Abstract
Background
Dietary linoleic acid (LA, 18:2n-6) lowering in rats reduces n-6 polyunsaturated fatty acid (PUFA) plasma concentrations and increases n-3 PUFA (eicosapentaenoic (EPA) and docosahexaenoic acid (DHA)) concentrations.
Objective
To evaluate the extent to which 12 weeks of dietary n-6 PUFA lowering, with or without increased dietary n-3 PUFAs, change unesterified and esterified plasma n-6 and n-3 PUFA concentrations in subjects with chronic headache.
Design
Secondary analysis of a randomized trial. Subjects with chronic headache were randomized for 12 weeks to: (1) average n-3, low n-6 (L6) diet; or (2) high n-3, low n-6 LA (H3-L6) diet. Esterified and unesterified plasma fatty acids were quantified at baseline (0 weeks) and after 12 weeks on a diet.
Results
Compared to baseline, the L6 diet reduced esterified plasma LA and increased esterified n-3 PUFA concentrations (nmol/ml), but did not significantly change plasma arachidonic acid (AA, 20:4n-6) concentration. In addition, unesterified EPA concentration was increased significantly among unesterified fatty acids. The H3-L6 diet decreased esterified LA and AA concentrations, and produced more marked increases in esterified and unesterified n-3 PUFA concentrations.
Conclusion
Dietary n-6 PUFA lowering for 12 weeks significantly reduces LA and increases n-3 PUFA concentrations in plasma, without altering plasma AA concentration. A concurrent increase in dietary n-3 PUFA for 12 weeks further increases n-3 PUFA plasma concentrations, but also reduces AA.
Keywords: Linoleic acid (LA), lowering, Eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), Arachidonic acid, omega-6 (n-6), omega-3 (n-3), polyunsaturated fatty acids, esterified, unesterified, plasma, lipids, fish, migraine
1. Introduction
Linoleic acid (LA, 18:2n-6) is a major constituent of the North American diet, accounting for approximately 7% of daily caloric intake and 20% of total dietary fatty acids (~16 g LA / day) [1]. This intake is more than three-fold higher than the historic norm of 2%, owing mainly to the increased consumption of seed oils containing 20–54% LA of total fatty acids [1]. The biochemical and health implications of this change are not fully understood.
The mammalian liver can convert LA to longer chain n-6 PUFAs, particularly arachidonic acid (AA, 20:4n-6), by elongation-desaturation via Δ5 and Δ6 desaturases and elongases-2 and -5. LA competes with alpha-linolenic acid (α-LNA, 18:3n-3) for elongation-desaturation enzymes that convert α-LNA to longer chain n-3 PUFAs including eicosapentaenoic acid (EPA, 20:5 n-3), n-3 docosapentaenoic acid (n-3 DPA 22:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) [2, 3], which have several putative health benefits [4–6]. LA and AA are precursors to bioactive LA oxidation products [7] and eicosanoids [8], respectively, which have been implicated in pathological conditions such as non-alcoholic steatohepatitis, Alzheimer disease and asthma [9–11]. By contrast, n-3 EPA, DPA and DHA can be converted into anti-inflammatory and pro-resolving lipid mediators [12, 5, 13, 14].
In rodents, dietary LA lowering has been shown to reduce the absolute concentration of AA (nmol per ml plasma or g tissue), and to increase EPA, DPA and DHA concentrations in plasma and numerous tissues [15, 2, 16, 17]. However, comparatively few human trials have evaluated the biochemical effects of lowering dietary LA. To our knowledge, there are no human data indicating that altering dietary LA changes circulating AA concentrations [18–20]. By contrast, dietary LA lowering in humans was reported to increase α-LNA conversion to EPA and DHA [21], to increase the EPA and DHA content of erythrocytes [19] and to increase EPA in plasma phospholipids [18]. In these human studies, data were expressed as percent composition (% of total fatty acids), which may not necessarily reflect changes in absolute concentrations because a change in the concentration of one fatty acid can reflect a change in the opposite direction of another [22, 23]. The effects of dietary n-6 PUFA lowering in humans on absolute n-3 and n-6 PUFA concentrations have not been reported for unesterified and esterified (phospholipids, triglycerides, cholesteryl esters) plasma lipid fractions.
We recently reported that the combination of increasing dietary n- 3 fatty acids with concurrent reduction in n-6 LA produced statistically significant, clinically relevant improvements in headache frequency, intensity and quality of life in chronic headache patients [24], a condition with reported elevations of AA-derived mediators in blood and saliva [25, 24, 26]. Blood collected from this trial provides a unique opportunity to evaluate the effects of targeted alterations of dietary n-3 and n-6 fatty acids on plasma esterified and unesterified fatty acid concentrations [19, 24], which could be used as biomarkers to relate efficacy in dietary treatment effects.
In the present study, we sought to evaluate the effects of dietary n-6 lowering with or without concurrent increases in dietary n-3 PUFA on unesterified and esterified plasma lipid fractions, using plasma samples from a completed dietary trial in patients with chronic headaches [19, 24]. We tested the following hypotheses: (1) an average n-3, low n-6 (L6) dietary intervention would increase n-3 PUFA and decrease n-6 AA absolute concentrations in esterified and unesterified plasma lipid pools; and (2) a high n-3, low n-6 LA (H3-L6) dietary intervention would produce significantly greater increases in circulating n-3 PUFA concentrations and reductions in AA concentrations.
2. Materials and Methods
2.1. Patients and dietary methods
A detailed description of the dietary methods and procedures of the main trial have been published [19, 24, 27]. The trial was conducted at The University of North Carolina at Chapel Hill (UNC) from April 2009 to November 2011. Subjects signed informed consent prior to participation. Trial procedures were approved by the UNC Institutional Review Board. This trial is registered under ClinicalTrials.gov (NCT01157208). In brief, sixty-seven subjects with chronic headaches were randomized to either a low n-6 PUFA (L6) diet or a high n-3 plus low n-6 PUFA (H3-L6) to be maintained for 12-weeks. Nutrient compositions of the two interventions are shown in Table 1. The interventions were designed to be equally credible and to provide equivalent: (1) amounts of study foods; (2) macronutrient and caloric intake; (3) interactions with the study investigators and dietitian; and (4) intensity and breadth of dietary advice and intervention materials [19]. A registered dietitian provided intensive counseling at randomization and at 2-week intervals. Foods meeting nutrient targets were provided to participants for two meals and two snacks per day. Detailed intervention-specific web-based materials were also provided to reinforce dietitian advice and complement the study food provision. To assess nutrient intakes six unannounced telephone-administered 24-hour recalls were administered for each participant– three during the baseline phase and three in the final four weeks of the intervention phase–as previously described [19].
Table 1.
