood was collected at baseline (day 0) and day 14 and plasma PC FA composition, total and HDL-cholesterol and triglyceride concentrations measured. Results Fourteen days supplementation with fish oil significantly (P < 0.01) increased, in a dose-dependent fashion, plasma PC EPA, DPA and DHA at all doses except 1 and 3 mL/day. For the combined group of women who consumed any fish oil there was a 16% (P < 0.01) decrease in plasma triacylglycerol concentrations after 14 days supplementation. Flaxseed oil supplementation for 14 day resulted in significant (P < 0.01) increases in ALA, EPA and DPA, whilst DHA remained unchanged. Conclusion Our data demonstrate plasma PC is a sensitive biomarker of n-3 FA intake and reflects changes within 14 days across a range of intakes. Introduction 3 LCPUFA intake in a dose–response manner [1–3]. However, for FA to be utilised as biomarkers to predict Plasma, erythrocyte and platelet phospholipids are the blood dietary intake, dose–response curves that cover a spectrum lipid fractions most abundant in n-3 long-chain poly- of FA intake are required and although assumed, it remains unsaturated fatty acids (LCPUFA). Dose–response studies, unclear that FA biomarkers of n-3 LCPUFA reflect a wide typically with only three ‘distinct’ levels of n-3 fatty acid range of intakes. Another important source of n-3 LCPUFA (FA), have shown blood phospholipids reflect changes in n- could come from the precursor alpha-linolenic acid (ALA). Longer-term studies have found the abundance of ALA, EPA and docosapentaenoic acid (DPA) increased in FA biomarkers with an increase in ALA intake with the data for * Leanne Hodson DHA being less clear [4–11]. leanne.hodson@ocdem.ox.ac.uk Although evidence from randomised controlled trials has not proven n-3 LCPUFA lowers cardiovascular disease Department of Human Nutrition, University of Otago, (CVD) risk [12], intervention studies have clearly demon- Dunedin, New Zealand strated n-3 LCPUFA (as fish oil or ethyl esters of EPA and Present address: Oxford Centre for Diabetes, Endocrinology and DHA) have a triglyceride-lowering effect [13]. A large Metabolism (OCDEM), University of Oxford, Churchill Hospital, Oxford OX3 7LE, UK proportion of these studies have been undertaken in middle- aged adults [13, 14]; it remains unclear whether n-3 Present address: Institute of Applied Health Research, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK LCPUFA have a similar effect in young adults. 1234567890();,: 1234567890();,: Effect of supplementation with flaxseed oil and different doses of fish oil for 2 weeks on plasma. . . 833 We sought to investigate the effect of short-term Biochemical and lipid analysis (14 days) supplementation with EPA and DHA (given as fish oil), across a range of doses, on plasma phosphati- On days 0 and 14 venous blood samples were collected dylcholine (PC) n-3 FA levels and plasma lipid concentra- from participants after an overnight fast and plasma isolated tions, along with the effect of short-term (14 days) ALA and stored [15]. Plasma total, high-density lipoprotein supplementation on plasma PC n-3 FA status in young, (HDL) cholesterol and triglyceride concentrations were healthy women. measured and plasma low-density lipoprotein (LDL) cho- lesterol concentrations calculated [16]. The analytical coefficient of variation for the measurement of total cho- Subjects and methods lesterol was less than 3% for plasma total cholesterol, HDL- cholesterol and triglyceride. Participants Plasma lipids were extracted, after the addition of a known amount of an internal standard (diheptadecanoyl Participants were recruited from an undergraduate nutri- [17:0] PC), according to the method of Bligh and Dyer [17]. tion course at the University of Otago and were eligible if Plasma PC was separated using thin-layer chromatography they were 18 years or older and were not allergic to fish or as we have previously described for erythrocyte PC [18] and nuts. Ethical approval was given for the study from The PC FAs converted to FA methyl esters (FAMEs). Separation Human Ethics Committee, University of Otago. All par- and quantitation of the plasma PC FAMEs was achieved ticipants gave informed written consent after receiving using a DB-225 megabore column (25 m × 0.53 mm internal both a verbal and written explanation of the study. The diameter; film thickness 0.25 µm; J & W Scientific) installed experiment was part of an undergraduate teaching and on an HP-6890 Series Gas Chromatograph (GC) with flame learning activity that was conducted annually for five ionisation detection [15, 19]. Students performed the lipid years from 2003 to 2007. extraction and thin-layer chromatography, under super- vision, whereas a qualified research technician performed Study design and supplementation the GC analysis of samples. Plasma FA were recorded as molecular percentages (mol%), defined as the number of Participants were randomised to receive fish oil or flaxseed molecules of the individual FA as a percentage of the total oil in capsule form and consumed the allocated daily dose, number of FA molecules. The concentrations (µmol/L) of with food for 14 days. Participants were asked to maintain FAs were calculated based on the area of the internal stan- their usual diet and physical activity; if they consumed fish dard (diheptadecanoyl [17:0] PC) peak. they were instructed to continue their usual pattern of consumption. MaxEPA (Seven Seas Health Company UK) Statistical analysis fish oil capsules were used in 2003 and 2004 and Omega-3 Salmon Oil capsules (Thompson’s, Auckland, New Zeal- Data were analysed using the statistical package STATA and) from 2005 to 2007; flaxseed oil was from Waihi Bush (version 11). Statistical differences in the plasma lipids and (Geraldine, New Zealand). Manufacturer’s information FA composition of PC between day 0 and day 14 were indicated that each capsule of MaxEPA contained 190 mg determined using a paired t-test. All comparisons were two- of EPA and 110 mg of DHA, each capsule of Omega-3 sided and changes were considered statistically significant Salmon Oil contained 180 mg of EPA and 120 mg of DHA, when the P-value was less than 0.01; this value was chosen and each capsule of flaxseed oil contained 600 mg of ALA. to reduce the chances of concluding erroneously that a The doses of fish oil used in the trials were 1, 2, 3, 4, 5, 6, difference existed between days 14 and 0. 10 and 15 capsules/day, not all doses were used in each year As subjects consumed EPA and DHA (given as fish oil), (due to participant numbers), whereas the dose of flaxseed across a range of doses (Supplementary Table 1) over a 14- oil was always 10 capsules/day. Intakes (g/d) of EPA and day period, this provided the opportunity to determine if DHA and ALA were estimated on the basis of manu- there was a dose–response effect. The change from day 0 to facturers’ information about the FA content of the fish oil 14 in plasma PC n-3 FA or plasma lipid concentration was and flaxseed oil and the number of capsules assigned to be calculated for each participant and these values used in the consumed (Supplementary Table 1). Compliance was regression analysis to test for a dose–response effect. The assessed in all years by change in FA composition of dose–response relation between the increase in EPA intake plasma PC and in 2005, 2006 and 2007 (but not 2003 and and change in EPA composition of plasma PC was esti- 2004) compliance was also assessed by a daily diary of mated using regression analysis, adjusting for year of the capsule consumption. study and baseline (i.e., day 0) FA composition. The dose–response relation was expressed as the incremental 834 L. Hodson et al. Table 1 Long-chain n-3 polyunsaturated fatty acid composition (mol%) of plasma phosphatidylcholine at before and after 14 days of consuming the oil supplement Intake (g/d) n Eicosapentaenoic acid (µmol/L) Docosapentaenoic acid (µmol/L) Docosahexaenoic acid (µmol/L) a a b a a b a a b EPA DHA EPA+DHA Day 0 Day 14 Difference Day 0 Day 14 Difference Day 0 Day 14 Difference 0.19 0.14 0.33 15 1.0 (0.5) 1.3 (0.3) 0.4 (−0.1, 0.8) 0.7 (0.2) 0.8 (0.2) 0.1 (−0.1, 0.3) 3.0 (1.0) 3.3 (0.9) 0.3 (−0.6, 1.2) c c c 0.38 0.28 0.66 12 0.8 (0.2) 2.2 (0.7) 1.5 (0.7, 2.2) 0.6 (0.2) 0.9 (0.3) 0.3 (0.2, 0.5) 2.7 (0.8) 3.7 (0.8) 1.0 (0.5, 1.5) c c 0.44 0.28 0.72 19 1.0 (0.4) 1.6 (0.6) 0.8 (0.1, 1.5) 0.7 (0.2) 0.9 (0.3) 0.2 (0.0, 0.4) 3.6 (1.2) 3.6 (1.0) 0.0 (−0.8, 0.9) c c c 0.55 0.42 0.99 12 1.2 (1.0) 3.1 (1.1) 1.9 (1.1, 2.6) 0.7 (0.3) 1.0 (0.4) 0.3 (0.1, 0.5) 3.1 (1.4) 4.3 (1.3) 1.2 (0.2, 2.2) c c c 0.76 0.56 1.32 12 0.8 (0.2) 3.5 (0.7) 2.6 (2.1, 3.2) 0.7 (0.2) 1.1 (0.2) 0.4 (0.3, 0.6) 3.1 (0.8) 4.5 (0.9) 1.4 (0.8, 2.0) c c 0.88 0.56 1.44 13 1.0 (0.3) 2.5 (1.0) 1.6 (0.8, 2.4) 0.7 (0.2) 1.1 (0.3) 0.3 (0.1, 0.5) 3.4 (0.9) 4.6 (1.1) 1.2 (−0.2, 2.6) c c c 0.95 0.55 1.50 47 1.0 (0.5) 3.7 (1.3) 2.7 (2.3, 3.2) 0.8 (0.3) 1.2 (0.3) 0.4 (0.3, 0.5) 3.4 (1.1) 4.6 (1.2) 1.3 (0.9, 1.7) c c c 0.95 0.70 1.65 7 1.3 (1.0) 4.3 (0.7) 3.0 (1.2, 4.7) 0.7 (0.2) 1.1 (0.3) 0.3 (0.0, 0.7) 3.4 (0.7) 4.7 (1.1) 1.3 (0.0, 2.6) c c 1.32 0.84 2.16 10 1.0 (0.6) 3.1 (1.5) 2.2 (0.5, 3.8) 0.8 (0.2) 1.1 (0.3) 0.3 (0.1, 0.6) 3.3 (0.7) 4.4 (1.4) 1.1 (−0.7, 2.9) c c c 1.90 1.10 3.00 48 1.0 (0.5) 5.6 (2.0) 4.6 (3.8, 5.4) 0.8 (0.2) 1.4 (0.3) 0.6 (0.5, 0.7) 3.5 (1.0) 5.2 (1.3) 1.8 (1.3, 2.2) c c 1.90 1.40 3.30 8 1.3 (0.7) 5.8 (2.0) 4.5 (2.0, 7.0) 1.0 (0.2) 1.6 (0.3) 0.6 (0.1, 1.0) 4.1 (1.1) 5.6 (1.5) 1.6 (−0.2, 3.4) c c c 2.20 1.40 3.60 12 1.1 (0.5) 4.5 (2.3) 3.4 (1.3, 5.5) 0.8 (0.3) 1.4 (0.3) 0.6 (0.2, 0.9) 3.3 (0.8) 5.3 (1.2) 1.9 (1.3, 2.6) c c c 2.85 1.65 4.50 10 1.0 (0.4) 7.0 (2.5) 6.1 (3.4, 8.7) 0.7 (0.3) 1.7 (0.4) 1.0 (0.5, 1.5) 3.7 (1.1) 6.4 (1.0) 2.7 (1.4, 4.1) Values are mean (SD) Values are mean (99% CI) Day 14 significantly different from day 0, paired t-test P < 0.01 change (95% CI) in mol% per g/d or