Diet fatty acid composition:
| Changes pre-post diet intervention | ||||||||
|---|---|---|---|---|---|---|---|---|
| L6 diet (n=28) | H3-L6 diet (n=27) | Between Diets | ||||||
| Pre | Post | Pre | Post | Pre | Post | |||
| Variable | Median (25%, 75%) | Median (25%, 75%) | P value | Median (25%, 75%) | Median (25%, 75%) | P value | P value | P value |
| Total energy (kcal) | 1997 (1487, 2253) | 1859 (1411, 2199) | 0.52 | 1707 (1377, 1880) | 1596 (1293,1959) | 0.89 | 0.03 | 0.09 |
| Total protein (en%) | 15.7 (13.8, 16.8) | 15.2 (13.7, 17.0) | 0.13 | 16.1 (13.5, 19.6) | 17.2 (15.1, 20.0) | 0.25 | 0.62 | 0.01 |
| Total fat (en%) | 33.6 (29.6, 40.1) | 30.4 (26.8, 34.3) | 0.05 | 33.4 (29.1, 36.4) | 30.7 (27.3, 34.0) | 0·08 | 0.38 | 0.84 |
| LA 18:2 (en%) | 7.4 (5.7, 9.6) | 2.4 (2.0, 2.9) | <0.001 | 6.4 (5.3, 7.4) | 2.5 (2.2, 3.9) | <0.001 | 0.03 | 0.15 |
| α-LNA 18:3 (en%) | 0.7 (0.6, 0.9) | 0.7 (0.6, 0.9) | 0.96 | 0.6 (0.5, 0.9) | 1.6 (1.3, 2.0) | <0.001 | 0.32 | <0.001 |
| AA 20:4 (mg) | 106 (57, 159) | 48* (18, 74) | <0.001 | 110 (66, 176) | 114* (69, 195) | 0.75 | 0.64 | <0.001 |
| EPA + DHA (mg) | 43 (25, 73) | 76* (19, 264) | 0.32 | 47 (20, 71) | 1482* (374, 2558) | <0.001 | 0.96 | <0.001 |
| Total SFA (en%) | 10.5 (9.1, 11.9) | 14.0 (12.0, 17.2) | <0.001 | 10.5 (9.8, 11.7) | 12.9 (9.9, 14.5) | 0.16 | 0.99 | 0.06 |
| Total Trans (en%) | 0.9 (0.7, 1.2) | 0.6 (0.5, 0.84) | 0.005 | 1.1 (0.9, 1.4) | 0.5 (0.3, 0.7) | <0.001 | 0.07 | 0.06 |
| Total MUFA (en%) | 11.8 (10.1, 13.2) | 10.4 (8.0, 12.6) | 0.008 | 12.1 (11.0, 13.5) | 9.3 (8.4, 11.4) | <0.001 | 0.82 | 0.51 |
| Total PUFA (en%) | 8.1 (6.4, 10.6) | 3.5 (3.1, 4.3) | <0.001 | 7.4 (5.9, 8.5) | 6.0 (5.1, 7.1) | 0.1 | 0.05 | <0.001 |
Adapted from [19]. Diet fatty acid composition pre and post intervention, expressed as energy % (en%) or mg.
SFA, saturated fatty acids; Trans, trans fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids; LA, linoleic acid; α-LNA, α-linolenic acid; AA, arachidonic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid.
Pre-to-post intervention comparisons were tested with the Wilcoxon Signed-Rank test for matched pairs. A Mann Whitney U test was used to compare differences in fatty acid concentrations between the two groups at baseline and at 12 weeks.
Fifty-six of the 67 randomized participants completed the 12-week intervention phase, with 55 providing pre- and post-intervention plasma samples (28 in the L6 group and 27 in the H3-L6 group). Baseline demographics and clinical characteristics were comparable in the two groups (Table 2); 87% of randomized subjects were female. At baseline, participants averaged 23 headache days per month and 10 headache hours per day, and reported taking an average of six different headache-related medications per subject.
Table 2.
Baseline characteristics of 67 patients with chronic headaches
| H3-L6 diet n = 33 |
L6 diet n = 34 |
|
|---|---|---|
| Age, y, mean (SD) | 41 (13.4) | 42 (11.1) |
| Female, n (%) | 28 (84.8) | 30 (88.2) |
| White race, n (%) | 28 (84.8) | 30 (88.1) |
| Married, n (%) | 19 (57.6) | 19 (55.9) |
| Education, n (%) | ||
| High school or college | 20 (60.6) | 19 (59.3) |
| Master's degree or higher | 13 (40.6) | 13 (39.4) |
| Employment, n (%) | ||
| Employed/student | 26 (78.8) | 23 (69.7) |
| Retired/Caretaker | 3 (9.1) | 3 (9.1) |
| Disabled/unemployed | 4 (12.1) | 7 (20.0) |
| Headache days per month, mean (SD) | 23.3 (20.9, 25.8) | 23.2 (20.2, 25.8) |
| Headache hours per day, mean (SD) | 10.2 (8.4, 12.3) | 9.8 (8.1, 11.8) |
| Number of different headache-related medications, mean (SD) | 6.4 (3.4) | 5.6 (3.3) |
Adapted from reference [24]., number of subjects;%, proportion of subjects; SD, standard deviation.
2.2. Sample collection
Fasting whole blood, drawn at baseline and again after 12 weeks of dietary intervention, was collected into ethylenediaminetetraacetic acid (EDTA) tubes. Samples were immediately centrifuged at 2000g for 15 min at room temperature, and plasma aliquots were stored in a −80°C freezer until analysis. Sample preparation and analyses were performed by investigators who were blinded to the study protocol and clinical data.
2.3. Analysis of plasma esterified and unesterified fatty acids
Total lipids were extracted from 200 µl of plasma in 3 ml of 2:1 chloroform / methanol following the addition of unesterified heptadecaenoic acid (17:0) as an internal standard (0.14 nmol/µl) for unesterified fatty acids. KCl (0.5 M, 0.75 ml) was then added to separate the aqueous phase. The bottom chloroform layer was separated and re-extracted with 2 ml chloroform. The pooled chloroform extracts containing total lipids were dried down and separated into neutral lipid subclasses (cholesteryl esters, triacylglycerol, unesterified fatty acids, and total phospholipids) using silica gel-60 thin layer chromatography plates (EM Separation Technologies, Gibbstown, NJ, USA), in a heptane: diethylether: glacial acetic acid (60:40:3, by vol) solvent system [28]. Authentic standards of neutral lipids and phospholipid classes were run on separate lanes on the plates to identify lipid bands under ultraviolet light, after spraying with 0.03% 6-p-toluidine-2-naphthalene sulfonic acid in 50 mM Tris-HCl buffer (pH 7.4) (w/v). The bands were scraped into test tubes and methylated with 1% H2SO4 in methanol for 3 h at 70°C [29]. Before methylation, di-17:0 PC was added to each tube as an internal standard for phospholipids, triglycerides and cholesteryl esters. The prepared fatty acid methyl esters (FAMEs) were analyzed by a gas-chromatography system (6890N, Agilent Technologies, Palo Alto, CA, USA) equipped with an SPTM-2330 fused silica capillary column (30 m × 0.25 mm i.d., 0.25 µm film thickness) (Supelco, Bellefonte, PA, USA) and a flame ionization detector as previously described [30]. Fatty acid concentrations were calculated by proportional comparison of peak areas of samples to the area of the 17:0 internal standard.