µmol/L per g/d from 2005 through 2007 completed a daily diary of capsule increase in intake of EPA. A simple plot of the results consumption; self-reported compliance indicated that 97% suggested a curvilinear relation between increasing intake of the assigned capsules were consumed. of EPA and change in mol% and µmol/L EPA in plasma PC; therefore, a quadratic term was included in the model. Effect of fish oil supplementation on plasma PC n-3 The same procedure was used to explore the dose–response FA relation between DHA intake and DHA composition of plasma PC; however, a quadratic term was dropped from Daily supplementation with different amounts of fish oil the model because it was not statistically significant (P = (EPA+DHA) significantly (P < 0.01) increased plasma PC 0.150). A similar approach was used to calculate the dose EPA mol% and µmol/L at all doses of fish oil intakes except response relation between EPA+DHA intake and plasma the one mL dose (Tables 1 and 2). The mean increase, lipid concentrations (mmol/L), with the dose response unadjusted analysis, in EPA mol% and µmol/L between expressed as the incremental change (95% CI) per g/d days 0 and 14 by dose of EPA is shown in Fig. 1a, c; the increase of EPA+DHA. increase was dose-dependent and did not differ by year of study (P = 0.988, interaction between dose and year). According to this association, the dose response, over Results 14 days, in plasma PC EPA was 1.5 mol% or 76 µmol/L per g EPA (Fig. 1b, d). Notably, daily supplementation with Baseline characteristics fish oil significantly increased the abundance (as mol% and µmol/L) of DPA in plasma PC, in most, but not all doses of Three hundred and three women participated in the sup- fish oil (Tables 1 and 2). plementation trials between 2003 and 2007, of which we Daily supplementation with fish oil also significantly (P have complete data for 294. The participants’ (n = 294) < 0.01) increased plasma PC DPA and DHA mol% at all were aged 22.1 years (4.0) (mean (SD)) with a BMI of doses of fish oil intake except one and three mL (Table 1) 22.6 kg/m (2.9); mean plasma lipid concentrations were but changes occurred at fewer fish oil doses when expressed 4.45 (0.80) mmol/L for plasma total cholesterol, 2.51 (0.68) as a concentration (µmol/L) (Table 2). The mean increase, mmol/L for plasma LDL-cholesterol, 1.51 (0.35) mmol/L unadjusted analysis, in DHA mol% and µmol/L between for plasma HDL cholesterol, and 1.11 (0.40) mmol/L for day 0 and 14 by dose of fish oil intake is shown in Fig. 2a,c; plasma triglycerides. The characteristics of women assigned the increase was linearly dose-dependent and did not differ to the fish or flaxseed oil groups did not differ. The number by year of study (P = 0.524, interaction between dose and of women enrolled each year and randomised to the dif- year). The predicted mean (95%CI) increase, over 14 days, ferent fish oil groups or the flaxseed oil group is shown in in plasma PC DHA mol% and µmol/L across the range of Supplementary Table 1. Ninety-four percent of participants daily supplemental DHA intake is shown in Fig. 2b,d. Effect of supplementation with flaxseed oil and different doses of fish oil for 2 weeks on plasma. . . 835 Table 2 Long-chain n-3 polyunsaturated fatty acid composition (µmol/L) of plasma phosphatidylcholine at before and after 14 days of consuming the oil supplement Intake (g/d) n Eicosapentaenoic acid (µmol/L) Docosapentaenoic acid (µmol/L) Docosahexaenoic acid (µmol/L) a a b c a b a a b EPA DHA EPA+DHA Day 0 Day 14 Difference Day 0 Day 14 Difference Day 0 Day 14 Difference 0.19 0.14 0.33 15 43 (19) 72 (51) 29 (−15, 72) 31 (10) 43 (32) 12 (−11, 34) 144 (92) 180 (167) 37 (−89, 163) c c 0.38 0.28 0.66 12 41 (10) 122 (55) 81 (37, 125) 29 (8) 49 (24) 20 (3, 37) 151 (75) 198 (68) 47 (−2, 96) c c 0.44 0.28 0.72 19 42 (22) 78 (45) 36 (6, 65) 31 (15) 45 (24) 13 (0, 27) 157 (92) 177 (105) 20 (−61, 101) c c c 0.55 0.42 0.99 12 54 (36) 168 (79) 113 (62, 164) 35 (20) 53 (24) 18 (5, 32) 147 (80) 227 (88) 80 (28, 132) c c 0.76 0.56 1.32 1 32 (11) 147 (67) 115 (58, 171) 27 (8) 48 (20) 21 (4, 39) 121 (42) 193 (90) 72 (−3, 148) 0.88 0.56 1.44 13 53 (29) 106 (43) 52 (10, 94) 39 (19) 43 (9) 3 (−13, 19) 201 (131) 186 (30) −15 (−123, 93) c c c 0.95 0.55 1.50 47 39 (23) 136 (60) 97 (75, 119) 29 (14) 42 (19) 13 (7, 19) 126 (57) 168 (75) 42 (17, 66) c c c 0.95 0.70 1.65 7 54 (38) 174 (40) 120 (66, 173) 29 (9) 42 (9) 12 (3, 22) 141 (38) 184 (32) 43 (14, 72) 1.32 0.84 2.16 10 43 (36) 117 (37) 74 (17, 130) 32 (11) 42 (12) 10 (−3, 23) 148 (71) 176 (65) 28 (−73, 129) c c c 1.90 1.10 3.00 48 38 (18) 200 (88) 162 (128, 196) 29 (12) 49 (19) 21 (13, 28) 129 (50) 185 (70) 56 (36, 76) c c 1.90 1.40 3.30 8 56 (32) 284 (124) 228 (88, 367) 42 (8) 74 (29) 32 (2, 62) 175 (60) 274 (114) 98 (−16, 213) c c c 2.20 1.40 3.60 12 45 (23) 229 (145) 183 (61, 305) 32 (12) 72 (32) 40 (14, 66) 133 (44) 282 (135) 149 (48, 250) c c c 2.85 1.65 4.50 10 36 (15) 298 (138) 262 (121, 403) 28 (11) 74 (31) 46 (15, 77) 143 (62) 272 (106) 129 (37, 221) Values are mean (SD) Values are mean (99% CI) Day 14 significantly different from day 0, paired t-test P < 0.01 According to this association, the dose response, over 3.60 g EPA+DHA/day for 14 day; they were also sig- 14 days, in plasma PC DHA was 1.1 mol% or 61 µmol/L nificantly lower (by 15%, P < 0.001 day 14 vs day 0) when per g intake of DHA (Fig. 2b,d). results were combined for all fish oil consumers (Table 4). There was no significant dose-response relation between Effect of flaxseed oil supplementation on plasma PC total n-3 LCPUFA intake (i.e., EPA+DHA) and total n-3 FA cholesterol (P = 0.235), LDL-cholesterol (P = 0.955), or HDL-cholesterol (P = 0.440) concentrations (Table 4)or Daily supplementation with flaxseed oil for 14 days with plasma triglyceride concentrations. Consuming 10 mL increased plasma PC ALA by 0.7 mol% (95% CI, 0.6–0.9; of flaxseed oil, containing 6 g of ALA, daily for 14 days P < 0.001) or 32 µmol/L (95% CI, 14–46, P <0.001) did not significantly alter plasma lipid concentrations (Table 3). The proportions and concentrations of plasma (Table 4). PC FA as EPA and DPA were significantly (P <0.01) increased, with no change in DHA, after 14 days (Table 3). The changes in FA composition with flaxseed oil con- Discussion sumption did not differ by year in which the study was conducted (P > 0.6). As the usefulness of plasma PC as a biomarker of n-3 FA intake has not been extensively examined, we assessed Effect of fish oil and flaxseed oil supplementation changes in plasma PC n-3 FAs before and after supple- on plasma lipids mentation with fish oil (across a range of doses) or sup- plementation with ALA. Our results clearly demonstrate the Daily fish oil supplementation for 14 days did not sig- FA composition of plasma PC to be a sensitive biomarker of nificantly alter plasma total cholesterol concentrations n-3 LCPUFA intake, with small increases in EPA and DHA within any one group of fish oil intake (Table 4); however, intake (from fish oil) being reflected within 14 days, in a when the results for all participants who received fish oil dose-dependent fashion. The increase in EPA and DHA were combined (n = 234), mean plasma total and LDL abundance with increasing dose of fish oil, and the increase cholesterol concentrations were significantly (P < 0.001) in ALA abundance with flaxseed oil supplementation higher at day 14 compared with day 0. For this combined indicate a high degree of compliance over the 14-day per- group, mean intake of fish oil, weighted for the proportion iod. EPA and DPA, two FA formed by the metabolic of participants in each dose group, was 6.1 mL/d, or 1.8 g of interconversion of ALA, were also increased with flaxseed EPA+DHA (Table 4). Plasma HDL-cholesterol concentra- oil supplementation, whilst DHA content remained tions were unaltered by fish oil consumption (Table 4). unchanged. Mean plasma triglyceride concentrations were significantly Blood lipid n-3 FAs have been extensively utilised as (P < 0.01) lowered by consuming 1.50, 1.65, 3.00 and biomarkers of n-3 intakes as they are predominantly derived 836 L. Hodson et al. A C Fig. 1 Effect of fish oil supplementation on a the molecular percent (mol %) and b 300 concentration (µmol/L) of EPA in plasma phosphatidylcholine (PC). Values are unadjusted means (95% CI) of the change, from days 0 to 14, in the mol % and µmol/L of EPA in plasma 1 PC and are shown by dose of 0 0 EPA intake and by year in which 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Eicosapentaenoic acid intake (g per day) the study was conducted. The Eicosapentaenoic acid intake (g per day) point estimates occur at the particular doses used in each year. Values are predicted means (95% CI) calculated by regressing the dose of EPA on B D Dose response: Dose response: the change, from days 0 to 14, in 76 μmol/L per g intake of EPA 1.5 mol% per g intake of EPA the mol % and µmol/L of EPA in plasma PC with adjustment for year of study and baseline EPA composition of plasma PC. The 150 point estimates shown on the graph were calculated for each dose of EPA intake (g/d) on the c mol % and d µmol/L 0.0 0.5 1.0 1.5 2.0 2.5 3.0 0.0 0.5 1.0 1.5 2.0 2.5 3.0 Eicosapentaenoic acid intake (g per day) Eicosapentaenoic acid intake (g per day) A C Fig. 2 Effect of fish oil 7.0 350 supplementation on a the 6.0 molecular percent (mol %) and b concentration (µmol/L) of DHA 5.0 250 in plasma phosphatidylcholine 4.0 (PC). Values are unadjusted 3.0 150 means (95% CI) of the change, 2.0 from days 0 to 14, in the mol % 1.0 50 and µmol/L of DHA in plasma 0.0 PC and are shown by dose of 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 DHA intake and by year in Docosahexaenoic acid intake (g per day) Docosahexaenoic acid intake (g per day) which the study was conducted. The point estimates occur at the particular doses used in each