2.4. Data Analysis
Non-parametric analyses were employed due to the presence of non-normal distributions. Pre-to-post intervention comparisons were tested with the Wilcoxon Signed-Rank test for matched pairs. A Mann Whitney U test was used to compare differences in fatty acid concentrations between the two groups at baseline and at 12 weeks. Statistical significance was accepted at P ≤ 0.05.
3. Results
3.1. Baseline fatty acid concentrations
As shown in Tables 3 to 6, absolute baseline fatty acid concentrations in plasma phospholipids, triglycerides, cholesteryl esters and unesterified fatty acids did not differ significantly between the groups (P > 0.05 by Mann-Whitney U test). Baseline absolute concentrations for esterified and unesterified fatty acids are comparable to a previous report in humans exposed to similar North American intakes of LA [31]. The percent composition in the various lipid pools are presented in Supplementary Tables 1 to 4, and are comparable to values in other studies [32, 33].
Table 3.
Pre- and post-intervention phospholipid fatty acids concentrations (nmol/ml)
| Changes pre-post diet intervention | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| L6 diet (n=28) | H3-L6 diet (n=27) | Between Diets | ||||||||
| Pre | Post | Pre | Post | Pre | Post | |||||
| Variable (nmol/ml) |
Median (25%, 75%) | Median(25%, 75%) | % Change |
P value | Median(25%, 75%) | Median (25%, 75%) | % Change |
P value | P value | P value |
| LA | 838.58 (765.18,984.43) | 742.13 (643.09,820.44) | −11.50 | <0.001 | 763.38(721.40,919.94) | 620.52 (550.79,753.91) | −18.71 | <0.001 | 0.178 | 0.030 |
| AA | 382.21 (306.80,440.84) | 333.04 (280.54,445.66) | −12.87 | 0.088 | 356.18 (292.04,421.84) | 286.64 (255.68,365.97) | −19.53 | <0.001 | 0.960 | 0.043 |
| DTA | 13.63 (9.69,15.40) | 11.85 (9.26,15.18) | −13.06 | 0.072 | 11.80 (9.81,14.31) | 8.44 (7.06,9.68) | −28.49 | <0.001 | 0.439 | 0.001 |
| n-6 DPA | 10.79 (6.08,12.83) | 8.00(6.46,11.47) | −25.82 | 0.007 | 9.03 (6.01,10.64) | 4.48* (3.67,5.68) | −50.43 | <0.001 | 0.201 | <0.001 |
| α-LNA | 7.57 (6.19,9.04) | 9.06 (6.62,13.01) | 19.73 | 0.020 | 6.92 (5.39,10.32) | 9.65 (5.75,13.42) | 39.47 | 0.038 | 0.769 | 0.853 |
| EPA | 20.35 (11.78,25.18) | 27.09 (20.31,35.68) | 33.10 | 0.001 | 17.28 (13.44,22.40) | 64.13 (37.50,80.09) | 271.08 | <0.001 | 0.400 | <0.001 |
| n-3 DPA | 21.81 (18.18,26.82) | 26.39 (18.88,31.81) | 21.01 | 0.021 | 19.99 (14.61,22.81) | 25.06 (20.78,33.78) | 25.34 | 0.001 | 0.110 | 0.814 |
| DHA | 76.84 (56.10,98.64) | 83.86 (66.91,113.49) | 9.13 | 0.011 | 71.18 (61.61,109.20) | 150.72 (112.18,181.80) | 111.74 | <0.001 | 0.920 | <0.001 |
| Palmitic acid | 1134.30 (939.56,1326.62) | 1172.08 (1046.50,1293.27) | 3.33 | 0.524 | 1121.47 (927.88,1352.70) | 1131.08 (940.30,1379.20) | 0.86 | 0.325 | 0.853 | 0.933 |
| Oleic acid | 334.48 (289.70,396.40) | 365.06 (307.76,425.62) | 9.14 | 0.036 | 332.96 (279.61,407.08) | 354.82 (284.22,409.46) | 6.57 | 0.904 | 1.000 | 0.274 |
| Total fatty acids | 3700 (3254,4162) | 3681 (3252,4008) | −0.52 | 0.733 | 3495.03 (3153,3971) | 3504 (3110,3953) | 0.26 | 0.456 | 0.556 | 0.409 |
Plasma phospholipid fatty acid concentrations (nmol/ml) pre and post intervention.
LA, linoleic acid; AA, arachidonic acid; DTA, docosatetraenoic acid; n-6 DPA, n-6 docosapentaenoic acid; α-LNA, α-linolenic acid; EPA, eicosapentaenoic acid; n-3 DPA, n-3 docosapentaenoic acid; DHA, docosahexaenoic acid.
Pre-to-post intervention comparisons were tested with the Wilcoxon Signed-Rank test for matched pairs. A Mann Whitney U test was used to compare differences in fatty acid concentrations between the two groups at baseline and at 12 weeks.
Table 6.
Pre- and post-intervention unesterified fatty acid concentrations (nmol/ml)
| Changes pre-post diet intervention | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| L6 diet (n=28) | H3-L6 diet (n=27) | |||||||||
| Pre | Post | Pre | Post | Between Group | ||||||
| Variable (nmol/ml) |
Median (25%,75%) | Median (25%,75%) | %Change | P | Median (25%,75%) | Median (25%,75%) | %Change | P | Pre | Post |
| LA | 57.99 (36.51, 78.58) | 54.91 (45.33, 73.23) | −5.30 | .964 | 57.89 (29.73, 80.62) | 52.47 (40.33, 65.35) | −9.36 | 0.325 | 0.736 | 0.400 |
| DGLA | 2.19 (1.55, 2.74) | 2.05 (1.34, 2.53) | −6.53 | .501 | 1.70 (1.05, 2.94) | 1.79 (1.37, 2.28) | 5.61 | 0.829 | 0.413 | 0.400 |
| AA | 1.82 (1.59, 2.72) | 2.05 (1.53, 2.80) | 12.75 | .767 | 2.14 (1.42, 2.87) | 2.22 (1.70, 2.62) | 3.73 | 0.773 | 0.749 | 0.724 |
| DTA | 3.91 (3.66, 4.45) | 4.00 (3.58, 4.36) | 2.10 | .509 | 3.74 (3.28, 4.17) | 3.93 (3.59, 4.42) | 5.08 | 0.195 | 0.035 | 0.711 |
| n-6 DPA | 0.23 (0.14, 0.31) | 0.22 (0.18, 0.26) | −4.94 | .975 | 0.20 (0.18, 0.29) | 0.18 (0.15, 0.21) | −8.03 | 0.125 | 0.947 | 0.114 |
| EPA1 | 0.22 (0.16, 0.32) | 0.29 (0.21, 0.36) | 29.60 | .039 | 0.18 (0.16, 0.25) | 0.53 (0.37, 0.82) | 198.52 | 0.001 | 0.303 | 0.001 |
| n-3 DPA | 0.38 (0.19, 0.57) | 0.36 (0.22, 0.55) | −4.94 | .639 | 0.35 (0.23, 0.45) | 0.41 (0.35, 0.63) | 18.68 | 0.007 | 0.560 | 0.187 |
| DHA | 0.90 (0.70, 1.70) | 1.10 (0.75, 1.32) | 22.77 | .354 | 0.77 (0.60, 1.54) | 2.47 (1.30, 3.39) | 220.73 | 0.001 | 0.594 | 0.001 |
| Palmitic acid | 90.84 (68.31, 111.64) | 88.14 (70.80, 119.86) | −2.97 | .616 | 99.22 (59.46, 115.91) | 96.93 (71.12, 118.07) | −2.31 | 0.981 | 0.762 | 0.827 |
| Oleic acid | 114.74 (92.16, 172.58) | 135.61 (103.45, 182.08) | 18.19 | .246 | 115.78 (65.72, 168.16) | 133.33 (96.25, 160.89) | 15.16 | 0.904 | 0.775 | 0.400 |
| Total fatty acids | 335.99 (260.61, 446.12) | 356.64 (275.96, 478.34) | 6.14 | .480 | 369.52 (214.80, 440.40) | 366.26 (274.80, 418.77) | −0.88 | 0.962 | 0.814 | 0.567 |
Plasma unesterified fatty acid concentrations (nmol/ml) pre and post intervention.
Unesterified α-LNA is not reported because it was not detected in most samples.
LA, linoleic acid; AA, arachidonic acid; DTA, docosatetraenoic acid; n-6 DPA, n-6 docosapentaenoic acid; α-LNA, α-linolenic acid; EPA, eicosapentaenoic acid; n-3 DPA, n-3 docosapentaenoic acid; DHA, docosahexaenoic acid.
Pre-to-post intervention comparisons were tested with the Wilcoxon Signed-Rank test for matched pairs. A Mann Whitney U test was used to compare differences in fatty acid concentrations between the two groups at baseline and at 12 weeks.
3.2. Phospholipid fatty acid concentrations
Table 3 shows median phospholipid fatty acid concentrations. Compared to baseline, dietary n-6 lowering (L6 group) decreased absolute concentrations (nmol / ml) of LA (12%) and n-6 DPA (22:5n-6; 26%), and increased concentrations of oleic acid (18:1n-9; 9%), α-LNA (20%), EPA (33%), n-3 DPA (22:5n-3; 21%) and DHA (9%). In the H3-L6 group, absolute concentrations of LA, AA, docosatetraenoic acid (DTA, 22:4n-6) and n-6 DPA were reduced significantly compared to baseline by 19%, 20%, 28% and 50%, respectively, whereas α-LNA, EPA, n-3 DPA and DHA concentrations were increased by 39%, 271%, 25% and 112%, respectively. The changes in LA, AA, EPA, DTA, n-6 DPA and DHA concentrations relative to baseline were higher in the H3-L6 than the L6 group, as evidenced by the statistically significant differences between the L6 and H3-L6 groups at 12 weeks (P < 0.05 by Mann-Whitney U test, final column in Table 3).
3.3. Triglyceride fatty acid concentrations
Within triglycerides, only the LA concentration was changed significantly (−29%) compared to baseline in the L6 group (Table 4). The H3-L6 intervention produced a comparable reduction in triglyceride LA concentration (−24%), with concurrent reductions in n-6 DPA and AA (−25 to −29%), and increased α-LNA, EPA, n-3 DPA and DHA concentrations (+57 to 216%). At 12 weeks, EPA and DHA concentrations in the H3-L6 group were significantly higher than in the L6 group (P < 0.05 by Mann-Whitney U test, final column in Table 4).
Table 4.
Pre- and post-intervention triglyceride fatty acids concentrations (nmol/ml)
| Changes pre-post diet intervention | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| L6 diet (n=28) | H3-L6 diet (n=27) | |||||||||
| Pre | Post | Pre | Post | Between Diets | ||||||
| Variable (nmol/ml) |
Median (25%, 75%) | Median (25%,75%) | % change |
P | Median(25%, 75%) | Median (25%, 75%) | % change |
P | Pre | Post |
| LA | 613.48 (346.72, 784.84) | 436.37 (315.04, 652.69) | −28.87 | 0.002 | 471.49 (304.00, 689.71) | 356.24 (259.04, 504.20) | −24.44 | 0.002 | .201 | 0.130 |
| AA | 37.20 (21.98, 51.70) | 28.20 (20.20, 43.64) | −24.18 | 0.210 | 38.63 (24.79, 53.45) | 28.90 (20.44, 34.58) | −25.18 | 0.024 | .840 | 0.686 |
| DTA | 5.33 (4.02, 7.45) | 4.78 (3.15, 7.32) | −10.38 | 0.648 | 5.59 (3.57, 6.95) | 4.96 (3.43, 5.62) | −11.28 | 0.191 | .986 | 0.880 |
| DPAn6 | 2.30 (1.79, 3.75) | 2.26 (1.89, 4.52) | −1.57 | 0.545 | 2.79 (1.76, 3.80) | 1.98 (1.65, 2.66) | −29.05 | 0.035 | .670 | 0.138 |
| α-LNA | 36.52 (18.22, 52.86) | 32.87 (22.45, 42.24) | −10.00 | 0.316 | 21.71 (14.98, 46.36) | 34.01 (19.27, 56.43) | 56.66 | 0.034 | .219 | 0.419 |
| EPA | 4.57 (2.70, 6.69) | 5.71 (3.53, 7.11) | 24.99 | 0.080 | 3.94 (2.71, 6.52) | 12.44 (5.99, 22.62) | 216.01 | 0.000 | .703 | 0.000 |
| DPAn3 | 4.84 (4.08, 8.06) | 5.73 (3.72, 8.66) | 18.29 | 0.486 | 3.85 (2.44, 5.89) | 7.77 (4.70, 11.23) | 101.95 | 0.000 | .058 | 0.132 |
| DHA | 7.56 (3.85, 11.56) | 8.91 (4.42, 13.77) | 17.84 | 0.187 | 6.45 (3.11, 10.96) | 20.40 (14.17, 45.56) | 216.31 | 0.000 | .614 | 0.000 |
| Palmitic acid | 786.35 (384.74, 992.81) | 720.41 (501.79, 1089.82) | −8.39 | 0.387 | 587.49 (449.54, 1013.38) | 584.31 (421.25, 988.54) | −0.54 | 0.564 | .662 | 0.337 |
| Oleic acid | 985.83 (554.29, 1159.58) | 945.73 (668.89, 1239.75) | −4.07 | 0.295 | 816.74 (495.32, 1203.88) | 766.35 (472.01, 1223) | −6.17 | 0.564 | .449 | 0.207 |
| Total fatty acids | 2887 (1689, 3617) | 2696 (1876, 3482) | −6.61 | 0.750 | 2359. (1582, 3269) | 2106 (1514, 3353) | −10.74 | 0.349 | .480 | 0.232 |
Plasma triglyceride fatty acid concentrations (nmol/ml) pre and post intervention.
LA, linoleic acid; AA, arachidonic acid; DTA, docosatetraenoic acid; n-6 DPA, n-6 docosapentaenoic acid; α-LNA, α-linolenic acid; EPA, eicosapentaenoic acid; n-3 DPA, n-3 docosapentaenoic acid; DHA, docosahexaenoic acid.