year. Values are predicted means (95%CI) calculated by B D regressing the dose of DHA on 3.5 Dose response: Dose response: the change, from days 0 to 14, in 1.1 mol% per g intake of DHA 61 μmol/L per g intake of DHA 3.0 the mol % and µmol/L of DHA 2.5 in plasma PC with adjustment for year of study and baseline 2.0 DHA composition of plasma 1.5 PC. The point estimates shown 1.0 on the graph were calculated for 0.5 each dose of DHA intake (g/d) 0.0 on the c mol % and d µmol/L 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6 1.8 Docosahexaenoic acid intake (g per day) Docosahexaenoic acid intake (g per day) from dietary intake [1, 20]. This relationship was reported Patterson et al. [3] undertook a dose–response study and by Lands et al. [21] who described an empirical association systematic review to investigate the relationship between between the maintenance of plasma phospholipid LCPUFA diet and blood n-3 FA. They found the abundance of whole and the dietary intakes of n-6 and n-3 FAs. More recently, blood, erythrocyte and plasma phospholipid EPA+DHA to Day 0 to day 14 difference in Docosahexaenoic acid (mol%) Day 0 to day 14 difference in Eicosapentaenoic acid (mol%) docosahexaenoic acid (mol%) eicosapentaenoic acid (mol%) Day 0 to day 14 difference in Docosahexaenoic acid (μmol/L) Day 0 to day 14 difference in Eicosapentaenoic acid (μmol/L) docosahexaenoic acid (μmol/L) eicosapentaenoic acid (μmol/L) Effect of supplementation with flaxseed oil and different doses of fish oil for 2 weeks on plasma. . . 837 Table 3 N-3 polyunsaturated Mol% µmol/L fatty acid composition of plasma a a b a a b phosphatidylcholine before and Fatty acid Day 0 Day 14 Difference Day 0 Day 14 Difference after 14 days of consuming c c flaxseed oil (10 mL/day) 18:3n-3 0.4 (0.2) 1.1 (0.5) 0.7 (0.6, 0.9) 14 (7) 46 (31) 32 (23, 42) c c 20:5n-3 1.0 (0.4) 2.0 (1.3) 1.0 (0.7, 1.4) 39 (19) 89 (82) 49 (24, 75) c d 22:5n-3 0.8 (0.2) 1.0 (0.3) 0.2 (0.1, 0.3) 30 (13) 42 (33) 12 (2, 23) 22:6n-3 3.3 (1.0) 3.4 (1.4) 0.1 (−0.4, 0.5) 132 (51) 148 (122) 16 (−24, 56) Values are mean (SD), n = 69 Values are mean (99% CI), n = 69 Day 14 significantly different from day 0, paired t-test P < 0.001 Day 14 significantly different from day 0, paired t-test P < 0.01 increase in a linear manner with dietary intakes up to 1 g/d unexpected, not least as supplementation was short-term EPA+DHA. In the present study, we found plasma PC EPA (14 days) and participants were young, healthy females with and DHA to increase in a linear manner with intakes up to relatively low baseline plasma triglyceride concentrations. 4.5 g/d EPA+DHA. As the participants in the present study In contrast, Flock et al. [28] reported no change in plasma consumed fish oil supplements for 14 days, the gen- triglyceride concentrations in healthy, young individuals eralisability of our estimates of the dose–response relation consuming up to 1.80 g of EPA plus DHA/day for between intake of n-3 LCPUFA and change in abundance 5 months. The discrepancy in findings between studies may of plasma PC EPA or DHA depend on whether this period be partly due to the higher doses of EPA plus DHA in the is sufficient for maximum changes in FA composition to present study. Supplementation with ALA did not decrease occur. Browning et al. [2] estimated the time to maximal plasma triglyceride concentrations, consistent with some [4] change in plasma PC EPA content ranged from 5 to 18 days but not all [7] previous reports. depending on the dose, with the majority of change Evidence for changes in plasma total, LDL- and HDL- occurring in the first few days of supplementation. Thus, cholesterol concentrations after n-3 LCPUFA supple- our estimate of the dose–response for EPA is unlikely to be mentation are less consistent, although it appears increases underestimated to a significant degree. In contrast, Brown- in LDL-cholesterol are more marked in individuals with ing et al. [2] found the time to peak changes in plasma PC hyper-triglyceridaemia [13, 14]. We found a small but DHA composition were longer, ranging between 12 and significant increase in plasma total cholesterol, which can 32 days, which is in-line with others [22]. Thus, it is most be explained by an increase in LDL-cholesterol after n-3 likely our estimate for dose–response for DHA under- LCPUFA supplementation, with no effect of ALA supple- estimates maximum incorporation. mentation as previously reported [14, 29]. A proposed Our findings of significant increases in plasma PC ALA, mechanism for the increase in LDL-cholesterol concentra- EPA and DPA, with no change in DHA after 14 days tions is via an increased conversion rate of VLDL to LDL supplementation with flaxseed oil are in line with previous [29]. work [4–10]. Although, Hennebelle et al. [11] reported Our study has some limitations. We used MaxEPA for ALA supplementation for 4 weeks increased total plasma two and salmon oil for three of the trials; however, as