Pre-to-post intervention comparisons were tested with the Wilcoxon Signed-Rank test for matched pairs. A Mann Whitney U test was used to compare differences in fatty acid concentrations between the two groups at baseline and at 12 weeks.
3.4. Cholesteryl ester fatty acid concentrations
In cholesteryl ester, n-6 lowering for 12 weeks increased palmitic acid, oleic acid, α-LNA, EPA and DHA concentrations by 7–45%, and decreased LA concentration by 9% compared to baseline (Table 5). The H3-L6 intervention significantly reduced AA by 13% (but not LA) and increased palmitic acid, oleic acid, α-LNA, EPA and DHA concentrations by 21%, 90%, 365% and 99%, respectively (P < 0.001), compared to baseline. At 12 weeks, α-LNA and EPA were significantly higher in the H3-L6 compared to the L6 group (P < 0.05 by Mann-Whitney U test, final column in Table 5).
Table 5.
Pre- and post-intervention cholesteryl ester fatty acid concentrations (nmol/ml)
| Changes pre-post diet intervention | ||||||||||
|---|---|---|---|---|---|---|---|---|---|---|
| L6 diet (n=28) | H3-L6 diet (n=27) | |||||||||
| Pre | Post | Pre | Post | Between Group | ||||||
| Variable (nmol/ml) |
Median (25%, 75%) | Median (25%, 75%) | % change | P | Median (25%, 75%) | Median (25%, 75%) | % change | P | Pre | Post |
| LA | 1618.47 (1457.38, 1922.56) | 1467.89 (1301.19, 1711.10) | −9.30 | 0.001 | 1570.69 (1281.42, 1706.78) | 1429.18 (1166.21, 1628.61) | −9.01 | 0.118 | 0.096 | 0.297 |
| AA | 199.99 (149.98, 278.79) | 172.10 (144.89, 252.91) | −13.95 | 0.088 | 195.71 (142.57, 268.79) | 169.98 (148.48, 211.88) | −13.15 | 0.017 | 0.960 | 0.533 |
| α-LNA | 15.63 (12.74, 18.82) | 22.67 (18.19, 31.76) | 45.06 | 0.001 | 14.55 (9.62, 18.09) | 27.70 (22.63, 44.07) | 90.33 | 0.000 | 0.184 | 0.049 |
| EPA | 14.85 (9.47, 20.21) | 21.27 (15.16, 29.41) | 43.18 | 0.004 | 12.34 (8.74, 17.09) | 57.37 (28.41, 82.16) | 365.03 | 0.000 | 0.259 | 0.000 |
| DHA | 13.59 (7.47, 82.53) | 17.60 (10.77, 80.28) | 29.52 | 0.018 | 11.22 (8.01, 73.77) | 22.33 (19.60, 98.22) | 99.06 | .0002 | 0.699 | 0.106 |
| Palmitic acid | 351.34 (306.49, 429.57) | 376.40 (309.70, 433.11) | 7.13 | .0210 | 344.34 (305.11, 403.35) | 404.04 (308.79, 455.38) | 17.34 | 0.000 | 0.590 | 0.490 |
| Oleic acid | 492.68 (427.56, 592.96) | 556.90 (499.60, 652.53) | 13.03 | 0.045 | 491.97 (425.77, 555.70) | 594.52 (453.05, 680.24) | 20.85 | 0.000 | 0.544 | 0.724 |
| Total fatty acids | 2808 (2549, 3507) | 3140 (2532, 3433) | 11.83 | 0.400 | 2827 (2460, 3162) | 2903 (2598, 3414) | 2.69 | 0.118 | 0.391 | 0.775 |
Plasma cholesteryl ester fatty acid concentrations (nmol/ml) pre and post intervention.
LA, linoleic acid; AA, arachidonic acid; α-LNA, α-linolenic acid; EPA, eicosapentaenoic acid; DHA, docosahexaenoic acid.
Pre-to-post intervention comparisons were tested with the Wilcoxon Signed-Rank test for matched pairs. A Mann Whitney U test was used to compare differences in fatty acid concentrations between the two groups at baseline and at 12 weeks.
3.5. Unesterified fatty acid concentrations
Compared to baseline, the EPA concentration within unesterified fatty acids was significantly increased (+30%) in the L6 group at 12 weeks (Table 6). This increase was much higher in the H3-L6 group (+199%; P < 0.001). n-3 DPA and DHA were significantly higher by 19% and 221%, respectively, in the H3-L6 group at 12 weeks compared to baseline (P<0.01). Statistical comparison of the medians at 12 weeks by the Mann-Whitney U test indicated that EPA and DHA were significantly higher in the H3-L6 compared to the L6 group.
4. Discussion
Dietary n-6 PUFA lowering (the L6 intervention) for 12 weeks did not significantly alter the AA concentration (nmol/ml) in any esterified or unesterified plasma lipid fraction of headache patients, but did increase the n-3 PUFA concentration of both esterified and unesterified plasma lipids. By contrast, dietary LA lowering with concurrent increase in dietary EPA and DHA (the H3-L6 intervention) significantly reduced plasma AA concentration, and produced significantly greater increases in n-3 PUFA concentrations in esterified and unesterified plasma lipids.
4.1. The L6 intervention
Findings in the L6 group are consistent with previous reports showing that dietary LA lowering for 8–12 weeks in humans without concurrent increases in dietary n-3 PUFA significantly increased n-3 EPA, DPA and DHA concentrations in various circulating lipid pools [34, 18, 24]. Increases in plasma esterified EPA, n-3 DPA and DHA concentrations produced by the L6 intervention could reflect increased hepatic synthesis-secretion from α-LNA, because dietary LA lowering might enhance the elongation-desaturation of α-LNA by reducing LA substrate availability and subsequent competition between LA and α-LNA for liver conversion into their respective longer-chain PUFAs [35]. This interpretation is consistent with evidence of increased hepatic desaturase and elongase transcription [35] and increased plasma and tissue EPA, n-3 DPA and DHA concentrations in rats fed a low LA diet [36, 16]. Since dietary LA also competes with n-3 EPA and DHA for esterification within liver phospholipids [37], reduced competition for esterification also may have contributed to the observed increase in n-3 PUFA concentration of phospholipids, triglycerides and cholesteryl esters in the L6 group.
The failure of the L6 intervention to significantly reduce the AA concentration in any plasma lipid pool is consistent with our erythrocyte assays from the same trial showing no change in AA percent composition [19, 24]. Interestingly, this L6 intervention produced significant reductions in the absolute concentrations of DTA and n-6 DPA (products of AA elongation / desaturation) in plasma phospholipids, despite the lack of reduction in AA. This discrepancy suggests that a diet-induced reduction in AA synthesis may be offset by homeostatic mechanisms that maintain AA concentrations, such as increased adipose mobilization of AA by hormone-sensitive lipase [38] or reduced metabolic conversion of AA into bioactive mediators [24]. Reduced AA metabolism is consistent with our report that the L6 intervention significantly reduced several bioactive hydroxy-eicosatetraenoic acid (HETE) derivatives of AA in plasma, without altering plasma or erythrocyte AA content [19, 24].
Plasma reductions in esterified AA concentration in the L6 group might become evident during a study sufficiently long to reduce adipose tissue AA stores, which may take years in view of 1–2 year fatty acid half-lives in human adipose tissue [39]. The disconnect between diet-induced changes in AA and AA-derived bioactive mediators suggests that simply measuring fatty acid concentrations may not adequately reflect metabolic state.
4.2. The H3-L6 intervention
The more marked increase (>10-fold) in esterified n-3 PUFAs in the H3-L6 diet compared to the L6 diet group was likely due to competition between dietary (preformed) n-3 EPA and DHA and other n-6 PUFAs for esterification. Since hepatic synthesis of EPA and DHA from α-LNA can be inhibited by dietary EPA and /or DHA supplementation in humans and rodents [3, 40, 41], the contribution of α-LNA to the increased long-chain plasma n-3 PUFA concentrations in the H3-L6 group likely was small compared to that of preformed dietary EPA and DHA. Consistent with this interpretation, clinical trials of α-LNA supplementation for up to one year did not change plasma DHA concentrations [42, 43].
The decrease in AA concentration in plasma phospholipid, triglyceride and cholesteryl esters in the H3-L6 group compared to the L6 group likely reflects reduced AA synthesis-secretion, since the consumption of EPA and DHA likely reduces the activity of elongase and desaturase enzymes involved in both AA and EPA synthesis [3, 41]. The observed reduction in AA concentration may also reflect competition of ingested EPA and DHA with endogenous AA for incorporation into phospholipids, triglycerides or cholesteryl esters within liver [44].
Adipose tissue unesterified fatty acid release and circulating esterified fatty acids secreted by the liver are the two principle sources of circulating unesterified fatty acids, the species that are preferentially incorporated into the brain [45, 46]. In this study, unesterified plasma EPA was increased in the L6 group, and unesterified plasma EPA, n-3 DPA and DHA were increased in the H3-L6 group, consistent with the increases in their respective esterified concentrations. By contrast, unesterified n-6 PUFA concentrations did not change in either group, despite being reduced within their esterified pools. LA presently accounts for 14–18% of total adipose fatty acids in the North American population [47], compared to about 6% in 1961 [48, 49], suggesting that a prolonged period of LA-lowering may be necessary to sufficiently reduce adipose stores and decrease LA in the unesterified fatty acid pool. Clearly, a different and more rapid homeostatic state was attained for n-3 PUFAs, which are much less abundant in adipose tissue (< 2%) compared to n-6 PUFAs (~20%) [47].
4.3. Potential implications for brain PUFA metabolism and neuroinflammation
In rats, dietary LA lowering from 3.5% to 0.3% energy for 15 weeks increased n-3 PUFA concentrations and decreased plasma unesterified and esterified LA and AA concentrations [17]. These changes were accompanied by decreased expression and activity of AA-releasing calcium-dependent phospholipase A2 (cPLA2) IVA and AA-metabolizing cyclooxygenase-2 (COX-2), and increased expression and activity of DHA-releasing calcium-independent phospholipase A2 (iPLA2 VIA) [50]. Since cPLA2 and COX-2 are upregulated during neuroinflammation, and increased iPLA2 VIA reflects increased DHA metabolism into bioactive pro-resolving metabolites, it is possible that that the H3-L6 intervention (and to a lesser extent the L6 intervention) would attenuate neuroinflammation. This may explain the efficacy of the H3-L6 diet in reducing headache frequency in patients with chronic migraine [24]. Future trials are needed to determine the efficacy of n-6 PUFA lowering and n-3 supplementation on brain PUFA metabolism and neuroinflammation in humans.
4.4. Limitations
The present trial was conducted in a population with high n-6 LA and low n-3 EPA+DHA consumption at baseline. The magnitudes of changes in plasma lipids may differ in populations with different dietary characteristics. Since this study was conducted in a predominantly female patient population with chronic headaches, the observed biochemical changes are not necessarily generalizable to men or neurologically healthy individuals.
4.5. Conclusion
In conclusion, 12 weeks of dietary n-6 lowering increased concentrations of n-3 PUFAs in plasma esterified and unesterified lipids, and a combination of dietary LA lowering with concurrent increases in EPA and DHA further increased circulating n-3 PUFA levels and reduced esterified AA and other n-6 PUFA concentrations in a patient population with chronic headaches.
Supplementary Material
Acknowledgement
The authors declare that they have no conflicting interest. The authors thank the patients who participated in the trial, Dr. Stanley Rapoport for reviewing the manuscript and the following individuals for their research assistance: Susan Gaylord, Marjorie Busby, Meg Mangan, Chanee Lynch, Rebecca Coble, David Barrow, Clarence Mayo, Jim Howerton, Derrick Williams, Olafur Palsson, Beth Fowler, Carol Carr, Regina McCoy and Tim McCaskill. This research was funded by Mayday Fund (primary source); the North Carolina Clinical and Translational Sciences Institute (grant UL1RR025747, NCRR, NIH); the UNC Nutrition Obesity Research Center, CHAI Core and Nutrition Epidemiology Core (grant DK056350, NIDDK, NIH); the UNC Research Fellowship in Complementary and Alternative Medicine (grant T32-AT003378, NCCAM, NIH); and the Intramural Research Program of the National Institute on Aging and the National Institute on Alcohol Abuse and Alcoholism, NIH. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the Mayday Fund or the National Institutes of Health.