there EPA and DHA abundance in older (73y) but not younger were only small compositional differences between the oils, (25y) adults. The synthesis of ALA to EPA, DPA and DHA we combined the dose of fish oil but used actual intake of occurs primarily in liver endoplasmic reticulum where there supplemental EPA and DHA in the statistical calculations of is competition between n-6 and n-3 FA for the same elon- the dose-response relationships. Our study participants were gase and desaturase enzymes [23]. The conversion of ALA young, healthy females who were undergraduate students may be downregulated by increased availability of con- studying nutrition. We did not assess participants’ back- version products; consumption of a fish—compared to a ground diet or lifestyle habits at baseline or over the course beef-based diet decreased conversion of DPA to DHA [24]. of the study. Although they were instructed to make no Additional factors that may influence the synthesis of DHA changes other than taking the oil supplements, it is possible from ALA include smoking tobacco [25], alcohol con- that alterations to diet and lifestyle occurred during the sumption [26] and hormonal status [27]. course of the study. Nor did we control for hormone status/ Numerous studies have demonstrated the hypo- phase of menstrual cycle or use of oral contraceptives. As triglyceridaemic effect of n-3 LCPUFA (from fish or sup- participants undertook a component of the analytical work, plements) [13, 14]. The significant decrease in plasma tri- on their own samples, and although clear instructions were glyceride concentrations in the present study was provided and they were supervised, the variation between 838 L. Hodson et al. Table 4 Plasma lipid concentrations before and after 14 days of consuming the oil supplement Intake n Total cholesterol (mmol/L) LDL cholesterol (mmol/L) HDL cholesterol (mmol/L) Triacylglycerol (mmol/L) (g/d) a b a b a b a b EPA DHA EPA Day 0 Day Difference Day 0 Day Difference Day 0 Day Difference Day 0 Day Difference a a a a +DHA 14 14 14 14 0.19 0.14 0.33 15 4.29 (0.52) 4.40 0.11 (−0.11, 2.50 (0.51) 2.59 0.10 (−0.13, 1.40 (0.20) 1.37 −0.02 0.86 (0.17) 0.95 0.08 (−0.06, (0.61) 0.33) (0.59) 0.32) (0.24) (−0.14, 0.10) (0.29) 0.23) 0.38 0.28 0.66 12 5.15 (0.90) 5.35 0.20 (−0.41, 2.74 (0.74) 3.01 0.27 (−0.37, 1.74 (0.36) 1.77 0.03 (−0.13, 1.48 (0.43) 1.25 −0.23 (−0.80, (1.09) 0.80) (1.01) 0.91) (0.33) 0.19) (0.51) 0.35) 0.44 0.28 0.72 19 4.20 (0.66) 4.31 0.11 (−0.12, 2.56 (0.54) 2.72 0.16 (−0.09, 1.47 (0.33) 1.42 −0.05 1.07 (0.34) 1.05 −0.01 (−0.17, (0.75) 0.34) (0.59) 0.41) (0.32) (−0.19, 0.10) (0.32) 0.14) 0.55 0.42 0.99 12 4.15 (0.77) 4.41 0.26 (−0.11, 2.27 (0.67) 2.58 0.31 (−0.06, 1.42 (0.26) 1.47 0.04 (−0.16, 1.02 (0.30) 0.80 −0.22 (−0.56, (0.93) 0.63) (0.81) 0.69) (0.27) 0.25) (0.20) 0.13) 0.76 0.56 1.32 12 4.25 (0.39) 4.51 0.26 (−0.26, 2.34 (0.39) 2.64 0.30 (−0.15, 1.48 (0.35) 1.46 −0.02 0.96 (0.20) 0.91 −0.05 (−0.42, (0.67) 0.77) (0.63) 0.76) (0.35) (−0.25, 0.21) (0.36) 0.32) 0.88 0.56 1.44 13 4.64 (0.85) 4.55 −0.09 2.74 (0.91) 2.67 −0.07 1.73 (0.38) 1.74 0.00 (−0.14, 1.06 (0.28) 0.92 −0.14 (−0.32, (0.92) (−0.51, 0.33) (0.86) (−0.48, 0.35) (0.44) 0.15) (0.27) 0.03) 0.95 0.55 1.50 47 4.50 (0.92) 4.61 0.11 (−0.08, 2.36 (0.61) 2.59 0.23 (0.04, 1.51 (0.35) 1.58 0.06 (−0.03, 1.20 (0.51) 1.00 −0.20 (−0.33, (0.84) 0.30) (0.63) 0.42) (0.35) 0.16) (0.44) −0.07) 0.95 0.70 1.65 7 3.81 (0.73) 4.09 0.28 (−0.20, 2.14 (0.58) 2.44 0.30 (−0.16, 1.19 (0.37) 1.31 0.12 (−0.06, 1.04 (0.40) 0.75 −0.29 (−0.43, (0.90) 0.77) (0.82) 0.75) (0.32) 0.29) (0.41) −0.16) 1.32 0.84 2.16 10 4.64 (0.80) 4.39 −0.25 3.08 (0.68) 2.85 −0.23 1.35 (0.43) 1.39 0.03 (−0.14, 1.27 (0.39) 0.93 −0.34 (−0.71, (0.80) (−0.74, 0.24) (0.68) (−0.73, 0.27) (0.39) 0.21) (0.30) 0.03) 1.90 1.10 3.00 48 4.67 (0.90) 4.73 0.06 (−0.12, 2.62 (0.81) 2.81 0.19 (−0.03, 1.51 (0.34) 1.54 0.03 (−0.07, 1.15 (0.33) 0.91 −0.24 (−0.38, (0.87) 0.24) (0.83) 0.41) (0.38) 0.13) (0.37) −0.10) 1.90 1.40 3.30 8 4.07 (0.48) 4.45 0.38 (−0.29, 2.12 (0.29) 2.50 0.37 (−0.07, 1.49 (0.42) 1.60 0.12 (−0.12, 0.99 (0.28) 0.75 −0.25 (−0.49, (0.57) 1.04) (0.37) 0.82) (0.48) 0.35) (0.24) −0.01) 2.20 1.40 3.60 12 4.29 (0.39) 4.34 0.05 (−0.32, 2.46 (0.47) 2.59 0.13 (−0.23, 1.65 (0.39) 1.61 −0.04 1.07 (0.25) 0.83 −0.23 (−0.37, (0.52) 0.42) (0.50) 0.48) (0.35) (−0.24, 0.17) (0.23) −0.10) 2.85 1.65 4.50 10 4.39 (0.78) 4.82 0.43 (−0.04, 1.38 (0.50) 1.27 −0.11 (−0.62, (0.92) 0.91) (0.53) 0.40) All doses of fish oil 225 4.46 (0.81) 4.58 0.12 (0.04, 2.51 (0.67) 2.68 0.17 (0.09, 1.51 (0.36) 1.53 0.02 (−0.02, 1.13 (0.39) 0.96 −0.17 (−0.24, c c c (0.84) 0.20) (0.71) 0.26) (0.36) 0.06) (0.38) −0.11) Flaxseed oil 10 69 4.44 4.49 0.05 (−0.06, 2.55 2.63 (0.73) 0.08 (−0.02, 1.50 1.50 (0.32) 0.00 (−0.04, 1.03 0.96 (0.35) −0.07 (−0.14, (0.73) (0.88) 0.16) (0.68) 0.17) (0.30) 0.04) (0.36) −0.01) Mean difference (99% CI) between days 14 and 0 for all doses of fish oil Paired t-test day 14 compared with day 0 Day 14 significantly different from day 0, paired t-test P < 0.01 Results for combined group of all participants consuming fish oil Effect of supplementation with flaxseed oil and different doses of fish oil for 2 weeks on plasma. . . 