Abbreviations
- AA
arachidonic acid
- cPLA2
calcium-dependent phospholipase A2
- COX-2
cyclooxygenase
- DHA
docosahexaenoic acid
- DPA
docosapentaenoic acid
- EDTA
ethylenediaminetetraacetic acid
- EPA
eicosapentaenoic acid
- HETE
hydroxyeicosatetraenoic acid
- iPLA2
calcium-independent phospholipase A2
- LA
linoleic acid
- α-LNA
α–linolenic acid
- PUFA
polyunsaturated fatty acid
Footnotes
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References
- 1.Blasbalg TL, Hibbeln JR, Ramsden CE, Majchrzak SF, Rawlings RR. Changes in consumption of omega-3 and omega-6 fatty acids in the United States during the 20th century. Am J Clin Nutr. 2011;93:950–962. doi: 10.3945/ajcn.110.006643. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 2.Bazinet RP, Douglas H, Cunnane SC. Whole-body utilization of n-3 PUFA in n-6 PUFA-deficient rats. Lipids. 2003;38:187–189. doi: 10.1007/s11745-003-1050-8. [DOI] [PubMed] [Google Scholar]
- 3.Domenichiello AF, Chen CT, Trepanier MO, Stavro PM, Bazinet RP. Whole body synthesis rates of DHA from alpha-linolenic acid are greater than brain DHA accretion and uptake rates in adult rats. J Lipid Res. 2014;55:62–74. doi: 10.1194/jlr.M042275. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 4.Carter JR, Schwartz CE, Yang H, Joyner MJ. Fish Oil and Neurovascular Reactivity to Mental Stress in Humans. Am J Physiol Regul Integr Comp Physiol. 2013 doi: 10.1152/ajpregu.00031.2013. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 5.Dalli J, Colas RA, Serhan CN. Novel n-3 immunoresolvents: structures and actions. Sci Rep. 2013;3:1940. doi: 10.1038/srep01940. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 6.Yurko-Mauro K, McCarthy D, Rom D, et al. Beneficial effects of docosahexaenoic acid on cognition in age-related cognitive decline. Alzheimers Dement. 2010;6:456–464. doi: 10.1016/j.jalz.2010.01.013. [DOI] [PubMed] [Google Scholar]
- 7.Ramsden CE, Ringel A, Feldstein AE, et al. Lowering dietary linoleic acid reduces bioactive oxidized linoleic acid metabolites in humans. Prostaglandins Leukot Essent Fatty Acids. 2012;87:135–141. doi: 10.1016/j.plefa.2012.08.004. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 8.Toborek M, Malecki A, Garrido R, Mattson MP, Hennig B, Young B. Arachidonic acid-induced oxidative injury to cultured spinal cord neurons. J Neurochem. 1999;73:684–692. doi: 10.1046/j.1471-4159.1999.0730684.x. [DOI] [PubMed] [Google Scholar]
- 9.Feldstein AE, Lopez R, Tamimi TA, et al. Mass spectrometric profiling of oxidized lipid products in human nonalcoholic fatty liver disease and nonalcoholic steatohepatitis. J Lipid Res. 2010;51:3046–3054. doi: 10.1194/jlr.M007096. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 10.Mabalirajan U, Rehman R, Ahmad T, et al. Linoleic acid metabolite drives severe asthma by causing airway epithelial injury. Sci Rep. 2013;3:1349. doi: 10.1038/srep01349. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 11.Yoshida Y, Yoshikawa A, Kinumi T, et al. Hydroxyoctadecadienoic acid and oxidatively modified peroxiredoxins in the blood of Alzheimer's disease patients and their potential as biomarkers. Neurobiol Aging. 2009;30:174–185. doi: 10.1016/j.neurobiolaging.2007.06.012. [DOI] [PubMed] [Google Scholar]
- 12.Bazan NG. The docosanoid neuroprotectin D1 induces homeostatic regulation of neuroinflammation and cell survival. Prostaglandins Leukot Essent Fatty Acids. 2013;88:127–129. doi: 10.1016/j.plefa.2012.08.008. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 13.Orr SK, Palumbo S, Bosetti F, et al. Unesterified docosahexaenoic acid is protective in neuroinflammation. J Neurochem. 2013;127:378–393. doi: 10.1111/jnc.12392. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 14.Tjonahen E, Oh SF, Siegelman J, et al. Resolvin E2: identification and anti-inflammatory actions: pivotal role of human 5-lipoxygenase in resolvin E series biosynthesis. Chem Biol. 2006;13:1193–1202. doi: 10.1016/j.chembiol.2006.09.011. [DOI] [PubMed] [Google Scholar]
- 15.Alvheim AR, Malde MK, Osei-Hyiaman D, et al. Dietary linoleic acid elevates endogenous 2-AG and anandamide and induces obesity. Obesity (Silver Spring) 2012;20:1984–1994. doi: 10.1038/oby.2012.38. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 16.Igarashi M, Gao F, Kim HW, Ma K, Bell JM, Rapoport SI. Dietary n-6 PUFA deprivation for 15 weeks reduces arachidonic acid concentrations while increasing n-3 PUFA concentrations in organs of post-weaning male rats. Biochim Biophys Acta. 2009;1791:132–139. doi: 10.1016/j.bbalip.2008.11.002. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 17.Igarashi M, Kim HW, Chang L, Ma K, Rapoport SI. Dietary n-6 polyunsaturated fatty acid deprivation increases docosahexaenoic acid metabolism in rat brain. J Neurochem. 2012;120:985–997. doi: 10.1111/j.1471-4159.2011.07597.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 18.Liou YA, King DJ, Zibrik D, Innis SM. Decreasing linoleic acid with constant alpha-linolenic acid in dietary fats increases (n-3) eicosapentaenoic acid in plasma phospholipids in healthy men. J Nutr. 2007;137:945–952. doi: 10.1093/jn/137.4.945. [DOI] [PubMed] [Google Scholar]
- 19.Macintosh BA, Ramsden CE, Faurot KR, et al. Low-n-6 and low-n-6 plus high-n-3 diets for use in clinical research. Br J Nutr. 2013;110:559–568. doi: 10.1017/S0007114512005181. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 20.Rett BS, Whelan J. Increasing dietary linoleic acid does not increase tissue arachidonic acid content in adults consuming Western-type diets: a systematic review. Nutr Metab (Lond) 2011;8:36. doi: 10.1186/1743-7075-8-36. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 21.Hussein N, Ah-Sing E, Wilkinson P, Leach C, Griffin BA, Millward DJ. Long-chain conversion of [13C]linoleic acid and alpha-linolenic acid in response to marked changes in their dietary intake in men. J Lipid Res. 2005;46:269–280. doi: 10.1194/jlr.M400225-JLR200. [DOI] [PubMed] [Google Scholar]
- 22.Taha AY, Cheon Y, Ma K, Rapoport SI, Rao JS. Altered fatty acid concentrations in prefrontal cortex of schizophrenic patients. J Psychiatr Res. 2013;47:636–643. doi: 10.1016/j.jpsychires.2013.01.016. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 23.Taha AY, McIntyre Burnham W. Commentary on the effects of a ketogenic diet enriched with omega-3 polyunsaturated fatty acids on plasma phospholipid fatty acid profile in children with drug-resistant epilepsy. Epilepsy research. 2007;76:148–149. doi: 10.1016/j.eplepsyres.2007.07.008. discussion 150-141. [DOI] [PubMed] [Google Scholar]
- 24.Ramsden CE, Faurot KR, Zamora D, et al. Targeted alteration of dietary n-3 and n-6 fatty acids for the treatment of chronic headaches: a randomized trial. Pain. 2013;154:2441–2451. doi: 10.1016/j.pain.2013.07.028. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 25.LaMancusa R, Pulcinelli FM, Ferroni P, et al. Blood leukotrienes in headache: correlation with platelet activity. Headache. 1991;31:409–414. doi: 10.1111/j.1526-4610.1991.hed3106409.x. [DOI] [PubMed] [Google Scholar]
- 26.Tuca JO, Planas JM, Parellada PP. Increase in PGE2 and TXA2 in the saliva of common migraine patients. Action of calcium channel blockers. Headache. 1989;29:498–501. doi: 10.1111/j.1526-4610.1989.hed2908498.x. [DOI] [PubMed] [Google Scholar]
- 27.Ramsden CE, Mann JD, Faurot KR, et al. Low omega-6 vs. low omega-6 plus high omega-3 dietary intervention for chronic daily headache: protocol for a randomized clinical trial. Trials. 2011;12:97. doi: 10.1186/1745-6215-12-97. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 28.Skipski VP, Good JJ, Barclay M, Reggio RB. Quantitative analysis of simple lipid classes by thin-layer chromatography. Biochim Biophys Acta. 1968;152:10–19. doi: 10.1016/0005-2760(68)90003-9. [DOI] [PubMed] [Google Scholar]
- 29.DeMar JC, Jr, Ma K, Bell JM, Rapoport SI. Half-lives of docosahexaenoic acid in rat brain phospholipids are prolonged by 15 weeks of nutritional deprivation of n-3 polyunsaturated fatty acids. J Neurochem. 2004;91:1125–1137. doi: 10.1111/j.1471-4159.2004.02789.x. [DOI] [PubMed] [Google Scholar]
- 30.Cheon Y, Park JY, Modi HR, et al. Chronic olanzapine treatment decreases arachidonic acid turnover and prostaglandin E(2) concentration in rat brain. J Neurochem. 2011;119:364–376. doi: 10.1111/j.1471-4159.2011.07410.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 31.Sublette ME, Bosetti F, DeMar JC, et al. Plasma free polyunsaturated fatty acid levels are associated with symptom severity in acute mania. Bipolar Disord. 2007;9:759–765. doi: 10.1111/j.1399-5618.2007.00387.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 32.Hodson L, Skeaff CM, Fielding BA. Fatty acid composition of adipose tissue and blood in humans and its use as a biomarker of dietary intake. Prog Lipid Res. 2008;47:348–380. doi: 10.1016/j.plipres.2008.03.003. [DOI] [PubMed] [Google Scholar]
- 33.Lands WE. Biosynthesis of prostaglandins. Annu Rev Nutr. 1991;11:41–60. doi: 10.1146/annurev.nu.11.070191.000353. [DOI] [PubMed] [Google Scholar]
- 34.Clark KJ, Makrides M, Neumann MA, Gibson RA. Determination of the optimal ratio of linoleic acid to alpha-linolenic acid in infant formulas. J Pediatr. 1992;120:S151–S158. doi: 10.1016/s0022-3476(05)81250-8. [DOI] [PubMed] [Google Scholar]
- 35.Tu WC, Cook-Johnson RJ, James MJ, Muhlhausler BS, Gibson RA. Omega-3 long chain fatty acid synthesis is regulated more by substrate levels than gene expression. Prostaglandins Leukot Essent Fatty Acids. 2010;83:61–68. doi: 10.1016/j.plefa.2010.04.001. [DOI] [PubMed] [Google Scholar]
- 36.Guesnet P, Lallemand SM, Alessandri JM, Jouin M, Cunnane SC. alpha-Linolenate reduces the dietary requirement for linoleate in the growing rat. Prostaglandins Leukot Essent Fatty Acids. 2011;85:353–360. doi: 10.1016/j.plefa.2011.08.003. [DOI] [PubMed] [Google Scholar]
- 37.Friesen RW, Innis SM. Linoleic acid is associated with lower long-chain n-6 and n-3 fatty acids in red blood cell lipids of Canadian pregnant women. Am J Clin Nutr. 2010;91:23–31. doi: 10.3945/ajcn.2009.28206. [DOI] [PubMed] [Google Scholar]
- 38.Gavino VC, Gavino GR. Adipose hormone-sensitive lipase preferentially releases polyunsaturated fatty acids from triglycerides. Lipids. 1992;27:950–954. doi: 10.1007/BF02535570. [DOI] [PubMed] [Google Scholar]
- 39.Katan MB, Deslypere JP, van Birgelen AP, Penders M, Zegwaard M. Kinetics of the incorporation of dietary fatty acids into serum cholesteryl esters, erythrocyte membranes, and adipose tissue: an 18-month controlled study. J Lipid Res. 1997;38:2012–2022. [PubMed] [Google Scholar]
- 40.Gibson RA, Neumann MA, Lien EL, Boyd KA, Tu WC. Docosahexaenoic acid synthesis from alpha-linolenic acid is inhibited by diets high in polyunsaturated fatty acids. Prostaglandins Leukot Essent Fatty Acids. 2013;88:139–146. doi: 10.1016/j.plefa.2012.04.003. [DOI] [PubMed] [Google Scholar]
- 41.Pawlosky RJ, Hibbeln JR, Lin Y, et al. Effects of beef- and fish-based diets on the kinetics of n-3 fatty acid metabolism in human subjects. Am J Clin Nutr. 2003;77:565–572. doi: 10.1093/ajcn/77.3.565. [DOI] [PubMed] [Google Scholar]
- 42.Brenna JT, Salem N, Jr, Sinclair AJ, Cunnane SC. alpha-Linolenic acid supplementation and conversion to n-3 long-chain polyunsaturated fatty acids in humans. Prostaglandins Leukot Essent Fatty Acids. 2009;80:85–91. doi: 10.1016/j.plefa.2009.01.004. [DOI] [PubMed] [Google Scholar]
- 43.Dodin S, Cunnane SC, Masse B, et al. Flaxseed on cardiovascular disease markers in healthy menopausal women: a randomized, double-blind, placebo-controlled trial. Nutrition. 2008;24:23–30. doi: 10.1016/j.nut.2007.09.003. [DOI] [PubMed] [Google Scholar]
- 44.Cazeils JL, Bouillier-Oudot M, Auvergne A, Candau M, Babile R. Lipid composition of hepatocyte plasma membranes from geese overfed with corn. Lipids. 1999;34:937–942. doi: 10.1007/s11745-999-0443-z. [DOI] [PubMed] [Google Scholar]
- 45.Chen CT, Ma DW, Kim JH, Mount HT, Bazinet RP. The low density lipoprotein receptor is not necessary for maintaining mouse brain polyunsaturated fatty acid concentrations. J Lipid Res. 2008;49:147–152. doi: 10.1194/jlr.M700386-JLR200. [DOI] [PubMed] [Google Scholar]
- 46.Rahman T, Taha AY, Song BJ, et al. The very low density lipoprotein receptor is not necessary for maintaining brain polyunsaturated fatty acid concentrations. Prostaglandins Leukot Essent Fatty Acids. 2010;82:141–145. doi: 10.1016/j.plefa.2009.11.003. [DOI] [PubMed] [Google Scholar]
- 47.Garland M, Sacks FM, Colditz GA, et al. The relation between dietary intake and adipose tissue composition of selected fatty acids in US women. Am J Clin Nutr. 1998;67:25–30. doi: 10.1093/ajcn/67.1.25. [DOI] [PubMed] [Google Scholar]
- 48.Kingsbury KJ, Heyes TD, Morgan DM, et al. The effect of dietary changes on the fatty acid composition of normal human depot fat. Biochem J. 1962;84:124–133. doi: 10.1042/bj0840124. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 49.Kingsbury KJ, Paul S, Crossley A, Morgan DM. The fatty acid composition of human depot fat. Biochem J. 1961;78:541–550. doi: 10.1042/bj0780541. [DOI] [PMC free article] [PubMed] [Google Scholar]
- 50.Kim HW, Rao JS, Rapoport SI, Igarashi M. Dietary n-6 PUFA deprivation downregulates arachidonate but upregulates docosahexaenoate metabolizing enzymes in rat brain. Biochim Biophys Acta. 2011;1811:111–117. doi: 10.1016/j.bbalip.2010.10.005. [DOI] [PMC free article] [PubMed] [Google Scholar]
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