839 participants and across years is likely to be higher than if the References analytical work had been undertaken by more experienced 1. Hodson L, Skeaff CM, Fielding BA. Fatty acid composition of technicians. Finally, we only studied young, healthy adipose tissue and blood in humans and its use as a biomarker of females so we can only speculate that the dose–response dietary intake. Prog Lipid Res. 2008;47:348–80. incorporation of EPA and DHA into plasma PC would be 2. Browning LM, Walker CG, Mander AP, West AL, Madden J, similar in young, healthy men. This seems likely, given the Gambell JM, et al. Incorporation of eicosapentaenoic and doc- osahexaenoic acids into lipid pools when given as supplements results of a population survey reporting men and women providing doses equivalent to typical intakes of oily fish. Am J (25–44 years) had similar-3 LCPUFA status [30]. Clin Nutr. 2012;96:748–58. Taken together, our data describe, the dose-response 3. Patterson AC, Chalil A, Aristizabal Henao JJ, Streit IT, Stark KD. relationship between EPA and DHA intake and plasma PC Omega-3 polyunsaturated fatty acid blood biomarkers increase linearly in men and women after tightly controlled intakes of 0.25, EPA and DHA mol% and µmol/L after 14 days of sup- 0.5, and 1 g/d of EPA+DHA. Nutr Res. 2015;35:1040–51. plementation, in young healthy women. We found that per g 4. Barcelo-Coblijn G, Murphy EJ, Othman R, Moghadasian MH, intake per day increase in EPA results in a 1.5 mol% or 76 Kashour T, Friel JK. Flaxseed oil and fish-oil capsule consump- µmol/L increase in plasma PC EPA abundance whilst per g tion alters human red blood cell n-3 fatty acid composition: a multiple-dosing trial comparing 2 sources of n-3 fatty acid. Am J intake per day increase in DHA results in an increase of Clin Nutr. 2008;88:801–9. 1.1 mol% or 61 µmol/L in plasma PC DHA abundance. 5. Goyens PL, Spilker ME, Zock PL, Katan MB, Mensink RP. These data highlight that even modest changes in dietary fat Conversion of alpha-linolenic acid in humans is influenced by the intake are reflected rapidly by plasma PC n-3 FA. In long- 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. term studies where n-3 FA biomarkers are only measured at 6. Mantzioris E, James MJ, Gibson RA, Cleland LG. Dietary sub- the study beginning and end to determine compliance, it stitution with an alpha-linolenic acid-rich vegetable oil increases may prove difficult to distinguish true compliers from non- eicosapentaenoic acid concentrations in tissues. Am J Clin Nutr. compliers if measuring plasma lipid pools [15, 31, 32]; 1994;59:1304–9. 7. Schwab US, Callaway JC, Erkkila AT, Gynther J, Uusitupa MI, however, the proportion of erythrocyte DHA has been Jarvinen T. Effects of hempseed and flaxseed oils on the profile of reported to characterise adherence to EPA and DHA intakes serum lipids, serum total and lipoprotein lipid concentrations and in long-term interventions [31]. Finally, our data highlight haemostatic factors. Eur J Nutr. 2006;45:470–7. that short-term supplementation with ALA is reflected 8. Wallace FA, Miles EA, Calder PC. Comparison of the effects of linseed oil and different doses of fish oil on mononuclear cell rapidly by plasma PC and has only a modest, if any effect function in healthy human subjects. Br J Nutr. 2003;89:679–89. on n-3 LCPUFA status in this cohort. 9. Wilkinson P, Leach C, Ah-Sing EE, Hussain N, Miller GJ, Millward DJ, et al. Influence of alpha-linolenic acid and fish-oil on Acknowledgements We thank Margaret Waldron and Maggie Oakley, markers of cardiovascular risk in subjects with an atherogenic Research Nurses who assisted with the blood collection; Ashley lipoprotein phenotype. Atherosclerosis. 2005;181:115–24. Duncan, and Michelle Harper, Laboratory Technicians, who advised 10. Zhao G, Etherton TD, Martin KR, West SG, Gillies PJ, Kris- and assisted with the laboratory analyses; and the participants, without Etherton PM. Dietary alpha-linolenic acid reduces inflammatory whom this study would not have been possible. and lipid cardiovascular risk factors in hypercholesterolemic men and women. J Nutr. 2004;134:2991–7. Funding The University of Otago funded the study. LH is British 11. Hennebelle M, Courchesne-Loyer A, St-Pierre V, Vandenberghe Heart Foundation Senior Research Fellow in Basic Science. C, Castellano CA, Fortier M, et al. Preliminary evaluation of a differential effect of an alpha-linolenate-rich supplement on ketogenesis and plasma omega-3 fatty acids in young and older Compliance with ethical standards adults. Nutrition. 2016;32:1211–6. 12. Rizos EC, Ntzani EE, Bika E, Kostapanos MS, Elisaf MS. Conflict of interest The authors declare that they have no conflict of Association between omega-3 fatty acid supplementation and risk interest. of major cardiovascular disease events: a systematic review and meta-analysis. JAMA. 2012;308:1024–33. Open Access This article is licensed under a Creative Commons 13. Harris WS. n-3 fatty’ acids and serum lipoproteins: human studies. Attribution 4.0 International License, which permits use, sharing, Am J Clin Nutr. 1997;65:1645S–1654S. adaptation, distribution and reproduction in any medium or format, as 14. Balk EM, Lichtenstein AH, Chung M, Kupelnick B, Chew P, Lau long as you give appropriate credit to the original author(s) and the J. Effects of omega-3 fatty acids on serum markers of cardio- source, provide a link to the Creative Commons license, and indicate if vascular disease risk: a systematic review. Atherosclerosis. changes were made. The images or other third party material in this 2006;189:19–30. article are included in the article’s Creative Commons license, unless 15. Hodson L, Eyles HC, McLachlan KJ, Bell ML, Green TJ, Skeaff indicated otherwise in a credit line to the material. If material is not CM. Plasma and erythrocyte fatty acids reflect intakes of saturated included in the article’s Creative Commons license and your intended and n-6 PUFA within a similar time frame. J Nutr. use is not permitted by statutory regulation or exceeds the permitted 2014;144:33–41. use, you will need to obtain permission directly from the copyright 16. Hodson L, Skeaff CM, Chisholm WA. The effect of replacing holder. To view a copy of this license, visit http://creativecommons. dietary saturated fat with polyunsaturated or monounsaturated fat org/licenses/by/4.0/. on plasma lipids in free-living young adults. Eur J Clin Nutr. 2001;55:908–15. 840 L. Hodson et al. 17. Bligh DG, Dyer WJ. A rapid method of total lipid extraction and 26. Pawlosky RJ, Hibbeln JR, Herion D, Kleiner DE, Salem N Jr. purification. Can J Biochem Physiol. 1959;37:911–7. Compartmental analysis of plasma and liver n-3 essential fatty 18. Hodson L, Skeaff CM, Wallace AJ, Arribas GL. Stability of acids in alcohol-dependent men during withdrawal. J Lipid Res. plasma and erythrocyte fatty acid composition during cold sto- 2009;50:154–61. rage. Clin Chim Acta. 2002;321:63–7. 27. Giltay EJ, Gooren LJ, Toorians AW, Katan MB, Zock PL. Doc- 19. Holub BJ, Skeaff CM. Nutritional regulation of cellular phos- osahexaenoic acid concentrations are higher in women than in phatidylinositol. Methods Enzymol. 1987;141:234–44. men because of estrogenic effects. Am J Clin Nutr. 20. Serra-Majem L, Nissensohn M, Overby NC, Fekete K. Dietary 2004;80:1167–74. methods and biomarkers of omega 3 fatty acids: a systematic 28. Flock MR, Skulas-Ray AC, Harris WS, Etherton TD, Fleming JA, review. Br J Nutr. 2012;107(Suppl 2):S64–76. Kris-Etherton PM. Determinants of erythrocyte omega-3 fatty acid 21. Lands WE, Libelt B, Morris A, Kramer NC, Prewitt TE, Bowen P, content in response to fish oil supplementation: a dose-response et al. Maintenance of lower proportions of (n - 6) eicosanoid randomized controlled trial. J Am Heart Assoc. 2013;2:e000513. precursors in phospholipids of human plasma in response to added 29. Barcelo-Coblijn G, Murphy EJ. Alpha-linolenic acid and its dietary (n - 3) fatty acids. Biochim Biophys Acta. conversion to longer chain n-3 fatty acids: benefits for human 1992;1180:147–62. health and a role in maintaining tissue n-3 fatty acid levels. Prog 22. Katan MB, Deslypere JP, van Birgelen AP, Penders M, Zegwaard Lipid Res. 2009;48:355–74. M. Kinetics of the incorporation of dietary fatty acids into serum 30. Crowe FL, Skeaff CM, Green TJ, Gray AR. Serum n-3 long-chain cholesteryl esters, erythrocyte membranes, and adipose tissue: an PUFA differ by sex and age in a population-based survey of New 18-month controlled study. J Lipid Res. 1997;38:2012–22. Zealand adolescents and adults. Br J Nutr. 2008;99:168–74. 23. Arterburn LM, Hall EB, Oken H. Distribution, interconversion, 31. Patterson AC, Metherel AH, Hanning RM, Stark KD. The per- and dose response of n-3 fatty acids in humans. Am J Clin Nutr. centage of DHA in erythrocytes can detect non-adherence to 2006;83:1467S–1476S. advice to increase EPA and DHA intakes. Br J Nutr. 24. Pawlosky R, Hibbeln J, Lin Y, Salem N Jr. n-3 fatty acid meta- 2014;111:270–8. bolism in women. Br J Nutr. 2003;90:993–4. discussion 994-5 32. Skeaff CM, Hodson L, McKenzie JE. Dietary-induced changes in 25. Pawlosky RJ, Hibbeln JR, Salem N Jr. Compartmental analyses of fatty acid composition of human plasma, platelet, and erythrocyte plasma n-3 essential fatty acids among male and female smokers lipids follow a similar time course. J Nutr. 2006;136:565–9. and nonsmokers. J Lipid Res. 2007;48:935–43.

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European Journal of Clinical NutritionSpringer Journals

Published: May 30, 2018

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